André Emilie

; Passirani

Seijo Begona

; Sanchez

Montero-Menei Claudia
email: claudia.montero-menei@univ-angers.fr

C Montero-Menei

; Pensado

Rosa Da Costa

MODELS OF HUNTINGTON'S DISEASE 3. EX VIVO MODELS OF HUNTINGTON'S DISEASE 4. EXPERIMENTAL TREATMENT IN HUNTINGTON'S DISEASE

Keywords: MSC, mesenchymal stem cells, RNAi, interference RNA, siRNA, short interfering RNA, miRNA, micro-RNA, NPs, nanoparticles, REST, repressor element-1 silencing transcription factor, PLGA, pol ylactide -co-gl ycolide, BDNF, brain-derived neurotrophic factor, ECM, extracellular matrix

4.1 Tissue engineering and regenerative medicine 4.2 Potential therapeutic use of the neurotrophic factor BDNF 4.3 Potential therapeutic use of scaffolds 4.4 Potential therapeutic use of stem cells 4.5 Potential therapeutic of small interfering RNAs for MSC differentiation 5. NANOCARRIERS 5.1 Different types of nanocarriers 5.2 Lipid nanocapsules 5.3 Solid span nanoparticle

I would like to cordiall y and sincerel y thank Associate Professor Claudia MONTERO-MENEI who welcomed me for the thesis' adventure . I thank you for the trust that you gave me during these three years. I enjoyed working autonomousl y and learning from you . While enjoying your availabilit y you provided me advices when needed, this was a clever mixture closed to the perfection. I also would like thank you about your management: you g ave me a lot of your time, your knowledge and your scientific background. These three years with you have been very pleasant and educative, professionall y and humanel y speaking. Thank you for this unique experience , which I will remember for long . I am very delight to continu e one more year with you. I would like to sincerel y thank Professor Alejandro SANCHEZ -BARREIRO who welcomed me in Spain during one year . When I was in Spain, I learned so much things such how to manage a project with a company , about patents and nanoparticles' process. I know that maybe I did not always live up to your expectations, but nevertheless I hope you appreciate m y work. From m y side, these three years with you have been very interesting and I learned so much . Thank you for this great experience; I would like to continue to work with you and your team. I would like honestly thank ing Professor Catherine PASSIRANI for your availabilit y and your humanit y during m y thesis. I reall y appreciated to work with you on the formulation part. Thank you for your scientific convers ation, and especiall y your advices and suggestion s during some experiments. I wish to thank you dear Associate Professor Begona SEIJO . I'm sincerel y sorry that you will not be present during my "viva voice". I appreciated so much to work with you. Thank you for all: for your confidence, for your management, for your enthusiasm, your French conversation and memorize s that you have shared ACKNOWLEDGMENT with me. Thank you for your time, for your patience, your sympathy and especially your kindness. I will remember you for long time.

I thank the Associate Professor Ana GRENHA , from the Universit y of Algarve and the Research Director Isabelle LOUBINOUX, from the Universit y of Toulouse, to make me the honor to evaluate this thesis as reviewers . I would like to sincerel y thank all the MONTERO -MENEI Team: The Doctor Nicolas DAVIAUD, the technician Laurence SINDJI and the PhD student Saikrishna KANDALAM from the unit Inserm U1066. Laurence, thank you so much for your kindness!! Thank you for your time, for your patience, your s ympathy and especiall y your kindness. I owe you all the technical skills that I have learned during all these years. Than k you for listening me during the last month, and helping me with experiments, so thank you! Krishna, I loved working with you! You are very professional. I also appreciated who you are, thank you for dinners, thank you for magic tricks, and you concern; You will be the next one, so good luck for the end of your thesis. I would like to visit India, let us go after your thesis! I would like to express m y sincere appreciation to m y Thesis Monitoring Committee, the Professor Anselm PERRIER and the Associate Professor Laurent DAVID, for their guidance and encouragement s. Popcorns with bacon and maple syrup, beers in the JJ and our best memory, the London trip!!! Please don't sing again with Hélène! You know how much I h ate the rain. You are sharing a "work space" with me; I know how much I was difficult. You will finish very soon and I hope all the best for you! I hope that we will subscribe to "La France a un incroyable talent"; presenting our choreography.

We should wi n! Marion, you did it!!! I'm so proud of you; I would like to thank you for your support during these moments, also for all nights in the Baroque café, drinking Mojito! Fabienne, thank you to be here, I was very happy to share "the day the most important o f your life!!" I very hope that finally everything will be good and you enjoyed your honeymoon. Hélène, I would like to thank you to welcome me in your flat when I get back from Spain. Please don't sing again, lol! You are sharing a "work space" with me; I know how much it was difficult. You will finish very soon and I hope all the best for you! Emilie, please tell me the name of your baby!!! You have the entire things that every girls desire, a lovely house, an interesting job, but it is nothing compare wi th the fact that you will become a mother! I hope all the best for you and Fabien! I also hope continuing the squash with you for long time! Thominette, thank you for all , thank you for listening to me, to play squash with me, and your support when I was c omplaining about a lot of things! I learnt with you r patience and to be more attentive and mature in m y comments. I hope that you will obtain your graduation.

Fabien, Noémie and Jules!!! I would like to address a special thank to Fabien who helped me with this work. We learnt from each other during those 3 years, thank you for all the dinner with cook fish… I hope to share a lot of beers with you, and eliminate with squash! Noémie, I don't know you so much but I reall y appreciate discussing with you. The pr egnancy suits you so well. Kiss kiss to m y Jules.

I would like to thank Mrs Marie PARIS, the Associate Professor Daniel NOEL, and the Professor John DEVOS who gave me support throughout these years.

I would like to state m y deepest gratitude for m y closest friends over the years and the distance: Romain (Thank Roro for the windsurf, you are t he best teacher!!), Ana (You reall y performed windsurf with pump?!) , Benjamin and Aurianne (Now we are available for some poker dinner) , Laura, Laurenn, Pauline Therond Alias Popo (Thank you so much to be on my side since all those years! We were graduated at the same time, and look now! I will always remember your little kindl y notes on m y fridge and the O'bar night , Maelle Aguilar (out of sight out of mind, I believe d in this expression before you). After living with you, sharing so much memories and laugh ing with you, I can clearly said how much t his sentence is wrong! I will co me to visit you very soon, I prom ise, furthermore I write it! I want to see your house, your life and maybe a little Maelle! T'as promis que je serais la tata ) Delphine and Manu (I'm not drunk ahahaha you favorite sentence m y Delphine -dophin …You are m y best memories from the Unit u646.

Manu thank you for your support during "the London Trip" and Delphine's move…), RoRo and Alex (Squash?! We share a lot of good memories. Alex, you were the first person to teach me cell culture! Thanks for that, and to be always on my side! Roro, you always were here to play squash, drink beers and helping me with the RT-qPCR. You finall y moved with your Nounou in Bordeaux and pacsed together! I 'm so glad for you ), Ma petite Aude and her famil y, Madenn and Cyrille (I would like a suspended bed barrel… I hope that you will have your house, I'm very exciting to visit you.)…Anais, Marion Peyressatre, Marie, Benoit, Crapaud (Cycy and Iris) (I am so impatient to meet the last little one on your famil y m y crapaud!) , Sylvianne, Alicia, Raphaël and Jenifer and Damien.

Because a deep and caring love provides an unconditional aid, thank you so much, Kevin, for these last months and during those 2 years . You know following a doctoral thesis implies difficult times, and you demonstrated selflessness and kindness. I hope onl y one thing: our relationship keeps going on those foundations. Dear Kevin, I will always be grateful ( Yes, promised on Sunda y morning, you will have French toast!!) Je souhaite finir ces remerciements en Francais (j'espère que vous m'excuserez pour cela) à ma Famille, Maman, Papa, Mamie, Papi et Titou. Je tenais à vous remercier pour être toujours à mes côtés, pour me supporter malgré mon mauvais caractère ! On se chamaille, on s'époudraille et je vous l'ai probablement jamais assez dit mais je vous aime ! Ce travail, je l'ai réalisé grâce à vous, à votre confiance. Avec tout mon amour. This PhD thesis has as main objective to offer an innovative tissue engineering strategy for Huntington's disease by combini ng nanocarriers delivering siRNA in mesenchymal stem cells and microcarriers releasing therapeutic proteins . This project is part of a general strategy of the laboratory INSERM U1066 "Biomimetic Micro and nanomedicine" from Angers and the department "Pharmacy and Pharmaceutical Technology " from Santiago de Compostela proposing an alternative and safe treatment in neurodegenerative disorders .

Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disorder with pr evalence in Europe of about 10 per 100,000 births (Figure 1.) [1,2]. HD appears in mid -life leading to death 15 -20 years later and involves the triad signs and symptoms: involuntary movement disorders called Huntington's chorea, cognitive impair ment and ps ychiatric manifestations. Worldwide estimates of the prevalence of HD. Overall, the prevalence of HD is much higher in European populations than in East Asia [3].

HD is one of the nine neurodegenerative disorders caused by the expansion of cytosine -adenine-guanine (CAG) triplet repeat sequence [4].

HUNTINGTON'S DISEASE.

This disorder is characterized by an unstable repetition of triplet cytosineadenine-guanine (CAG) of the Huntingti n (htt) gene in exon 1 of chromosome 4. It is translated at the protein level by a pol yglutamine expansion at the NH2-terminal part of the protein huntingtin ( HTT) [5]. The htt gene is considered as normal when it contains less than 27 CAG repeats and generall y more than 40 repeats defines the adult -onset HD. The age of HD onset is inversel y correlated with the length of the expansion, with variable agedependent penetrance between 36 and 39 CAG repeats, but full penetrance at 40 or more repeats. In other words, people with 36 -39 CAG repeats are at a risk of developing all the HD symptoms [6] and conversel y, a larger number of repeats is usuall y associated with an earlier onset of signs and symptoms [7].

The HTT protein has ubiquitous roles in apoptosis [8], regulating microtubule-based transport [9] and scaffolding of cytoskeletal molecules at s ynapses [10]. Therefore, mutant HTT (mHTT) primaril y affects the central nervous s ystem (CNS). The translated wild-t ype huntingtin protein is a 350 -kDa protein containing a pol ymorphic stretch of between 6 and 35 glutamine residues in its N -terminal domain [11]. For the length superior to 35 CAG repeat in the htt gene, the accumulation of pol yglutamine in the protein le ads to its aggregation in specific area s in the brain such as: striatum, cortex, thalamus, hypothalamus and th e substancia nigra pars compacta. mHTT has a toxic gain of function that causes cell death vi a very different mechanisms, which still remain unclear . However, it is known to result in transcriptional dysregulation as well as mitochondrial dysfunction and energy deficits (for review see [1,12]). The accumulation of the mutant htt protein progressivel y compromises survival and normal neuronal functioning, primaril y in the striatum (caudate/putamen). The mutant htt lead s also to proteosomal dysfunction, induction of autophaghy, release of Calcium from intrace llular stores and excitotoxicit y at extrasynaptic NMDA receptors. It particularl y affects the GABAergic neurons, called medium spiny neurons (MSN) situated in the striatum, which have axonal projections to the globus pallidus and substantia nigra. They express Dopamine-and cAMP -Regulated neuronal PhosphoProtein of 32kDa ( DARPP32). The progressive loss of these neurons is accompanied by a corresponding ventricular enlargement and gliosis (Figure 2). The disease progresses with the degeneration of cortical pyramidal neurons, mainl y projecting to the caudate/putamen [13]. Many of the s ymptoms of HD result from the loss of inhibitory connections from the striatum to other structures such as the globus pallidus (Figure 3). The cause of such specific regional and sub -population neuronal loss and the absence of cell loss in other tissues remain uncertain . Brain pathological hallmarks leading to 25% of brain weight loss in HD develop well before evident s ymptoms appear. Brains' comparison by MRI between healthy and Huntington's disease subjects [14].

The most visible symptom of HD is the presence of involuntary jerk y movements named chorea. During the first stage of the disease, the chorea dominates and w hen the disease progress, dystonia, rigidity and bradykinesia are also observed. Cognitive impairments also progressivel y appear as well as emotional disturbances marked in the most case by anxiety, memory loss, dementia, depression and psychosis [15]. They frequentl y lead to considerable distress and psychologic difficult y for patients , which have more prevalence than the general population to commit suicide. The unequivocal presence of chorea in a person with a famil y history or genetic confirmation of risk for HD forms the basis for clinical diagnosis. Recentl y, the Ameri can Academ y of Neurology published guidelines to evaluate the motor and cognitive function, behavioral symptoms and functional capacit y based on the Unified Huntington's Disease Rating Scale -Total Motor Score [16]. Different areas involved in HD explaining symptoms. The cortico-striatal system is essential for the execution of movements (left). In patients with HD, progressive degeneration of neurons in the striatum, and later also in cerebral cortex, disrupts function in the cortico-striato-pallidal circuit and induces severe impairments in both motor and cognitive functions. The striatal GABAergic projection neurons provide an inhibitory control of two major striatal output structures, globus pallidus and pars reticulata of the substantia nigra (not shown). Loss of these neurons, in animals with striatal lesions or in HD patients, results in disinhibition of pallidal outflow (right) [17].

There is no effective treatment for the progressive neurodegenerative process underl ying HD, and man agement includes pharmacological s ymptomatic control of the movement disorder and psychiatric features, as well as non-pharmacological treatments, such as parenteral feeding and therapy services [18]. When the physiotherapy is not enough, tetrabenazine is the first choice of medication for uncomplicated chorea. Tetrabenazine is acting to decrease dopamine levels and can be helpful to reduce movement disorder but present s various side effects like the increase of depression or ps ychiatric disorder s. Neuroleptics or benzodiazepines can also be prescribed and mood stabilizers such as anti-depressants and anti -anxiet y reducing ps ychological dysfunctions are also proposed [18]. Unfortunatel y, many of these medications have adverse side effects that can worsen HD symptoms. In order to help to define the best treatment The Unified Huntington's Disease Rating Scale-Total Motor Score classified the level of evidence for drugs to reduce chorea based on a review of randomized clinical trials [19,20].

Nowadays, two approaches are under pre-clinical evaluation: disease modifying treatment s, more particularl y by reducing pol yQ repeats and more recentl y by the suppression of the mRNA of the HTT gene with interference RNA (iRNA) [21]. Another approach consists in recent tissue engineering strategies to replace lost neurons by new one s obtained in vitro from stem cells. These metho ds need to be further improved and developed in order to be validated in the in vivo models of HD [18].

Most animal models of HD fall into two broad categorie s, genetic and non-genetic. Historicall y, non -genetic models have dominated the field of HD research. Although George Huntington first described HD in 1872, researchers did not identify the actual genetic mutation responsible for the disease until 1993, wh ich delayed the development of appropriate genetic models until the last decade [22].

The emergence of genetic and molecular technology allow ed the development of animal models expressing a truncated [23] or full length [24,25] form of mutant htt (mhtt) (Table 1). Animal models are divided in to two genetic categories: transgenic or knock -in. Transgenic models result from the random insertion of mutated human htt. The R6/1 and R6/2 transgenic mouse models were the first characterized [26] (Table 1). These mice express onl y mutant exon 1 of the human htt gene with di fferent length repeats. These principal models containing onl y the truncated human mhtt gene are still the most used nowadays, together with yeast artificial chromosome's (YAC) models [24]. The latter consist on the cloning of an artificial yeast vector that contains the entire human mhtt with differ ent expanded CAG repeats (YAC 46, YAC 72, YAC 128) which are then integrated into the rodent genome [27] (Table 1). Alternativel y, the knock -in models result by the insertion in the htt mouse genome of the CAG repeats of human mhtt, which are then within the context of the rodent mhtt gene [28,29] (Table 1). In all of these animal models the pathophysiology of the disease appears in adult age as for the HD patients.

IN VIVO MODELS OF HUNTINGTON'S DISEASE

In any case none of these models exactl y reproduce the human pathology. Moreover, the existence of various genetic animal models did not allow predicting HD symptoms. Indeed, we can believe that the progression is correlated with the number of CAG repeat length, but the mechanism of the disease seems to be more complicated. Moreover, it has been shown that therapeutic success in animal models is not always paralleled by clinical success in patients.

with glutamatergic targets such as kainic acid (KA) [35], ibotenic acid (IA), quinolinic acid (QA) [36] [37] and N-methyl-d-aspartate (NMDA) [38]. Indeed, glutamatergic excitotoxicit y is involved in the pathophysiology of the disease. These lesions induced death of striatal medium spiny neurons similar to the neuropathology present in HD patients. The symptoms most commonl y associated with the diseased state induced by the neurotoxins are loss of weight, possible tremor or seizures, eventual paral ysis, recumbence, and often death representing the later stages of the disease. But these mode ls do not allow investigation of disease progression or the mechanism of neuropathology because the htt protein d oes not present the mutation. In addition, genetic and non -genetic models need proper care and animals must be dail y monitor ed. Immediatel y after toxin administration or in later stages in genetic models, euthanasia may be n ecessary for moribund animals. To reduce the number of animal experiments ex vivo models represents an excellent compromise between single cell cultures and animal studies.

Another way for modelling HD consists in using organotypic brain slices, which have been also reported for modelling Parkinson's disease and cerebral ischemia. Brain slices are kept alive during several weeks in culture and represent a simple method to model the neurodegeneration and evaluate potential treatments before in vivo studies.

Brain slice models offer unique advantages over other in vitro platforms in that they can replicate many aspects of the in vivo context. Slices preserve largel y the tissue architecture of the brain regions that they originated from and maintain neuronal activities with intact functional local synaptic circuitry [39]. Nevertheless, brain slices remain fragile, they can be easil y distorted, and often flatten during the culture. In other word, organotypic brain sl ices are delicate and frequentl y become damaged during the preparative stages, so brain slice preparation and culture need experience. Functional outcome of a therapeutic strategy cannot be evaluated with this technique , but as brain slices can be maintained for a few weeks in culture they offer the possibilit y to screen a large quantit y of therapeutic strategies. P harmacological s [40],

EX VIVO MODELS OF HUNTINGTON'S DISEASE

gene manipulation strategies with the goal to reduce the CAG repeat on HD [41] or injection of growth factors or cells can be evaluated for their neuroprotective or neurorestorative ability of damaged structures (Figure 4 ). Furthermore, organotypi c slides represent a powerful tool for understanding the interaction between grafted cells and resident cellular matrix, or for comprehending the mechanisms of experimental treatments in HD [42]. As in vivo models, organot ypic brain slices of HD are divided in 2 categories: genetic and non -genetic models. Genetic models can be derived from adult animals already described above, presenting the MSN degeneration, or by transfecting the mutated htt within the organot ypic culture. However, these organot ypic cultures are difficult to generate due to the diminished neuronal plasticit y and the fragilit y of these brains . Nongenetic models can be generated from pups or young animals by neurotoxin administration directl y in the organotypic slice culture. The first HD organot ypic model was developed in 1986 by injecting KA in the striatal organot ypic slices [44]. Over the years, brain slice culture s have been successfull y established with QA and/o r 3-NPA, and KA after addition in the media of striatal or ganot ypic slices to mimic the disease. In order to study later stage of onset disease all these neurotoxins have been injected in the frontal cortex, hippocampus or caudate nucleus for modeling HD in rat [45][46][47] (Table 2). But the use of neurotoxins lead s to a heterogeneit y in the results obtained, which must be taken in consideration. Furthermore, with that type of model, onl y the cellular aspect of HD can be studied, they can not take into account the genetic component of the pathology. Advantages are the speed and low-cost associated to this model when normal rodents are used [42]. During the past years, many preclinical studies initiall y reported the efficacy of human fetal striatal tissue to replace and provide functional recovery in a variety of rodent and non -human primate models of HD. Some teams demonstrated the feasibilit y and the safet y of this therapeutic strategy [53][54][55][56][57] and functional improvements were obtained in the study led by Bachoud-Lévi. They reported graft survival, which contained striatal projection neurons and interneurons, and received host -derived afferents [53,54]. But the survival is still very poor and the comparisons of these clinical trials are very difficult because of the heterogeneit y in their design and the lack of controls . Nevertheless, the reported improvements in these trials appear to be modest and transient (for review see [58]). Moreover, the use of human fetal brain for striatal transplantation derived from elective abortions is limited by th e lack of standardization inevitabl y correlated with the use of such a source as well as ethical, practical, and regulatory concerns and is dependent upon availabilit y of donor tissue [59]. Stem cell -based

EXPERIMENTAL TREATMENT IN HUNTINGTON'S DISEASE

therapies can provide a limitless source of cells due to their self -renewal capacit y and their neuronal differentiation potential, but good cell engraftment remains a drawback. On this basis, the development of cell replacement strategies for regenerative medicine and more particularl y tissue engineering has been under light the last decade for HD.

TISSUE ENGINEERING AND REGENERATIVE MEDICINE

Tissue engineering and regenerative medicine are fields which have a unique tactic to solve clinical problems aforementioned by combini ng the principles of engineering, clinical medicine, biology and materials science [60]. Tissue engineering, according to the National Institute of Health (NIH), is a broad field which involves " biomaterials development and refers to the practice of combining scaffolds, cells, and biologicall y active molecules into functional tissues " [61] and regenerative medicin e as the development of " therapies to restore lost, damaged, or aging cells and tissues in the human body". Those approaches may include, but is not limited to, th e use of soluble molecules [62], gene therapy, stem cell s transplantation [63], reprogramming of cells. The strategic introduction of these bioactive and soluble molecules, as well as stem cells into the human body is directed not onl y to replacing tissue but also at inducing regeneration and revascularization by host tissue .

Various cells can be considered in tissue engineering , stem or modified cells to replace lost neurons and somatic cells for t heir neuroprotective properties (Figure 4). Biomaterial scaffolding is often employed to provide a supporting spatial and biomolecular environment for transplanted cells. This approach named "top-down" in which cells are seeded onto a scaffold with biocompatible and biodegradable propertie s is the most used (Figure 4). Regenerative medicine for brain stem cell therapy has been primaril y developed with neuronal cell lines (PC12 cells), due to their availabilit y and ease of expansion, or with cultured dorsal root ganglion (DRG) neurons and fetal NSCs, owing to their natural abilit y to integrate and differentiate within the brain.

The scaffold can also release bioactive molecules able to act as neuroprotectors. In the case of HD, two ne urotrophic factors have been used for regenerative medicine: ciliary neurotrophic factor (CNTF) and brainderived neurotrophic factor (BDNF). Clinical trials were performed using CNTF-producing cells with stage 1 and 2 HD pat ients [64,65]. During this phase 1 study, subjects received one capsule implanted into the right lateral ventricle, and the capsule was exchanged every 6 months during 2 years.

While the CNTF-induced sparing of s triatal neurons and maintenance of intrinsic circuitry in animal models was impressive, the effect in human was less than that seen in rodents. Finall y, human clinical trials did not present relevant positive effects, and progressivel y the supplementation of CNFT in human has been given up. To our kno wledge, no clinical trial ha s been conducted with BDNF.

POTENTIAL THERAPEUTIC USE OF THE NEUROTROPHIC FACTOR BDNF

Neurotrophic factors a re essential for the survival of the central nervous s ystem neurons and d emonstration of their reduced availabilit y in HD indicates that they may play an important role in this disorder. Indeed, the reduction of BDNF in HD contributes to the disease onset and or progression [66,67]. BDNF is essential in sustaining the physiological process es of normal intact adult brain [68] and more particularl y for GABAergic striatal neurons (Figure 5). Indeed, although widel y expressed in the adult mammalian central nervous system, BDNF is particularl y abundant in the hippocampus and cerebral cortex where it is anterogradel y transported to its striatal targets via the corticostriatal afferents [69]. Several evidences demonstrate the role of BD NF in the maturation of striatal neurons and how BDNF promotes the survival of DARPP -32 positive neurons [70,71]. The supplementation of BDNF in the case of HD has been evaluated and promising results show that BDNF treatment prevent striatal degeneration in a chronic model of HD. [73]. More interestingl y, the supplementation of BDNF protein increase s the survival of enkephalin-immunoreactive striatal neurons, reduces striatal interneuronal loss and improve s motor function in HD animal models [74][75][76]. Despite these data, the BDNF supplementation raises a number of problems such as the stability of BDNF as it cannot cross the blood-brain-barrier. Moreover, an uncontrolled BDNF administration may interfere with neuronal plasticit y and even give rise to serious s ide effects [77]. One way to avoid this limitation is the encapsulation of BDNF within the scaffold necessar y for tissue engineering.

POTENTIAL THERAPEUTIC USE OF SCAFFOLDS

To overcome the poor cell surv ival and engraftment usually observed after transplantation, several strategies have been developed and among them, two methods seem particularl y promising: in situ controlled drug delivery and implantation of cells adhered on biomaterial -based scaffolds. Such scaffolds should provide an adequate 3D support for transplanted cells, thereby increasing cell survival and even guiding cell differentiation and fate in vivo [78,79]. However, delivery of cells with scaffolds to the damaged brain still remains challenging due to practical limitations of delive ry [80]. Ideal properties of a scaffold for brain tissue engineering are biocompatibilit y, very small size, controlled biodegradabilit y with non -toxic derivative products, and three-dimensional (3D) matrices with appropriate mechanical p roperties to mimic the extracellular matrix [81,82].

An innovative scaffold for tissue engineering combining the se ideal properties with a biomimetic 3D approach and the release of bioactive molecules has been developed in our laboratory. This scaffold or microcarriers named Pharmacologically activ e microcarriers (PAMs) are constituted of a synthetic pol ymer based on pol y(lactic-co-gl ycolic acid) (PLGA) [78]. They are obtained by a non -denaturing solid-in-oil-in-water (s/o/w) emulsion evaporation/extraction t echnique. The protein is first nanoprecipitated with poloxamer 188 a biocompatible hydrophilic pol ymer that protects the protein against irreversible denaturation [83]. PAMs are biodegradable and biocompatible with a mean size of 60 µm , covered by a biomimetic surface providing a 3D support for the cells and delivering a therapeutic protein in a prolonged manner . All these combined properties stimulate the survival and differentiation of the transported cells [84]. Their small size allows their implantation into the brain through a needle or catheter and after the complete degradation of the pol ymer the cells may integrate the parenchyma . These PAMs have been successfull y employed for different tissue engineering strategies, in neurodegenerative disorder s, cerebral ischemia, myocardial infarction, and cartilage re pair (Figure 6) [85,78,86,83,87] . For these strategies the appropriate growth factor and extracellular matrix protein have been combined to either progenitors or stem cells. In this work we will use stem cells combined with PAMs presenting a biomimetic surface of laminin , which stimulates neuronal differentiation [88] and delivering BDNF as a therapeuti c strategy for HD. Concept of PAMs: the biomimetic surface of PAMs is obtained by coating their surface with extracellular matrix proteins that can favor cell adhesion. During their formulation, the encapsulation of neurotrophic factor is performed.

. POTENTIAL THERAPEUTIC USE OF STEM CELLS

During the last decade, several preclinical experiments have used cell replacement strategies in order to restore MSN using HD animal models.

Different human stem cell sources are being activel y explored for potential cell replacement therapy including embryonic stem cells (ESCs), induced pluripotent or neural stem cells (iPSC or NSC), fetal and adult neural precursors and mesenchymal stem cells (MSCs). Both ESC, iPSC have been successfull y committed into MSN in vitro and then grafted into rodent models of HD [37,89,90]. But after human ESC transplantation into rat brains , tumor formation has been reported, which was not the case for iPSC -MSN-derived cells, which were further committed with in this lineage. In addition, the ethical issues related to the use of ESCs and the lack of availabilit y of fetal neural precursors drive us to focus in other cell sources. To find the best way to obtain the most important benefits with less ethical and pr actical constraints, mesenchymal stem cells have been investigated.

Human MSCs, as Friedenstein reported, are capable of differentiating into cells deriving from the mesodermal layer, such as osteoblasts, chondrocytes and adipocytes. Along with their self -renewal propert y, MSC s secrete tissue repair factors, such as growth factors, which affect the surrounding microenvironment to promote angiogenesis, decrease inflammation, and enhance tissue repair [91]. In this way, MSCs are being widel y evaluated in many clinical trials for cell therapy showing the feasibilit y of this approach. Recent advances in s tem cell biology hold great promise in the development of MSCs-based therapy for tissue engineering . It was also demonstrated that these cells could differentiate to an ectodermal neural/neuronal phenot ype, particularl y under the influence of specific fact ors [85] [79,92] enabling their use for cell therapy for neurodegenerative disease including HD. The principal limitation is that MSCs are a heterogeneous population with cells presenting different differentiation properties. To avoid these limitations a homogenous subpopulation of MSCs, named "Marrow-Isolated Adult Multilineage Inducible" (MIAMI), which present an unique genetic profile expressing several pluripotency markers (Oct4, Sox2, Nanog, SSEA4) and secrete many varied cytokines (Figure 7) are interesting for the treatment of neurodegenerative disorders [93,94]. They are able to generate cells derived from all three embryonic germ layers and cultured on fibronectin; they are capable of differentiating into neuron-like cells under treatment with various factors. After treatment, the cells show neurites, express neuronal factors and present some electrophysiological characteristics similar to those observed in mature neurons [95]. Recentl y, in a rat model of Parkinson's disease (PD), striatal implantation of MIAMI cells pre -committed towards the dopaminergic phenot ype adhered to microsphere releasing bio active molecules improved stem cell survival and show ed dopaminergic differentiation. This led to the protection/repair of the nigro -striatal pathway and to functional recovery of the PD rats. Furthermore, implantation of pre -treated MIAMI cells also induced functional recovery in PD rats, probabl y due to the release of glial cell line derived neurotrophic factor (GDNF) [88]. But MIAMI cells' abilit y to differentiate into neurons, although better than the simple MSC needs to be improved.

POTENTIAL THERAPEUTIC OF SMALL INTERFERING RNAS FOR MSC

DIFFERENTIATION

The conventional methods of generating neurons from MSC s, through bolus supplementation of small molecules or neurotrophic factors (growth factors: GF), still lack in efficiency in neural conversion and lineage selection. One possible reason may be the inadequacy of GFs to control gene expression. Cell differentiation may be achieved by RNA interference (RNAi) strategies and more particularl y by small interfering RNA (siRNA), which selectivel y knock -down the expression of onl y a few pivotal genes. Indeed, siRNAs are synthetic duplex of 21 -23 nucleotides, approximatel y 7.5 nm long and 2 nm in diameter , which are capable of specificall y target ing one gene and silencing it in a post-transcriptional way. SiRNA are rapidl y taken up into an enz yme complex, RNA induced silencing complex (R ISC), that degrades the mRNA through guidance to a specific target mRNA resulting in specific gene silencing. At R ISC one siRNA strand is t aken into the effector complex, the catal ytic subunit Argonaute2, and then serves as a template, guiding the hydrolysis of complementary or near complementary mRNA sequences [96]. Initiall y siRNA emerged as a potential therapeutic treatment for cancer. Although current applications in stem cells remain largel y restricted to studies on molecular pathways and s ignalling, RNAi can be used as a biomedical strategy to direct li neage-specific differentiation of stem cells for therapeutic purposes [97]. One key factor that can p ossibl y be adapted into the siRNA strategy for directing ne uronal differentiation of neural stem cells, is the repressor element 1 (RE -1) silencing transcription factor (REST) [98]. In most differentiated non -neuronal cells and uncommitted neural stem cells, REST functions as a transcriptional repressor fo r a m yriad of neuronal specific genes such as ion channels, synaptic vesicles proteins, and neurotransmitter receptors by binding to a highl y conserved DNA sequence known as RE-1. During neurogenesis, REST is rapidl y down regulated in embryonic stem cells (ESCs) and neural stem cells upon differentiation into neurons [99] (Figure 8). (Figure 8) and when applied to MSCs, cross -lineage differentiation to neurons was observed [102,103]. Although the knockdown of REST holds great potential, its therapeutic applications in neuronal differentiation is hindered by poor cellular uptake of siRNA molecules and their rapid enz ymatic degradation [104]. siRNAs' molecular weight ( ∼13 kDa) and strong anionic charge due t o the presence of aphospho -diester backbone ( ∼40 negative phosphate charges), make them incapable of freel y crossing the cell membrane. The electrostatic repulsion from the anionic cell membrane surface results in the failure of siRNA to passivel y diffuse through the cell membrane (Figure 9). Moreover, the synthetic siRNA molecules show low stabilit y in physiological fluids, poor tissue/cell specificit y, and rapid clearance [105]. Therefore, successful siRNA therapeutics requires effective and safe carrier systems to overcome the inherent limitations of siRNA and achieve maximum gene silencing effect. In the last decade, two different approaches for siRNA delivery have been developed: viral and non -viral vectors. In particular, the advantages of non -viral vectors are their low immunogenicit y, their relativel y low production cost and reproducibilit y potentiall y. These reasons make them promising carriers for siRNA deliver y [106].

DIFFERENT TYPES OF NANOCARRIERS

Nanocarriers (NCs), including nanoparticles and nanocapsules were first developed for the potential delivery of therapeutic factors such as

NANOCARRIERS

chemotherapeutic agents to tumors or, when combined to stem cells, mostl y for stem cell imaging [108]. First, the use of nanocarriers aims to protect an active ingredi ent against a potential degradation , and secondly to modify the natural distribution of the active substance in the body and in cells. It is theoreticall y possible to accumulate the active ingredient to the desired site of action and away from undesirable sites to limit side effects. NCs ranging from 1 to 1000 nanometer sizes are divided in to 2 main categories:

-Organic NCs which include liposomes, lipid nanoparticles, solid nanoparticles and dendrimers .

-Inorganic NCs with quantum dots, carbon nanotubes, iro n or gold nanoparticles [109].

The organic NCs and more particularl y lipid based NCs are interesting to transfect cells because lipid based nanoparticles can contain lipids present in the biological membranes which help the entry of nanoparticles. Cationic charges contained in some lipids are able to int eract with nucleic acid. In addition, the risk of undesirable immunogenic reactions to lipids is also relativel y lower than most of the pol ymeric materials which generall y have higher molecular weights [110]. Furthermore, some clinical trials have been conducted with siRNA and lipids based nanoparticles [111,112]. To our knowledge, clinical trials with nanoparticles and MSCs have not been performed in HD. When compared with liposomes, lipid -based nanoparticles such as solid lipid nanoparticles generally have solid, lipophilic core regions so it is inherentl y difficult to trul y encapsulate the hydrophilic, pol y -anionic RNA molecules. As a result, there are relativel y few lipid nanoparticles for RNA delivery [113,114].

LIPID NANOCAPSULES

Lipid nanocapsules (LNC) are nanocarriers developed and recentl y patented. These nanocapsules are constituted by oil y core of tryglicerides and a shell made of surfactants particularl y pol yethylene gl ycol hydrox ystearate .

They are obtained by a phase inversion temperature dependent process. This solvent-free process requires little energy and allows easy large-scale transposition [115].

The formulation is based on a simple process, named emulsion's phase inversion, developed and patented in 2002. This is realized by oil in water emulsion (O / W ) using the various constituents described above. This emulsion is subject ed to an increase of temperature which induces a change in the hydrophilic / lipophilic balance. Several temperature cycles (between 50 and 90°C) are produced and the addition of cool water final stablizes and solidifies LNCs.

Previous studies demonstrated the possibilit y to encapsulate plasmid DNA within the LNCs to develop a gene therapy strategy [116]. For this, the DNA is complexed with cationic lipids by electrostatic interactions leading to formation of complexes called lipoplexes which are added to other components of the LNC. Moreover, the phase inversion temperature was reduced to avoid degradation of the plasmid [116]. This strategy ha s demonstrated the capacit y of LNC s to transfect in the in vivo models of gliobastoma [117][118][119].

SOLID SPAN NANOPARTICLES

Solid span nanoparticles have been recentl y developed and patented [120]. These nanocarriers are based o n sorbitan esters, which are components widel y used in the pharmaceutical industry due to its non -ionic surfactant properties at low concentrations . These nanoparticles can be prepared using a have been specifical ly adapted to provide effective DNA association [122].

They have the capacit y to provide higher in vivo transfection levels than adeno-associated viral vectors (AAV2 ). Indeed, it has been recentl y patented their use in gene transfer or gene deliv ery and described an in vivo proof of concept in a disease animal model showing the safet y and clinical efficacy of a gene therapy approach based on these nanocarriers [123].

The evaluation of a regenerative medicine strategy may be easil y performed in a simple ex vivo model before pre -clinical study in vivo.

Although, the transplantation of MSCs showed promising results the differentiation state needs to be improved. In order to ameliorate this point, the homogenous more pluripotent population of MIAMI cells seem to be a good option. Although MIAMI cells can be committed into neural/neuronal precursors their maturation still needs to be safel y improved. Consequentl y, the transfection of siREST with nanoparticles seems to be a safe way to help induce a neuronal differentiation process. However, in order to control the behaviour of the cells after transplantation and enhance/maintain their differentiation their combination to PAMs delivering BDNF seems appropriate. Furthermore, the delivered BDNF may also induce the neuroprotection of the damaged tissue. In consequence, this study aims to develop an innovative and safe regenerative medicine strategy combining siREST nanoparticle -engineered MSCs, combined to PAMs with a laminin biomimetic surface and delivering BDNF for HD (Figure 10).

The principal objectives of this study are:

-The development of a simple ex vivo HD model to evaluate a tissue engineering strategy.

OBJECTIVES OF THIS THESIS

-The development, the understanding and the optimisation of two nanocarriers (LNC and SP) transporting a siRNA (siREST).

-The evaluation of their efficacy to deliver this siRNA into MSCs and the selection of the best nanocarrier for this application.

-The evaluation of the capacit y of si-REST to induce a neuronal To direct stem cell fate, a delicate control of gene expression through RNA interference (RNAi) is emerging as a safe epigenetic approach. RNAi allows selecting specific knock-down the expression of mRNAs by degrading them.

This epigenetic modificati on is quite simple, does not need genetic manipulation, is transitory and is now quite well understood. Nonetheless, nucleic acids need to be vectorized to be protected and to be able to cross biological membranes. Thus, the RNAi used for gene suppression strategies in many cell models are conventionall y mixed with cationic lipids . Their toxicit y limits their use and thus many nanocarriers have been designed to carry RNA inside cells.

A bibliographic research work has been undertaken here to identify new tissue engineering strategies currentl y under evaluation for HD. The first part of this study is focused on the possible source of cell for tissue engineering, presenting thei r advantages and disadvantages. A detailed review of the different formulations ava ilable for RNAi transport within stem cells, their mode of action and some examples of their use to control cell behavior follow.

In the last part, innovative tissue engineering strategies using stem cells, biomaterials and epigenetic cell regulation are r eported and discussed.

This work is submitted for publication in Biomaterials

Revisions demanded

REVIEW INTRODUCTION 29
The potential treatments for neurodegenerative disorders will be revolutionized by t he transplantation of stem cells or neuronal progenitors derived from these cells. It is however crucia l to better monitor their proliferation, improve their survival and differentiation and hence ameliorate their engraftment after transplantation. To direct stem cell fate, a delicate control of gene expression through RNA interference (RNAi) is emerging as a safe epigenetic approach. The development of novel biomaterials (nano and microcarriers) capable of delivering proteins, nucleic acids and cells, open the possibilit y to regulate cell fate while achieving neuroprotection and neurorepair. This review fir st provides an overview of stem cell therapy for the neurodegenerative disorder Huntington 's disease. Within that context, an integrative discussion follows of the control of stem cell behaviour by RNAi delivered by different nanocarriers in vitro prior to their transplantation. Finall y, combined in vivo strategies using stem cells, biomaterials and epigenetic cell regulation are reported. Stem cells; nanoparticles; microcarriers; tissue engineering; RNAi HD, Huntington's disease; MSN , medium spiny neurons ; ESCs, embryonic stem cells; iPS, induced pluripotent stem cells; NSC, neural stem cells;

HUNTINGTON'S DISEASE

Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative dis order with a general prevalence of about 10 per 100,000 births [1][2][3]. HD appears in middle life leading to death 15 -20 years later and involves the triad signs and symptoms: involuntary movement disorders called Huntington's chorea, cognitive impairment and ps ychiatric manifestations. This disorder is characterized by an unstable repetition of triplet cytosine -adenineguanine (CAG) of the Huntingtin gene, translated at the protein level by the pol yglutamine expansion at the NH2 -terminal part of the protein hunt ingtin (HTT) [4]. The gene is considered as normal when it contains less than 27 CAG repeats and generally more than 40 repeats defines the adult -onset HD, with people developing the disease at 30 -40 years of age. However, people with 36 -39 CAG repeats are at a risk of developing all the HD symptoms [5].

Conversel y, a larger number of repeats is usuall y associated with an earlier onset of signs and symptoms [6]. Aggregation of the mutated htt results in transcriptional dysregulation as well as mitochondrial dysfunction and energy deficits (for review see [1,7]). The accumulation of the mutant htt protein is excitotoxic, therefore it progressive l y compromises survival and normal neuronal functioning, primaril y in the striatum (caudate/putamen). It particularl y affects the GABAergic neurons, called medium spiny neurons (MSN), which have axonal projections to the globus pallidus and substantia nigra. They express Dopamine -and cAMP-Regulated neuronal PhosphoProtein of 32kDa (DARPP32). The progressive loss of these neurons is accompanied by a corresponding ventricular enlargement and gliosis. The disease progresses with the degeneration of cortical p yramidal neurons, mainl y projecting to the evidence for drugs to reduce chorea based on a review of randomized clinical trials using the Unified Huntington's Disease Rating Scale -Total Motor Score (UHDRS-TMS) to choose the best treatment. Tetrabenazine, acting to decrease dopamine levels, is the most prescribed treatment but in some cases, antips ychotics can help to reduce chore a. Anti-depressants and anxiol ytics can be prescribed to reduce psychological dysfunctions. Unfortunatel y, many of these medications have adverse side effects that can worsen HD symptoms.

During the past years, many preclinical studies initiall y reported t he efficacy of human fetal striatal tissue to provide functional recovery in a variet y of rodent and non -human primate models of striatal neuronal loss. On this basis, some clinical trials then assessed the potential of fetal neural transplants for the treatment of HD. In this review, we will briefl y outline the emergence of fetal neural therapy replacement and its limitations. We will continue by describing the pre-clinical studies performed with different stem cells, which represent an alternative cell source, and we will comment on their limitations, the most important one being their limited engraftment. We will further provide an integrative description and discussion of nanoparticles transporting interference RNA therapeupic (RNAi) to initiate cell dif ferentiation and increase survival in order to avoid some of the limitations described above. Finall y, in order to improve their engraftment within the brain parenchyma, increase neuroprotection and neuro -repair, we will present combining approaches with cell modified with RNAi therapeutics nanoparticles and drug delivery devices.

CELL TRANSPLANTATION

To replace the degenerating neurons in HD patients, some teams explored the transplantation of fetal human brain tissue. They demonstrated the feasibilit y and the safet y of this technique [10][11][12][13][14]. Functional improvements were obtained in the study led by Bachoud -Lévi. They reported graft survival, which contained striatal projection neurons and interneurons, and receive d hostderived afferents [10,11]. Comparisons of these clinical trials are very difficult because of the heterogeneit y in their design, lack of controls, unblended nature, and different methods used to assess clinical and motor outcome in each.

Nevertheless, the reported improvements in these trials appear to be modest and transient (for review see [15]). Indeed, limitation of cell therapies resulted from the extent of d amage affecting HD patients. Moreover, the use of human fetal brain for striatal transplantation derived from elective abortions is limited by ethical, practical, and regulatory concerns and is dependent upon availabilit y of donor tissue [16]. Besides the limited suppl y of human fresh fetal tissue, the strategy of striatal transplantation is further complicated by the lack of standardization inevitabl y correlated with the use of such a source.

One of the challenges is to ident ify an alternative cell source able to differentiate into MSN such as pluripotent stem cells (PSC), which are currentl y under investigation, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC). Adult neural stem cells (NSCs) and mesenchymal stem cells (MSCs) are also alternative candidates for regenerative medicine (Figure 1).

PLURIPOTENT STEM CELLS FOR CELLULAR THERAPY

PSC are defined by their capacit y of self -renewal thus offering the possibilit y of an unlimited suppl y of cel ls and by their pluripotency that is their abilit y to differentiate into all three germ layers. In vitro differentiation of pluripotent stem cells into GABAergic MSNs generall y follows a multi -step process: i) induction of neural lineages; ii) regional pat terning and the differentiation of neural progenitor cells; and iii) specialization of a mature neuronal subt ype. GABAergic MSNs can be generated using several culture methods, including co -culture on feeder cells and suspension culture as embryoid bodies. Additionall y, in vitro differentiation of pluripotent stem cells into MSNs requires cytokine signalling, controlled timing, duration, and concentration of exposure to developmental factors and reliable markers to identify mature MSNs capable to acquire el ectrophysiology properties.

Embryonic stem cells

Human ESCs (hESCs) derive from the inner cell mass of blastocyst stage embryos created by in vitro fertilization. Aubry et al., were the first ones to describe the differentiation of hESC into MSNs [17]. The authors proposed a novel 3 step -protocol to obtain striatal progenitors using 2 major proteins involved in the striatum ontogenesis: Sonic Hedgehog (SHH) and Dickkopf -related protein 1 (DKK1). The caudate/putamen or striatum derives principall y from the lateral ganglionic eminence (LGE) [18], with DLX2 -positive cells differentiating into MSNs, the most important population in the striatum. The injection into an adult rat model of HD (quinolinate -lesioned adult rats) of an earl y striatal differentiation stage of these hESCs presented massivel y overproliferating neural pr ogenitor cells. However, later stage striatal progenitors engrafted into the quinolinate -lesioned striatum and proved the feasibilit y of this cellular therapy [17]. The optimal time and dosage of SHH pathways in these cells was further determined as well as a major improvement to obtain the optimal commitment stage of the cells for transplantation [19]. When these

LGE-like ESC -derived cells were induced to terminal GABAergic differentiation in vitro, the majority of cells expressed GABA (90%) and DARPP32 (81%) and presented appropriate neuronal characteristics as determined by HPLC and whole-cell patch-clamp. In addition, there was no sign of massive overgrowth or tumor formation up to 16 weeks after transplantation. The graft -derived GABAergic projection neurons were integrated into the host ne ural circuitry, receiving dopaminergic inputs from the midbrain and glutamatergic inputs from the cortex while projecting fibres to the substantia nigra [20].However, ESC research is laden with ethical concerns, particularl y regarding the ideas of personhoo d and justice toward humankind that arise from dealing with human life in one of its earliest forms, the embryo. It is a controversial international issue, and many governing bodies have either banned the research altogether or placed restrictions on what may be done with embryos and ESCs, limiting their use for cell therapy.

Induced pluripotent stem cells

Initiall y, iPSCs are human somatic cells that are reprogrammed with four retroviral -incorporated specific transcription factors to a pluripotent stem -like state. IPSCs gained immediate international attention for their apparent similarit y to embryonic stem cells after their successful creation in 2006 by the group of Yamanaka and in 2007 by Thomson and collaborators [21,22]. Since, many methods have been reported to obta in these cells including high reprogramming efficiency, introducing non -viral and non-integrating methods of cell reprogramming, and using novel gene editing techniques for generating geneticall y corrected lines from patient -derived iPSCs, or for generatin g mutations in control cell lines [23,24]. Moreover, striatal neurons derived from iPSCs obtained from HD patient s, provide an in vitro disease model of HD.

IPSC grafts avoid the ethical issues intrinsic to hESC work whilst also allowing autografts to be performed (for review see [25] ). This latter strategy is intuitivel y mo re attractive, but does bring with it concerns, particularl y in the case of HD patients, that the disease being treated may develop in the grafted tissue given it is derived from the patient themselves. iPSCs may appear to solve the controversy over the de struction of embryos in ESC research, by involving onl y the genetic reprogramming of somatic cells. However, further anal ysis of this approach and its subsequent technical and ethical issues such as low reprogramming efficacy, genetic instabilit y, oncogeni c potential, conservation of the somatic cell's epigenetic origin, reprogramming process creating totipotent stem cells, could potentiall y lessen some advantages iPSCs seemingl y hold over ESC [26].

Concerning the use of iPSCs in vitro for striatal differentiation, the most recent protocol coupled neural induction via Bone Morphogenic Protein (BMP)

and Transforming growth factor beta (TGF-β), and ventral telencephalic specification with exposure to the developmental factors SHH and DKK -1, followed by terminal GABAergic differentiation. They obtained a neuronal population monitored by the appearance of progenitors of the LGE and able to mature into GABA-and DARPP -32-positive cells mimicking the striatum ontogenesis in 80 days. These cells presented electrophysiological properties expected for full y functional MSNs. The authors did not explore the iPSC transplantation but studied the outcome of hESC transplantation with their protocol [27]. Results indicated cell survival and extensive axonal projections, suggesting integration of the donor cells into the neuronal network of the host brain with the reduction of motor asymmetry.

For both ESCs and iPSCs, a bonafide differentiation protocol, relies on the identification of region and stage specific marker gene determinants of brain development. From a practical standpoint, the general aim is to recapitulate in vitro the human foetal development up to the specific and committed neuron precursors of the desired region. The transplanted cell population for HD will most probabl y consist of committed LGE progenitors as it has been shown that the highest proportion of s triatal-like cells in humans is obtained from grafts comprising the LGE expressing DLX2 under SHH pathways. Further challenges consist in defining the proportion of specific phenot ypes that will give the best therapeutic outcome after transplantation, and whether it will consist or not of a population of pure MSN progenitors [28].

ADULT STEM CELLS

Neural stem cells

An alternative source of cell transplantation in HD would be neural stem cells (NSCs) that participate i n normal central nervous system (CNS) development and differentiate into regionall y appropriate cell t ypes in response to environmental factors [29]. In this way, previous studies have shown that NSCs isolated from fetal or adult mammalian CNS can be propagated in vitro [30] and subsequently intracerebrall y implanted i n animal models of human neurological disorders including HD [31,32]. In the latter case, some cells differentiated in vivo into DARPP32+ neurons replacing neurons primaril y targeted in this disorder [31]. Also, geneticall y modified NSCs producing neurotrophic factors have been used to protect striatal neurons against excitotoxic insults [33]. NSCs derived from human brain exhibited ext ensive migration in the rat brain [34,35] and adult rats receiving intrastriatal transplantation of human NSCs prior to striatal damage induced by 3 -NP toxin exhibited significantly improved motor performan ce and increased resistance to striatal neuron damage compared with control sham injections [36]. The neuroprotection provided by the proactive transplantation of human NSCs in the rat model of HD appears to be mediated by brain -derived neurotrophic factor (BDNF) secreted by the transplanted human NSCs. Many studies have also demonstrated that BDNF could block neuronal injury under pathological conditions in animal models of HD [37,38]. Recentl y, human striatal NSCs were treated with a hedgehog agonist to generate DARPP -32 cells and transplanted in R6/2 HD transgenic mouse brain. The results were disappointing as the outcome was the same as a vehicle control injection. This is the onl y study using human NSCs for cell therapy in a HD genetic animal model [39].

Mesenchymal stem cells

Since the discovery by Friedenstein, of colony forming unit fibroblast cells (CFU-F) [40], MSCs never ceased to amaze by the many advantages they have in terms of regenerative medicine. The mechanisms through which MSCs exert their therapeutic potential in tissue repair, although not full y defined, might rel y on some fundamental key properties of these cel ls: i) their abilit y to secrete soluble factors capable to stimulate survival and functional recovery of injured cells; ii) the abilit y to home to sites of damage; iii) the abilit y to modulate immune responses and iv) their easy accessibilit y and potential for autologous transplantation. Although these cells differentiate to mesodermal lineage (chondrocytes, adipocytes, osteoblasts) differentiation of MSCs toward nonmesodermal cells has also been reported (for review see [41]).

MSCs may be expanded in vitro with varying degrees of additional differentiation towards neuronal lineages [41,42] enabling their use for cell therapy for neurodegenerative disease in cluding HD. MSCs are a heterogeneous population with cells presenting different differentiation properties and capable of expressing specific embryonic markers. A homogenous subpopulation of MSCs, named "Marrow -Isolated Adult Multilineage Inducible" (MIAMI ), which express several pluripotency markers (Oct4, Sox2, Nanog, SSEA4) are able to generate cells derived from all three embryonic germ layers [43]. They are capable of different iating into neuronal -like cells with electrophysiological properties of immature neurons under the influence of a sequential addition of specific growth factors and respond to dopaminergic inducers acquiring this neuronal phenot ype [44,45]. Moreover, they also show some degree of in vivo neuronal differentiation and they can secrete high amounts of tissue repair factors, some of which are involved in the protection/induction of blood vessels ex vivo [46]. Recently, adult human somatic cells, such as MSCs and dermal fibroblasts were shown to contain a small number of se vere stress -tolerant pluripotent stem cells, named ''multilineage differentiating stress enduring'' (Muse) cells [47]. These cells expressing pluripotency markers such as Nanog, Oct3/4, and Sox, can be isolated from MSCs or from fibroblasts after severe stress as stage-specific embryonic antigen-3-positive cells (a marker for human ES cells). Most importantl y, they possess specific properties like self -renewal, and pluripotency as they are able to generate cells representative of all three germ layers from a single cell. One li mitation to the use of Muse cells is linked to their low yield (with the range between 1% -5%), but their stress -resistance makes of them an interesting cell therapy candidate [47].

These cells are implanted either non -differentiated or after a predifferentiation stage, as p recursors, rel ying on the host environment to drive selective functional differentiation for cell replacement. However, the lack of consistent neuronal differentiation of transplanted MSCs has limited their therapeutic efficacy in animal models of HD (Table 1). In vitro and ex vivo characterizations of MSCs have revealed the presence/secretion of many growth repair factors, including BDNF, Glial cell -derived neurotrophic factor ( GDNF), vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) [48][49][50]. Researchers have recentl y explored the secretome of MSCs, with the aim of identifying all secreted molecules, which, in turn, m ay provide insight into the mechanisms of MSC benefits (for review see [51]). Indeed, intra striatal transplanted MSCs integrated in the host brain and exerted neurotrophic effects that correlated with increased levels of laminin, von Willebrand factor (VWF), stromal cell -derived factor -1 (SDF-1) α, and its receptor in the damage d striatum [52]. Moreover, after MSCs transplantation into the QA model of HD, rats presented a significant improvement in apomorphine -induced rotation tests, which correlated with a reduced lesion volume and a lower number of apoptotic striatal cells compared to control animals [53]. In the R6/2 mouse HD model MSCs transplanted mice showed improved motor functions compared to untreated R6/2 controls, although the overall performance continued to decline. Interestingl y, mice, which received MSCs transplantation, displayed a significant longer survival time than untreated R6/2 mice [54,55]. The improvement may be imputed to BDNF secreted by MSCs into the striatum.

Moreover, the secretion of BDNF has been associated with recruitment of forebrain neural progenitors [56] [57] . MSCs pre-differentiated towards a neuronal phenot ype, in order to better engraft within the brain parenchyma, may also be able to release the required repair factors involved in neuroprotection.

One study showed that the specificatio n of MIAMI cells towards a neural/earl y neuronal phenot ype still enabled the secretion of neurotrophic factors by these cells [45], but the appropriate degree of commitment before transplantation needs to be investigated.

Table 1.

Human mesenchymal stem cells based treatment of Huntington's disease in preclinical animal's model.

Induced neuron cells

It has recentl y been shown that mouse dermal fibroblasts can be directl y programmed to functional neurons by forced expression of a set of neural lineage transcript ional factors, named induced Neurons cells (iNs) (for review see [61]). The authors initiall y tested a pool of 19 transcription factors from which three (Ascl1, Brn2 and Myt1l, abbreviated hereafter as BAM factors) were found sufficient to generate functional neuronal phenot ype from fibroblasts [62]. Several different groups, after reprogramming fibroblast from

Bone marrow MSC producing BDNF

YAC 128 mice

Both BDNF-modified and nonmodified MSCs had significant effects in reducing the behavioral defects in an HD mouse model.

Improved rotared performance

Reduced hindlimb clasping [60] mouse, reported the generation of human iN cells (hiNs) [63][64][65]. None of the groups were successful in producing functionall y mature hiN cells using the same three transcription factors (BAM) that worked in mouse cells [65]. A major limitation of direct conversion to a te rminall y differentiated mature cell type is the inabilit y to expand the programmed cells in sufficient quantit y for various applications [66]. Therefore, direct conversion to expandable neural stem/progenitor cells (NSCs) is desirable in practical applications that demand large amount of cells.

As described, cells may be "manipulated" to control their proliferation, to improve their survival and differentiation and therefore ameliorate their engraftment after transplantation. This can be achieved by a previous in vitro genetic or epigenetic manipulation of the transplanted cells or by cotransplanting them with transcriptiona l and/or trophic factors that can be delivered by vectors and biomaterials. The latter being moreover able to provide a 3D template of extracellular matrix (ECM) contributing to shape the cells into a functional tissue and increase their engraftment [17,45,19,27] . The real challenge consists in controlling stem cell survival and differentiation by monitoring intracellular levels of relevant biomolecules while replacing lesioned tissues with these cells. Moreover, if possible, they should maintain their neuroprotective effects, when present. Typicall y, cell programming (differentiation within the same lineage) or cell reprogramming (reversal to pluripotency) consists in overexpression or knocked -down expression of genes involved in stem cell differentiation or transdifferentiation (p assage to a different lineage) in a specified manner. The control of gene expression may be achieved by RNAi, by selectivel y knocking down the expression of onl y a few pivotal genes. This epigenetic modification is quite simple, does not need genetic manipulation, is transitory and is now quite well understood.

RNAi were first used in 2003 to promote differentiation to the m yogenic lineage from pluripotent P19 teratocarcinoma [67]. Differentiation to the neuronal lineage is one of the most popular areas in cell therapy due to the poor regenerative potential of the nervous system. In fact, a large number of studies

EPIGENETIC MANIPULATION

have been dedicated to identifying potential knockdown targets to facilitate neurogenesis in ESCs, NSCs and MSCs. For example, the use of a lentiviralmediated RNAi vector that down -regulates the expression of REST (repressor element-1 silencing transcription factor) was shown to promote MSC differentiation into neuronal cells [68]. The unrestricted potential of RNAi has encouraged strategies for large scale silencing of protein encoding genes in the human genome. RNAi can be triggered by three different pathways: 1) a RNAbased approach where the effectors siRNAs are delivered to target cells as preformed 21 base dup lexes; or 2) a DNA -based strategy in which the siRNA effectors are produced by intracellular processing of longer RNA hairpin transcripts; 3) a RNA -based approach, similar to siRNAs, produced endogenousl y by the cells, the pre -miRNAs stem -loops which are then spliced to release the mature miRNA duplex (Figure 2). Inspired from Cullen, nature genetics 2005, we represent the intracellular trafficking of siRNA delivery systems [69]. Internalization of delivery systems can be performed by several mechanisms as clathrin, caveole pathways or lipid rafts. Two modalities are observed in this phenomenon, a receptor mediated endocytosis and a non-receptor mediated endocytosis. RNAi is delivered into the cytoplasm and produces its inhibitor effect.

In vitro, RNAi does not readil y bind to or cross the cell membrane, so RNAi must be introduced in cells by innovate delivery systems. In the case of RNAi, the siRNA or miRNA must be delivered to the RNA -induced silencing complex (RISC ) in the cytoplasm (Figure 2). The most common delivery system relies on recombinant virus, but although very effective, viral methods for RNAi delivery have been associated with immunogenicit y and tumorigenicit y [70].

Non-viral delivery systems are traditionall y less effective but can be designed to avoid issues t ypical of viruses. They have considerable advantages over viralbased vectors due to the control of their molecular composition, their simplified manufacturing, modification and anal ysis, and their tolerance for cargo sizes, while displaying a relativel y low immunogenicit y [71]. Non-viral gene delivery utilizes chemical reagents including lipids, cationic pol ymers and nanoparticles or physical means such as electroporation. More interestingl y, some nanocarriers can secure the control of cellular behaviour. Indeed, materials with unique nanotopographical characteristics and size offer properties producing similar effects than growth factors [72]. They can be used to ind uce specific biological responses. However, before RNAi reaches its target in vitro and in vivo when co-transplanted with delivery vectors , it faces a number of significant barriers. More importantl y, cellular and local delivery strategies have to deal with the need for internalization, release, and distribution in the proper subcellular compartment (Figure 2). Many earl y efforts at RNAi delivery used materials that were already well studied for DNA delivery and can be applicable, with some limitations, to the RNAi delivery. Development of different strategies to encapsulate and deliver RNAi has been described in literature [73] (Figure . 3). We will first discuss the properties of these nanocarriers, which make them suitable for nucleic acid delivery in general and then describe their utilit y for overcoming barriers spe cific to RNAi delivery and their use in stem cells for controlling neuronal differentiation or survival .

[Attirez l'attention du lecteur avec une citation du document ou utilisez cet espace pour mettre en valeur un point clé. Pour placer cette zone de texte n'importe où sur la page, faites-la simplement glisser.] Methods of RNAi complexation with delivery systems. The RNAi localization into nanocarriers differs according to structure and organization of the delivery system. RNAi can be incorporated into a matrix structure and allow a global repartition in the entire volume of the sphere (cluster), with polymers components. Other strategies consist in either associating by electrostatic or Van der Walls interactions the RNAi on the surface of delivery systems. The chemical cross-link is also a possibility. Last organization concerns shell/core structure observed for liposomes and some nanoparticles. [74].

NANOCARRIERS TO CONTROL STEM CELL FATE

The use of nanocarriers, also named nanoparticles (NPs) ranging in size from 1nm to 1000nm to cont rol stem cell fate after intracellular incorporation is a novel approach. NPs were first developed for the potential delivery of therapeutic factors such as chemotherapeutic agents to tumours or, when combined to stem cells, mostl y for stem cell imaging [71] . The nanocarriers most commonl y used in stem cell research are organic (liposomes, lipid nanoparticles , solid nanoparticles and dendrimers) and inorganic nanocarriers (quantum dots, carbon nanotubes, iron or gold nanoparticles) (Figure 4). These last materials with unique characteristics such as carbon nanotubes, gold nanoparticles, and gold nanorods have attracted attention as innovative carriers for RNAi (for review see [75]) but have not yet been used for cell programming in vitro combined with RNAi. However, inorganic nanoparticles seem to possess certain properties stimulating neuronal differentiation. Iron oxide nanoparticles incorporated within rat PC12 cells induced extensive changes in genes related to the cytoskeleton, signalling molecules, receptors for growth hormones and ion channels, all required and expected to occur during neuronal differentiation [76]. One explanation for the effect on gene expression is that several inorganic particles, including iron oxide nanoparticles, have the potential to release inorganic ions. In the case of manganese, the ions play an important role in neuronal differentiation by activating cell adhesion molecules, which interact

Chemical cross-link or absorption

with the extracellular matrix and direct cell binding and signalling. Neuronal differentiation is known to be influenced by the amount and subcellular distribution of integrin clusters, i.e. cell adhesion [77]. However, the mechanism by which metal ions affect gene expression is still unclear.

The encapsulation of hydrophilic drugs such as nucleic acids and also proteins has been develop ed from different formulations. A large variet y of s ynthetic materials or pol ymers, such as pol ylactide -co-gl ycolide (P LGA), pol ycaprolactone, and natural materials, such as chitosan, collagen, pullulan, and pol y-arginine can be used to formulate nanopart icles for medical applications. Different types of nanocarriers able to control the stem cell fate. Nanoparticles are commonly defined as objects with a rank of dimensions of 1-1000nm, which includes micelles, reversed micelles, nanoparticles, nanogels, and nanofibers. We represent here the morphology of the most commonly particles used ones for siRNA complexation and regenerative medicine.

LIPID-BASED NANOCARRIERS

Lipid-based nanocarriers are the most widel y used biomaterials for nanoparticulate RNAi delivery. Of over 20 siRNA phase I clinical trials, nearl y half use NPs as the delivery vehicle, and almost all of these are lipid -based [78]. Although lipid-based nanoparticles have been historicall y designed for lipophilic drug delivery, the idea of using cationic lipids with their positive charged head group to efficientl y bind negativel y cha rged RNAi became rapidl y evident.

Liposomes

Transfection t ypically involves the use of packaging particles called liposomes to facilitate the cellular uptake of RNAi. Cationic lipids in the liposomes mimic the physical characteristics of natural phospholi pids that represent the individual components of a cellular membrane. Liposomes possess long hydrophobic chains and a positivel y charged head group, allowing the formation of nano -sized complexes with negativel y charged RNAi (lipoplexes) that is encapsulated by a lipid bilayer. Since first being used for gene therapy in 1990, numerous commercial cationic lipids (also called cytofectins, lipofection, oligofection reagents) have been synthesized and used for delivery in cell culture [79]. Lipoplexes offer protection to RNAi from enzymatic degradation and efficient endocytosis of RNAi by the cell. Lipid complexation with the payload (i.e. RNAi) simpl y involves mixing and incubation [71]. Neutral lipids such as dioleylphosphatidylethanolami ne (DOPE) and cholesterol are usuall y incorporated in the liposomal formulations, where they serve as helper lipids: increasing the transfection efficiency of the gene or molecule -containing liposome by facilitating membrane fusion and aid in the destabili zation of the plasma membrane [80]. Liposomes can escape from the endosome through a so called 'proton sponge' effect where positivel y charged cationic lipids cause influx of protons and water leading to endosome swelling and eventuall y disruption to release the lipoplexes to cytoplasm. Safe and e fficacious delivery in vivo is however rarel y achieved due to toxicit y, nonspecific uptake, and unwanted immune response.

Very few studies however transfect human iPS or ESC cells with RNAi to drive neuronal differentiation because limited transfection ef ficiencies have been reported in this t ype of cells and transitional action reported with these materials is not sufficient on their own to have an effect. Indeed, in vitro transfection efficiency is affected by the t ype of cell, proliferative activit y of the cell, the t ype of culture media and cell plating density. It appears that lipoplexe transfection performed well in human MSCs [81,82], but produced more inferior outcomes in NSCs [83] and ESCs [84,85]. These results suggest that conventional lipoplexe transfection is an efficient RNAi delivery means for human MSCs but not for human ESCs. In order to determine guidelines to transfect human ESCs while inducing their differentiation, two groups have attempted to knock down Oct4, a pluripotent stem cell gene, in ESCs with transfection efficiencies of onl y ∼60% [84] and 69.8% [85] compared to Lin28, another pluripotent marker. This low efficiency for Oct -4 suggests that the t ype of target molecule will also affect the transfection outcome. Interestingl y, the transfection efficiencies and the resulting silencing efficiencies were not strictl y proportional to each other; with a more efficient Oct4 knock down being obtained by the less effective transfection. This is probabl y attributed to autoregulation mechanisms of the levels of Oct4, which were moreover quickl y restored after 1 day, indicating the need f or additional boosters to further increase the silencing efficiency to >90%. Cationic lipids were the first non -viral vectors demonstrating their capacit y to easil y transfect mouse embryonic stem cells in vitro with microRNAs (miRNA) in order to induce n euronal commitment [86]. In order to determine the role of miRNA -125b mimic in human MSC during in vitro neuronal differentiation, the cells were transfected or lentiviral infected with miRNA-125b resulting in the increased percentage of TH -positive neu rons. With the two methods used, similar results were obtained confirming its role in dopaminergic differentiation. The miRNA -181* also increases the number of TH-positive neurons, intriguingl y, just one of the strands allows this differentiation [87]. Furthermore, the role between miRNA 9 and REST to promote neuronal commitment was determined. A mutual down -regulation between REST and miRNA -9 was observed, which may contribute to the maintenance of a neuronal differentiation program [88]. More interestingl y, MSCs after liposomal transfection with miRNA -124 and miRNA-145, are able to deliver exogenous miRNAs to human NSC in co -culture with astrocytes via exosomes. Moreover, the delivered miRNAs altered gene expression in th e recipient neural cells and impacted their phenot ype and function. The delivered miRNA124 increased the expression of the glutamate transporters, EAAT1/EAAT2 in NSC and astrocytes and down-regulated Sox9 expression which increased the expression of β 3-tubulin, a marker of neuronal precursors [89]. Similar results were obtained with MSCs transfected with pre -miRNA-124.

MSCs can therefore be easil y transfected with exogenous miRNA to further induce neuroprotection or the differentiation of neuronal progenitors after transplantation.

The transfection e fficiency with these delivery vehicles in vitro is, in part, determined by their stabilit y and particle size. For example, serum proteins can decrease transfection efficiency by neutralization of the positive zeta potential [90] of the complexes. Toxicit y may, in part, result from the large size of the complexes, and the high positive zeta potential required for their uptake [91]. The toxicit y is normall y closel y associated with the charge ratio between the cationic lipid species and the nucleic acids, as wel l as the dose of lipoplexes administered. So although cationic lipid formulations have been used to deliver RNAi in vitro and in vivo, cell toxicit y caused by cationic lipid is still a major concern. To bypass problematic toxicit y, particle instabilit y and to maximize siRNA delivery, fragments of liposomes or lipoplexes can be encapsulated into nanoparticles.

Lipid based nanoparticles

Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) consisting of spherical solid lipid particles in t he nanometer range are an interesting alternative to liposomal toxicit y. They are comparativel y stable colloidal carrier systems in which melted lipids are dispersed in an aqueous surfactant by high-pressure homogenization, solvent injection, solvent emulsification or micro-emulsification [92]. The lipids which are used in their production are solid at room temperature, and most of them have an approved status, e.g. GRAS (Generall y Recognized As Safe), due to their low toxicit y [93]. Although cationic lipids are an inherent part of SLN formulations, it is unclear to which extent their presence bears relevance to the mechanism by which S LNs deliver their cargo. Whether their p ol ymorphic properties play a role in destabilizing cellular membranes, as discussed above for cationic lipidbased nanocarriers, remains to be determined. In vitro, the comparison of cell time retention of siRNA between S LN, lipofectamine ® and liposomes showed that the siRNA is present during five to nine days for SLN and three days for commercial reagents or liposomal formulations. This propert y is quite interesting for maintaining a differentiated cellular state over time for cell therapy applications. Ho wever, the percentage of siRNA loading is quite low around 35% and still needs to be improved [94]. Nanostructured lipid carriers possess the advantage of offering increas ed hydrophilic drug loading and decreased drug leakage from the NPs.

Lipid nanocapsules (LNCs) came into the spotlight as a strategy to overcome positivel y charged lipoplexe toxicit y encapsulated within the carriers, while enhancing cell uptake and protec ting the nucleic acid from l ysosome degradation. Our research group has reported innovative LNCs, consisting of a lipid liquid core of trigl ycerides and a rigid shell of lecithin and pol yethylenegl ycol. The simple solvent -free formulation process based o n the phase inversion of an emulsion makes them an ideal nanocarrier for translational studies. They are highl y stable (more than 3 months) [74] in comparison to SLNs [95] and, in function of their composition and size, can escape the endolysosomal compartment [96]. These LNCs can mediate highl y efficient gene transfer not onl y in vitro but also in vivo [97,98]. They were recentl y modified to encapsulate siRNA, complexed with DOTAP/DOPE lipids forming lipoplexes (Fig. 3), and showed efficient encapsulation and protein inhibition in a glioma cell-line [98]. Interestingl y, the cellular uptake mechanism with siRNA -LNCs seems to be membrane fusion. The study of this mechanism by Fluorescence confocal spectral imaging demonstrated the degradation of this nanocarrier and the release of siRNA in the cytoplasm explained beside by the presence of DOPE lipids in the formulation [99]. LNC-siRNA transfection for neuronal commitment and resulting protein inhibition capacit y is currentl y being evaluated in MSCs.

Recentl y, our research group has reported a novel lipid nanocarrier based on sorbitan mono oleate, more commonly known by the commercial name of Span 80 (SP80); a surfactant widel y used in the pharmaceutical industry and generall y recognized as safe (GRAS). However, instead of using span as a surfactant, this component was used as a lipophilic m ain ingredient of these nanos ystems which have demonstrated different component loading capacities (Sanchez et al., WO2013068625A1) [100]. This new system can incorporate cationic components such as oleylamine or pol y-arginine to complex RNAi by electrostatic interaction at the surface (Fig. 3). The capacit y to load a variet y of pol ymers into the shell offers an adaptable system for all the t ypes of cells and the possible incorporation of na tural pol ymers can reduce the cytotoxicit y.

POLYMER BASED NANOCARRIERS

Pol ymer based nanocarriers are a very promising class of biomaterials for the delivery of nucleic acids and are an active subject of ongoing research.

Because many of the pol ymers used for RNAi delivery were originall y investigated as DNA delivery materials, generalities about DNA delivery can be extended to RNAi delivery. Pol ymers used for RNAi delivery can be divided into two categories: (i) those with synthetic components, such as den drimers, pol yethylenimines (PEI), etc (ii) those with biodegradable natural components, such as collagen and pullulan. We will briefl y describe most synthetic pol ymers used for RNAi delivery for stem cell neuronal differentiation and then we will present novel nanoparticles with natural pol ymers.

Synthetic components

PEIs, presented in branched or linear forms, available in a broad range of molecular weights are widel y used for gene delivery. In the physiological medium, PEI is positivel y charged due to pro tonation of the amine groups and thus can be used to condense nucleic acids. [101]. Cationic pol yplexes, formed by PEI and nucleic acids, t ypicall y retain a net positive charge promoting interactions with negativel y charged pol ysaccharides on the cell surface followed by endocyt osis . The pol yplexe escape from the endosome by the same 'proton sponge' effect described for cationic liposomes. PEI is known to mediate highl y efficient gene transfer, but also exhibits significant toxicit y during the transfection process [102]. Therefore, many studies have reduced the cytotoxicit y caused by PEI-mediated delivery by conjugating PEI to synthetic or natural pol ymers, such as hyaluronic acid [103]. In this way, several studies have shown that PEI-modified pol ymers can be used to deliver genes to regulate stem cell differentiation usuall y with DNA but also in combination with siRNA [104]. In this regard, combined transfection of a Sox 9-bearing plasmid and Cbfa-1 siRNA complexed with PEI-PLGA nanoparticles were found to enhance hMSC chondrogenic differentiation [105]. To our knowledge, no neuronal stem cell differentiation study has yet been performed with these pol ymers.

Nevertheless, the inhibition of ROCK (with PEG-PEI/siROCK2) increased the neuroprotection against an external agent of primary cortical neurons [106].

Despite these advances in PEI-based gene delivery, it remains a challenge to balance the transfection efficiency and cytotoxicit y of PEI -based delivery s ystems.

Dendrimers have attracted a great deal of interest in different areas including nucleic acid delivery applications for cell differentiation due to their uniform, well-defined, three dimensional structures [107]. The name "dendrimer" originated from the greek words which describe a structure consisting of a central core molecule that acts as a root, from which a number of highl y branched, tree -like arms originate in a well -ordered and symmetrical manner [107]. The commercial dendrimers named PAMAM transfection reagents (Superfect and Pol yfect) are mainl y indicated for plasmid DNA and RNAi transfection. Amine-terminated PAMAM dendrimers develop highl y positive charged densities at their surfaces when they are at physiological pH or dissolved in water. However, double stranded RNA including siRNA and miRNA are less flexible than pDNA, which can lead to the incomplete encapsulation or the formation of undesirabl y large complexes. Highl y branched dendrimers have thus been developed for siRNA efficient delivery [108,109].

Different strategies have been further developed to reduce the toxicit y of dendrimers, such as the neutralization o f the surface charge with PEG, the attachment of peptides at the surface [110], or the association with natural components such as collagen [111]. PAMAM, with a reported abilit y to trigger a proton sponge endosomal escape, showed an efficient delivery of RNAi in neurons in vitro and in vivo (intracranial injection in rab bits) with very low toxicit y levels [112]. Interestingl y, the silencing of high mobilit y group box -1 (HMGB1, a novel cytokine -like molecule) in primary cortical cultures successfull y reduced both basal and H 2 O2-or NMDA-induced neuronal cell death [113]. A similar approach could be tested with neur onal committed stem cells to increase their survival after transplantation in HD paradigm.

Nowadays, the combination between siRNA and morphogens are effective for stem cell differentiation. [START_REF] Shah | Single Vehicular Delivery of siRNA and Small Molecules to Control Stem Cell Differentiation[END_REF] developed cyclodextrin-modified dendritic pol yamine construct (termed DexAM) f or effective stem cell differentiation [114]. DexAM present the abilit y to simultaneousl y deliver nucleic acids (siRNA against Sox9) and hydrophobic small molecules (Retinoic acid) to achieve a synergistic enhancement in stem cell differentiation. When rat NSCs were transfected with siSox9 using these nanoparticles, they differentiated into neurons, wherein more than 70% expressing the neuronal precursor marker B3 -tubulin [114].

Natural components:

Recentl y, siRNA -grafted natural pol ymers have been proposed as a promising strategy for siRNA delivery. They can be divided into two main categories: (i) pol ymers obtained strictly from natural sources, (ii) and semis ynthetic pol ymers obtained from natural sources but which are rationall y modified with the aim of adapting their properties according to the delivery requirements of the therapeutic molecules.

Chitosan has been widel y described in the formulation with RNAi because of its cationic n ature, low toxicit y, biocompatibilit y, and degradabilit y in the human body and has been designated as safe (GRAS) by the FDA [115]. This linear pol ysaccharide of randoml y distributed N-acet ylglucosamine and glucosamine units is protonated in slightly acidic conditions, which permits an efficient complexation of nucleic acids into NPs. Recentl y, a commercial DNA transfection reagent based on a chitosan oligomer has been developed (Novafect, Novamatrix). Regarding siRNA delivery, various studies describe the importance of chitosan characteristics to achieve good efficiency in vitro, including the degree of deacet ylation and molecular weight (MW) of the chitosan, charge ratio of amine (chitos an) to phosphate (RNA), serum concentration, pH and cell t ype [116]. However, this system has a significant limitation, owing to its low transfection efficiency [117].

Spermine had been recentl y shown to enhance transfection when grafted in the anionicall y modified pullulan exhibiting excellent blood compatibilit y and in vitro transfection. More interestingl y a complexation to pullulan -spermine achieved success ful in vitro neuronal gene delivery irrespective of the negativel y charged complexes with no measurable cytotoxicit y at up to 20 g/ml DNA [118]. Actually, the tendency to improve transfection efficiency and reduce the cytotoxicit y is mixing naturals components with lipids, or inorganic components to obtain hybrid systems [119].

CELL AND TROPHIC FACTOR RELEASING MICROCARRIERS

Transplanted s tem and progenitor cells can promote the survival of host cells by releasing neuroprotective trophic factors. In addition, many studies have demonstrated the preclinical feasibilit y of encapsulation as a means of

COMBINED STRATEGIES FOR CELL TRANSPLANTATION

delivering factors to the CNS, and more par ticularl y, geneticall y engineered cells secreting trophic factors for HD [120]. In general, these studies demonstrate that encapsulated cells can be protected and remain viable for extended periods of time to produce significant neuroprotective and behavioral benefits. Clinical trials have been conducted on evaluating the benefits of encapsulated cells deliver ing ciliary neurotrophic factor for HD [121]. In this case, the implanted cells were safel y tolerated without serious adverse events, justifying further clinical evaluation. However, the relativel y modest cell survival and the heterogeneit y of the results in these studies need to be improved. Schematic illustrating technologies to spatially control stem cell fate. During microcarriers' process, nanocarriers containing RNAi or neurotrophic factors can be integrated directly into these biomaterials. All possibilties for combined strategies for stem cells transplantation have been presented. The first consideration for microcarriers tailoring in brain is size: microcarriers should necessarily be small enough to be easily implanted into the striatum, either via stereotactic implantation or under neuro-navigation. Moreover, small-sized scaffolds render repeated implantations possible, with no need for open-surgery [122].

Another way to deliver a growth factor is after enc apsulation within pol ymeric microcarriers that in their turn may transport cells on their surface (for review [123]). In this sense, our group has developed the pharmacologicall y active microcarriers (PAMs) combining these two approaches. These PAMs are biodegradable and biocompatible PLGA microspheres conveying cells on their biomimetic surface, therefore providing an adequate 3D microenvironment in vivo. Moreover, the controlled delivery of a trophi c factor in combination with a biomimetic surface acts synergistically to stimulate the survival and/or differentiation of the grafted cells toward a specific phenot ype, therefore enhancing their engraftment after their complete degradation [124]. Finall y, it should be noted that the delivered molecule might also affect the host microenvironment allowing the integration of the grafted cells and/or stimulating the lesioned brain repair capacities. The efficacy of PAMs for cell therapy of Parkison's disease in a clinical paradi gm was demonstrated using GDNF-releasing PAMs, conveying a small number of embryonic ventral mesencephalon dopaminergic cells [44]. Similarl y, PAMs with a biomimetic laminin surface delivering Neurotrophin factor -3 and conveying adult stem cells showed a neuroprotective and repair effect leadin g to an ameliorated behavior in an animal model of Parkinson's disease [125]. This technology can be adapted for HD, delivering stem cells and neurotrophic factors able to drive GABAergic commitment such as BDNF. However, as described, external factors (media and growth factors) are insufficient to significantl y control stem cell fate on their own; the ideal strategy relies on their combination with the modification of gene expression by RNAi (Fig. 5) .

Another study demonstrated enhancement of neuronal differentiation of

NPCs by nanofibrous carriers -mediated release of BDNF and retinoic acid (RA).

In that study, nanofibrous were constructed with the copol ymers of caprolactone and ethyl ethylene phos phate. BDNF and RA were then encapsulated in the carrier. Neuronal differentiation of NSCs was enhanced due to the s ynergistic effects of nanofibrous topography and sustained delivery of BDNF and RA [126]. These cell -loaded capsules can be implanted into the damaged brain area favori ng the local, targeted and long -term release of drugs or proteins.

RNAI LOADED NANOCARRIERS AND MICROCARRIERS FOR REGENERATIVE MEDICINE

Conventional approaches using cationic pol ymers or lipids usuall y onl y permit transient gene expression in stem cells af ter transfection [127]. To overcome the limitation of gene delivery using onl y cationic substances, porous 3D carriers made of biodegradable polymers, such as sponges, particles, o r hydrogels, can be employed as gene carriers and depots for prolonged gene delivery. Immobilization or incorporation of gene (DNA, RNA) complexes with cationic pol ymers or lipids in the carriers may facilitate sustained delivery to stem cells cultured on the scaffolds, leading to prolonged transgene expression in the stem cells and extended control of stem cell differentiation [128].

MSCs osteogenic differentiation was enhanced by decorated nanostructured scaffold, composed by pol y-ε-caprolactone capable of retaining and delivering siRNA, with broad applications for controlling stem cell differentiation in vitro and in vivo. [129]. Recentl y, a simple and efficient siRNA delivery system based on nanotopography-mediated reverse transfection was developed. The authors deposited a self -assembled silica nanoparticle monolayer on a glass cover slip and then coated extra cellular matrix proteins and siRNA on top of the nanoparticle monolayer [128]. This platform allowed highl y efficient transfection of Sox9 siRNA into rat NSCs due to nanotopographical cues for stimulating endocyt osis and intracellular gene transfer, which enhanced neuronal differentiation of NSCs while inhibiting glial differe ntiation [128]. In vitro cell culture models are of particular importance in Neuroscience Research. While single cell cultures are widel y used, the organot ypic brain slice culture model is the closest to an in vivo situation. Indeed, brain slices contain all the cells present in the brain and maintain its three dimensional architecture [39]. Brain slice cultures allow maintaining the survival of all cell t ypes, the functionalit y of synapses and neuronal properties. In other words, the basic cellular and connective organization of the donor brain regions are well preserved, thus the s lice cultures provide an easil y accessible platform to study: neurodegeneration, mechanisms of disease (Alzheimer, Parkinson's disease, Huntington's disease), and test different treatments such as pharmacological treatments, cells replacement or tissue eng ineering treatments [42,124].

The first description of organot ypic brain slices have been described for the culture of dorsal cerebellum in 1970 [125,126]. Then the culture of brain slice model was perfectl y developed in 1982 using spinal cord -dorsal root ganglia [127]. The characterization and the improvement of this unique ex vivo model was realized by Gähwiler and Hefti (1984) with roller tube cultures [128].The technique was modified as a permanent culture model on membrane inserts [129,130] and is nowadays used by several research groups [START_REF] Ostergaard | Rat ventral mesencephalon grown as organotypic slice cultures and co-cultured with striatum, hippocampus, and cerebellum[END_REF][START_REF] Gähwiler | Organotypic slice cultures: a technique has come of age[END_REF][START_REF] Daviaud | Modeling nigrostriatal degeneration in organotypic cultures, a new ex vivo model of Parkinson's disease[END_REF].

Slices can be cultured as single slices from a respective brain area or as a coculture where two functional related brain slices are connected. Since then ex vivo models of Parkinson's disease, Alzheimer, cerebral ischemia, HD have been developed (For a review see [42])

In the case of HD, organot ypic model development dates back to 1986 with the study of the effects of KA administration on glial cells, both in the animal model of HD (which uses this drug to da mage GABAergic neurons) and in striatal organot ypic cultures [44]. Since then other genetic and non -genetic models have been de veloped to model HD.

Genetic models of HD derive principall y from animal models expressing a truncated [23] or full length [24,25] form of mutant htt (mhtt). This model mimics the pathology including the genetic aspect of HD and as a consequence

INTRODUCTION

CHAPTER I leads to the MSN death. The model acquirement does not demand specific knowledge but may be more expensive in a long term as genetic mice need to be purchased. The R6/2 and knock -in Q175 mouse are mostl y used, from mice of12 weeks old until 1 year or more [START_REF] Ferrante | Mouse models of Huntington's disease and methodological considerations for therapeutic trials[END_REF][START_REF] Wójtowicz | Reduced tonic inhibition in striatal output neurons from Huntington mice due to loss of astrocytic GABA release through GAT-3[END_REF][START_REF] Indersmitten | Altered excitatory and inhibitory inputs to striatal medium-sized spiny neurons and cortical pyramidal neurons in the Q175 mouse model of Huntington's disease[END_REF]. So usuall y, organot ypic slices deriving from genetic models are made with adult brain. These brain slices are more difficult to prepare as th e cranium is unbending and the culture of adult brain is more complicated. Indeed, most brain slice culture systems have used the perinatal rodent brain, taking advantages of its higher viabilit y relative to the older one. The survival of adult brain slice s did not exceed 1 week in cultures [START_REF] Wilhelmi | Organotypic brain-slice cultures from adult rats: approaches for a prolonged culture time[END_REF] with serum and 3 -4 weeks without serum [START_REF] Kim | Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology[END_REF].

Another way to obtain genetic models of HD brain slices is the transfection of HD-pol yQ-GFP plasmids directl y onto the slice. Three different non-viral transfection methods were tested on mouse cerebellum organot ypic slices [START_REF] Murphy | Gene Transfer Methods for CNS Organotypic Cultures: A Comparison of Three Nonviral Methods[END_REF]. And then authors proposed an innovate model obtained in 72h with a transfected mutant huntingtin gene which when expos ed to malonate produced HD-like lesions and provided a new model of HD, conserving the correlation between CAG repetitions and aggregation length [START_REF] Murphy | A single-chain Fv intrabody provides functional protection against the effects of mutant protein in an organotypic slice culture model of Huntington's disease[END_REF]. This new model is powerful because in 72h they obtained a genetic model of HD, but even with the best transfection methods the maximum of cells containing the plasmid were around 35%. Slice transfection requires high technology equipments and a skilled operator, which make them difficult to use in routine.

Non genetic ex vivo models of HD have been performed using neurotoxins (KA [44], IA, QA [48], NMDA [52], 3-NPA [46]) added into the media, which then mainl y affect the striatum, the cortex and the hippocampus, inducing neuronal loss [36,49,[START_REF] Heyes | Regional brain and cerebrospinal fluid quinolinic acid concentrations in Huntington's disease[END_REF]. Striatal degeneration of HD can also be caused in vivo by 3-NPA, a mitochondrial inhibitor. This model allows understanding the neurotoxicit y and the excitotoxicit y mechanisms [46]. Although onl y the cellular aspect of HD can be studied, they present many advantages: they are eas y and rapid to develop and do not involve high economic impact as normal rodents are used. For these models, usuall y newborn or one day old rodent are sacrificed and cultured during 3 -4 weeks and then brain slice cultures are exposed for 48h to neurotoxins. In thi s way, a model is obtained in 1 month, which is quite some time. Moreover, the heterogeneit y of the results after treatment evaluation prompts to focus on another system.

In this work, we propose a novel and simple approach to model HD. We developed a coronal organot ypic culture model obtained just by vibrosectioning of the brain. This brain slice model allows selecting the area that we want to study. The main areas involved in HD are obtained in a unique slice without neurotoxins to induce the GABAergic MSNs depopulation. The aim was to induce progressive striatal MSNs degeneration in a single step while preparing the slices, in order to obtain a simple reproducible HD ex vivo model. To our knowledge, this is the first time that vibrosection was investiga ted for modeling HD. We have already demonstrated the interest of vibrosectioning the brain for developing a full y characterized ex vivo PD model. Interestingl y, we obtain ed all the areas involved in PD : cortex, striatum, ventral pallidum the visualization of the fibers present in the medial forebrain bundle and the substantia nigra [START_REF] Daviaud | Modeling nigrostriatal degeneration in organotypic cultures, a new ex vivo model of Parkinson's disease[END_REF]. Based on the same method, we elaborated this unique model of HD.

Publication in process for Neuroscience methods

André E. 1,2 ; Daviaud N. 1,2 ; Sindji L.

A METHOD FOR A SIMPLE IN VITRO HUNTINGTON'S MODEL: SLICE CULTURE FOR MODELING THE DISEASE AND STUDY THE REPAIR OF THE GABAERGIC NEURONS

BACKGROUND: Organot ypic brain slice cultures represent an excellent compromise between single cell cultures and animal. They preserve brain threedimensional architecture, synapti c connectivit y and brain cells microenvironment. This model has allowed researchers to observe cellular interactions and mechanisms through a simple and rapid method. Moreover, slice culture model systems provide a unique opportunity to monitor the circuits' repair in a dish after cells transplantation without any concern about the possible interaction between neurotoxins and cells grafted.

NEW METHODS:

We aim to develop a novel model of Huntington's disease. We have generated a coronal slice from rats' br ains that includes all the areas involved in HD in a single slice preparation, without using neurotoxins to induce the lesion.

RESULTS:

After investigated different axis to cut the rats' brains, we determined that coronal brain slices can be cultured durin g approximativel y 3 weeks with a preservation of normal cyto -architecture except in the striatum. After 1 week, we observed a reduced volume of the striatum associated with the decrease of GABAergic medium spiny neurons markers mimicking HD cellular hallmarks.

COMPARISON WITH EXISTING METHODS:

This organot ypic model involves GABAergic MSNs degeneration simpl y by cutting the tissue with a specific axis. Inversel y, the use of neurotoxins such as quinoleic acid to model Huntington's disease lead to heterogen eity in the results obtained, which must be taken into consideration.

CONCLUSIONS:

This unique model presents a new approach for modeling Huntington's disease in vitro, and provides a useful innovative method for screening new potential therapies for neurodegenerative diseases .

ABSTRACT:

Keywords:

Abbreviations:

Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disorder with a general prevalence of about 10 per 100.000 births [1,2]. This high CAG triplet repetition is localized in the IT -15 gene of the chromosome 4: the huntingtin gene (HTT). This repeti tion will lead, at the protein level, to a pol yglutamine repetition at the NH 2 -terminal part of the huntingtin protein [3]. The accumulation of the mutant htt protein is excitotoxic, therefore it progressivel y compromises survival and normal neuronal functioning, primarily in the striatum or caudate/putamen and pr ogresses with the degeneration of cortical pyramidal neurons, mainl y projecting to the striatum [4]. Striatal GABAergic medium spiny neurons (MSN), which constitute the majorit y of cells in this region are affected by this pathology. They extend axonal projections to the external segment of the globus pallidus (GP), and to the substantia nigra pars reticulata. The progressive loss of these neurons is accompanied by a corresponding ventricular enlargement and gliosis [5].

Clinicall y, this neurodegenerative disorder lead s to involuntary movements , cognitive impairment and psychiatric manifestations [6]. Medications are available to help managing the symptoms of Huntington's disease, but treatments can't prevent the physical, mental and behavioral progressive decline associated with the disease.

Novel drug and cell therapy approaches in development require extensive evaluation before use in humans [7]. Considering the role of mHtt in the pathogenesis of HD, this protein has been transfected in different cell lines for the study of HD in vitro. [8] Recent studies have explored the use of embryonic stem cells and induced pluripotent stem cells as cellular models for disease research and the development of biomedical applications. Significant work being done in the field is the establishment of human iPSC lines from patients with HD mutations [9]. Although these models are relevant to study important hallmarks of HD, allowing the investigation of key intrace llular mechanism involved in the disease, as well as the identification of novel pharmacological targets, in vitro models do not reflect the complexity of the disease. The effect of the microenvironnement, and the functional aspects of the disease can onl y be

INTRODUCTION

provided by in vivo models.These models can be broadl y divided into genetic models presenting the mutated htt gene in various forms or into neurotoxic models showing a quite specific GABAergic MSN degeneration [10]. These models have been designed to elucidate the pathogenesis, cell death mechanisms and to evaluate therapeutic strategies for HD [11]. But in vivo studies require high technical and financial resources and they do not allow to simultaneousl y test several conditions in the same animal [12][13][14]. For innovative cell therapy or tissue engineering studies it is very complicated and time consuming t o understand and elucidate all the interactions between grafted cells and host brain. Even more, generall y human cells are grafted into rats or mice models involving an immunological component that needs to be taken into account in these models.

Organot ypic brain slices, which can be maintained in culture for several weeks, are commonly used in brain disease research as they provide unique advantages over in vivo and in vitro platforms [15,16]. They largel y preserve tissue structures, maintain neuronal activities and synapse circuitry, and replicate many aspects of the in vivo context [17]. Further advantages of these brain cultures reside in their low -cost, rapid preparation and the use of many brain slices/animal allowing the study of an important number of conditions. Thus, gene functions and pathways can be easil y studied as in an in vitro s ystem.

Recentl y, organot ypic brain slice cultures have been used for HD modeling. Two major techniques can lead to this model. The first one uses organot ypic slices made directl y from transgenic mice expressing HD patterns, such as R6/2 transgenic mice [18,19]. But these models require the use of adult tissue, as mice develop the disease over time. This adult tissue is very difficult to manipulate in ex vivo conditions, the cranium is unbending and the organot ypic slices are not viable over time. The survival of adult brain slices did not exceed 1 week in cult ure with serum [20] and around 3 weeks without serum [21]. Indeed, most brain slice culture systems have used the perinatal rodent brain, taking advantages of its higher viabilit y relative to the older one.

In the second technique, organot ypic slices were prepared from normal rodent s and GABAergic neuron loss was obtained by injecting neurotoxic agents such as quinolic acid or 3-NPA, a mitochondrial inhibitor, added in the media. However, the use of neurotoxins leads to heterogeneit y in the results, which must be taken into consideration. Furthermore onl y the cellular aspect of HD can be studied, in detriment of the genetic component of the pathology [22][23][24]. Another solution is to transfect the slices with HD -pol yQ plasmids or with DNA constr ucts derived from the human pathological HTT gene [25][26][27]. However, slice transfection involves high technology equipment and a skilled operator as org anot ypic brain slices are delicate and frequentl y become damaged during the preparative stages [16]. Notwithstanding these obstacles, the introduced exogenous proteins into both neuronal cells have been consistently difficult to achieve.

In this study, we developed a novel approach to model HD. We developed a coronal organot ypic culture model that includes the main areas involved in HD in a unique slice that does not need neurotoxins to i nduce the GABAergic MSN depopulation. The aim was to induce progressive striatal MSNs degeneration in a single step while preparing the slices, in order to obtain a simple reproducible HD ex vivo model. We first studied and characterized different axis to cut the whole brain to determine the one that induces the degeneration of MSN. Then we explored the viabilit y of this unique slice containing all the areas affected by the disease and characterized the decrease of striatal GABAergic neurons visualized with DARPP32 and GAD67. Our study is the first one describing a cheap, simple and reproducible model of HD without neurotoxin.

PREPARATION OF ORGANOTYPIC SLIDES

Animal care and use were in strict accordance with the regulations of the French ministry of agriculture and all animal procedures were approved by the animal experimentation ethic committee of "Pays de la Loire". Every effort was made to minimize animal suffering and the number of animals used. Timed pregnant Sprague -Dawley rats were purchased from Janvier (St Berthevin, France), or from SCAHU (Service commun d'animalerie hospitalouniversitaire, Universit y of Angers, France). Postnatal 6 to 8 days old pups were

MATERIALS AND METHODS

used to prepare organot ypic slices according to the Stoppini metho d [28] recentl y modified [29] by our team. Before starting the experiment, solution 1 (Table 1) needs to be prepared and kept at 4°C and the membrane has to be hydrated with solution 1 for at least 30 min. Animals were rapidl y sacrificed after intraperitoneal injection of 80 mg/kg of ketamine ( Clorketam 1000, Vetoquirol, Lure, France) and 10 mg/kg of xylazine (Rompum 2 %, Bayer Health Care, Kiel Germany) (Figure 1A). Brain are removed and rapidl y dissected (Figure 1B) before being glued onto the chuck of a water -cooled vibratome. This gesture mu st be quick in order to preserve the brain, and immediatel y immersed in the buffer solution (Table 1) at 4°C (Figure 1C).

Under aseptic conditions, 400 µm slices were cut in different configurations in order to obtain a progressive degeneration of the GABA ergic MSNs. Finall y, cerebellum and olfactory bulbs/prefrontal cortex were cut off and brains were glued, onto the chuck of a water cooled vibratome (Motorized Advance Vibroslice MA752, Campdem instruments). Coronal sections were collected and placed in st erile ice-cold Grey's Salt Balanced Solution (GSBS) (Sigma Aldrich, St Louis, USA) supplemented with 6.5 mg/L of glucose and antibiotics (100 U/mL penicillin, 0.1 mg/mL streptom ycin, 0.25 μg/m L amphotericin B) (Sigma Aldrich, St Louis, USA) for one hour (Table 1).

Slices were cultured in two different media. From days 0 to 3, a serum containing medium was used: 50 % MEM (Minimum Essential Medium Eagle, Sigma Aldrich), 25 % Hank's (Hank's Balanced Salt Solution, Sigma Aldrich), 25 % of horse serum (decompl emented horse serum, Gibco), 6.5 mg/m L of glucose, 1 mM of L-glutamine (Sigma Aldrich, St -Louis, USA) and 1 % of antibiotics (Sigma Aldrich, St -Louis, USA) (Table 2). From days 3 to 18, a serum free medium was used: Neurobasal medium (Gibco, Life Technolog ies, Paisley, UK) supplemented with 6.5 mg/L of glucose, 1 mM of L -glutamine, 1X B27 supplements (Gibco, Life Technologies, Paisley, UK) and 1% of antibiotics (Table 2). The media was changed the first day after of culture and was then renewed every two da ys during the entire culture period. Typicall y, about 10 slices can be obtained per brain. The first two and the last two brain slices did not contain the main areas involved in the pathology and were discarded. Four slices per animals were next transferred to 30 mm diameter semipor ous membrane inserts (Millicell -CM, Millipore, Bedford, MA, USA) within a 6-well plate and incubated at 37ºC and 5 % of CO2. A total of about 20 rat pups and about 80 organot ypic slices were necessary to perform the whole characterization. For each conditi on, a minimum of three slices taken from three different rat pups were used. Description of the media needed during all the organotypic slices culture. These media should be prepared under aseptic conditions. Media should be renewed every 2 or 3 days during culture.

SLICE ANGLE SELECTION TO OBTAIN A HUNTINGTON'S DISEASE MODEL.

Brains were cut with different angles to obtain the most complete DARPP32 and GAD67-positive cell degeneration over time. For this anal ysis, three angles have been chosen: sagittal, coronal and transversal. For sagittal slices, brain hemisphere s were separated and glued onto the chuck of a watercooled vibratome and slices were cut alongside of the midline. Concerning coronal slices, cerebellum and olfactive bulbs/prefrontal cortex were cut off and brains were glued, on their dorsal side, onto t he vibratome chuck. At last, to perform transversal slices, the underside of the brain was glued on the vibratome chuck. For each condition, 400 µm slices were performed with razor blade angle of 14°.

HISTOLOGICAL STUDIES

At different times, ranging from 0 to 19 days, organot ypic slices were washed with phosphate-buffered saline (PBS) (Lonza, Verviers, Belgium), fixed with 4 % paraformaldehyde (PFA) (Sigma -Aldrich, St Louis, MO, USA) in PBS pH 7.4 for 2 h and then washed three times with PBS. Finall y, sli ces were removed from membrane inserts and stored at 4°C in PBS until use.

DARPP32 and GAD 67 Immunohistochemistry

A mouse anti GAD67 antibody (5 µg/mL, clone 1G10.2, Millipore SA, Guyancourt, France) and mouse anti -DARPP32 (250 µg/mL, clone 15, DB science, Le Pont de Claix, France) were used to observe striatal -globus pallidus GABAergic pathway. Slices were incubated 48 h with the primary antibody diluted into PBS, BSA 4 % at 4°C. After washes, slices were incubated with the biotinylated anti -mouse secondary antibody (7.5 µg/mL, Vector Laboratories, Burlingame, USA). Then slices were washed and quenching of peroxidase was performed with 0.3 % H 2 O2 (Sigma, St Louis, USA) in PBS , at RT for 20 min.

After PBS washes slices were incubated with Vectastain ABC r eagent (Vector Laboratories, Burlingame, USA) in PBS at RT for 1 h. Sections were then washed and revealed with 0.03 % H 2 O2, 0.4 mg/m L diaminobenzidine (DAB, Sigma, St Louis, USA) in PBS, 2.5 % nickel chloride (Sigma, St Louis, USA) and dehydrated before m ounting.

Quantification of DARPP32 and GAD 67 positive fibers and cells

GAD67-positive fibers and DARPP32 -positive neurons were quanti fied in the striatum at different time -points, from 0 until 19 days post -lesion, using the Metamorph® software from Molecular Devices. Results were presented as mean differences +/ -average deviation and were ca lculated from 6 independent pictures taken from 4 different rats for each group.

NeuN Immunohistochemistry

An antibody against neuronal nuclei (NeuN) (clone A60, Merck Millipore, Billerica, MA, USA) was used to observe the viabilit y of cells within the bra in organot ypic slices. Slices were incubated 48 h with the primary antibody diluted into PBS BSA 4 % (1/50) at 4°C. After washes, slices were incubated with the biotinylated anti -mouse secondary antibody (7.5 µg/mL, Vector Laboratories, Burlingame, USA). Then slices were washed and quenching of peroxidase was made with 0.3 % H 2 O2 (Sigma, St Louis, USA) in PBS -T, at RT for 20 min.

After PBS washes, incubation with Vectastain ABC reagent (Vector Laboratories, Burlingame, USA) in PBS was performed at RT for 2 h. Sections were washed and revealed with 0.03 % H 2 O2, 0.4 mg/mL diaminobenzidine (DAB, Sigma, St Louis, USA) in PBS, 2.5 % nickel chloride (Sigma, St Louis, USA) and dehydrated before mounting. were presented as mean differences ± average deviation.

Statistical analysis

Data are presented as the mean value of three independent experiments ± standard deviation (SD), unless otherwise stated. Significant differences between samples were determined using an anal ysis of variance (ANOVA) test, followed by a Scheffe post h oc test which indicates if conditions were significantl y different. P-value was set to 0.05, unless otherwise stated.

SLICE ANGLE SELECTION TO OBTAIN A HUNTINGTON'S DISEASE MODEL.

To determine the best model of GABAergic MSN neurodegeneration, mimicking one of the pathological hallmarks of HD, the neonatal brain was cut following three different axis to obtain the organot ypic slices (Figure 2A and2B). With the three types of sectioning planes the striatum, cortex and globuspallidus, the brain areas mainl y affected in HD were visible. The progressive degeneration of MSN was evaluated by DARPP32, expression. We observed that DARPP32 staining was present and observable in the whole striatum independentl y of the slice plane chosen (Figure 2C). Moreover, in horizontal and sagittal slices, DARPP32 positive cells were still highl y present in the striatum after 4 days and even after 7 days of culture (Figure 2C). In coronal sections (Figure 2C), the number of DARPP32 -positive cells was reduced by around 30% after 4 days and up to 70% after 7 days. As a quick degeneration of DARPP32-positive cells was desired, coronal sections were used for the rest of this study. Moreover, the sections can be kept as a whole therefore containing the striatum from the two h emispheres, providing a control in the same slice. If desired, and in order to increase the viabilit y of the slices they can also be cut along the midline. Slice angle selection to model HD. Rat's brain was cut in order to obtain saggital, coronal or horizontal brain slices (A). Which each different angles, we can vizualized all the areas involved in the HD (B) DARPP32 was detected in brain slices obtained by immunofluorescency at day 1, 4 and 7 after sagittal, coronal and horizontal (C) sections.

RESULTS

C

ORGANOTYPIC SLICES MORPHOLOGY AND VIABILITY OF CULTURED NEONATAL BRAIN

SLICES.

Culture conditions remarkabl y affect the organot ypic culture viabilit y. We already described a protocol using 2 specific media with and without serum [29].

Using these conditions, the whole c oronal HD organot ypic slices can be kept viable during at least 3 weeks in culture (Figure 3). We observed a decrease of striatum volume from 4 days onwards and a good conservation of organot ypic slice's morphology during 3 weeks (Figure 3A). No important distortion or flattening of the slices was detected. However, an important thinning of the striatum within 6 days was observed by bright field microscopy, predicting a reduction of viability of the striatal neurons (Figure 3A).

To confirm the viabilit y of organot ypic brain slices, neurons were visualized with an antibody against NeuN and quantified at day 0, 7, 11 and 19.

During the 3 weeks of culture the number of NeuN -positive cells decreased less than 20 % +/-4 % in the cortex and lateral septum compar ed to striatum, (Figure 3C and3D), underscoring the viabilit y of the neuronal cells in these areas.

However, onl y 20 % +/ -5 % of NeuN-positive cells were counted in the striatum, explaining the decrease of volume observed during the culture and confirming the neurodegeneration (Figure 3).

ORGANOTYPIC SLIDES CULTURES DISPLAY PROGRESSIVE DEGENERATION OF

GABAERGIC PATHWAY

In order to demonstrate the degeneration of medium spiny n eurons in the striatum, we followed by immunohistochemistry the progressive decrease of DARPP32 (Figure 4A) and GAD67 (Figure 4B), which are markers of GABAergic MSNs. These markers were perfectl y specific because we didn't find any positive cells in the c ortex (Figure 4A and4B). The number of DARPP32positive neurons and GAD67 -positive neurons decreased over time with around a 50% decrease during the first week when compared to day 0 (Figure 4A and4B). A quantification of the GABAergic marker densit y GAD 67 shows a progressive loss of 30 % at day 4, 45 % at day 7 and 70 % at day 11 when compared to day 0. It was almost complete by day 19 with an average of onl y 1 % staining left. However, the staining intensit y varied between slices due to the tissue damage (Figure 4D). Indeed, a more dramatic loss of DARPP32 staining was detected over time with an average of 25 % decrease at day 2, 50 % at day 4 and 75% at day 7 compared to day 0, and was complete by day 19 (Figure 4C).

Even though the average loss of DARP P32 staining was a bit more rapid the profile of these 2 graphs are similar, confirming the progressive loss of GABAergic neurons in the organot ypic slices. were obtained from 6 to 8 day old pups It is well known and established that tissue from embryonic or post natal donors survive better [30,31]. Slices postnatal donors is recommended because at this age, brain slices will conserved a better morphology, an increase survival and we will obtain a more stable models regarding to the lesions. Animals were sacrificed betwe en days 6 to 8 which provides a perfect time window for establishing brain slice cultures on membranes. More particularl y, for Parkinson's disease the slices are principall y harvested between 4 and 9 weeks after birth [32,33].

Hypothermia has been shown to improve the preservation of hippocampus brain slices [34][35][36]. In our study, in order to preserve brain slices we insured that the brain was kept at 4°C in solution buffer containing high percentage of glucose during the whole slice preparation process. We chose not to place the head in ethanol 70 % bath as some teams have reported [31] in order to maintain physiological conditions of organot ypic brain slices. Within the same line, the media containing horse serum was onl y used during the first 3 days even though horse serum diminishes tissue flattening, and promotes survival of neurons, astroglia or microglia in organot ypic brain slices [37]. In order to diminish the variabilit y between slices due to changes in serum batches we then used a serum -free media containing glucose, glutamine, antibiotics and B-27 ® . Indeed, serum increased the degeneration of neurons in brain slices when the media contain seru m for a long-time period [21]. In a previous study, we confirmed the benefice of using horse serum during a short period followed by "s ynthetic" media and accord ing to these results, we applied this method to culture organot ypic brain slices modeling HD. In this way, we are able to maintain the viability of brain ex vivo for at least 3 weeks, and obtained the HD model in 5 days. Other studies demonstrated the ben efit to culture organot ypic slices without serum such as our study, and brain slices from transgenic mouse or rat brain slice with injection of neurotoxins were principall y cultured with artificial cerebrospinal fluid or specific media containing HEPES (( 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid ) [38][39][40].

This organot ypic model involves GABAergic MSNs degeneration due to mechanical cutting of tissue between the striatum and substantia nigra, which leads to retrograde and anterograde neuronal degeneration in the substantia nigra and in the striatum, respectivel y. In this report, a complete characterization of the behavior of MSNs and GABAergic pathway was assessed by immunostaining through the visualization of the decrease of DARPP32 and GAD67, two specific markers of MSNs [5]. It revealed that a rapid loss of DARPP32-positive neurons occurred in the striatum followed by a decrease in GAD67 staining. A decrease of 50% and 30% of the DARPP32 and the GAD67 positive cells respectivel y was detected 4 days after slice preparation compared to day 0. This allows the development of an earl y model of th e disease, in which a window of time of at least 15 days was obtained to study new therapeutic approaches. One important reason to culture brain slices is to study and characterize the interaction between neuronal populations. The coronal section allows studying those parameters, thanks to the observation of the globus pallidus, the striatum and all the structures involved in the HD in one slice. We can easil y imagine testing different molecules with therapeutic effect such as growth factors directly onto s lices or in the media and observe their effect in all the MSNs afferents and projections [41,42]. [43]. Indeed, with the same method, we developed an innovative model of PD and we were able to evaluate new therapeutic approaches combining stem cells and biomaterials releasing growth factors in order to treat the disease or to reduce the related symptoms. [29] In summary, this model represents a promising tool to quickl y and efficientl y test innovating treatment in the HD such as regenerative medicine with stem cells and biomaterials. The development of simple an d non expensive model with precise characterization of neurodegeneration could allow to easil y study the therapeutic effect of treatment but also the role of microenvironment in the response of the treatment.

The use of organot ypic slices allowed the development of a new simple model of Huntington's disease. Indeed, GABAergic MSNs degeneration is observed after mechanical cutting necessary to obtain brain slices. In this way, no other supplementation with neurotoxins, quinoleic acid or 3 -NPA is needed for the progressive loss of striatal neurons, which reduces the risk of heterogeneous results. Usuall y, onl y some areas such as the striatum, the hippocampus or cortex and striatum are used in ex vivo models of HD [42] [ 44,52] Here we have all the regions involved in HD, the cortico -striatal-globus pallidus area of one hemisphere or of both hemisphere s allowing to use one side as control.

Nonetheless, the full characterization of this model is required. Indeed, we followed NeuN and GABAergic markers for onl y 19 days. We should know how much time brain slices survive and understand exactl y the mechanism of the decrease of DARPP32 and GAD67. DARPP-32 is a fundamental component of the dopamine-signaling cascade, and its expression is essential to the abilit y of dopamine to regulate the physiology of striatal neurons . In our study, the decrease of GAD67 is progressive and comparable to NeuN while DARPP32 decreased drasticall y from day 5. The results presented here suggest a selective impairment in several aspects of dopaminergic signaling. Indeed, dopaminergic signaling has been already described in the lit erature to be involved in pres ymptomatic defects and contribute to HD pathology [START_REF] Bibb | Severe deficiencies in dopamine signaling in presymptomatic Huntington's disease mice[END_REF] , and DARPP32 is the first marker decreasing. DARPP32 is also a mediator of signaling cascades in dopaminoceptive neurons which corroborate this hypothesis. So, our model seems to be severely impaired in GABAergic neurons' abilit y to regulate the physiological state of striatal neurons via dopamine. To confirm our hypothesis, we can imagine checking the dopamine -regulated ion channels by electrophysiological studies and determine if a reduction in the striat al-specific dopamine targets, PKA, DARPP -32, ARPP-16, and ARPP-21 is associated, with RT-qPCR or immunofluorescence.

DISCUSSION

The aim of this study has been to develop a simple model to screen potential treatment including cell replacement with MIAMI cells combined with PAMs. This model allows the development of an earl y model of the disease , in which a window of time of at least 7 days was obtained if we considered DARPP32 and 14 days if we considered GAD67 . The timing differences are evidenced when comparing this model with those in which organot ypic brain slices are cultured during 3 week s followed by the addition of neurotoxins in the media in order to mimic HD [44][45][46][47]. Thus, stem cell transplants coupled or not with MPA in this model enable the evaluation of the survival and differentiation of grafted cells and the study of the behavior of the host tissue in particular related to the evaluation of the secret ion of neurotrophic factors by stem cells that can be made during 14 days. We can also easil y imagine testing different molecules with therapeutic effect such as growth factors directl y onto slices or in the media and observe their effect in all the MSNs a fferents and projections [START_REF] Riley | A peptide preparation protects cells in organotypic brain slices against cell death after glutamate intoxication[END_REF][START_REF] Lynch | Brain-derived neurotrophic factor restores synaptic plasticity in a knock-in mouse model of Huntington's disease[END_REF]. In summarize, our model is so much faster, simpler and less expensive than the in vivo model.

Transplantation of stem cells f or the treatment of HD garnered much attention prior to the turn of the century. Several studies using MSCs have indicated that these cells have enormous therapeutic potentia l in this disease [START_REF] Lee | Slowed progression in models of huntington disease by adipose stem cell transplantation[END_REF][START_REF] Snyder | Human multipotent stromal cells (MSCs) increase neurogenesis and decrease atrophy of the striatum in a transgenic mouse model for Huntington's disease[END_REF]. In the case of HD, t he beneficial effect of stem cell transplantation should be provided by the generation of new graft -derived neurons and by the secretome of these cells [START_REF] Drago | The stem cell secretome and its role in brain repair[END_REF]. MSCs are not onl y able to differentiate into neuron-like cells but they also exert paracrine effects by modulati ng the plasticit y of damaged host tissues.

The trans-differentiation of MSCs into neural lineage has been achieved in vitro by culturing them with specific media. This media is often composed of growth factors or small molecules involved in neuronal linea ge differentiation.

The degree to which the cells will differentiate varies depending on the protocol and cells used [START_REF] Croft | Mesenchymal stem cells expressing neural antigens instruct a neurogenic cell fate on neural stem cells[END_REF]79,[START_REF] Huat | IGF-1 enhances cell proliferation and survival during early differentiation of mesenchymal stem cells to neural progenitor-like cells[END_REF] . In all cases, although the MSCs were committed to neuronal linage, the functionalit y at the end of the process was still lacking.

Attempting to understand how to improve the di fferentiation, REST also known as neural-restrictive silencing factor (NRSF) has been proposed as it functions as a master negative regulator of neurogenesis [START_REF] Schoenherr | The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes[END_REF]. REST is a zinc finger transcriptional repressor able to bound the RE -1 sequence in his target gene promoter which allows recruiting histone deacet ylases and methyltransferase to modulate the chromatin structure [START_REF] Ballas | The many faces of REST oversee epigenetic programming of neuronal genes[END_REF][START_REF] Ballas | REST and Its Corepressors Mediate Plasticity of Neuronal Gene Chromatin throughout Neurogenesis[END_REF]

INTRODUCTION

(Introduction and aims). We focused on two innovative systems: lipid nanocapsules and solid span nanoparticles.

Concerning LNCs, previous studies demonstrated the possibilit y to encapsulate plasmid DNA to develop a gene therapy strategy [116]. For this, theDNA is complexed with cationic lipids by electrostatic interactions leading to formation of complexes called lipoplexes , which are added to other components of the LNC. This strategy has demonstrated the capacit y of LNC s to transfect in vivo models of gliobastoma [117][118][119]. Strong of these results, the encapsulation of siRNA in LNCs was successfull y performed [START_REF] David | siRNA LNCs -A novel platform of lipid nanocapsules for systemic siRNA administration[END_REF] and this system was able to transfect melanoma and glioma cells in vitro [START_REF] Resnier | EGFR siRNA lipid nanocapsules efficiently transfect glioma cells in vitro[END_REF][START_REF] Resnier | Efficient in vitro gene therapy with PEG siRNA lipid nanocapsules for passive targeting strategy in melanoma[END_REF].

Nonetheless, the instabilit y over-time of this system needed to be improved. In this way, the optimization of LNC -siRNA had to be performed with two goals: improving the stabilit y over time of LNCs and protecting the siRNA from denaturation. In any case this system provides a p ositive surface, which can easil y interact with negativel y charged cell membranes. Although it is well known that nanoparticles with a positive surface charge transfect very well, the risk of toxicit y is higher with those systems. However, recent evidence proves that anionic nanocarriers can also be effectivel y internalized [START_REF] Resina | Physico-Chemical Characteristics of Lipoplexes Influence Cell Uptake Mechanisms and Transfection Efficacy[END_REF], and that the transfection efficiency is not onl y dependent on the surface charge but also on the particle size and composition [START_REF] Thierry | Characterization of liposomemediated gene delivery: expression, stability and pharmacokinetics of plasmid DNA[END_REF][START_REF] Migliore | Nanomaterials and neurodegeneration: Nanomaterials and Neurodegeneration[END_REF].

So, to determine the optimal nanocarrier to transfect MSCs, we chose a second s ystem: Solid span nanoparticles (SP) to compare with LNCs. SP based on sorbitan monooleate (Span® 80) have been specificall y adapted to provide effective siRNA association . Previous studies demonstrated the efficiency of SP to associate DNA by incorporating to their composition positivel y charged molecules like oleylamine (OA ) [START_REF] Martini | Effect of cationic lipid composition on properties of oligonucleotide/emulsion complexes: Physico-chemical and release studies[END_REF]. This fatt y amine is employed in the creation of emulsions and other delivery systems [START_REF] Borgheti-Cardoso | Self-assembling gelling formulation based on a crystalline-phase liquid as a non-viral vector for siRNA delivery[END_REF]. Pol y-L-arginine (PA), a cationic moiet y broadl y used in gene therapy approaches [START_REF] Tan | Layer-by-layer nanoparticles as an efficient siRNA delivery vehicle for SPARC silencing[END_REF] was also evaluated. OA and PA were used to provide a positive surface charge for the SP nanoparticles, thus enabling the association with the negatively charged , nucleic acids thus resulting in Span® 80-oleylamine nanosystems (SP -OA) or Span® 80pol y-L-arginine nanosystems (SP -PA), with a characteristic net negative surface charge [122].Their capacit y to provide higher in vivo transfection levels than adeno-associated viral vectors (AAV2) has been recentl y reported [123]. In this work, we conserved SP-PA to transfect MS Cs and further modified the SP nanoparticles in order to coat them with cationized pullulan (AP), a natural pol ysaccharide obtaining SP-AP, as a strategy to easil y associate siRNA by electrostatic interaction [START_REF] Cheng | Pullulan: biosynthesis, production, and applications[END_REF].

In this chapter, to improve human stem cell transfection and efficiency control of the stem cell fate, we have designed, optimized, characterized and evaluated two different siRNA nanocarriers transpo rting siR NA.

Accepted in International journal of Pharmaceutic s

ABSTRACT:

To direct stem cell fate, a delicate control of gene expression through small interference RNA (siRNA) is emerging as a new and safe promising strategy. In this way, the expression of proteins hindering neuronal commitment may be transientl y inhibited thus driving differentiation. Mesenchymal stem cells (MSC), which secrete tissue repair factors, possess immunomodulatory properties and may differentiate towards the neuronal lineage, are a promising cell source for cell therapy studies in the central nervous system. To better drive their neuronal comm itment the repressor element -1 silencing transcription (REST) factor, may be inhibited by siRNA technology. The design of novel nanoparticles (NP) capable of safel y delivering nucleic acids is crucial in order to successfull y develop this strategy. In this study we developed and characterized two different siRNA NP. On one hand, sorbitan monooleate (Span ® 80) based NP incorporating the cationic components pol y -L-arginine or cationized pullulan, thus allowing the association of siRNA were designed. These NP presented a small size (205 nm) and a negative surface charge ( -38 mV). On the other hand, lipid nanocapsules (LNC) associating pol ymers with lipids and allowing encapsulation of siRNA complexed with lipoplexes were also developed. Their size was of 82 nm w ith a positive surface charge of +7 mV. Both NP could be frozen with appropriate cryoprotectors. C ytotoxicit y and transfection efficiency at different siRNA doses were monitored by evaluating REST expression. An inhibition of around 60% of REST expression was observed with both NP when associating 250 ng/mL of siRNA -REST, as recommended for commercial reagents. Span NP were less toxic for human MSCs than LNCs, but although both NP showed a similar inhibition of REST over time and the induction of neuronal c ommitment, LNC -siREST induced a higher expression of neuronal markers. Therefore, two different tailored siRNA NP offering great potential for human stem cell differentiation have been developed, encouraging the pursuit of further in vitro and in vivo in studies.

GRAPHICAL ABSTRACT:

Nanocarriers, siRNA, mesenchymal stem cells, neuronal differentiation, REST.

MSC: mesenchymal stem cells; siRNA: small interfering RNA; SP: Span Mesenchymal stem cells (MSC) are multipotent stromal cells, capable of self-renewal and able to differentiate into a diverse set of cells within the mesoderm lineage [1]. They can also be an interesting source of cells for brain regenerative medicine because they secrete tissue repair factors, possess immunomodulatory properties and, in some conditions, may differentiate towards the neuronal lineage [2][3][4]. In order to safel y implant them in the brain parenchyma th ey should be pre-committed to a neuronal phenot ype. However, protocols onl y using external growth factors to drive MSCs into specific lineages seem to be insufficient to obtain high percentages of differentiated cells [5,6]. A lentiviral-mediated RNA interference vector that down -regulates the expression of REST/NRSF (Repressor Element -1 silencing Transcription /Neuron-Restrictive Silencing Factor -1) has been recentl y used to promote MSC differentiation into neuronal cells, which exhibited neuron -like morphology and function [7]. Indeed, REST/NRSF is a repressor transcription factor functioning as a master negative regulator of neurogenesis by binding to a specific DNA domain named RE1 motif [8,9]. In non-neural cells and in pluripotent stem cells, REST is abundant and represses neuronal genes [10] Consequentl y, the loss of REST in neural progenitors resulted in an acceleration of neuronal and oligodendrocyte differentiation [11].

KEYWORDS ABBREVIATIONS

INTRODUCTION

Nowadays, a transitory inhibition of targeted gene expression in cells may be obtained by small interference RNA s (siRNA) without affecting the genome.

These siRNA are able to produce a partial or total extinction of targeted messenger RNA (mRNA) and protein levels by degrading the mRNA. This epigenetic modification is quite simple, does no t need genetic manipulation, is transitory and is now quite well understood. Although current applications in stem cells remain largel y restricted to studies on molecular pathways and signaling, siRNA can be also used as a biomedical strategy to improve su rvival, direct lineage-specific differentiation of stem cells or help maintain a desired phenot ype [12,13]. Within this line, the inhibition of Noggin with siRNA was capable to induce new bone formation in vivo [14]. But the mode of delivery of these novel therapeutic agents is of crucial importance for an efficient ex vivo or in vivo therapeutic strategy.

In the last decade, two different approaches for siRNA delivery have been developed: vi ral and non -viral vectors. Non -viral vectors (also named nanoparticles (NP) or nanocarriers) have been formulated to associate and to efficientl y and safely deliver nucleic acids both in vitro and in vivo, opening many possible applications. In addition, t hey have many advantages and potentialities, including large -scale manufacture, low toxicit y and low immunogenicit y, and the possibilit y to customize them to target specific cell types [7,8]. Initiall y, NPs were designed to have a pos itive surface charge, in order to improve the interaction with the cell membrane that has a negative surface, and to avoid electrostatic repulsion [9]. However, recent evidence proves that anionic NPs can also be effectivel y internali zed [15], and that the transfection efficiency is not onl y dependent on the surface charge but also on the particle size and co mposition [16,17] . In this frame, our research group has reported a novel NP based on sorbitan monooleate (Span ® 80) (SP) that can be prepared using a simple, one -step and easil y scalable procedure [18]. SP is a component widel y used in the pharmaceutical industry (generall y recognized as safe (GRAS)) due to its non -ionic surfactant properties at low concentrations [19]. These NPs are able to transfect in vivo the retinal pigment epithelial cells with a similar efficiency as adeno -associated viral vectors [20] Transfection techniques currentl y use packaging particles such as liposomes to facilitate the cellular uptake of RNAi. They have s hown high biocompatibilit y and are available in the market such as lipofectamine ® , oligofectamine ® , ready to be used. However, they exhibit some disadvantages such as high positive zeta potentials destabilising cell membrane, low transfection efficiencies in human stem cells, and instabilit y immediatel y after complexation [21]. Moreover, transitional action reported with these liposomal carriers is not sufficient on their own to have an ef fect over-time. In vitro liposome transfection efficiency is also affected by the t ype of culture media and cell plating densit y, and does not provide an efficient protection against lysosomal nucleic acid degradation [22]. Lipid nanocapsules (LNCs) consisting of a lipid liquid core of trigl ycerides and a rigid shell of lecithin and pol yethylene gl ycol hydrox ystearate, are formulated by a simple and easil y industrialized solvent -free process based on the phase inversion of an emulsion [23,24]. They allow entrapping the liposomes within their core and were thus recentl y modified to encapsulate the siRNA complexed with DOTAP (1,2dioleyl-3-trimethylammoniumpropane) and DOPE (1,2 -dioleyl-sn-gl ycero-3phosphoethanolamine), protecting the siRNA from degradation [25]. DOTAP lipids presenting a cationic charge, allow the fixation of siRNA by electrostatic force and the interaction with the cell membrane improving the cellular uptake [26][27][28]. Moreover, DOTAP and DOP E are able to destabilize l ysosome's membranes by a proton sponge effect [29]. However, the stabilit y of these LNCs should be improved.

The transfection efficiency of the siRNA delivered by NP is often affected by the proliferative activit y of human stem cells, and varies widel y depending on the t ype of target cells [30]. To ameliorate the transfection of human stem cells and efficientl y induce a neuronal -like differentiation, we have designed a novel SP-based NP conveyi ng REST and further developed LNCs incorporating siREST with a good stabilit y over time. [20]. First, we have modified SP NPs with cationized pullulan (AP) as a strategy to easil y associate siRNA by electrostatic interaction thus obtaining a novel NP. Pullulan is a non -toxic natural pol ysaccharide presenting non -immunogenic properties [31]. This FDA approved component possesses a wide range of industrial applications including health care and pharmacy [31]. Secondl y, we further developed LNCs, and evaluated different formulations of LNCs incorporating siREST in order to obtain a better stabilit y. The siREST -NPs were characterized by dynamic light scattering, UV spectrophotometry and electron microscopic methods in order to improve the understanding of their organization and structure. After evaluation of the long-term storage and stabilit y of siRNA -NPs over time, we tested the cytotoxicit y of both systems on human (h)MSCs and, finall y, we evaluated their abilit y to transfect hMSCs and drive the commitment of hMSCs towards a neuronal phenot ype determined by expression of neuronal genes.

FORMULATION

Synthesis of pullulan derivate :

Pullulan was chemicall y modified in order to obtain a positiv el y charged derivative (ammonium pullulan, AP) by alkylation of the original pol ymer.

Synthesis was performed based on a previousl y described methodology [32]: an aqueous solution (5 mL) of KOH (0.504g, 9 eq), was prepared in a round bottom flask, under stirring, at 60 ºC, to which pullulan (500 mg) and 3.6 2 mL (4.09 g, 27 eq) of GTMAC were added. After 5 h, an equal amount of GTMAC was added to the mixture, which was allowed to react until the completion of 24 h. It was then diluted with 10 mL of miliQ water, allowed to cool down to room temperature, and neutralized with HCl (2M). The resulting solution was dial yzed for 3 days, the water being replaced every 24 h, and the modified pol ymer recovered by freeze drying.

SP-PA and SP-AP nanoparticles associated with siRNA

To produce the SP nanoparticles, a solut ion of 6.6 mg/ml of sorbitan monooleate (Span ® 80) (Sigma, Saint-Quentin Fallavier, France) in ethanol (organic phase) was prepared and it was subsequentl y added under magnetic stirring to an aqueous phase, leading to the spontaneous nanoparticle formation [18]. To produce SP -PA and SP -AP nanoparticles, Pol y-L-arginine (PA) or ammonium pullulan (AP) were dissolved in Mili -Q water at 0.16 mg/ml. Finall y, ethanol was removed under reduced pressure on a rotary evaporator and the nanoparticles were concentrated to a final volume of 10 ml. Nanopar ticles were isolated by filtration -centrifugation (Amicon  Ultra-0.5 centrifugal filter Devices, Merck Millipore, Ireland). The genetic materials (siRNA REST and Scramble) were associated to the nanoparticles' surface at different concentrations (from 0.1m g/ml to 0.5mg/ml) by incubation with nanoparticles at a 1:1 (v:v) ratio (100 -100 µl) under magnetic starring at room temperature during 2 hours in order to obtain siRNA -SP-AP and siRNA-SP-PA nanoparticles.

MATERIALS AND METHODS

Liposomes and lipoplexes

For liposome preparation, a cationic lipid DOTAP ( Lipoplexes were formulated by simple equivolumar mix of siRNA and liposomes. This complex is characterized by the charge ratio [27] corresponding to the ratio between positive charge of lipids and negative charge of nucleic acids at 5.

siRNA-LNCs

LNCs were formulated, as described previousl y [23] To obtain siRNA LNCs, the water introduced at the last P IT was replaced by lipoplexes, i.e. REST siRNA: (sense sequence: 5′ -CAG-AGU-UCA-CAG-UGC-UAA-GAA -3′; Eurogentec, Seraing, Belgium) and control (scrambled) siRNA (sense sequence: 5′-UCUACGAGGCACGAGACUU -3′; Eurogentec, Seraing, Belgium) complexed with cationic liposomes in a defined charge ratio as described above.

To obtain siRNA LNCs, the water introduced at the last P IT was replaced by lipoplexes, i.e. REST siRNA : (sense sequence: 5′-CAG-AGU-UCA-CAG-UGC-UAA-GAA -3′; Eurogentec, Seraing, Belgium) and control (scrambled) siRNA (sense sequence: 5′-UCUACGAGGCACGAGACUU -3′; Eurogentec, Seraing, Belgium) complexed with cationic liposomes in a defined charge ratio as described abo ve.

To avoid the possible denaturation of siRNA by the high temperatures, the addition of lipoplexes at 75°C or 40°C was tested within the classical formulation. Two other formulations were performed, one with reduce PIT by increasing the NaCl concentratio n to 8% in the formulation and the last one without lipoid ® .

CHARACTERIZATION OF BOTH NANOPARTICLES

Size, zeta potential, transmission electron microscopy and cryo-transmission electron microscopy

Size and zeta potential of LNCs and SP were measured by D ynamic Light Scattering (DLS) method using a Malvern Zetasizer ® apparatus (Nano Series ZS, Malvern Instruments S.A., Worcestershire, UK) at 25 °C, in triplicate, after dilution in a ratio of 1:100 with deionized water. These parameters were followed dail y during one month.

The morphological examination of siRNA -SP-AP was conducted by transmission electron microscopy (TEM) (CM 12 Philips, Eindhoven, The Netherlands). Samples were placed on copper grids with carbon films (400 mesh) (Ted Pella, USA) and staine d with 2% (w/v) phosphotungstic acid solution for TEM observation.

The morphological examination of siRNA -LNC for cryo -TEM imaging were prepared using a cryo -plunge cryo-fixation device (Gatan, Pleasanton USA) in which a drop of the aqueous suspension was deposited on a carbon -coated grid (Ted Pella Inc., Redding, USA). The TEM grid was then prepared by blotting the drop containing the specimen so that a thin liquid layer remained across the holes of the carbon support film. The liquid film was vitrified by rapidl y plunging the grid into liquid ethane cooled by liquid nitrogen. The vitrified specimens were mounted in a Gatan 910 specimen holder (Gatan, Pleasanton, USA), which was inserted into the microscope using a CT -3500-cryotransfer s ystem (Gatan, Pleasanton, USA), and cooled with liquid nitrogen. TEM images were obtained from specimens preserved in vitreous ice and suspended across a hole in the supporting carbon substrate

Encapsulation efficiency (EE)

Qualitative BET electrophoresis detection -The EE and the integrit y of siRNA molecules after the process of nanoparticle formulation were evaluated by agarose gel electrophoresis. A volume of nanoparticle suspension equivalent to 2.5 μg of siRNA before and after treatment with Triton® X100 (Sigma, Saint -Quentin Fallavier, France) was mixed with gel -loading solution (Sigma, Saint -Quentin Fallavier, France) and disposed in each well of 1% agarose gel containing ethidium bromide (Sigma, Saint -Quentin Fallavier, France) [12].

Free siRNA in solution corresponding to 2.5 μg constituted the controls.

Samples were let to migrate 40 min at 50 V and 10 min at 90V in a Tris -EDTA buffer (Sigma, Saint -Quentin Fallavier, France) .

Quantitative UV detection -A spectrophotometric method based on the study recentl y reported by David et al., (2012), was used to evaluate the EE for LNC. Briefl y, siRNA LNCs were mixed with chloroform and water to separate respectivel y hydrophilic and lipophilic components, sodium hydroxide was added to destabilize lipoplexes and finall y absolute ethanol to destroy LNCs [28]. After multiple centrifugations, four compartments were obtained: free siRNA, free lipoplexes (i.e. siRNA associated with liposomes), encapsulated siRNA and encapsulated lipoplexes within LNCs.

siRNA and SP -AP were separated from supernatant using Nanosep® Omega 30 kD microcentrifuge filters (Pall Corporation, Ann Arbor, USA) and siRNA was measured in the supernatant. To determine the concentration of siRNA, optical densit y of each sample was determined at 260 nm by UV spectrophotometer (UV -2600, Shimadzu, Noisiel, France) in triplicate conditions. The EE was determined by dividing the experimental drug loading by the theoretical drug loading. EE was followed every week during one month by these two methods.

Encapsulated siRNA stability

In order to determine the stabilit y of the encapsulated siRNA during the transfection, a spectrophotometric method based on the study recentl y reported by David et al., (2012), was used to evaluate the encapsulated si -RNA of LNCs.

Briefl y, siRNA LNCs were dissolved at a ratio of 1:10 in Opti -MEM ® media (Life technologies, France) to mimic the transfection condition at two temperatures: 4°C (to mimic the storage condition) and 3 7°C (to mimic transfection condition in the incubator). Methods used to separate the different phases were described earlier. The encapsulated siRNA was determined by dividing the experimental drug loading by the theoretical drug loading and taking into account the dilution. It was followed at different times: 0, 4, 8, and 12 h after formulation. To confirm results, the integrit y of siRNA was evaluated by agarose gel electrophoresis in the same conditions.

Freezing

The long-term storage assessment of siRNA -LNCs and siRNA -SP-AP was carried out by freezing at -20°C. The siRNA -LNCs and siRNA-SP-AP were frozen with phosphate buffered saline (PBS) (control solution) or with an aqueous solution of trehalose or of glucose as cryoprotectants in a 1:1 (v/v) ratio nanoparticles:cryoprotectants with a range from 1% to 15%. They were then anal yzed after 1 -month storage at -20°C by evaluating size and zeta potential measures by Dynamic Light Scattering (DLS) method using a Malvern Zetasizer® apparatus (Nano Series ZS, Ma lvern Instruments S.A., Worcestershire, UK) at 25 °C, in triplicate, after dilution in a ratio of 1:100 with deionized water. The integrit y of siRNA molecules after storage at -20°C of nanoparticle formulations was evaluated by agarose gel electrophoresis.

MENSENCHYMAL STEM CELL (MSC) ISOLATION AND EXPANSION:

Whole bone marrow was obtained from the iliac crest of 15 -yr-old (#34984) living male donor (Lonza, Walkersville, MD). Cells were seeded with -MEM : BE 12169F (LONZA) supplemented with 10% FBS, L -Glutamine 2mM (Sigmaaldrich, France, G7513) FGF 1 ng/ml, Heparin 5 µg/ml, 1% penicillinstreptom ycin (Sigma, Saint -Quentin Fallavier, France). The cells were maintained at 37°C with 5% CO 2 humidified atmosphere. Passage numbers between 5-7 were used for the following experiments.

MSC TRANSFECTION

Experiments were performed in 1mL of MSC Opti -MEM ® media (Life technologies, France). Oligofectamine ® (Life technologies, France), which served as a positive control, was used with 100 ng or 250 ng of siRNA. SiRNA -LNCs were filtered with a 0.2 μm filter (Acrodisc PALL GHP, VWR, Radnor, USA). siRNA-LNCs and siRNA -SP-AP were incubated with cells at 37 °C in a humidified atmosphere with 5 % CO 2 for 4h before serum addition, following Oligofectamine-siRNA protocol. Cells were harvested after appropr iate time and assayed for mRNA expression levels by RT -qPCR and protein expression levels by western blot.

IN VITRO VIABILITY:

Cytotoxicit y assays were performed using MTS For the AlamarBlue assay, 10% AlamarBlue assay was added in RPMI media without serum and phenol red. After 4h of incubation, the fluorescence was read using a SpectraMax fluorescence multi -well plate reader (Molecular Devices, Sunnyvale, CA) at 530/590 excitation/emission wavelengths. Three replicates were used for each treatment.

Furthermore, cell numbers were also counted after each transfection performed. Fort y-eight hours after transfection 200.000 cells were resuspended by trypsin (0.5 g porcin trypsin, 0.2 g EDTA; Sigma, Saint Louis, USA), washed twice with PBS and trypan blue staining was used to count the number of cells per well.

RNA EXTRACTION, RT-QPCR :

These experiments were performed following the guidelines of th e PACEM core facilit y ("Plate-forme d'Anal yse Cellulaire et Moléculaire", Angers, France). Sense and antisense desalted primer pairs (Eurofins MWG Operon, Ebersberg, Germany) were mixed in RNAse free water at a final concentration of 5 µM (Table 1). Total RNA of cells were extracted and purified using , where E is related to the primer efficiency (E=2 if the primer efficiency=100%). Relative quantities (Q) were normalized using the multiple normalization method described in Vandesompele et al [33]. Q normalized=Q/(geometric mean of the three most stable housekeeping genes Q).

The 2(-ΔΔCt) method was retained, using the housekeeping genes and gene of interest tested on control sample and treated sample [34]. 440CF. The image anal ysis was performed using the Kodak 1D 3.5 software (27). To confirm that equal amounts of protein were lo aded membranes were incubated with anti-β actin (Sigma-Aldrich, Ital y) and revealed in the same way.

Gene

STATISTICAL ANALYSIS:

Three independent biological replicates were performed for all experiments described in this paper. Comparisons between all groups, supposed with normal distribution, were performed using a classical anal ysis of variance (one -way ANOVA) followed by a Tukey's post-hoc anal ysis. The encapsulation efficiencies for modified LNCs and non -modified LNC were compared using a ttest. Statistical significance was ascribed to a threshold p -value of 0.05 (*p≤ 0.05; **p≤ 0.01; ***p≤ 0.001).

SP NANOPARTICLES: SELECTION OF SURFACE COMPOSITION AND SIRNA DOSE.

Using SP we can spontaneousl y form negativel y charged nanoparticles with a size of ab out 140 nm and a zeta-potential of -17 mV, being these nanoparticles stable during at least 1 month (Figures 1A and1B). However, the negative surface charge of this nanocarrier renders a stable interaction with the negativel y charged siRNA difficult to a ccomplish. In order to modify the surface charge of these SP nanoparticles and to easil y associate the siRNA to the nanoparticle surface we evaluated the effect of incorporating on their composition two different cationic molecules, pol y -L-ariginine (PA) a nd ammonium pullulan (AP) to the SP formulation. The developed SP -PA and SP-AP blank nanoparticles (no siRNA associated) showed a nanoparticle size of 180 nm and 140 nm, respectivel y, which did not vary much from the SP nanoparticles. They also presented a ppropriate positive surface charges of

RESULTS

+40mV and +38mV, respectivel y (Figure 1B). No change in terms of size or zeta potential were observed when NPs were conserved for 1 month at 4°C

(Figures 1A and1B). Once developed, we complexed each system with the siRNA-scramble and the siRNA REST by simpl y mixing them together. Similar sizes and zeta-potentials were observed with both siRNAs. At day zero, the increase in size and the decrease on surface charge for siRNA -SP-PA NPs (280 nm and -37 mV) and siRNA-SP-AP NPs (205 nm and -38mV) (Figures 1A and1B) indicated an effective electrostatic interaction between siRNA and the nanoparticles. On the contrary, when combining AP and siRNA to form AP -siRNA complexes as a control formulation a broad -range particle size distribution was observed, being characterized by the presence of aggregates rather than nanosystems. We also performed a stabilit y study with siRNA -SP-PA and siRNA-SP-AP during 30 days. siRNA -SP-AP and siRNA-SP-PA were both perfectl y stable in terms of size and in surface charge (Figures 1A and1B).

We selected SP -AP for the rest of experiments because pullulan is a non -toxic FDA approved natural pol ysaccharide presenting non -immunogenic properties with very interesting characteristics. Indeed, pullulan may be chemicall y modified in order to afford either hydrophobized or cationized derivatives, the former with the abilit y to carry hydrophobic molecules, and the latter showing high affinit y towards DNA and RNA [35].

To determine the most favorable siRNA co ncentration capable of interacting with these NPs, we have tested different concentrations of siRNA ranging from 0.1 mg/ml to 0.5 mg/ml and checked the size and zeta -potential for each one. Similar particle sizes were obtained in this concentration range (around 190 nm). However, the surface charge decreased when the siRNA concentration increased, demonstrating the high capacit y of AP to complex siRNA on the surface. In this case, we therefore speak of association efficiency.

Above 0.3mg/ml of siRNA, we obs erved a stagnation of the surface charge ( -45 mV) (Figure 1C) revealing a saturation of the system. This observation was confirmed by electrophoresis experiments performed to further assess the siRNA association abilit y of the developed SP -AP NPs. The absence of the t ypical bands of free siRNA in the formulations of NPs incubated with 0.1 and 0.3 mg/ml siRNA corroborated the effective association to the nanosystems in this concentration range (Figure 1D). At 0.3 mg/ml of siRNA the efficiency of association with the SP-AP was 33 % +/-6 and we selected this dose for the subsequent studies.

OPTIMISATION OF SIRNA-LNC

Initiall y, during siRNA -LNC formulation lipoplexes were added at the beginning of the last phase inversion temperature (PIT) (75°C), but an instabilit y of the system has been observed [33]. To improve the stabilit y and to avoid the possible denaturation of siRNA by the high temperatures this initial formulation was further modified. First, we increased the NaCl concentration to 8% thus obtaining a PIT at 60°C. In a second experime nt, a novel formulation (modification of some components recentl y patented -ref: FR 4185991, 24 Sept 2014) was developed and the lipoplexes were also introduced at lower temperatures (40°C). Blank LNCs were used as control because their size and zeta potential were perfectl y stable for 2 months ( Figures 2A and2B ). We first noticed a difference in size (increased size) and zeta potential (increased surface charge) when comparing blank -LNC and the siRNA -LNCs formulations developed under the different experim ental conditions (75°C, 60°C and 40°C), which suggest an effective encapsulation of lipoplexes in LNCs. The initial size (D0) of the siRNA -LNCs prepared at 75°C progressivel y decreased during a 2 months storage period from an initial value of 102 nm, where as an increase of zeta-potential can be observed. These results suggest and instabilit y and a rearrangement of the system ( Figures 2A and2B ). The same behavior was observed with the formulation having a PIT at 60°C. There was an increase in size followed by a sharp decrease at D60, as well as a slight decrease in zeta potential. The last formulation at 40°C had a size of 85 nm with a positive surface charge of +7 mV which was perfectl y stable over -time (Figures 2A and2B). To evaluate the encapsulation eff iciency of siRNA in LNCs electrophoresis experiments were performed. A low fluorescence band may be visible, indicating an incomplete encapsulation. The encapsulation yi eld determined by UV spectrophotometry was 43% +/ -7%, confirming that approximatel y ha lf of the siRNA is stabl y incorporated within the LNCs. As in the case of SP -AP nanoparticles, generall y no siRNA was visible probabl y due to free liposomes outside LNC able to complex and protect the siRNA.

The encapsulation efficiency was also evaluated after storage at 4°C under the conditions subsequentl y used in the transfection studies (37°C, 4h of incubation in Opti -MEM), in order to determine the stabilit y of the encapsulated siRNA. No differences were observed in both conditions at 0h and at 4h an d no significant release was observed at 37°C in the 4h and 12h period.

MORPHOLOGICAL COMPARISON BETWEEN BOTH NANOCARRIERS

The morphology of SP -AP and LNCs was observed by transmission electronic microscopy (TEM) and cryo -TEM, respectivel y. The images illustrate the homogeneit y of each nanocarrier population ( Figure 3) and are in accordance with the sizes obtained by the light scattering technique for both siRNA-SP-AP (200 nm) and siRNA -LNC (80 nm) ( Figures 3B and3D).

SIRNA-NANOPARTICLE FROZEN STORAGE.

Table 2 summarizes the NPs size, zeta potential and pol ydispersit y of the LNC and SP -AP formulations after 1 month storage at -20°C. The frozen NPs can be easil y resuspended by gentle shaking. However, in order to standardize the redispersion conditions, the particle size was determined after redispersing the frozen samples by vortex stirring. siRNA -LNCs were perfectl y conserved with 1% of glucose without size or zeta -potential modification and si-RNA-AP could be frozen independentl y with 5% of glucose or trehalose, allowing integrit y conservation of siRNA. The EE decreased of around 10% (from 43% to 33%) but the integrity of the siRNAs was still conserved for each nano carrier as the siRNAs were still visible by electrophoresis after freezing and both NPs were able to transfect efficientl y (data not show). 3. Physicochemical characterization of siRNA-SP-AP and siRNA-LNC after storage at -20°C. Formulations were stored at -20°C during 1 month with PBS (control), trehalose or glucose (cryoprotectors). Size and zeta-potential were determined using DLS (Zetasizer, Malvern) before and after storage. Most relevant results were presented here. Results (n=3) are expressed as mean measure ± standard deviation.

EVALUATION OF NANOCARRIERS TOXICITY AND SELECTION OF THE SIRNA DOSE FOR MSC TRANSFECTION:

In order to study the toxicit y of LNCs and SP -AP we evaluated th e cell viabilit y after 48 h exposure to different NPs concentrations expressed in terms of siRNA concentrations, ranging from 1µg/mL to 50ng/mL, that corresponds to a range of 60 µg/mL to 1000 µg/mL in the case of LNC nanosystems or 1 µg/mL to 20 µg/mL in the case of the SP -AP ones. A decrease in cell viabilit y correlated to an increase in concentration of NPs was observed for both nanocarriers. No difference between both formulations was observed for 100 ng/ml of siRNA. However, such differences can be app reciated at higher concentrations. Thus, in the case of LNCs, the most prominent decrease in cell viabilit y was observed at 500 μg/mL (corresponding to 500 ng/mL si -RNA). At 250 ng/mL and 500 ng/mL of siRNA, SP -AP are significantl y less toxic than LNCs (Figure 4A). On the basis of these results we selected siRNA doses of 100 and 250 ng for the initial transfection studies in human MSCs with siCtl and siREST (Figure 4B ). The RT-qPCR analysis revealed that the down -regulation of REST seems to be dose -dependent for SP-AP in contrast to the LNCs, which showed an important effect at the lower concentrations assayed. Concretel y, for LNC NPs a 77 % decrease of REST expression was observed using a siREST dose of onl y 100 ng, providing higher REST inhibition levels t han the Oligofectamine ® reagent that served as a positive control. A similar inhibition of REST expression of around 60% was observed with all the nanosystems when a concentration of 250 ng/m L of siRNA was evaluated. So, we selected this siRNA dose for further experiments. These experiments include the quantification of the SCG10 or, in other words, the expression of one direct target of REST. As we can appreciate in Figure 4C,a Nanosystems cytotoxicity and siRNA dose selection for transfection studies: cell survival for the SP-AP and LNC formulations assayed in human mesenchymal stem cells at increasing nanosystem concentrations (100% survival corresponded to 200 000 cells) (A), showing that SP-AP nanoparticles are the formulation with a lower toxicity at 250 ng/ml of siRNA. Quantification of REST inhibition by RT-qPCR 48 h after transfection with SP-AP and LNC, using the Oligofectamine reagent as positive control and si-Control (siCtrl) as negative control with each nanocarriers (REST expression was normalized to REST expression in MSC with siCtrl) (B), showing that REST was equally inhibited by both nanocarriers at a siREST dose of 250 ng/ml. Quantification of the overexpression of a direct target of REST (SCG10) (The SCG10 expression was normalized to SCG10 expression in MSC with siCtrl) (C). Cell counting 48h after transfection with the different formulations at a siRNA dose of 250ng/ml (D). Quantification of REST protein by western blot 56 h after transfection with the different formulations (250 ng/mL of siRNAs) to confirm the efficacy of REST knock-down (siCtrl was used as negative control and actin served as protein loading control) (mean REST expression ± standard error of mean SEM (n=3)) (E). Statistical analysis performed with ANOVA 1W, post-hoc Tukey, * p=0.05 **p=0.005.

MESENCHYMAL STEM CELL DIFFERENTIATION

In order to determine the efficacy of siREST associated to the different formulations to drive neuronal differentiation over time, we quantified the mRNA expression for REST, his direct target (SCG10 ) and two neuronal markers: β3-Tubulin (β3 -TUB) an earl y neuronal marker and a more mature neuronal marker, neurofilament medium pol ypeptide (NFM). MSCs were maintained in expansion media and not in differentiation media to exclusivel y evaluate the effect of REST inhibition.

The inhibition of REST was maximal at 48 h increasing progressivel y until day 9 for all nanocarriers (Figure 5). siREST-LNC was slightl y more efficient compared to SP -AP over time and quite comparable to Oligofectamine® reagent used a s positive control (Figure 5A), even when no statisticall y significant differences can be found.

On the other hand, although no real change was observed in SCG10 expression over -time (data not shown), the expression of β3 -TUB, which was already slightl y expressed without treatment increased with both nanocarriers conveying siREST, particularl y at D9 a fter transfection (un til 160 % with siREST-LNC vs siC trl-LNC), demonstrating the neuronal commitment induced by siREST. More interestingl y, NFM expression appeared at day 4 and was four times more expressed with siREST -LNC than with siCtl -LNC. Its expressi on was also higher in comparison with siREST SP-AP and oligofectamine at D9 (Figure 5C). For brain regenerative medicine, embryonic stem cells (ESC) and induced pluripotent stem cells (iPS) have been proposed for transplantation therapy in human neuronal diseases. But the use of ESC or iPS present the risk of overproliferation in the brain, in addition to the numerous ethical problems [34,35].

Research studies need to focus on resolving the choice of cell t ype. In order to preserve brain function in neuronal disorders, the graft of MSC is considered as an alternative therapeutic strategy. In this study we used. MSCs as they are easil y accessible, allow autologous grafting and present neuroprotective and tissue repair properties du e to their paracrine activit y [36]. Moreover, many clinical trials have been engaged worldwide in the last few years d emonstrating their safet y [37]. The versatile differentiation potentialities of MSCs have been demonstrated not onl y for various cells of mesodermal ori gin, but also for ectodermal origin such as neural/neuronal cells under defined culture conditions [36,3,38]. For brain protection and repair it is not necessary to obtain a mature neuronal phenot ype establishing functional synapses with the surrounding cells, as the grafted cells will m ainl y function via a paracrine effect. However, they should present a neuronal -like phenot ype to avoid any potential proliferation or non-desired mesodermal phenot ype. In this study we demonstrated that a transitory inhibition of siREST, without altering t heir genome, is enough to induce neuronal commitment in vitro of MSCs. These pre -committed cells may thus be safel y transplanted in the brain parenchyma and exert their tissue repair function [39].

Classicall y, growth factors and cytokines are added in the media to drive the differentiation of stem cells to neuronal -like progenitors [36,40]. The incorporation of a RNA interference approach to existing bio -chemical based differentiation protocols may provide an alternative synergistic approach to enhance the efficiency of directing stem cell fate. REST, a major negative regulator of several neuronal genes, thus repressing neuronal differentiation, is a target of choice for this approach [10]. Moreover, REST is differentiall y regulated throughout neuronal differentiation: highl y expressed in embryonic stem cells, reduced in neural stem cells and neural progenitors [41] and largel y

DISCUSSION

absent in mature neurons [8]. The inhibition of REST to improve neuronal differentiation has been previousl y evaluated by a permanent expression of siREST in mouse MSCs using a le ntivirus carrying siREST [7], or by combining its inhibition with a cocktail of growth factors inducing neuronal differentiation in MSCs [42,43]. Our results are in agreement with these studies showing that the knock-down of REST induced a neuronal differentiation of MSCs [8].

However, in our hands, the sole inhibition of REST, without any other differentiation media, induced the neuronal commitment of hMSCs. Moreover, these NPs effectively delivered siREST to human stem cells which are not easil y transfected and which generall y show a high mortalit y rate [30,44]. The development of these biocompatible, large -scale manufactured NP (SP -AP and LNC) delivering siREST to hMSCs and inducing their neuronal commitment allow us to envisage this approach in human neuroregenerati ve medicine.

In this work, we have selected SP based nanocarriers on the basis of their demonstrated high in vivo transfection efficac y, being even higher than that provided by adeno associated viruses (AAV2) vectors [20]. For effective association between SP NP and siRNA we incorporated to these nanocarriers' two different cationic moietes: pol y-l-arginine (PA)a synthetic pol yamino acid -and ammonium pullulan (AP) -a cationised pol ysaccharide -. Although PA was initiall y selected for its extensive use as a tool for gene delivery and due to its capacit y for binding siRNA, we rejected PA based SP NP due to the resulting large particle size. We chose pullulan, due to its non -immunogenic, non -toxic, non-carcinogenic and non -mutagenic nature. Moreover, pullulan has been proposed for regenerative medicine and as a nanocarrier component, on the basis of its potential in the fields of immunization, gene del ivery, and also in the design of imaging tools [45]. Although, pullulan is a promising pol ysaccharide for biomedical applications, the inherent neutral charge of this sugar does not allow its association with genetic materials [45]. To avoid this limitation we modified this pol ysaccharide and cationised pullulan (AP) was used as a component to be incorporated in SP nanocarriers [32] to efficientl y transfect hMSC. Our results show that u sing AP we can develop homogeneous populations of SP-AP, which can be easil y chemicall y modified in order to afford either hydrophobized or cationized derivatives, the former with the abilit y to carry hydrophobic molecules, and the latter showing high affi nit y towards DNA and RNA. SP -AP NP effectivel y associated with the siRNA showing a spherical morphology that seemed more compact than that corresponding to LNCs. We have now managed to ameliorate the EE by optimizing the formulation of SP -AP thus obtaining 70% of siRNA complexation.

Concerning LNCs, these NPs were previousl y used for the encapsulation of lipophilic compounds, such as paclitaxel [46] and with MSCs as vehicles to deliver drugs into the brain [47]. LNCs were more recently adapted to the encapsulation of hydrophilic compounds (DNA, siRNA) [27,24,28]. However, stabilit y over time for siRNA -LNC was not satisfying, especiall y in terms of size. In addition, due to their preparation requirements (phase inversion process at 75°C) the siRNA could be denatured during the as sociation to these nanocarriers. However, recent experiments confirm the possibilit y to optimize these parameters. The optimization of the process (patent ref: FR 4185991, 24 sept 2014) and the diminution of the temperature down to 40°C led to LNCs with a long stable profile. Furthermore, the addition of siRNA after the phase inversion at 40°C avoids their denaturation, while preserving the characteristic positive charge on the surface of these nanocarriers. Recently, a new method of detection has been deve loped allowing the measure of the EE of siRNA -LNC which is now of 75%.

In this work we decided to evaluate the potential of transfection of the above described two t ypes of nanosystems, which differ in the main properties affecting interaction with the bio logical media in general and with cells in particular (composition, particle size and surface charge). S urface charges pla y an important role in the internalisation within the cell [48]. Classicall y, NPs have been designed with a positive surface charge to interact favourabl y with the negativel y charged phospholipid components of the cell membrane, but this interaction causes membrane c ell damages and can be toxic [49]. This has been confirmed by this study, as the negatively charged span nanocarriers were onl y slightl y toxic, whereas the higher toxicity of LNCs can be explained by a better cell interaction, internalisation and low cell densit y. Previous studies using LNCs and cancer cells did not show a high toxicit y [28,33] because cell lines are more resistant than hMSCs and the densit y of cancer cells was ten times more concentrated than in this study. Moreover, in a previous publication, we showed that MSCs can be internalized with higher doses of LNCs without any toxicit y in 1h [50] and not in 4h as recommended in Oligofectamine's protocol. Indeed, in order to compare these NPs with a commercial reagent, we used the same protocol recommended in the Oligofectamine ® guidelines. In addition, it is noticeable that the negativel y charged SP -AP nanocarriers were able to transfect hMSCs with the same efficacy than the positiv el y charged commercial reagent but avoiding cytotoxic events. Until recentl y, the internalisation of negativel y charged NPs was controversial. Indeed, the internalisation of negativel y charged NPs is believed to occur through nonspecific binding and cluste ring of the particles on cationic sites on the plasma membrane [51]. We here confirm the transfection abilit y of the negativel y charged nanocarriers previousl y reported and discussed by our group [52]. Based on the literature we can suppose that SP-AP have the abilit y to undergo in ternalization via caveolae pathways, whereas cationic NPs such as LNC, commonl y use the clathrin pathways [53][54][55].

The abilit y of a nanoparticulate system to stabilize various cargos from degradation and aggregation during storage represents a major advantage for its application. It is quite well known that aqueous suspension of NPs have a tendency to aggregate during long term storage [56]. In order to avoid this, we showed that the proposed NPs (LNC and SP -AP) could be frozen and stored while preserving the integrit y of the associated siRNA. Nonetheless the NPs stabilit y and genetic material association can be negativel y influ enced during freezing due to the ice crystals formed [57]. A suitable stabilizer, usuall y sugars (glucose, trehalose,mannitol…) can be used in the formulation protecting the physico-chemical properties of NPs and genetic materials [58]. On this respect, we initiall y thought that the pullulan -based nanocarrier might be frozen without cryoprotectant, due to its natural pol ysaccharidic nature, but we found that the cationization treatment seems to modify the cryoprotecter capacit y of pullulan.

In any case, the siRNA -SP-AP were easily frozen with different sugars (glucose and trehalose), conserving intact the associated siRNA. On the contrary, siRNA -LNCs conserved their size and zeta -potential onl y with glucose. Moreover, the loading efficiency after freezing and storage in glucose decreased less than 10% allowing using practicall y the same concentration of NPs. The observed increase of the zeta-potential with trehalose was probabl y due to the loss of a fraction of the siRNA. Such features represent an advantage of LNC and SP -AP as compared to commercial transfection reagents. Indeed, several studies have demonstrated that lipoplexes (oligofectamine ® ) are not stable in liquid suspension for long-term storage [59,57], requiring their preparation immediatel y before use [60].

The two NPs effectivel y delivered siREST in a transient manner in hMSCs inducing a marked inhibition of REST resulting in a significant increase of neuronal markers (NFM and B3 -TUB), which was maximal at day 9. This induced commitment of hMSCs to a neuronal phenot ype in expansion media by an epigenetic approach using onl y synthetic Np is very encouraging. One other study report ed the transfection of mouse neural progenitors with siREST and showed comparable levels of B3TUB expression at day 5 which was further increased at 2 weeks when a second transfection was performed to maintain REST inhibition [43]. However, in the cited study they added retinoic acid to better induce a neural differentia tion. In our study we also observed with LNC -siREST a high increase of neurofilament levels, a mature neuronal marker, which was previously described for MSCs, but with a stable inhibition of REST using viral vectors [7]. The cell differentiation process occurs thr oughout time and requires a number of sequential events that lead from one cell differentiation state to another. Each new step is facilitated by the previous one, and our results show that onl y a transient inhibition of REST is necessary to engage the cells towards a neuronal -like phenot ype. However, we didn't observe any significant change of SCG10 expression which is a direct target of REST and correlates with neurite out -growth [61]. Studies showing neurite out -growth and morphological changes use a stable inh ibition of REST with lentivirus or shREST [7,42] which is not our case. The high level of NFM and B3 -TUB observed in this work with LNCs, could be explained by a slow release of siRNA from these NPs. Indeed, LNCs remain at least 7 days in MSCs [50] compared to commercial reagents which are able to release siRNA during onl y 3-5 days [62]. A very fast siRNA deliv ery could saturate the endogenous microRNA (miRs) processing mechanisms or enter in competition with them.

For example for the incorporation and retention in RNA-Induced Silencing Complex (R ISC), which is essential for the silencing mechanism of both interfering RNAs [63]. An over-expression of miR-124 increases neuronal (earl y and mature) markers in MSCs [64]. Furthermore, several miRs, such as miR -124, miR-132, miR-9, are essential for the neuronal differentiation and are direct targets of REST [65]. In our study, we can suppose that the slow release of siREST allows the cooperation with miRs to enhance the neuronal commitment. However, more experiments are necessarily to confirm this hypothesis.

For regenerative medicine s tudies, we can envisage the possibilit y to elicit a s ynergistic effect on neuronal commitment of stem cells by the combined delivery of a morphogen with a REST knock -down. This approach may be applied to SP -AP where siRNA is in the surface letting the core free. It could also be envisaged with LNCs, which have the capacit y to associate different morphogens, such as retinoic acid [66]. It could be a useful strategy for generating functional neurons for therapeutic purposes and drive differen tiation directl y into the brain in vivo.

In this study we designed and optimized two novel nanocarriers capable of safel y associating and delivering nucleic acids: LNC and SP -AP. These systems were perfectl y reproducible and could stand long t ime storage. We have demonstrated the capacit y of both systems to knock -down REST and differentiate human stem cells towards a neuronal phenot ype at least with the same efficacy of a commercial reagent. Therefore, these nanocarriers can be considered as promising platforms for the development of effective and safe gene based regenerative approaches.

The authors thank the SFR ICAT and PACEM core facilit y for RT -qPCR and especiall y Jérôme CAYON for his expertise in this field and Cedric GAILLARD for the cryo -TEM images. We are also grateful to Guillaume BASTIAT and Laurent LEMAIRE for their expertise in statistical anal ysis and in RMN, respectively. This project is supported by the "Education Audiovisual" and the cultural executive agency of the European Union through the NanoFar

CONCLUSION

ACKNOWLEDGEMENTS

DISCUSSION

The use of non -viral siRNA delivery seem s to be a promising strategy to induce a neuronal MSC differentiation prior to cell transplantation for a regenerative and safe therapeutic strategy in HD. In this study we showed that MSCs were onl y induced to neuronal progenitors with the inhibition of R EST which have already described in the literature to play a role in the neurogenesis [START_REF] Ballas | Regulation of neuronal traits by a novel transcriptional complex[END_REF]. Indeed, REST is highl y expressed in ES cells but it is down-regulated to a minimal level in neural stem cells and neural progenitors [99,[START_REF] Ballas | REST and Its Corepressors Mediate Plasticity of Neuronal Gene Chromatin throughout Neurogenesis[END_REF]. After the differentiation of these progenitors, REST remains present in oligodendrocytes [START_REF] Dewald | The RE1 binding protein REST regulates oligodendrocyte differentiation[END_REF] and largel y absent in neurons [START_REF] Ballas | REST and Its Corepressors Mediate Plasticity of Neuronal Gene Chromatin throughout Neurogenesis[END_REF]. REST is crucial for maintaining the self-renewal of neural progenitors and the ratio of neurons. In deed the loss of REST in neural progenitors resulted in an acceleration of neuronal and oligodendrocyte differentiation [START_REF] Covey | REST regulates the pool size of the different neural lineages by restricting the generation of neurons and oligodendrocytes from neural stem/progenitor cells[END_REF]. We demonstrated the neuronal commitment with the significant apparition of β 3-tubulin and NFM two days after transfection with siREST. Nonetheless, siRNA cannot cross cell membranes without modifications or carriers. So we have designed two nanocarriers LNC and SP-AP. In the design of such nanocarriers significant factors have been taking into account to overcome limitations associated with insufficient siRNA delivery, such as nanocarrier size, surface charge, shape, chemical composition, and stabilit y of f nanocarriers [START_REF] Murugan | Parameters and characteristics governing cellular internalization and trans-barrier trafficking of nanostructures[END_REF].

In order to compare these nanosystems, we chose to trans fect the same quantit y of siRNA, which correspond ed to different quantities of nanoparticles .

As siRNA-SP-AP were non toxic we can easil y imagine to transfect MSC s for a longer time, repeat the transfection or increase the dose to further induce the neuronal differentiation of these cells. These data are interesting because the major problem nowadays in transfection is the toxicit y [START_REF] Wang | Genetic engineering of mesenchymal stem cells by nonviral gene delivery[END_REF]. Indeed, MSCs are difficult to transfect without affecting their viabilit y, resulting in very low efficiencies of transfection with range from 2 to 35% [START_REF] Santos | Non-Viral Gene Delivery to Mesenchymal Stem Cells: Methods, Strategies and Application in Bone Tissue Engineering and Regeneration[END_REF].

While siRNA-LNC had a better capacit y to transfect MSC, siRNA -SP-AP had the same efficacy as a commercial reagent without any toxicit y. The difference in terms of size (less than 100 nm for LNC, more than 100 nm for SP -AP), the composition (both are lipid based nanocarriers but their components are different), the surface charge (posit ive for siRNA -LNC, and negative for siRNA-SP-AP), as well as the localization of siRNA (entrapped in liposomes for siRNA-LNC and on the surface for siRNA -SP-AP) could explain the results observed.

Indeed, we can imagine that the positively charged siRNA -LNCs favorabl y interacted with the negativel y charged phospholipid components o f the cell membrane, and that this interaction cause d membrane cell damages probabl y explaining their toxicit y [START_REF] Liu | Influence of surface functionalization and particle size on the aggregation kinetics of engineered nanoparticles[END_REF]. On the other hand, the negativel y charged span nanocarriers were only slightl y toxic . However, the better neuronal commitment with siREST-LNC can be explained by a better cell interaction and internalization demonstrated . Long-term conservation of those nanocarriers represents another advantage of LNC and SP -AP as compared to commercial transfection reagents or viral vectors [START_REF] Felgner | Improved cationic lipid formulations for in vivo gene therapy[END_REF][START_REF] Anchordoquy | Maintenance of Transfection Rates and Physical Characterization of Lipid/DNA Complexes after Freeze-Drying and Rehydration[END_REF]. Therefore, these nano carriers can be considered as promising platforms for the development of effective and safe gene based regenerative approaches.

Cell therapy strategies using mesenchymal stem cells in HD have been investigated for the last 20 ye ars because MSCs secrete many tissue repair factors favorable for neuroprotection. The paracrine effect of MSCs on HD was investigated by Lee et al. (2009) in cell culture experiments and in vivo in two different rodent HD models [START_REF] Lee | Slowed progression in models of huntington disease by adipose stem cell transplantation[END_REF]. Transplantation of MSCs into QA model of HD led to improvement in behavior and reduced the lesion volume. These beneficial effect could be explain ed by the secretion of neurotrophic factors including BDNF, CNTF, nerve growth factor, insulin -like growth factor 1 and epidermal growth factor [START_REF] Drago | The stem cell secretome and its role in brain repair[END_REF]. Then similar results were obtain ed in genetic models of HD, YAC128, N171 -Q82Q and BACHD, demonstrat ing the interest of the use of mesenchymal stem cells in this disease [START_REF] Lee | Slowed progression in models of huntington disease by adipose stem cell transplantation[END_REF][START_REF] Snyder | Human multipotent stromal cells (MSCs) increase neurogenesis and decrease atrophy of the striatum in a transgenic mouse model for Huntington's disease[END_REF][START_REF] Kwan | Bone marrow transplantation confers modest benefits in mouse models of Huntington's disease[END_REF] . More importantly, these studies reveal the stimulation of neuronal differentiation of endogenous neural stem cells by MSCs due to the paracrine effect [START_REF] Snyder | Human multipotent stromal cells (MSCs) increase neurogenesis and decrease atrophy of the striatum in a transgenic mouse model for Huntington's disease[END_REF].

However, the lacks of consistent neuronal differentiation of transplanted MSCs have limited their therapeutic efficacy in slowing the progression of HD -like s ymptoms in animal models of HD. MIAMI cells, which are a primiti ve and unique subpopulation of MSCs secreting more tissue repair factors than MSCs [94] seem to be a good alternative. MIAMI cells, have also been shown to have a neuroprotective effect after implantation in Parkinson's disease and we have already demonstrated their neuronal commitment using a pre -treatment with EGF (epithelial growth factor) and bFGF (Basic fibroblast growth factor) which i nduced a decrease in the expression of stem cells markers such as Oct4A, Notch1 and Hes5 and increased markers of neural precursors such as Nestin and β3 -tubuline.

Recentl y, we showed in a rat model of Parkinson's disease (PD), that striatal implantation o f EGF/bFGF pre-treated MIAMI cells had a neuroprotective effect leading to functional recovery. This effect was probabl y due to their secretion of GDNF, but also of VEGF as observed in an ex vivo model of PD [START_REF] Daviaud | Survival, differentiation, and neuroprotective mechanisms of human stem cells complexed with neurotrophin-3-releasing pharmacologically active microcarriers in an ex vivo model of Parkinson's disease[END_REF]. A similar neuroprotective effect was also observed for MIAMI cells and EGF/bFGF pre-treated MIAMI cells in an ex vivo and in an in vivo model of cerebral ischemia [START_REF] Garbayo | Neuroprotective properties of marrow-isolated adult multilineage-inducible cells in rat hippocampus following global cerebral ischemia are enhanced when complexed to biomimetic microcarriers: BMMs enhance MIAMI cell protection in cerebral ischemia[END_REF]. PAMs, which can stimulate the survival and

INTRODUCTION

differentiation of cells, were combined to MIAMI cells to evaluate their effect in these models. In both of the cerebral ischemia models as well as in the ex vivo model of PD, if the cells were adhered onto PAMs, an increased survival of the transported cells was observed after transplantation leading to an enhancement of their neuroprotective effects [84,[START_REF] Garbayo | Neuroprotective properties of marrow-isolated adult multilineage-inducible cells in rat hippocampus following global cerebral ischemia are enhanced when complexed to biomimetic microcarriers: BMMs enhance MIAMI cell protection in cerebral ischemia[END_REF].

In addition, MIAMI cells can be induced towards a neuronal phenot ype in a NT-3 dependant manner and into a dopaminergic phenot ype using specific inductors (SHH and FGF8) in vitro. Moreover, a laminin substrate favours the neuronal differentiation of EGF/bFGF pre -treated MIAMI cells. The implantation of the E/F MIAMI pre-committed towards the dopaminergic phenot ype and adhered onto laminin-coated PAMs (LM-PAMs) releasing NT-3 (LM-PAMs-NT-3) led to the protection/repair of the nigro -striatal pathway and to functional recovery of the PD rats. Moreover, t he evaluation of these cells in the Parkinson organot ypic slices, demonstrated the partial capacit y of the E/ F MIAMI cells adhered onto LM -PAM-NT-3 to differentiate into dopaminergic neurons [84]. Based on these promising results, we wanted to investigate the effect of PAMs delivering BDNF as neuroprote ctive strategy in HD. Indeed, BDNF has neuroprotective properties and sustain the physiological process es of normal intact adult brain [68] and more particularl y for GABAergic striatal neurons.

In this Chapter, we described a new strategy to induce neuronal commitment using siREST, media, growth factors to improve the differentiation.

Inspired from the literature, we tested different protocols to obtain GABAergic neurons. The implantation of cells adhered to PAMs releasing BDNF then has been realized into organot ypic brain slices modeling HD.

Publication in process

Clinical trials using mesenchymal stem cells in the central nervous syst em are now underway, and are focused on the safet y of these cells. Marrow -isolated adult multilineage inducible (MIAMI cells), a subpopulation of mesenchymal stem cells raises great interest for cell therapy in Huntington's disease (HD) because they possess immunomodulatory properties and tissue repair capacities. Moreover, they can be differentiated into neurons -like cells under specific conditions. In this study, we investigated the role of REST inhibition in the GABAergic differentiation of MIAMI cells. We designed an in vitro protocol combining siRNA -nanocapsules, substrates, media, and cytokines to push MIAMI to a GABAergic neuronal lineage. We further combined these cells to pharmacologicall y active microcarriers with a biomimetic coating of laminine and releasing brain derived neurotrophic factor (BDNF) to improve their survival and differentiation. These results show that siREST combined with media containing growth factors allowed differentiating MIAMI cells toward the neural lineage in vitro. These data are supported by the slow delivery during 6 days of siRNA on MIAMI cells due to the nanocapsules. The monitoring of gene expression of neuronal and GABAergic markers during the differentiation period suggested that GABAergic precursors and finall y GAB Aergic neuronal-like cells were obtained. Importantl y, those cells did not lose their paracrine effect. Indeed, the secretion of VEGFa and BDNF are interesting for the neuroprotection in HD. Huntington's disease (HD) is a genetic disor der caused by the abnormal repetition of CAG in the Huntingtin gene which results in a pathological expansion of a pol yglutamine (pol yQ) tract in the huntingtin (htt) protein [1,2]. This neurodegenerative disorder is characterized by aggregation of htt in the brain, and more specificall y in the striatum [3]. The neuropathological changes in HD are selective, and progressive degeneration of striatal GABAergic medium spiny projection neurons is observed [4]. Clinically, this results in involuntary movements , cognitive impairment and psychiatric manifestations [5].

Typicall y, HD eventuall y culminates in death around 15 -20 years after the onset of motor symptoms [6]. Currentl y, there is no proven medical therapy to alleviate the onset or progression of Huntington's disease [7].

Cell replacement therapies in neurodegenerative diseases have been investigated for the last 30 years. Although the procedures are theoreticall y feasible, some limitations of the therapy still give cause for concern. At the beginning, fetal striatal tissues were transplanted to modify HD progression in humans [8,9]. Nonetheless, some favorable effects have been demonstrated but the difficulties in tissue availabilit y and viabilit y remain the major concern.

Moreover, fetal tissue grafts do not alter the toxic effects of mutant huntingtin and has a high risk of rejection. There are also ethical arguments and concerns about contamination and heterogeneit y of the tissues [10]. Recentl y, embryonic and induced pluripotent stem cells (ESC and iPS) were shown to successfull y differentiate into medium spiny neurons [11][12][13]. But after human ESC transplantation into rat brains, tumor formation was reported. Mesenchymal stem cells (MSCs), have emerged for clinical transplantation studies due to their eas y availabilit y and their capacit y to release neurotrophic factors and create a neuroprotective microenvironment [14]. Clinical trials using MSCs in the central nervous s ystem (CNS) are now also underway for many neurological disorders and have shown the feasibilit y of this approach [8,10]. Pre-clinical studies with HD models have shown improvement in behaviour and reduced the lesion

INTRODUCTION

volume. These beneficial effects could be explained by the secretion of neurotrophic factors including BDNF, CNTF, nerve growth factor, insulin -like growth factor 1 and epidermal growth factor [15,16].

The trans-differentiation of MSCs into a neural/neuronal lineage is possible but their functional maturit y is still insufficient. The MSC must commit to a neuronal cell and maintain this phenot ype and if possible try to replace the damaged neurons. In order to induce a neural/neuronal differentiation of MSCs the silencing of a master gene involved in neural specification named repressor element 1 (RE -1) silencing transcription factor (REST) [17,18] was obtained by a recombinant lentivirus. Nanoparticles have been formulated to associate and to efficientl y and safely deliver siRNA both in vitro and in vivo. In particular, lipid nanocapsules (LNC) were recently modified to encapsulate the siRNA complexed to lipids into their core, thus protecting the siRNA from degradation.

LNCs consisting of a lipid liquid core of trigl ycerides and a rigid shell of lecithin and PEG are formulated by a simple and easil y industrialized solventfree process based on the phase inversion of an emulsion [19,20]. They have a high stabilit y and are able to destabilize l ysosome's membranes by a proton sponge effect [21]. Recentl y, we demonstrated that LNCs associated with siREST in MSCs were able to induce their neuronal commitment with a better efficiency than a commercial reagent (Publication submitted). MIAMI cells, which are a primitive and unique subpopulation of MSCs secreting more tissue repair factors than MSCs [22] seem to be a good alternative because they can be induced differentiated in all three germ layers [23]. The demonstration of the capacit y to MIAMI cells to differentiate into dopaminergic neurons presenting appropriate electrophysiological properties after commitment with EGF (epithelial growth factor) and bFGF (Basic fibroblast growth factor) have already done [24].

The major problem concerning cell therapy studies is the survival and engraftment of transplanted cells. Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor that is widel y used in brain regeneration applications due to its abilit y to support the survival of neurons [25,26] and promote neuronal differentiation [27]. Moreover, in the case of HD, several studies demonstrated that the expression of BDNF is reduced in the patient's brains. Promising results show that the BDNF supplementation increases the survival of en kephalin-immunoreactive striatal neurons, reduces striatal interneuronal loss and improves motor function in HD animal models [28][29][30]. Despite these promising results, the therapeutic delivery of human BDNF has raised a number of problems related to its, short in vivo half-life, poor availabilit y resulting from its degradation after injection, and an uncertain passage through the blood -brain barrier (BBB) when administered in the periphery [31]. The delivery of therapeutic proteins requires microcarriers able to protect them and control their release over-time. In this way, pharmacologicall y active microcarriers (PAMs) developed by our laboratory are carriers constituted of PLGA a "Food and Drug Administration" approved pol ymer [32] . PAMs are biodegradable and biocompatible P LGA microspheres covered with ECM molecules, t hus conveying cells on their biomimetic surface providing an adequate 3D microenvironment for the transplanted cells in vitro and in vivo. Recentl y, laminine substrate favours the neuronal differentiation of EGF/bFGF pre -treated MIAMI cells, and can integr ate to the PAMs surface.

Moreover, the encapsulation of protein during the formulation allows a controlled release of bioactive molecules. In this way, BDNF which is also involved in neuronal GABAergic differentiation and may maintain the differentiated p henot ype of the transported cells could be encapsulated in PAMs. So PAMs represented an ideal microcarrier able to release active proteins [33][34][35] and providing a microcarrier for M IAMI cells due to their biomimetic surface [36].

In this study, we propose an innovative strategy for cell replacement in HD. Inspired by recent studies; a simple protocol will be tested to differentiate MIAMI cells into GABAergic neurons -like. In order, to improve the commitment, the transfection of siREST will be performed with LNC. Finall y, we plan to combine PAMs releasing BDNF with pre -committed MIAMI cells and graft these complexes in an ex vivo model of HD to evaluate their impact on the regeneration of the lesioned striatal GABAergic cells.

LNC FORMULATION

Liposomes and lipoplexes

For liposome preparation, a cationic lipid DOTAP ( Lipoplexes were formulated by simple equivolumar mix of siRNA and liposomes. This complex is characterized by the charge ratio [37], correspondi ng to a ratio of 5 between positive charge of lipids and negative charge of nucleic acids.. To obtain siRNA LNCs, the water introduced at the last P IT was replaced by lipoplexes, i.e. REST siRNA: (sense sequence: 5′ -CAG-AGU-UCA-CAG-UGC-UAA-GAA -3′; Eurogentec, Seraing, Belgium) and control (scrambled)

MATERIALS AND METHODS

siRNA (sense sequence: 5′-UCUACGAGGCACGAGACUU -3′; Eurogentec, Seraing, Belgium) complexed with cationic liposomes in a defined charge ratio as described above.

CHARACTERIZATION OF SIRNA LNCS

Size and Zeta potential

The size and zeta potential of LNCs were measured by using the Dynamic Light Scattering (DLS) method using a Malvern Zetasizer® apparatus (Nano Series ZS, Malvern Instruments S.A., Worcestershire, UK) at 25 °C, in triplicate, after dilution at a ratio of 1:200 wit h deionized water.

Encapsulation efficiency

A spectrophotometric method was used to evaluate the encapsulation efficiency (EE %) as recentl y described [9]. Briefl y, siRNA LNCs were mixed with chloroform and water to separate hydrophilic and lipophilic components, respectivel y. Sodium hydroxide was added to destabilize lipoplexes, and finall y absolute ethanol was added to destroy the LNCs. After two cent rifugations, four fractions were obtained: free siRNA, free lipoplexes, encapsulated siRNA and encapsulated lipoplexes into LNCs. To determine the concentration of siRNA, the optical densit y of each sample was read at 260 nm (UV -2600, Shimadzu, Noisiel, France) in triplicate conditions and compared to a range curve to determine the ratio of the encapsulated siRNA per total siRNA detected in the formulation (EE%). [39][40][41] . Protein loading was 1 μg of protein and 5 μg of human serum albumin (HSA)/mg of MS. BDNF and HSA were nano -precipitated separatel y using a process previousl y described [39] but adapted to l yophilized BDNF. Briefl y the protein powder was first dissolved in a non -buffered aqueous solution of sodium chloride containing poloxamer and this solution was introduced into gl ycofurol. After 30 min at 4 °C, the nanoprecipitated proteins were recovered by centrifugation and dispersed in the organic phase (670 μL of 50 mg P LGA-P188-PLGA dissolved in a 3:1 methylene chloride:acetone solution). The suspension was then emulsified in a pol y(vinyl alcohol) aqueous solution (30 m L, 6% w/v at 1 °C) and mechanicall y stirred at 995 rpm for 1 min.

CELL

After addition of 33 mL of deionized water and stirring for 10 min, the emulsion was added to 167 mL deionized water and stirred for 20 min to extract the organic solvent. Finall y, the MS were filtered on a 5 μm High Volume Low Pressure (HVLP) t ype filter, washed and freeze -dried. MS without protein were prepared following the same process, and called blank -MS or blank-PAMs when covered with laminin.

LM-PAM preparation & characterization

PLGA-P188-PLGA MS were coated with LM and pol y-D-Lysine (PDL).

Coating solutions were prepar ed in Dulbecco's Phosphate -Buffered Saline DPBS. The concentration of the coating molecules was 6 μg/mL of LM and 9 μg/mL of PDL (corresponding to a 40:60 ratio of LM:PDL). 5 mg of MS was microscopy and scanning electron microscopy. Samples were prepared for scanning electron microscopy anal ysis as previousl y described [34].

INJECTION

CELLULAR UPTAKE AND LNC TIME RETENTION IN MIAMI CELLS

SiRNA delivery and cell time retention of LNCs was studied on MIAM I E/F cells by fluorescence confocal multispectral imaging, FCSI (Figure 1A). In order to follow both the LNC and the siRNA distribution, double -labelled LNCs were generated by loading them with the DiD and w ith the siRNA -Alexa488.

RESULTS

Lysosomes were observed with the l yzotracker. Directl y, after transfection, named Day 0, we observed a varying number of LNCs in the majorit y of MIAM I E/F cells (Figure 1A and1B). The heterogeneit y of LNC distribution in each cell disappeared at Day 2. (Figure 1A). The number of positive cells for the siRNA decreased progressivel y but we still observed 12% of positive cells at day 6 (Figure 1B). In general, there are more DiD tagged LNCs in the cells than fluorescent siRNA as indeed the encapsulation efficiency of siRNA -REST in LNC's is around 50%. Although many LNCs co -localized with l ysosomes, we clearl y observed that the siRNA did not co -localize, suggesting that it was released.

MIAMI CELL NEURONAL COMMITMENT

We previousl y described MIAMI E/F as neural precursors [24] . In order t o obtain neuronal precursors of the lateral ganglionic eminence like (LGE-like) that later give rise to the striatal GABAergic neurons, we used SHH as an inductor (Figure 2A). To investigate the role of REST silencing to better induce the neuronal commitment of MIAMI E/F, we transfected siREST and a siRNA control, named siCtl. Fourteen days after treatment, most of the MIAMI -siREST SHH cells exhibited a neuron -like morphological change, with long neurite -like structures (Figure 2B). The densit y of cells dur ing the first step is very important and if the confluence is superior to 30%, over -proliferation is observed (data not shown). No obvious morphological changes were observed in MIAMI-siCtl-SHH under these same culture conditions. There was a tendency to increase the cell surface (Figure 2C), and the majorit y of cells presented a total length of around 600µm (Figure 2C) due to the long neurite -like structures.

Thirt y percent inhibition of mRNA of REST was still observed fourteen days after transfection comp ared with siCtl expression (Figure 2D).

LGE progenitors express Gsx2, Dlx2, and Meis2 and to a lesser degree Pax6. To further characterize these cells, we performed RT -PCR anal ysis to detect the decrease of embryonic markers and the increase of neural/neu ronal differentiation markers. The expression of pluripotent markers has been quantified and some variations on results are observed (supplementary data).

The expression of Pax6 involved during the neurogenesis decreased slightl y fourteen days after the tr eatment as described in the literature (Figure 2D).

Dlx2, a major gene, characteristic from LGE progenitors is strongl y expressed in M IAM I E/F cells and decreased at the end of the commitment (Figure 2D).

β 3-tubulin protein increased during the differentia tion with siREST further suggesting obtaining LGE -like progenitors (Figure 2E). Furthermore, the immunofluorescence of neurofilament (NFM) protein revealed a high expression in MIAM I-siREST-SHH, which was not detected in MIAMI E/F cells (Figure 2E).

MIAMI CELL DIFFERENTIATION TO A GABAERGIC LIKE PHENOTYPE

The LGE-like progenitors were further exposed to Valproic acid (VPA) for 7 days and then BDNF until day 28 (named VPA/ BDNF) or to a mix of VPA and BDNF for 15 days (named VPA+BDNF) (Figure 3A). At this step, no further morphological change was observed. However, siREST increased the number of cells responding to the second protocol using VPA (10µM) and BDNF (30ng/m L) in t he same time treatment (Figure 3B). In other words, 45,09% +/ -3,4 of cells presented neu rite-like structures with siCtl and 59,63% +/-10,4 with siREST. At the end of the differentiation, onl y a slight expression of Dlx2 or REST was detected by RT -qPCR (Supplementary data). A high expression of β3-tubulin was observed in all conditions. Importantl y, the majorit y of the GABA-like cells were positive for dopamine -and cAMP-regulated phosphoprotein of 32 kDa (DARPP32) and glutamate decarboxylase (67kDA), a feature of MSNs. These two proteins appeared when VPA and BDNF were in combination (Figure 3C). With siREST and VPA/BDNF; β 3-tubulin, GAD67, DARPP32 and GAT1 protein were detected (Figure 3D) suggesting a GABAergic like differentiation enhanced by siREST. MIAMI E/F cells are very interesting because they secrete many tissue repair factors [24]. The expression of mRNA in vitro allowed detecting which factors may be secreted by cells alone. In all cases, VEGFa, BDNF and NGF were expressed demonstrating the potential neuroprotective effect of MIAMI differentiated cells in the HD (Figure 3C). Nonetheless, the mRNA of those factors were highl y present in MIAMI -siREST-VPA+BDNF when compared with the other conditions; two -fold higher than MIAMI-siREST-VPA/BDNF. These results need to be confirmed. Differentiation and Characterization of GABA neurons. Schematic procedure of GABAergic differentiation (A). The quantification of cell length superior to 600µm considered as positive response to the treatment, was performed fourteen days after the differentiation for MIAMI-siCtl-VPA/BDNF, MIAMI-siRest-VPA/BDNF, MIAMI-siCtl-VPA+BDNF, and MIAMI-siREST-VPA+BDNF (B). The characterization of the differentiation was realized by RT-qPCR and immunofluorescence. The expression of genes Dlx2, REST, β3 Tubulin, DARPP32 and GAD67 (C) was quantified at the end of the differentiation Results (n=1). Cytokines expression profile of differentiated cells was also determined by RTqPCR of cells alone at the end of the differentiation (C). For both cells VEGFA mRNA is the most expressed followed by BDNF mRNA and NGF. Results were in 2^-Delta Ct . Results (n=1). In vitro immunofluorescence against β3-tubulin, DARPP32, GAD67, GAT1 on MIAMI-siREST-VPA+BDNF (D) was performed. Scale bar = 50 μm.

CARACTERIZATION OF PAM/CELLS COMPLEXES

The particle size of PAMs measured using a Multisizer Coulter Counter was around 30µm (data not shown). Observation of the microspheres and PAMs by bright field microscopy and with scanning electron microscope was performed to ensure the qualit y of the formulation (Figure 4A and4C) .

Microspheres were perfectl y spherical with a smooth surface, and no pores on their surface (Figure 4C). During the commitment and the differentiation, no serum and no antibiotics were used. In order to respect these conditions different media for cell adherence were tested with MIAMI SHH cells (Figure 4A). Unfortunatel y, we observed a high proportion of cells adhered to the plastic or cells alone with B27 and N2 media, whi ch is not the case for 3% of serum (Figure 4A). PAMs with LM surface allowed MIAMI SHH cells adherence on their surface in 4hours (Figure 4B and4D). The percentage of cells adhered onto PAMs' surface at the end of the cell attachment protocol was about 95% (Figure 4E). The optimal attachment could be explained by the homogenous coating of laminin observed with confocal microscopy (Figure 4F). Adherence of MIAMI siRNA-SHH on PAMs. Different media for adherence were tested (A). We selected the media with 3% of serum for the rest of experiments. In this condition, we observe the adherence onto PAMs by brightfield microscopy at time 0 and 4H after cells and PAMs contact (B). Observation of blank PAMs (C) and cells/PAMs complexed (D) by scanning electronic microscopy. Cells number after 4h adherence onto PAMs was quantified by Cyquant ® Reagent (E). Immunofluorescence of laminin on PAM was observed by confoncal microscopy (F).

INJECTION OF PAM/CELLS IN ORGANOTYPIC SLICES HD

Cells (MIAMI siREST-SHH) alone or com plexed to Blank-PAMs or BDNF-PAMS were grafted in organot ypic slices at day 5 which represent 30% of GABAergic striatal cell degeneration. Immunofluorescence against human Mitochondria was used to visualize MIAMI cells in rat brain, 7 days after grafting (Figure 5). PAMs clearl y improved survival of cells. Indeed, immunofluorescent staining was faint with cells alone, suggesting that some cells died. Blank -PAMs improved the survival compared to cells alone but the intensit y of fluorescence was higher with B DNF-PAMs (Figure 5).

Immunofluorescence against DARPP32 was used to determine MSN -like differentiated MIAMI cells 7 days after grafting in the HD organot ypic slices.

Very few cells were positive for DARPP32 and onl y with BDNF -PAMs was there some immunofluorescence clearl y different from the background staining, suggesting the expression by MIAMI cells of DARPP32 with this condition (Figure 5). Nonetheless, these observations have been realized onl y seven days after grafting, and the mRNA of DARPP32 appea red fourteen days after treatment in vitro. The combination of mesenchymal stem cells and pol ymeric bioactive scaffold to enhance cell survival and differentiation needs to be developed to improve therapeutic approaches for HD. In this study we showed that a subpopulation of MSCs, the MIAMI cells were able to differentiate towards the neuronal GABAergic lineage by an epigenetic ARN interfering approach inhibiting REST expression combined to GABAergic inducers. Moreover, these cells committed towards this phenot ype cells could be transplanted with PAMs delivering BDNF in an ex vivo model of HD, survive and maintain this GABAergic neuronal precursor phenot ype, particularl y in response to BDNF.

It has been shown that transplantation of MSCs into QA model of HD le d to improvement in behavior and reduced the lesion volume. These beneficial effects could be explained by the secretion of neurotrophic factors including BDNF, CNTF, nerve growth factor, insulin -like growth factor 1 and epidermal growth factor [42]. Within this line, previous studies have shown that MIAM I cells release more tissue repair factors than MSCs [22]. Moreover, in a PD model and after stroke their tissue repair capacit y allowed neuroprotection of these damaged cells [14,34,35]. Indeed, in this study MIAMI cells conserved their paracrine effect even at the end of the differentiation with high level of VEGFA, BDNF and NGF mRNA. Although, it was previousl y re ported that the addition of GDNF on organot ypic slices cultures media enhanced the expression of DARPP32 in striatal neurons [30], the low level of GDNF should not increase by itself DARPP32 in our model. However, the paracrine effect may be indirect and increase the survival not onl y of the transplanted cells but also of the issue parenchyma by an angiogenic effect. Indeed, it has been previousl y shown in an ex vivo model of PD that release of VEGF by MIAMI cells increased the vascularization around the graft site probabl y participating to the neuroprotective effects observed in this paradigm [35]. Secretion of neurotr ophic factors have not been reported for ESC and iPS cells, which show the advantage of M IAM I cells for a neuroprotective treatment of HD. The release of these factors by the MIAMI cells alone or combined to PAMs needs to be further investigated. However, the lacks of consistent neuronal differentiation of transplanted MSCs have limited their therapeutic efficacy in slowing the

DISCUSSION

progression of HD -like symptoms in animal models of HD. Indeed, their capacit y to differentiate into neuronal like cells is still i nsufficient. MIAMI cells have already demonstrated their capacit y to differentiate into dopaminergic neurons, with electrophysiological properties of immature neurons [14,24]. Based on the literature, we developed and tested a simple protocol with different steps in order to obtain GABA -like neurons [11][12][13]. During the first step we decided to inhibit REST and drive the commitment with sonic hedgehog. Indeed, we already demonstrated that REST inhibition induced s pontaneousl y neural differentiation, increasing the mRNA of neurofilament (NFM). In particular, LNC siREST had a better effect on the differentiation than a commercial reagent (lipofectamine ® ) with siREST (paper in press). In order to better comprehend thi s effect, we followed LNC and siRNA retention time in MIAMI E/F cells during 6 days. The internalization of LNC seems to be by endocytosis because we observed concentrated spots exactl y as those observed after endocytosis phenomenon such as with calcium ph osphate particles recentl y developed [43]. More interestingl y, LNC properties proved their capacit y to destabilized the l ysosomal compartment [21]. In this study, LNCs co -localized with l ysosomes while this was not the case for th e siRNA. The observation in confocal microscopy showed a high proportion of siRNA -positive cells during two days, and a progressive decrease of positive cells until day 6 where we found 15% of cells still presenting siRNA. Conversel y, in vitro cell time retention of siRNA with lipofectamine and liposomes showed that the siRNA was present during onl y three days [44]. The progressive siRNA delivery in our study suggests that t his LNC propert y is quite interesting for maintaining a differentiated cellular state for cell therapy applications.

The effect of siREST on the neuronal commitment to obtain lateral ganglionic eminence was promising. Through 14 days of differentiation o f MIAMI E/F-siREST under commitment culture conditions [12], these cells graduall y stretched out long neurite -like structures. The morphological similarit y of these cells with neurons suggests that REST knock -down allows MIAMI cells to engage into the neuronal lineage. Furthermore, the cells presented some similarities to LGE precursor cells with the decrease of Pax6 (neural stem cell marker), the slight increase of β3-tubulin (neuronal precursor marker) and high expression of NFM (immature and mature neuron marker).

However, the expression of Dlx2, a marker of GABAergic progenitors [45],was more important in MIAMI E/F or with the siCtl and the high expression of NFM suggests that the cells could be further committed in the GABAergic differentiation lineage. At the end of the diffe rentiation, the knock -down of REST seemed to have a limited influence on the appearance of DARPP32 markers or GAD67 (mRNA and proteins). However, it led to an increased response to the best treatment, which was the combination of VPA with BDNF during the l ast stage of the differentiation protocol.

Pre-clinical trials with mesenchymal stem cell transplantation suggest that cell therapy is a potential promising option for HD [16,46]. Unfortunatel y, transplanted cells have low survival rate. In this study, we used carriers providing a biomimetic support of laminin and the delivery of BDNF, the PAMs, combined with MIAMI cells to maximize the resulting protective/reparative effects on HD model. This effect should be double, drive the MIAMI cell survival and differentiation as well as increase the neuroprotective effect that we hope to observe with LGE -like cells obtained from MIAMI -siREST committed cells. Indeed, the secretion of BDNF by MIAMI cells and the release by PAMs should have a potent neuroprotective action. As reported earlier, PAMs may improve the integration of PC12 cells and embryonic dopaminergic cells within the brain parenchyma, after transplantation in hemi -parkinsonian rats, by improving their survival and differentiation. This work clearl y confirmed previous studies. Cells alo ne or complexed to LM -PAMs were grafted at day 5, when 30% of degeneration was obtained. A high detection of human Mitochondria was observed when the cells were complexed to BDNF -PAMs, which suggests that scaffold and BDNF improved the survival. More interestingl y, slight expression of DARPP32, demonstrated their potential to differentiate into MSN in onl y seven days, probabl y driven by the released BDNF. In order to confirm that, secretome anal yses with cells complexed to Blank or BDNF-PAMs should be perf ormed. In any case, these data provide encouraging results arguing for an enhancement of the neuronal differentiation of MIAM I cells towards a GABAergic phenot ype and a neuroprotective effect for HD with this innovative nano and micromedicine safe combinat orial strategy.

In this study, we tested an innovate strategy combining stem cells and biomaterials in ex vivo model of HD. We demonstrated in this way, the capacit y to our model to screen new therapy. We proved, in vitro, the capacit y to MIAMI cells to differentiate into GABAergic -like neurons by REST inhibition and appropriate media. After graft, the survival of MIAMI cells combined with LM -BDNF-PAMs seems to be increased, probabl y due to the synergic effect between the 3D support and the rele ase of BDNF. Nonetheless, the biological effect of committed MIAMI-LM-BDNF-PAMs on the HD environment needs to be characterized and understood.

In this study we showed that MIAMI cells could be induced towards the GABAergic phenot ype in vitro and that when transplanted as GABAergic precursors in an ex vivo model of HD they survived better when attached to PAMs delivering or not BDNF compared to cells alone. Through this study, we also showed that the ex vivo model developed for modelling HD, can be used for evaluating different strategies in the treatment of this disease.

We first determined that LNCs were able to release the siRNA during 6 days explaining the persistent effect of siREST to induce neuronal commitment already observed in normal MSCs. MIA MI cells were first treated with EGF and bFGF as these factors induced their specification towards the neural/neuronal phenot ype. Based on Ma et al., 2013 discovery, we decided to further treat MIAMI cells with sonic hedgehog [89] after transfection to obtain LGE -like progenitors. Indeed, the inhibition of REST before the differentiation allowed obtaining functional neurons [2]. In our case, the inhibition of REST during the first step of the differentiation had a favourable effect during al l our protocol. Even if siREST is not able to silence REST during fourteen days during the LGE commitment, we clearl y observed an increase of cells responding to the treatment during the GABAergic differentiation. Moreover, some evidences suggested that we overpassed the LGE stage with the inhibition of REST. We observed the protein expression of NFM at the end of the commitment, which was not reported previousl y with SHH treatment. A better characterization should be performed with for example the evaluati on of the CTIP2 expression in the LGE-like progenitors obtained. Indeed, in a recent study, CTIP2 was reported as the only transcription factor tested that was able to directl y reprogram dermal fibroblasts into DARPP32 + neurons, suggesting that it a key regulator for GABAergic differentiation [START_REF] Arber | Activin A directs striatal projection neuron differentiation of human pluripotent stem cells[END_REF].

As part of the therapeutic strategy relies on the neuroprotective effect of LM-PAMs-MIAMI-committed cells, we also checked the neuroprotective effect by RT-qPCR in vitro. The high expression of VEGFa at the end of the treatment permits to think that these GABAergic -like neurons still retain some characteristics from MIAMI cells, and will be able to have a beneficial effect on DISCUSSION brain slices. It has already been demonstrated that VEGFa possesses angiogenic capacities, and maintained the blood vessels in the in brain slices [84].

Furthermore, during the differentiation VEGFa enhances the gammaaminobut yric acid (GABA) synaptic activit y in embryonic spinal motoneurons [START_REF] Guérit | VEGF modulates synaptic activity in the developing spinal cord[END_REF]. We can easily imagine that VEGFa will induce an improvement of the differentiation of MIAMI cells by stimul ating GABAergic synapses. But VEGF a was not the onl y one secreted by MIAMI derived GABA -like cells, because BDNF and GDNF were also dete cted in those cells. MSCs secreting BDNF and GDNF have been already described to improve the behavior of rats with HD [START_REF] Lee | Slowed progression in models of huntington disease by adipose stem cell transplantation[END_REF][START_REF] Rossignol | Mesenchymal stem cell transplantation and DMEM administration in a 3NP rat model of Huntington's disease: Morphological and behavioral outcomes[END_REF][START_REF] Lin | Human Mesenchymal Stem Cells Prolong Survival and Ameliorate Motor Deficit through Trophic Support in Huntington's Disease Mouse Models[END_REF]. In this way, we can suppose that LM -PAM-LGE-like progenitors will reduce the degeneration. But the major advantage of MIAMI cells is their capacit y to express DARPP32, a marker of GABAergic neurons, when grafted attached to LM -PAMs-BDNF. These results are very surprising because when ES cells differentiated into striatal progenitors were grafted in quinolinc acid treated rats, they exhibited a 15% of DARPP32 positive neurons after 13 weeks in situ [37], while a LGE -like progenitors grated reveal a 50% of DARPP32 expression [89]. The advantage of the prolonged release of BDNF by the PAMs should be further investigated. With MIAMI cells we have not quantified yet the pourcentage of DARPP32 expression which could be very interesting in future studies

GENERAL DISCUSSION AND CONCLUSION

Since the discovery of Huntington' disease in 1872, pharmacological treatment or other therapeutic approaches are still insufficient to cure the disease [16]. Progressivel y, cell therapy became one of the most promising treatments [58]. However, despite many pre -clinical and clinical trials showing functional benefits with this strategy for CNS disorders, it is still not used routinel y. In the last twent y years, the implantation of fetal cells for the treatment of HD has demonstrated the possibilit y of this approach, but shows an important diversit y of results in clinical trials due to the h eterogeneit y in protocols [START_REF] Dunnett | Striatal Transplantation in a Transgenic Mouse Model of Huntington's Disease[END_REF]55]. Moreover, the use of ESC or iPS present the risk of overproliferation in the brain, in addition to the numerous ethical problems previousl y mentioned [37,90]. Research studies need to focus on resolving the choice of cell t ype, the number of injected cells and the time of injection.

Moreover, the optimization of their survival and differentiation need to be improved before imagining using them in patients. In consequence, in this work, we evaluated an innovative and safe strategy based on regenerative medicine for HD with modified MSCs and appropriate biomimetic scaffold.

In regenerative medicine, an innovative approach is the association of modified cells with therapeutic molecules, morphogens, materials or biomaterials with different physicochemical properties to enhance their biological effects and optimize cell therapy. Howe ver, the use of adult stem cells showed numerous limits with a significant death of the implanted cells quickl y detected. In addition, relativel y low rates of implanted cells differentiate into mature neurons. The capacit y of MSCs to undergo functional differentiation into neurons has been questioned over the years and remains controversial [START_REF] Chen | Coaxing bone marrow stromal mesenchymal stem cells towards neuronal differentiation: progress and uncertainties[END_REF]. Despite the initial skepticism regarding the capacit y of MSCs to differentiate into neurons or glial cells, it was discovered that MSCs express a considerable repertoire of neural genes, which l ikel y contributes to the contested neurogenic predisposition of these cells [START_REF] Blondheim | Human Mesenchymal Stem Cells Express Neural Genes, Suggesting a Neural Predisposition[END_REF][START_REF] Deng | Mesenchymal stem cells spontaneously express neural proteins in culture and are neurogenic after transplantation[END_REF]. In addition, bone marrow MSCs express higher neuronal markers [START_REF] Tondreau | Bone marrowderived mesenchymal stem cells already express specific neural proteins before any differentiation[END_REF] compared to MSCs derived from other origins.

In this work, we decided to use a homogenous subpopulation of bone marrow mesenchymal stem cells, MIAMI cells, which have already demonstrated their capacit y to differentiate into dopaminergic neurons with appropriate electrophysiological properties of immature neurons [95,[START_REF] -R. Delcroix | EGF and bFGF pre-treatment enhances neural specification and the response to neuronal commitment of MIAMI cells[END_REF]88].

Nonetheless, in order to improve the differentiation and their integration in the parenchyma, we chose to knock -down REST. The approach used here is slightl y different from strategies generall y used for reprogramm ing MSCs into neurons.

Indeed, recent studies have shown that over -expression of transcription factors can serve to promote neuronal differentiation and maintain a neuronal phenot ype. Based on the same model to obtain induced Neurons cells (iNs) by forcing the expression of a set of neural lineage transcriptional factors [START_REF] Kim | Direct lineage reprogramming to neural cells[END_REF],

the differentiation of MSCs towards a neuronal lineage has been achieved with Neurogenin1, LMX1 and/or PDX1 (For a review see [START_REF] Barzilay | Introducing transcription factors to multipotent mesenchymal stem cells: making transdifferentiation possible[END_REF]). Very few studies using the opposite strategy to knock -down and not over-express transcripti on factors have been performed to improve neuronal commitment [102,[START_REF] Trzaska | Loss of RE-1 silencing factor in mesenchymal stem cell-derived dopamine progenitors induces functional maturity[END_REF]. In those studies, knock -down of REST was effective with lentivirus and plasmids allowing a stable inhibition of REST.

In this study, we preferred to safel y modify the MSCs and focused on transitory inhibition with siREST carried by non -viral vectors to boost the differentiation. We thus designed and characterized two different nanocarriers LNC and SP-AP to efficientl y deliver siRNA. Normall y, epigenetic manipulation of stem cells is a delicate and complex task. Their remarkable capacit y of proliferation makes them poor candidates for non -viral vectors, electroporation and nucleofection, and most stem cells are sensitive to antibiotic selection; hence, attempts to stabl y establish transf ected stem cells are rarel y successful [START_REF] Conrad | Genetically engineered stem cells for therapeutic gene delivery[END_REF]. But, in our hands both nanocarriers were perfectl y able to delivery the siRNA with a varying degree of toxicit y. We also tried to reduce the off-target effect usuall y described in the literature with cationic lipids for siRNA delivery [START_REF] Jackson | Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application[END_REF] . These effects are obtained due to silencing of genes other than the intended one and thus compromise the use of a siRNA to study gene function and can even cause cell death. Furthermore, it has been observed that even scrambled sequences, which theoreticall y do not target any mRNA, can have a moderate to high impact on the cell viabilit y, depending on the cell line and siRNA concentration [START_REF] Fedorov | Off-target effects by siRNA can induce toxic phenotype[END_REF][START_REF] Mendonça | Transferrin receptortargeted liposomes encapsulating anti-BCR-ABL siRNA or asODN for chronic myeloid leukemia treatment[END_REF]. In order to avoid this, particular care was provided, in this study and in previous ones, to design siRNA nanocarriers with liposomes representing onl y 20% of the system for the LNCs or without any cationic lipids in the case of SP-AP nanocarriers [120,[START_REF] David | siRNA LNCs--a novel platform of lipid nanocapsules for systemic siRNA administration[END_REF]. Moreover, viabilit y assays were performed with both siREST and siCtle and no difference in terms of toxicit y or not differential expression of 2 housekeeping genes was observed which permits us to suppose that the off -target effect is reduced with these nanocarriers.

Both of these systems were recentl y further optimized and developed. The optimization of the formulation of SP -AP allowed obtaining 70% of siRNA complexation. Furthermore, a new method of detection has been performed to measure the encapsulation efficiency of siRNA -LNC which is no w of 75%. Due to their characteristics, the use of other morphogens or therapeutic molecules together with siRNA -nanocarriers can be envisaged. Indeed, during this thesis, a collaborative study allowed determining that the core of LNCs is not used [START_REF] Resnier | Efficient in vitro gene therapy with PEG siRNA lipid nanocapsules for passive targeting strategy in melanoma[END_REF] and the loading of different morphogens such as re tinoic acid, [START_REF] Gonnet | Evaluation in vitro et in vivo de la stabilité et de la biodisponibilité de molécules liposolubles encapsulées et incluses dans une matrice modele[END_REF] in order to obtain GABAergic precursors, could be added during the formulation. In this way a s ynergistic effect on neuronal commitment of stem cells could be obtained. In the same way the siRNA of SP -AP nanocarriers is on the surface letting the matrix core free f or addition of growth factors. In other words, combining delivery of a morphogen or a growth factor with a REST knock -down may be a useful strategy for generating functional neurons for therapeutic purposes and easil y applied to LNCs or SP -AP. To our knowledge, the codelivery of siRNA and growth factors with nanoparticles has not been investigated until now in regenerative medicine approaches, but it has already been performed for cancer treatment, demonst rating the possibilit y to use this approach [START_REF] Gandhi | Nanocarrier mediated delivery of siRNA/miRNA in combination with chemotherapeutic agents for cancer therapy: current progress and advances[END_REF]. determine the t ypes of neurons and glial cells they produced [START_REF] Álvarez-Buylla | Sonic hedgehog signaling in the postnatal brain[END_REF]. Using these conditions LGE progenitors were obtained [89] (Figure 12). Nonetheless, in our work, we concluded that we over -passed this stage with the high expression of NFM and β3-tubulin as well as the decrease of DLX2 at the end of the commitment. In order to better understand those results the mechani sm and signaling forced by the inhibition of REST must be elucidated.

REST expression is very high in ESCs compared to most other cell types [START_REF] Ballas | REST and Its Corepressors Mediate Plasticity of Neuronal Gene Chromatin throughout Neurogenesis[END_REF], and is thought to be involved in their self -renewal. REST is regulated directl y by the pluripotent factors Oct4/Sox2 and Nanog, maintaining a high expression of REST, but its function in the self -renewal transduction network is unclear [START_REF] Singh | REST maintains selfrenewal and pluripotency of embryonic stem cells[END_REF][START_REF] Jørgensen | Is REST required for ESC pluripotency?[END_REF][START_REF] Yamada | Rest promotes the early differentiation of mouse ESCs but is not required for their maintenance[END_REF]. In this work, the inhibition of REST without any other differentiated media was enough to induce neuronal commitment of MSCs, which is not surprising due to the functionalit y of REST [START_REF] Ballas | The many faces of REST oversee epigenetic programming of neuronal genes[END_REF][START_REF] Ballas | The many faces of REST oversee epigenetic programming of neuronal genes[END_REF]. But, at the end of this period we still observed a slight diminution of REST. This inhibition is not onl y explained by the prolonged release of REST with LNCs, because we did not observe any siRNA after 6 days (data not shown). We thus assume that siREST helped and accelerated the commitment as described in mouse previousl y [START_REF] Yamada | Rest promotes the early differentiation of mouse ESCs but is not required for their maintenance[END_REF][START_REF] Abrajano | Differential deployment of REST and CoREST promotes glial subtype specification and oligodendrocyte lineage maturation[END_REF] and during neurogen esis in vivo [START_REF] Chen | NRSF/REST is required in vivo for repression of multiple neuronal target genes during embryogenesis[END_REF] However, various other explanations can be advanced. One explanation relates to the levels of pluripotent factors such as Nanog. Indeed, the level of Oct4 and Nanog is lower in EGF/bFGF pre-treated MIAMI cells compared to non pre -treated MIAM I cells [START_REF] -R. Delcroix | EGF and bFGF pre-treatment enhances neural specification and the response to neuronal commitment of MIAMI cells[END_REF] and they should continue to decrease during the differentiation. In this way, they are unable to maintain or re -activate high levels of REST within the cell. The microenviro nnement is also very important for REST regulatio n. In a recent publication, Shing et al., investigated the role of laminin on pluripotency and demonstrated the reduction of self -renewal of ESCs on laminin coated surfaces [START_REF] Singh | Dynamic status of REST in the mouse ESC pluripotency network[END_REF]. In this way, we chose to transfect MIAMI cells after laminin adherence because laminin coated surfaces also play a modest role in the loss of pluripotency [START_REF] Singh | Dynamic status of REST in the mouse ESC pluripotency network[END_REF]. Moreover, in vivo reports describe the role of laminin in axonal regeneration of the CNS [START_REF] Grimpe | The critical role of basement membrane-independent laminin gamma 1 chain during axon regeneration in the CNS[END_REF]. In our study, proliferation seems to diminish after transfection with siREST and some neurites appeared during the commitment step with SHH but in order to confirm those data the possible interaction between REST and kinase or cyclin proteins involved in the cell cycle needs to be explored. Finall y, REST activit y represses the expression of GluR2 in neurons destined to die, indicating that REST might play an important role in insult -induced death of neurons [203] [204].

The final differentiation step allowed obtaining GABAergic -like neurons in vitro. The best condition was a 4 week s treatment with the supplementation of Valproic acid and BDNF at the same time (Figure 12). Those results will be confirmed with repeated experiments, but they established the choice of BDNF as the growth factor to be delivered by the PAMs for the grafting of committed MIAMI cells and PAMs. Accordingl y, laminine was confirmed as the best molecule for functionalizing the surface of the PAMs. In this way we chose to graft the EGF/bFGF MIAMI cells after commitment towards GABAergic precursors with siREST LNCs and SHH with or without laminine PAMs delivering or not BDNF. We already successfull y showed the regenerative effect of EGF/bFGF MIAMI cells and PAMs releasing NT -3 in a PD model [84,88,[START_REF] Garbayo | Neuroprotective properties of marrow-isolated adult multilineage-inducible cells in rat hippocampus following global cerebral ischemia are enhanced when complexed to biomimetic microcarriers: BMMs enhance MIAMI cell protection in cerebral ischemia[END_REF]. In addition, the transduction pathway allowing NT -3 to induce differentiation into a neuronal cell type was studied in vitro and it was demonstrated that the fixation of NT -3 to its receptor stimulated the kinase pathway MEK / ERK. Moreover, the action of Rac1b on this pathway to block proliferation and stimulate the development of neurites was also elucidated [START_REF] Curtis | Rac1b regulates NT3-stimulated Mek-Erk signaling, directing marrow-isolated adult multilineage inducible (MIAMI) cells toward an early neuronal phenotype[END_REF]. So, strong of those results, we could intend a similar strategy for HD. This ex vivo model will allow screening the best strategy for HD and further comprehending the mechanisms involved in the regeneration of the GABAergic neurons as perf ormed with an ex vivo model of PD by our group [START_REF] Daviaud | Modeling nigrostriatal degeneration in organotypic cultures, a new ex vivo model of Parkinson's disease[END_REF]. For example, we can interrogate the amount of cells to be injected and if they should be injected as a bolus or repeated injections. Furthermore, the evaluation of the behavior of stem cells after grafting must be performed at long-term. Therefore, we are going to follow NeuN and GAD67 over -time until In this way, we can screen the different regenerative medicine strategies and select the best approach showing benefits in terms of neuroprotection, not onl y in the striatum but also in the cortex and the globus pallidu s, which are involved in HD.

In adult PD rat brain, we have first shown that the 60 µm size and P LGA -PAMS could be stereotacticall y implant ed in precise areas of the brain without causing damage to the surrounding tissue. Nonetheless in organot ypic slices , we thought that 30 µm could be better. So, in this study, the triblock copol ymer poloxamer P188 ((pol y (ethylene oxide) (PEO)-pol y(propylene oxide) (PPO) -PEO) linked to PLGA to form this PLGA -P188-PLGA co-polymer was used to formulate PAMs with 30 µm si ze. Indeed, PLGA based implantable devices are FDA approved [START_REF] Tatard | Combining Polymeric Devices and Stem Cells for the Treatment of Neurological Disorders: A Promising Therapeutic Approach[END_REF] but the efficient and sustained release of proteins from PLGA-PAMs remains a challenge, mainly due to protein instabilit y (adsorpti on, aggregation, and denaturation) during the formulation process or the release period. Therefore this pol ymer was used to formulate the PAMs in this study.

Interestingl y, P LGA-P188-PLGA 60 µm microsphere releasing TGFb3, developed for cartilage tissue en gineering, resulted in a prolonged an d almost complete TGFb3 release [START_REF] Morille | New PLGA-P188-PLGA matrix enhances TGF-β3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells[END_REF]. They offered the advantage to limit protein adsorption but also to stabilize the pH inside the microsphere thus preventing protein unfolding and denaturation. M oreover, an increase in MSC number was observed after 7 days in culture on fibronectin -covered PLGA -P188-PLGA PAMs compared to fibronectin -covered PLGA PAMs.

In our study the 3D support offered by the scaffold allowed cell retention within the striatum and , probabl y cell survival as seen by the increased number of human cells with the PAMs compared to cells implanted alone. However, BDNF-PAMs also seemed to drive cell differentiation of pre -committed MIAM I cells, demonstrated by the increase of DARPP32 mark ers compared to Blank -PAMs. These results are in accordance with previous reports showing that BDNF increases DARPP32 expression in neurons of the striatum [START_REF] Snyder | Human multipotent stromal cells (MSCs) increase neurogenesis and decrease atrophy of the striatum in a transgenic mouse model for Huntington's disease[END_REF][START_REF] Hathorn | Nicotinamide improves motor deficits and upregulates PGC-1α and BDNF gene expression in a mouse model of Huntington's disease[END_REF].

Moreover, it appears to induce significant neuroprotection of NMS neurons in in vivo models of Huntington's disea se [START_REF] Reiner | BDNF may play a differential role in the protective effect of the mGluR2/3 agonist LY379268 on striatal projection neurons in R6/2 Huntington's disease mice[END_REF][START_REF] Samadi | Relationship between BDNF expression in major striatal afferents, striatum morphology and motor behavior in the R6/2 mouse model of Huntington's disease: BDNF expression, behavior and striatum morphology[END_REF]. A collaborative work allowed determining the release of BDNF from PAMs, which corresponded perfectl y to the amount (15 ng/day) needed by the cells for their differentiation [37,89,90] Nonetheless, we don't know if the protective effect on MIAMI cells is due to the BDNF released from PAMs or BDNF secreted by MIAMI cells. The neuroprotective effect of MIAMI on the GABAergic neurons is also another important part of this project. In the present study, in preliminary results, we observed in vitro that MIAMI cells expressed BDNF and GDNF as well as a very high level of hVEGFA. BDNF and GDNF have both been reported as neuroprotective factors whose effects were, in part, mediated by autophag y regulation [START_REF] Chen | Neuroprotective effect of brain-derived neurotrophic factor mediated by autophagy through the PI3K/Akt/mTOR pathway[END_REF] and oxidative stress diminution [START_REF] Ebert | Ex vivo delivery of GDNF maintains motor function and prevents neuronal loss in a transgenic mouse model of Huntington's disease[END_REF][START_REF] Tasset | The molecular bases of Huntington's disease: the role played by oxidative stress[END_REF], respectivel y. The neuroprotective effect of BDNF and GDNF has been evaluated in pre -clinical studies in HD [START_REF] Pérez-Navarro | Intrastriatal grafting of a GDNF-producing cell line protects striatonigral neurons from quinolinic acid excitotoxicity in vivo[END_REF][START_REF] Alberch | Neuroprotection by neurotrophins and GDNF family members in the excitotoxic model of Huntington's disease[END_REF][START_REF] Silva | Overexpression of BDNF and Full-Length TrkB Receptor Ameliorate Striatal Neural Survival in Huntington's Disease[END_REF]. In this way, pre-committed MIAMI cells are good candidates for neuroprotection. hVEGFA is playing a dual role, indeed, hVEGFA can increase grafted cells survival by creating new blood vessels around the graft enhancing oxygen and nutrients suppl y and a neuroprotective effect. Indeed, in a more recent study, the delivery of hVEGFA via injectable hydrogels, to rats receiving striatal injection of quinolinic acid to mimic neuronal loss and behavioral deficits characteristic of HD, was shown to be neuroprotective at 3 weeks [START_REF] Emerich | Injectable hydrogels providing sustained delivery of vascular endothelial growth factor are neuroprotective in a rat model of Huntington's disease[END_REF]. As hVEGFA is well -known to have proangiogenic effect, a quantification of blood vess els in the brain slices should be performed. Complete anal yses of the secretome of MIAMI cells are going on in order to better characterize proteins secreted by LGE -like progenitors. However, a final assessment of this strategy includes the behavioral evaluation in an appropriate in vivo model. In this work, intracranial injection of MIAM I cells is envisaged and we think it is the best option as the cells and PAMs need to b e close to their site of action to avoid undesired side-effects. We can also consider delivery of siREST by implantation of the si -REST nanocarriers together with the cell graft. Indeed, REST expression is increased in HD [START_REF] Bithell | Transcriptional dysregulation of coding and non-coding genes in cellular models of Huntington's disease[END_REF][START_REF] Soldati | Dysregulation of RESTregulated coding and non-coding RNAs in a cellular model of Huntington's disease[END_REF] and its association with HTT in the cytoplasm is disrupted in HD due to the mutated HTT. This leads to increased nuclear REST and concomitant repression of several neuronal -specific genes, including BDNF [67]. The findin g described indicates that inhibition of REST expression may offer a new therapeutic avenue for treatment of HD. Nowadays, innovative strategies including the use of RNAi to reduce REST expression [START_REF] Conforti | In vivo delivery of DN: REST improves transcriptional changes of REST-regulated genes in HD mice[END_REF] or decoy oligonucleotides or synthetic peptide nucleic acid (PNA) oligomers to seque ster REST away from its genomic sites [START_REF] Zuccato | Widespread Disruption of Repressor Element-1 Silencing Transcription Factor/Neuron-Restrictive Silencer Factor Occupancy at Its Target Genes in Huntington's Disease[END_REF][START_REF] Fang | Cationic shell-cross-linked knedel-like (cSCK) nanoparticles for highly efficient PNA delivery[END_REF] are underway. All of these approaches lead to reduce interactions of REST with its target genes; however, translation of this into a useful therapeutic strategy requires safe and efficient delivery of these tools to the brain of HD patients. This is no easy task and LNCs or SP -AP could be used for that, but this strategy needs to be care full y anal yzed. More particularl y, a full characterization of this combined strategy will be evaluated during a post -doctoral contract.

Emilie ANDRE

Title Combination of nano and microcarriers for stem cell therapy of Huntington's disease: new regenerative medicine strategy Résumé La combinaison de biomatériaux et cellules souches, a pour but de protéger des cellules endommagées et de ralentir la progression des maladies neurodégénératives, comme la maladie de Huntington (MH). Les cellules souches mésenchymateuses et particulièrement une sous-population, les cellules MIAMI, ont déjà démontré leur efficacité dans la maladie de Parkinson. Il est cependant essentiel d'améliorer leur différenciation neuronale, leur survie et évaluer leur sécrétome. L'objectif principal de ce travail fut de proposer une stratégie innovante de médecine régénératrice pour la MH associant cellules souches, nano et micro médecines. Pour l'évaluer, un nouveau modèle animale ex vivo de la MH a été mis en place. Nous avons ensuite développé et optimisé deux nano-vecteurs, des nanocapsules lipidiques et des nanoparticules solides de SPAN, et les avons associés à un inhibiteur de REST qui est un facteur de transcription qui empêche la différenciation neuronale. La transfection de ce siREST a montré une amélioration du phénotype neuronal. Ces cellules ainsi modifiées furent ensuite induites vers un phénotype GABAergic grâce à des facteurs de croissance. Puis elles ont été associées à un support 3D, les microcarriers pharmacologiquement actif (MPA) permettant une meilleure intégration des cellules après greffe. Les MPA sont des microsphères ayant une surface biomimétique de laminine et libérant de façon contrôlée un facteur trophique le « brain derived neurotrophic factor » (inducteur d'un phénotype neuronal et neuro-protecteur). Des résultats prometteurs ont été obtenus, encourageant à continuer l'évaluation de cette stratégie in vivo dans des modèles génétiques de la MH.

Mots clés

Médecine régénérative, cellules cellules souches mesenchymateuse, Maladie de Huntington, siREST, nanoparticules, microcarriers pharmacologiquement actif

Abstract

The combination of biomaterials and stem cells aims to protect damaged cells and slow the progression of neurodegenerative diseases such as Huntington's disease (HD). Mesenchymal stem cells, particularly a subpopulation known as MIAMI cells, have already demonstrated their effectiveness in Parkinson's disease. However, it is essential to improve their neuronal differentiation, survival, and to assess their secretome. The main objective of this work was to propose an innovative regenerative medicine strategy for HD by combining stem cells, micro and nano medicines. To perform this assessment, a new ex vivo animal model of HD has been set up. We then developed and optimized two nanovectors, lipid nanocapsules and solid SPAN nanoparticles, carrying an inhibitor of REST a transcription factor, which prevents neuronal differentiation. The transfection of this siREST showed an improvement in the neuronal phenotype. These modified cells were then induced into a GABAergic phenotype through growth factors. They were then associated with a 3D support, the pharmacologically active microcarriers (PAM) allowing a high rate of engraftment. The PAM are microspheres which have a biomimetic surface of laminin and release a trophic factor BDNF, brain derived neurotrophic factor (inducer of a neural phenotype and neuroprotective) in a controlled manner. Promising results were obtained, further encouraging continuing the evaluation of this strategy in vivo in genetic models of HD.

Key Words

Regenerative medicine; mesenchymal stem cells, Huntington's disease, siREST, nanoparticles, pharmacologically active microcarriers.

Firstl

  y, I would like to thank Professor Jean -Pierre BENOIT, Director of the INSERM U1066, and Professor Francisco OTERO ESPINAR , Director of the department of Pharmacy and Pharmaceutical Technology , for wel coming me in their laboratory during m y thesis.

I

  thank the Professor Eduardo FERNANDEZ-MEGIA, from the Universit y of Santiago de Compostela, to make me the honor to evaluate this thesis as examiner. I also sincerel y thank Associate Professor Marie MORILLE , from the Universit y of Montpellier for agreeing to be m y thesis examiner. Than k you for your help during my Master 2 with m y C .V. and for giving me the contact of Associate Professor Claudia MONTERO -MENEI. You kindly advised me when I was lost. I would like to thank you again for giving me the honor of evaluating this thesis. M y thanks also go out to the different work teams wh ich so kindl y participated in these researches and make this work possible : Doctor Luis BRAZ and Doctor Ana ROSA DE COSTA from the University of Algarve, Faro (Portugal), who gave me the pullulan. I also have special thanks for the Doctor Andrea PENSADO BELEN and PhD student Ines FERNANDEZ-PINEIRO from the Universit y of Santiago de Compostela who teach me a lot about nanoparticles (Spain). The Professor Paul SCHILLER and the Associate Professor Gaëtan DELCROIX, from the Universit y of Miami Miller School o f Medicine, for your help during the last experiments (USA).

I

  would also like to express m y heartfelt gratitude for their time and expertise generousl y offered to Jérôme CAYON from the PCR platform, Rodolphe PERROT from the Service Commun d'Imageries et d'Analyses Microscopiques (SCIAM), Pierre LEGRAS and Jérôme ROUX from the Service Commun d'Animalerie Hospitalo -Universitaire (SCAHU), and Dr Laurent LEMAIRE , Dr Guillaume BASTIAT, Pr Franck BOURY, Dr Jé rémie RIOU, Dr Anne CLAVREUL, Edith GRELEAU and all t he persons from the laboratory in Angers and in Santiago de Compostela . Immeasurable appreciation for the moral support and the wonderful time spent with: -M y office coworkers in France and in Spain : Drs UMERSKA Anita and CORDONNIER Thomas, Chantal alias Chacha, Hélène, Rose-Monde (ex co-workers), Jesus, Joaquin, Michaela, Eli, Diana from spain . -M y other lab mates: Drs Anne-Claire Groo, Fabienne Danhier, Pauline Resnier, Emilie Roger and Fabien Violet, Marion Pittore, Nada Matougi, Angélique Montagu, Thomas Briot and Aurélien Contini ; -My NanoFar colleagues: Ana, Lu, Emma, Floja, Be, Zeynep , Krishna, Subaash, and all NanoFar students. Chacha, Emilie, Fabienne, Hélène, Marion, Nada and Thomas, I would not just include you in this liste, I would like to sincery thank you, for supporting me during this thesis! ChaCha you had share a flat with me, good and bad time!

FinallFigure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 9 .Figure 10 .Figure 11 .Figure 12 .Figure 13 .

 12345678910111213 Figure 1. Worldwide estimates of the prevalence of HD

Figure 1 .

 1 Figure 1.Worldwide estimates of the prevalence of HD. Overall, the prevalence of HD is much higher in European populations than in East Asia[3].

Figure 2 .

 2 Figure 2.Brains' comparison by MRI between healthy and Huntington's disease subjects[14].

Figure 3 .

 3 Figure 3.Different areas involved in HD explaining symptoms. The cortico-striatal system is essential for the execution of movements (left). In patients with HD, progressive degeneration of neurons in the striatum, and later also in cerebral cortex, disrupts function in the cortico-striato-pallidal circuit and induces severe impairments in both motor and cognitive functions. The striatal GABAergic projection neurons provide an inhibitory control of two major striatal output structures, globus pallidus and pars reticulata of the substantia nigra (not shown). Loss of these neurons, in animals with striatal lesions or in HD patients, results in disinhibition of pallidal outflow (right)[17].

Figure 4 .

 4 Figure 4. The use of organotypic slices for the evaluation of innovative treatment in HD [43].

Figure 5 .

 5 Figure 5.Schema of the three pillars of regenerative medicine. To bring tissue engineering into reality, it is crucial to sufficiently advance and combine the three. Currently, there is increasing recognition of the importance of cell culture methods, scaffolds supporting cells and drug delivery of signaling molecules.

Figure 6 .

 6 Figure 6. Role of BDNF in developing, adult, and HD striatum. (A) A proposed model showing that BDNF and NT3 anterogradely transported from mesencephalic dopaminergic neurons regulate survival of immature neurons in the indirect and direct pathways, respectively. Ctx, cerebral cortex; Stm, striatum; SN, substantia nigra. (B) Cortical BDNF in the adult striatum mediates dendritic complexity and spine number and morphology. (C) Mutant htt reduces BDNF-TrkB signaling by inhibiting BDNF gene transcription, axonal transport of vesicles containing BDNF, retrograde dendritic transport of TrkB-positive endosomes to the cell body [72].

Figure 7 .

 7 Figure 7.Concept of PAMs: the biomimetic surface of PAMs is obtained by coating their surface with extracellular matrix proteins that can favor cell adhesion. During their formulation, the encapsulation of neurotrophic factor is performed.

Figure 8 .

 8 Figure 8. Soluble factors secreted by MIAMI cells involved in the tissue repair. [93,94]

Figure 9 .

 9 Figure 9. REST expression during the neurogenesis [100].

Figure 10 .

 10 Figure 10. The benefits and limitations of synthetic siRNA application. The representation of the limitations involved in the siRNA delivery. [107]

Figure 11 .

 11 Figure 11.The tissue engineering strategy envisaged in this work.

Figure 1 .

 1 Figure 1. The origin, isolation, & specialization of stem cell to produce neurons in vitro. The induction of neuronal commitment can be realized by external and internal factors. Recently the combinations of both methods obtain best results.

Figure 2 .

 2 Figure 2.Inspired from Cullen, nature genetics 2005, we represent the intracellular trafficking of siRNA delivery systems[69]. Internalization of delivery systems can be performed by several mechanisms as clathrin, caveole pathways or lipid rafts. Two modalities are observed in this phenomenon, a receptor mediated endocytosis and a non-receptor mediated endocytosis. RNAi is delivered into the cytoplasm and produces its inhibitor effect.

Figure 3 .

 3 Figure 3.Methods of RNAi complexation with delivery systems. The RNAi localization into nanocarriers differs according to structure and organization of the delivery system. RNAi can be incorporated into a matrix structure and allow a global repartition in the entire volume of the sphere (cluster), with polymers components. Other strategies consist in either associating by electrostatic or Van der Walls interactions the RNAi on the surface of delivery systems. The chemical cross-link is also a possibility. Last organization concerns shell/core structure observed for liposomes and some nanoparticles.[74].

Figure 4 .

 4 Figure 4.Different types of nanocarriers able to control the stem cell fate. Nanoparticles are commonly defined as objects with a rank of dimensions of 1-1000nm, which includes micelles, reversed micelles, nanoparticles, nanogels, and nanofibers. We represent here the morphology of the most commonly particles used ones for siRNA complexation and regenerative medicine.

  Although many efforts have been made to drive cells into specific lineages, maturit y and functionalit y remains a problem for regenerative medicine. Most probabl y, two dimensions (2D) culture do not authorize a complete and functional differentiation. Number of publications reporting benefits on three dimensions (3D) culture is increasing, and 3D supports have been demonstrated as improving cell survival after grafting. Micro -and nano-scale chemical and physical cues from the ECM environment control and direct various key cell behaviours including their adhesion, proliferation, migration and differentiation. Strategies to achieve brain protection in HD, repair and recovery include the delivery of neuroprotective compounds to prevent cellular degeneration, cell transplantation to replace lost cells, approaches using tissue engineering, and methods to enhance plasticit y by promoting the intrinsic capacit y of the brain to regenerate and reorganize. The latter strategy still has poor control of cellular growth processes, differentiation and migration to the appropriate location in vivo. Cell-based therapies have encountered poor cell survival and integration in the host. Moreover, it is necessary to recapitulate the sophisticated and precise architecture and functional wiring present in the cellular and molecular environment of the brain. Th erefore, combined in vivo strategies using stem cells, biomaterials, growth factors and epigenetic control of gene expression with different vectors are nowadays being investigated (Figure. 5).

Figure 5 .

 5 Figure 5.Schematic illustrating technologies to spatially control stem cell fate. During microcarriers' process, nanocarriers containing RNAi or neurotrophic factors can be integrated directly into these biomaterials. All possibilties for combined strategies for stem cells transplantation have been presented. The first consideration for microcarriers tailoring in brain is size: microcarriers should necessarily be small enough to be easily implanted into the striatum, either via stereotactic implantation or under neuro-navigation. Moreover, small-sized scaffolds render repeated implantations possible, with no need for open-surgery[122].

  Low et al. (2010) used nanofibrous carriers -mediated siRN A delivery to enhance neuronal differentiation of stem cells. By using a mussel -inspired coating with strong adhesive properties, siRNA complexed with cationic transfection reagent (Lipofectamine RNAimax) was immobilized on the electrospun PCL nanofibrous scaffolds. Application of this system resulted in significant enhancement of neuronal differentiation of mouse neural progenitor cells due to the synergistic effects of 3D nanofibrous topographical cues and carriers -mediated knockdown of the REST[130].In the treatment of HD, so far there is a limited success rate in modifying disease s ymptoms by traditional pharmacological agents. Nowadays advances in drug delivery and cell/ tissue engineering open the possibilities to achieve, beyond s ymptomatic relief in HD, neuroprotection and neurorepair. RNAi engineered nanotherapeutics has emerged during these years as an innovate strategy to control stem cell fate in vitro and in vivo. Nano and microcarriers for nucleotide delivery offer numerous benefits over lipoplexe transfection, electroporation and lentiviral transduction. Nano and microcarriers based delivery of cells, growth factors and RNAi may be among the best means for controlling the differentiation and survival of the delivered cells while stimulating neuroprotection and repair. This project is supported by the Education Audiovisual and cultural excetutive agency of the Europ ean Union throught the NanoFar Erasmus Mundus join Doctoral program and financed by the Angers Loire Métropol e.

Figure 1 .

 1 Figure 1. Protocol to obtain organotypic slices. Rats' anesthesia must be realized with an intraperitoneal injection of ketamine and xylazine, in a solution of NaCl 0,9 %. (A) The head must be dissected quickly and carefully to preserve brain structure. Cortex and cerebellum should be cut (B). Brain is glued onto the chuck of a water-cooled vibratome and plunged in a cold solution containing high level of glucose. Under aseptic conditions, 400µm thick whole brain sections are cut and collected (C) The brain slices are disposed in a 0.4 µm membrane insert with media 1 solution and incubate in 5% CO2 and observation of the coronal slice. Cortex (Cx), striatum (St), globus pallidus (GP) and Corpus Callosum (CC) can be easily observed (D).

2. 3 . 4

 34 Quantification of NeuN-positive neurons The survival of tot al neurons in certain organot ypic slice regions was estimated by immunohistochemestry against NeuN. NeuN -positive neuron was calculated, from days 0 to 19 using the Metamorph® software. At each timepoint, six pictures taken from three different slices showing cortex, striatum and lateral septum were used. NeuN -positive neuron in these areas was expressed as a percentage of NeuN staining at day 0 considered as positive control. Results

  Figure 2.Slice angle selection to model HD. Rat's brain was cut in order to obtain saggital, coronal or horizontal brain slices (A). Which each different angles, we can vizualized all the areas involved in the HD (B) DARPP32 was detected in brain slices obtained by immunofluorescency at day 1, 4 and 7 after sagittal, coronal and horizontal (C) sections.

Figure 3 .

 3 Figure 3. Morphology and viability of organotypic slices. Observation of coronal brain slices was realized by bright field microscopy (A). Immunohistochemistry against neuronal nuclei (NeuN) showing three brain regions: cortex, striatum and lateral septum at day 0, 11 and at day 19 (B) at the bottom. Immunohistochemistry against neuronal nuclei (NeuN) showing the striatum (C) Quantification of NeuN-positive cells in cortex, striatum and SN shows no important loss of staining in the cortex after 19 days in culture (D). While NeuN expression decreased by 80 % and 27 % in the striatum. * : Significantly different results with n = 3 and P-value = 0.05. Pictures about organotypic slides until day 19.

Figure 4 .

 4 Figure 4. Modelisation of specific MSN markers. Immunohistochemistry against striatal A) DARPP32 positive neurons and B) GAD67 positive neurons at day 0, 7, 11 and 19. C) Striatal DARPP32 positive neurons decreased progressively until day 11 and became total at day 19 in comparison with day 0 which represents 100% represented with SD in doped lines. D) Striatal GAD67 positive neurons quantification with SD in doped lines. A 10% decreased was detected at day 2 which reached 30 % at day 4, 45 % at day 7 and reached 100 % at day 19, holding the fact that day 0 represent 100 %. N=4

  80 ® ; PA: pol y-Arginine; AP: ammonium pullulan / cation ized pullulan; NP: nanoparticles; DOPE: 1,2 -dioleoyl-sn-gl ycero-3-phosphoethanolamine; DOTAP: : (Repressor Element -1 silencing Transcription /Neuron -Restrictive Silencing Factor-1)

  1,2 -dioleyl-3trimethylammoniumpropane) (Avanti® Polar Lipids Inc., Alabaster, AL, USA), solubilized in chloroform, wa s weighted at a 1/1 molar ratio with the neutral lipid DOPE (1,2 -dioleyl -sn-gl ycero-3-phosphoethanolamine) (Avan ti® Polar Lipids Inc., Alabaster, AL, USA) to obtain a final concentration of 30 mM of cationic lipid, (considering the number of charges per lipid molecule), i.e 1 for DOTAP. After chloroform evaporation under vacuum, deionized water was added to rehydrat e the lipid film overnight at 4 °C which was sonicated the day after during 30 min.

( 3 -

 3 (4,5-dimethylthiazol -2yl)-5-(3-carbox ymethoxyphenyl) -2-(4-sulfophenyl)-2H tetrazolium) (Promega,Madison, USA). and AlamarBlue R (resazurin) (Invitrogen). Twent y-five thousand cells, cells were transfected in a 24 -well plate with the siRNA -REST and siRNA scramble with increasing concentrations of siRNA (0.05, 0.1, 0.25, 0.5 and 1 mg/m L). To determine the IC 50, cells were subsequentl y cultured during two days and 20 µL of MTS/well was added and plates incubated 2.5 h at 37 °C in a humidified atmosphere with 5 % CO 2 . The OD was evaluated by Mutliskan Ascent (Labsystems, Fisher Scientific , Wilmington, USA) at 492 nm.

  RNeas yMicrokit (Qiagen, Courtaboeuf, France), and treated with DNase (10 U DNase I/µg total RNA). RNA concentrations were determined using a ND -2000 NanoDrop (Thermo Fisher Scientific, Wilmington, Delaware USA ) and used for normalization of the input RNA in the RT. First strand complementary DNA (cDNA) s ynthesis was performed with a SuperScriptTM II Reverse Transcriptase (Invitrogen), in combination with random hexamers, according to the manufacturer's instruct ions. Following first -strand cDNA synthesis, cDNAs were purified (Qiaquick PCR purification kit, Qiagen, Courtaboeuf, France) and eluted in 40 µL RNAse free water (Gibco). 2,5ng of cDNA was mixed with MaximaTM SYBR Green qPCR Master Mix (Fermentas) and pri mer mix (0.3 µM) in a final volume of 10µ L. Amplification was carried out on a Chromo4 thermocycler (Biorad) or LightC ycler 480 (Roche) with a first denaturation step at 95°C for 10 min and 40 cycles of 95°C for 15 s, 60°C for 30 s. After amplification, a melting curve of the products determined the specificit y of the primers for the targeted genes. Several housekeeping genes, Gl yceraldehyde -3phosphate dehydrogenase (Gapdh), Beta -2-microglobulin (B2M), Beta actin (Actb), and Heat shock 90 kDa protein 1 be ta (Hspcb) were tested for normalization.The GeNormTM freeware (http://medgen.ugent.be/jvdesomp/genorm/) was used to determine that GAPDH and ACTB were the two most stable housekeeping genes. The relative transcript quantit y (Q) was determined by the del ta Cqmethod Q=E(Cq min in all the samples tested -Cq of the sample)

Figure 1 .

 1 Figure 1. Characterization of SP nanoparticles with different surface composition and siRNA dose: (A ) Size of blank nanoparticles based on SP, SP and poly-arginine (SP-PA) or SP and cationized pullulan (SP-AP), siRNA-SP-PA and siRNA-SP-AP at days 0 and 30 after storage (D0 and D30): (B) zeta-potentials at days 0 and 30 after storage; (C) Size and zeta-potentials of nanoparticles associating siRNA doses ranging from 0.1 to 0.5 mg/ml; (D) Electrophoresis experiments performed with free siRNA (Line 1) and siRNA-SP-AP associating different siRNA at different concentrations, such as (2): 0.1 mg/mL of siRNA; (3): 0.3 mg/mL; (4): 0.4 mg/mL and (5): 0.5 mg/mL. Free siRNA can be observed in the lines 4 and 5 illustrating the saturation on the association efficiency of the nanocarrier. Results are expressed as mean ± standard deviation (n=3). No difference was demonstrated on stability of SIRNA-SP-AP using ANOVA 1W, posthoc Tukey.

Figure 2 .

 2 Figure 2. Optimization of different siRNA-LNCs. Size (A) and zeta potential (B) at different storage times (D0, D15, D30 and D60) showing a long-term stability for siRNA LNCs 40°C. EE evaluation by UV spectrophotometry of siRNA in LNCs in the Opti-MEM ® at 4 and 37°C, showing no release during the first 4h (C), further confirmed in the electrophoresis experiments (D). Results (n=3) are expressed as mean measure ± standard deviation and the encapsulation efficiencies were compared using a t-test.

Figure 3 .

 3 Figure 3. Morphological comparison between SP-AP and LNC observed by transmission electronic microscopy (TEM) for Blank-SP-AP (A) and siRNA-SP-AP (B). and by cryotransmission electronic microscopy (cryo-TEM) for Blank-LNC (C) and siRNA-LNC (D) showing homogeneous populations of spherical nanocarriers of around 200 nm (SP-AP based nanosystems) or 80 nm (LNC nanosystems).

  slight increase of SCG10 expression can be observed at 48 hrs after transfecti on when using both nanos ystems at 250ng. The comparison of cell number 48 h after transfection with SP-AP, LNC and the Oligofectamine ® reagent revealed the same profile with the LNCs and Oligofectamine (53 % of cell death) ( Figure 4 D), while with SP-AP we observed onl y a 25 % of cell death. These results confirmed that SP -AP NPs are less toxic for hMSC.To confirm the down regulation of gene expression provided by the different NPs we determined the protein knock -down by western blot anal ysis 36 h after transfection at a siRNA dose of 250 ng. In Figure4Ewe can appreciate that REST protein was strongl y inhibited with Oligofectamine ® and LNC confirming the RT -qPCR data.

Figure 4 .

 4 Figure 4.Nanosystems cytotoxicity and siRNA dose selection for transfection studies: cell survival for the SP-AP and LNC formulations assayed in human mesenchymal stem cells at increasing nanosystem concentrations (100% survival corresponded to 200 000 cells) (A), showing that SP-AP nanoparticles are the formulation with a lower toxicity at 250 ng/ml of siRNA. Quantification of REST inhibition by RT-qPCR 48 h after transfection with SP-AP and LNC, using the Oligofectamine reagent as positive control and si-Control (siCtrl) as negative control with each nanocarriers (REST expression was normalized to REST expression in MSC with siCtrl) (B), showing that REST was equally inhibited by both nanocarriers at a siREST dose of 250 ng/ml. Quantification of the overexpression of a direct target of REST (SCG10) (The SCG10 expression was normalized to SCG10 expression in MSC with siCtrl) (C). Cell counting 48h after transfection with the different formulations at a siRNA dose of 250ng/ml (D). Quantification of REST protein by western blot 56 h after transfection with the different formulations (250 ng/mL of siRNAs) to confirm the efficacy of REST knock-down (siCtrl was used as negative control and actin served as protein loading control) (mean REST expression ± standard error of mean SEM (n=3)) (E). Statistical analysis performed with ANOVA 1W, post-hoc Tukey, * p=0.05 **p=0.005.

Figure 5 .

 5 Figure 5. Mesenchymal stem cells differentiation: Transfection of hMSC was performed with 250 ng/ml of siControl and siREST. The expression of genes REST (A), β3 Tubulin, (B) NFM (C) was quantified at D0, D2, D4 and D9 after transfection with SP-AP, LNC and Oligofectamine ® . Results were normalized with the expression of each gene with siControl. Results (n=3) are expressed as mean measure ± standard deviation. Statistical analysis performed with ANOVA 1W, post-hoc Tukey, * p=0.05 **p=0.005.

  Abbreviations:

2. 1 .

 1 2 siRNA-LNC LNCs were formulated, as describ ed previousl y[19] by mixing 20 % w/w Labrafac® W L 1349 (caprylic -capric acid trigl ycerides, Gatefossé S.A. Saint-Priest, France), 1.5 % w/w Lipoid S75 -3® (Lecithin, Ludwigshafen, Germany), 17 % w/w Kolliphor® HS 15 ( Pol yethylene gl ycol -15-Hydroxystearate HSPEG BASF, Ludwigshafen, Germany), 1.8 % w/w NaCl (Prolabo, Fontenay -sous-Bois, France) and 59.8 % w/ w water (obtained from a Milli -Q system, Millipore, Paris, France) together under magnetic stirring. Briefl y, three temperature cycles between 60 and 95°C were performed to obtain phase inversions (PI) of the emulsion. A subsequent rapid cooling and diluti on with ice cooled water (1:1.4) at the last phase inversion temperature (P IT) led to blank LNC formation.

2. 1 . 3

 13 Fluorescent siRNA-LNC-DID To formulate fluorescent siRNA LNCs, a solution of DiD (1,1′dioctadecyl -3,3,3′,3′-tetramethylindodicarbocyanine perchlorate; em. = 644 nm; exc. = 665 nm) (Invitrogen, Cergy-Pontoise, France) solubilized in acetone at 25 mg/mL was prepared. DiD concentration was fixed at 200 µg/m L of LNC suspension or corresponding to 1,36 mg of DiD per grams of Labrafac ® [38]. The adequate volume of DiD I solubilized in acetone was incorporated in Labrafac ® and acetone was evaporated at room temperature. The formulat ion process was unchanged and formulation was stored at 4°C, protected from light. For siRNA fluorescent LNCs, a fluorescent Alexa488 siRNA (em. = 488 nm; exc. = 524 nm, Eurogentec) was used.

Figure 1 .

 1 Figure 1. Cellular uptake and retention time of siRNA-LNC in MIAMI E/F cells. After 4 hours' incubation, confocal microscopy was performed on MIAMI cells with siRNA LNCs. Cells werefixed on glass slide and nucleus staining was performed with DAPI (blue). Double fluorescent probes were used to follow siRNA LNCs: lipophilic DiD (yellow) and Alexa488 siRNA (green). Analysis confirmed the internalization of siRNA LNCs and it presence until day 6 (A) Positif siRNA cells were counting using imageJ. 6 images per conditions in X10 objective were selected (B) Scale bar represents 10µm.

Figure 2 .

 2 Figure 2. Specification of MIAMI siREST committed into LGE-like progenitors. For neuronal commitment, a simple protocol has been designed using sonic Hedgehog protein. Transfection of MIAMI E/F was performed with 250 ng/ml of siControl and siREST-LNCs (A). During the culture period of 14 days, MIAMI-siCtl-SHH cells show very few morphological changes. In contrast, MIAMI-siREST-SHH stretched out short neurite at 7 days, while at 14 days later, almost all of the MIAMI-siREST-SHH exhibited a neuron-like morphology (B). The quantification of surface area and the measure of cell lenght was performed fourteen days after transfection for MIAMI E/F, MIAMI-siCtl-SHH and MIAMI-siREST-SHH (C) the characterization of the differentiation was realized by RT-qPCR and immunofluorescence. The expression of genes

Figure 3 .

 3 Figure 3.Differentiation and Characterization of GABA neurons. Schematic procedure of GABAergic differentiation (A). The quantification of cell length superior to 600µm considered as positive response to the treatment, was performed fourteen days after the differentiation for MIAMI-siCtl-VPA/BDNF, MIAMI-siRest-VPA/BDNF, MIAMI-siCtl-VPA+BDNF, and MIAMI-siREST-VPA+BDNF (B). The characterization of the differentiation was realized by RT-qPCR and immunofluorescence. The expression of genes Dlx2, REST, β3 Tubulin, DARPP32 and GAD67 (C) was quantified at the end of the differentiation Results (n=1). Cytokines expression profile of differentiated cells was also determined by RTqPCR of cells alone at the end of the differentiation (C). For both cells VEGFA mRNA is the most expressed followed by BDNF mRNA and NGF. Results were in 2^-Delta Ct . Results (n=1). In vitro immunofluorescence against β3-tubulin, DARPP32, GAD67, GAT1 on MIAMI-siREST-VPA+BDNF (D) was performed. Scale bar = 50 μm.

Figure 4 .

 4 Figure 4.Adherence of MIAMI siRNA-SHH on PAMs. Different media for adherence were tested (A). We selected the media with 3% of serum for the rest of experiments. In this condition, we observe the adherence onto PAMs by brightfield microscopy at time 0 and 4H after cells and PAMs contact (B). Observation of blank PAMs (C) and cells/PAMs complexed (D) by scanning electronic microscopy. Cells number after 4h adherence onto PAMs was quantified by Cyquant ® Reagent (E). Immunofluorescence of laminin on PAM was observed by confoncal microscopy (F).

Figure 5 .

 5 Figure 5. Ex vivo GABAergic MSN differentiation of grafted cells. Immunofluorescence against Human mitochondria and human DARPP32 for MIAMI cells alone, seven days after grafting of cells or complexes with blank or BDNF PAMs. Scale bar is 150µm.

A

  previous PhD student (Nicolas Daviaud) demonstrated a limited effect of EGF/bFGF pre-treated MIAMI cells in terms of neuroprotection or differentiation in the ex vivo HD model. After injecting these MIAMI cells precommitted towards the GABAergic lineage, he described only 10-15% of stem cells expressing β3-tubulin, very few committed MIAMI cells expressed DARPP32 and the expression of GAD67 was undetected even when cells were adhered onto LM -PAMs. Based on the literature[37,89,90] we investigated a new 2-step protocol and we characterized the cells obtained four weeks after differentiation in vitro. The first step consisted on t he neuronal commitment in order to obtain LGE progenitors, from which MSN derive, and the second one on the final differentiation to obtain these GABAergic -like neurons. Specific media with Sonic Hedgehog was used during fourteen days after siREST transfection. SHH is a pleiotropic factor for the development of CNS driving proliferation, specification, and axonal targeting in multiple sites within the forebrain, hindbrain, and spinal cord. Studies in embryonic CNS have shown how gradients of this morphogen are translated by neuroepithelial precursors to

Figure 12 .

 12 Figure 12. The different steps observed during in vitro differentiation

30 and 60

 60 days to evaluate the long -term viabilit y of the slices and follow the degeneration of the GABAergic neurons. A recent study allowed observing few axonal projections (0.017%) after 9 wee ks injection of differentiated ESC and more axonal projections after in utero implantation, suggesting integration of the donor cells into the neu ronal network of the host brain[START_REF] Delli Carri | Developmentally coordinated extrinsic signals drive human pluripotent stem cell differentiation toward authentic DARPP-32+ medium-sized spiny neurons[END_REF]. But the poor quantit y of axonal projection demonstrated the diff iculty to hope replace GABAergic neurons. Although transplanted cells integrated and were able to reduce the motor asymmetry in the QA -lesioned model, long -term studies are needed to assess circuit reconstruction and behavioral recovery. With our model, we can obtain cortico -striatal brain slices or the whole coronal section.

Figure 13 .

 13 Figure 13.Schematic strategy finally used during this thesis and some advantages.

Table 1 .

 1 Most used of genetic model of rodent animals to model HD available nowadays

Table 2 .

 2 Non genetic models for modeling Huntington with brain slices

	INTRODUCTION

Table 2 . Non genetic models for modeling Huntington with brain slices.

 2

	Neurotoxins	Ex vivo models	Cells modification	Ref
	Kainic acid (KA)	Striatal organotypic	Enlargement of mitochondria,	[44]
		model	dilation of rough endoplasmic
			reticulum,	presence	of
			numerous	vacuoles,	glial
			fibrillary changes
	Quinoleic acid (QA)	Corticostriatal	Excitotoxic damages, presence	[48,49]
		organotypic model	of numerous vacuoles
	α-Amino-3-hydroxy-propionic acid 5-methylisoxazole-4-	organotypic model Corticostriatal	of numerous vacuoles Excitotoxic damages, presence	[50]
	hydrate
	3-nitropropionic acid (3-NPA)	Striatal and organotypic model corticostriatal	impaired mitochondrial function is No described but reductions of complex II-III Activity,	[46]
	QA + 3-NPA	Striatal organotypic	No described.		[51,52]
		model

1,2 and Montero-Menei CN. 1,2 *

	1 PRES LUNAM -University of Angers, F-49933 Angers, France
	2 INSERM U1066 -Micro et Nanomédecines Biomimétiques, 4 rue larrey, F-49933
	Angers, France
	* Corresponding author
	C. MONTERO-MENEI
	INSERM U1066, IBS-IRIS, 4 rue Larrey, 49 933 Angers Cedex 9, France
	Tel : +33 244 688 536, Fax : +33 244 688 546 : E-mail : claudia.montero-menei@univ-
	angers.fr

Table 2 . Description of the buffer solution needed during the first step of organotypic slice preparation.

 2 This solution must be prepared under aseptic conditions.

	Products	Quantity		Special instructions
	Gey's balanced salt	50 mL		Must be at 4°C
	solution
	D-glucose	6.5 mg/mL		Must be filtrated at 0.22
				µm
	Antibiotics	1 %		Streptomycine and
				penicilline
	Solution	Products	Quantity	Special
				instructions
	Media 1	Minimum Essential	50 %	sterile
		Medium Eagle
		(MEM) Alpha
	(From day 0 to	Modifications
	day 3)	Hanks balance	25 %	sterile
		salt solution
		Horse serum	25 %	Must be
				inactivated
		D-glucose	6.5 mg/mL	Must be filtrated
				at 0.22µm
		L-glutamine	1 mM	Must be filtrated
				at 0.22µm
		Antibiotics	1 %	Streptomycine
				and penicilline
	Media 2

(From day 3 to the end of the experiment)

	Neurobasal		sterile
	media
	D glucose	3.25 mg/mL	Must be filtrated
			at 0.22µm
	L-glutamine	1 mM	Must be filtrated
			at 0.22µm
	B27	2 X
	Antibiotics	1 %	Streptomycine
			and penicilline

Table 1 .

 1

Table 2 . Sequence of primers validated in RT-qPCR 2.7 WESTERN BLOT:

 2

	Total proteins were isolated by sonication of cells in l ysis buffer composed
	of 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, pH 8, 2.5 mM EGTA,
	pH 7.4, 0.1% Tween 20, 10% gl ycerol, 0.1 mM sodium orthovanadate, 1 mM
	sodium fluoride, 10 mM β -gl ycerophosphate, 0.1 mM phenylmethylsulfonyl
	fluoride (PMSF), and complete protease inhibitor cocktail (Roche). The
	quantification of protein used was performed according to Bradford method
	(26).
	Twent y μg of protein extracts were separated by SDS -10% PAGE and
	transferred to PVDF membranes (GE Healthcare, VWR, Milan, Ital y). The
	membranes were incubated overnight with rabbit anti -REST antibody (1/200)

(Abcam, Cambridge, UK), washed with Tris -buffered Saline and incubated with anti-rabbit (1/5000) (32460, Thermo scientific) for 6 hours. Immunostaining was revealed by the Immuno -Star HRP substrate (BioRad, Segrate (MI), Ital y) according to manufacturer's instructions and quantified by Kodak Ima ge Station

Table

	Simples		Size (nm) before	Zeta-Potential	Size	(nm)	Zeta-Potential
			freezing	(mV) before	after		(mV)	after
				freezing	freezing		freezing
	siRNA-LNC PBS	81,7 +/-2,45	4,929 +/-0,82	6000		ND
	siRNA-LNC	1%	83,9 +/-3,15	5,35 +/-1,56	95 +/-4,72	17,2 +/-6,18
	trehalose
	siRNA-LNC	1%	82,34 +/-1,87	7,23 +/-2,27	80,6 +/-0,59	6 +/-0,25
	glucose
	siRNA-SP-AP PBS	195,64 +/-3,90	-36,3 +/-1,22	275 +/-7,40	-14,56 +/-5,3
	siRNA-SP-AP 5%	202,65 +/-2,39	-41,2 +/-0,74	199 +/-1,98	-27,1 +/-1,04
	trehalose
	siRNA-SP-AP 5%	202,12 +/-2,83	-36,7 +/-3,2	179 +/-2,57	-27,4 +/-0,88
	glucose

Table 1 . Different media tested for the differentiation of MIAMI cells 2.5 REVERSE TRANSCRIPTION AND REAL TIME QUANTITATIVE PCR

 1 Roche) with a first denaturation step at 95°C for 10 min and 40 cycles of 95°C for 15 s, 60°C for 30 s. After amplificat ion, a melting curve of the products determined the specificity of the primers for the targeted genes.

	CULTURE
	2.3.1 MIAMI E/F
	MIAMI cells were expanded in vitro from passage 4 -5 on fibronectin
	(Sigma Aldrich, St Louis, USA) coated flasks at 125 cells/cm² in low ox ygen
	tension in Dulbecco's Modified Eagle Medium -low glucose (DMEM, Gibco, Life
	Technologies, Paisley, UK), supplemented with 3% of serum, 30µg/ml ascorbic
	acid and a mixture of lipids (w orking concentration of 510nM lipoic, 70nM
	linolenic and 150nM linoleic acid, all from Sigma). Then a 10 day treatment
	with an addition of 20ng/mL of EGF and 20ng/mL of bFGF (both from R&D
	s ystems, Lille, France) and 5µg/ml of Heparin (Sigma Aldrich, St -Louis, USA)
	is conducted to enhance neuronal specification. Cells were fed every 3 days by
	changing half of the medium, and split every 5 days.
	2.4 MIAMI CELL DIFFERENTIATION
	2.4.1 MIAMI cell transfection
	MIAMI E/F cells were seeded at 3000 cells per cm² coated with laminine
	(2µg/cm², Sigma Aldrich, St -Louis, USA). Experiments were performed in
	MIAMI Opti -MEM ® media (Life technologies, France). SiRNA -LNCs were
	incubated with cells at 37 °C in a humidified atmosphere with 5 % CO 2 for 4h
	before serum addition. Cells we re harvested at appropriate times after culture
	and assayed for mRNA expression levels by RT -qPCR or protein expression by
	immunofluorescence.
	2.4.2 LNC cell time retention
	MIAMI cells were seeded on glass at 3000 cells per cm² coated with
	laminine (2µg/cm², Si gma Aldrich, St-Louis, USA). Experiments were
	performed in MIAMI Opti -MEM ® media (Life technologies, France). SiRNA
	fluorescent LNC and 100 n LysoTracker Red (Molecular Probes, Eugene, OR,
	U.S.A.) were incubated with cells at 37 °C in a humidified atmosphe re with 5 %

Table 2 . Sequence of primers validated in RT-qPCR 2.6 IMMUNOFLUORESCENCE

 2

	After treatment, cells were fixed by addition of 1ml of ice cold 4%
	paraformaldehide (PFA, Sigma, St Louis, USA) in PBS (Lonza, Verviers,
	Belgium) pH 7.4 during 15min. Then cells were washed in DPBS three ti mes.
	Non-specific sites were blocked with DPBS, Triton 0.1% (PBS -T, Triton X -100,
	Sigma, St Louis, USA), bovine serum albumin 4% (BSA, Fraction V, PAA Lab,
	Austria), normal goat serum 10% (NGS, Sigma, St Louis, USA) during 45 min at
	RT. A mouse anti human β3-tubulin (2ng/ml, clone SDL.3D10, Sigma, St Louis,
	USA), a mouse anti human neurofilament medium (NFM, 1:50, clone NN18,
	Sigma Aldrich, USA), a monoclonal rabbit anti human dopamine -and cAMP -
	regulated neuronal phosphoprotein (DARPP32, 1:400, clone EP721 Y, Abcam,
	Paris, France), A mouse anti glutamic acid decarbox ylase -67 antibody (GAD67,
	5µg/ml, clone 1G10.2, Millipore SA, Guyancourt, France), were used to
	characterize cell differentiation. Cells were incubated overnight with the
	primary antibody diluted in PBS-T, BSA 4% at 4°C. After washes, slices were

OF PAMS-CELLS IN ORGANOTYPIC SLICES

	left in place for 5 min to avoid the cells being expelled from the organot ypic
	slices.
	2.8.3 Histological study
	2.8.1 Organotypic slices preparation
	Seven days after injection , organot ypic slices were fixed by addition of
	Six to eight days Sprague Dawley rat pups were sacrificed after 5ml of ice cold 4% PFA (Sigma, St Louis, USA) in PBS (Lonza, Verviers,
	anaesthetic and brains were removed. Cerebral hemispheres were separated and Belgium) pH 7.4 during 2 hours. Then slices were washed in PBS three times.
	glued on the vibratome plate on their central side. 400µm coronal slices were Nonspecific sites were blocked with PBS, Triton 1% (PBS -T, Triton X-100,
	obtained using a vibratome (mo torized vibroslice, Campben instruments, Sigma, St Louis, USA), BSA 4% (Fraction V, PAA Lab, Austria), NGS 10%
	Loughborough, England) °. Cortico -striatal slices were then dived into Grey"s (Sigma, St Louis , USA) during 4 hours at RT under agitation (expect for CD31
	Salt Balanced Solution (Sigma Aldrich) supplemented with 6,5mg/ L of glucose study, in this case, 0.05% Triton is employed).
	(Sigma Aldrich, St -Louis, USA) and antibiotics (Sigma Aldrich, St Louis, USA).
	Three to four slices per hem isphere were next transferred to 30 mm diameter MIAMI cells were detected using a mouse anti -human mitochondria
	semi-porous membrane inserts (Millicell -CM, Millipore, Bedford, MA, USA) antibody (10ng/ml, clone MTCO2, Abcam, Paris, France). A monoclonal rabbit
	within a 6-well plate and put in culture at 37ºC, 5% CO2. From day 0 to day 3, a anti human dopamine-and cAMP -regulated neuronal phosphoprotein (DARPP32,
	serum containing medium was used: 50% MEM (Minimum Ess ential Medium 1:400, clone EP721Y, Abcam, Paris, France), antibody was used to characterize
	Eagle, Sigma Aldrich), 25% Hank"s (Hank"s Balanced Salt Solution, Sigma grafted cells differentiation. Slices were incubated 48h with the primary
	Aldrich), 25% of horse serum (decomplemented horse serum, Gibco), 6.5mg/ml antibody diluted in PBS -T, BSA 4% at 4°C. After washes, slices were incubated
	of glucose, 1 mM of L-glutamine (Sigma Aldrich, St -Louis, USA) and with the corresponding biotinylated mouse or biotinylated rabbit secondary
	antibiotics (Sigma Aldrich, St-Louis, USA). From day 3 to day 16, a serum free antibody (7.5 µg/ml, Vector Laboratories, B urlingame, USA). Then slices were
	medium was used: neurobasal medium (Gibco) supplemented with 6.5mg/L of washed and incubated with Streptavidin Fluoroprobes R488 or R547H
	glucose, 1mM of -glutamine (Sigma Aldrich, St Louis, USA), B27 supplements (Interchim, Montluçon, France) diluted 1:200 in PBS for 2 hours before
	(50x, B27 supplements, Gibco) and antibiotics (Sigma Aldri ch, St-Louis, USA) mounting with a fluorescent mounting medium. Isot ypic controls and/or
	this media was changed every two days. omission of the primary antibody were performed to assess the specificit y of the
	immunostainings. Slices were observed with a fluorescence microscope
	2.8.2 Injection of stem cell/ PAM complexes into organotypic slices (Axioscop, Carl Zeiss, LePecq, France).
	Five days after organot ypic slice preparation, treatments were injected
	into the striatum using a 22 -gauge needle (Hamilton, Bonaduz, Switzerland)
	connected to a micromanipulator. Three experimental groups were tested: (1)
	MIAMI siREST SHH cells, (2) MIAMI siREST SHH LM -PAM, and (3) MIAMI
	siREST SHH LM -PAM-BDNF. Total injection volume consisted of 4µl of
	culture media containing approxim atel y 100.000 cells alone or adhered to 0.2
	mg of PAMs. Injections were done at 0.5µl/minute infusion rate. The needle was

ANNEXES

The authors thank the SCAHU and more particularl y Pierre LEGRAS and Jérôme Roux for animal care. We are also grateful to Jerem y R IOU for his expertise in statistical a nal ysis. This project is supported by the "Education Audiovisual" and the cultural executive agency of the European Union through the NanoFar Erasmus Mundus joint Doctoral program, by Angers Loire Métropole. ACKNOWLEDGEMENTS BIBLIOGRAPHY CHAPTER I We thank the SCIAM ("Service Commun d' Imagerie et d'Anal yse Microscopique") of Angers for conf ocal microscopy images , more particularl y Rodolphe PERROT and MEB images as well as the PACEM core facilit y ("Plateforme d'Analyse Cellulaire et Moléculaire", Angers, France) of Angers for the use of PCR facilities. We also wanted to thanks Professor Pau l C. Schiller and Gaëtan Delcroix for helpful critiques and experiments. This project is supported by the "Education Audiovisual" and the cultural executive agency of the European Union through the NanoFar Erasmus Mundus Joint Doctoral program, by Angers Loire Métropole, ACKNOWLEDGMENTS BIBLIOGRAPHY

Competitiveness of Spain (MAT2013 -47501-C2-2-R) and Xunta de Galicia (Competitive Reference Groups, FEDER Fu nds, Ref. 2014/043), as well as National Portuguese funding through FCT -Fundação para a Ciência e a Tecnologia, project PEst -OE/QUI/UI4023/2011. "La science a fait de nous des Dieux, avant même que nous soyons dignes d'être des hommes."

PUBLICATION N°1

In this study we developed and cha racterized a relevant ex vivo model of HD using membrane techniques culture and specific media. This new model can be use to study the earl y stage of the pathology because we observed the reduction of striatum such as in the HD in 4 days. This represents a perfect model to study the relevance of new and innovative treatment.

PUBLICATION N°3

André E. 1,2 ; Kandalam S. 1,2 ; Sindji L.