J-R Gaëtan

Delcroix

Kevin Curtis
email: kevinmcurtis@gmail.com

Paul C Schiller
email: pschille@med.miami.edu

Claudia N Montero- Menei

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

pre-treatment enhances neural specification and the response to neuronal commitment of M I A M I cells

Keywords: Mesenchymal stromal cell, epidermal growth factor, basic fibroblast growth factor, neuronal development, cell therapy

Aims. Multipotent mesenchymal stromal cells raise great interest for regenerative medicine studies.

Some MSC subpopulations have the potential to undergo neural differentiation, including MIAMI (Marrow Isolated Adult Multilineage Inducible) cells, which differentiate into neuron-like cells in a multi-step neurotrophin 3-dependent manner. Epidermal and basic fibroblast growth factors are often used in neuronal differentiation protocols for MSCs, but with a limited understanding of their role. In this study, we thoroughly assessed for the first time the capacity of these factors to enhance the neuronal differentiation of MSCs. Materials & Methods. We have characterized MIAMI cell neuronal differentiation program in terms of stem cell molecule expression, cell cycle modifications, acquisition of a neuronal morphology and expression of neural and neuronal molecules in the absence and presence of an EGF-bFGF pre-treatment. Results. EGF-bFGF pre-treatment down-regulated the expression of stemness markers Oct4A, Notch1 and Hes5, whereas neural/neuronal molecules Nestin, Pax6, Ngn2 and the neurotrophin receptor tyrosine kinase 1 & 3 were up-regulated. During differentiation, a sustained Erk phosphorylation in response to NT3 was observed, cells began to exit from the cell cycle and exhibit increased neurite-like extensions. In addition, neuronal -tubulin and

Neurofilament expression was increased, a slight preoligodendrocyte engagement was noted, and no default neurotransmitter phenotype was observed. Overall, mesodermal markers were unaffected or decreased, while neurogenic/adipogenic PPAR 2 was increased. Conclusion. EGF and bFGF pretreatment enhances neural specification and the response to neuronal commitment of MIAMI cells, further increasing their potential use in adult cell therapy of the nervous system.

Abbreviation list: bFGF: basic fibroblast growth factor; BSA: bovine serum albumin; DMEM:

-high glucose; DPBS: Dulbecco's phosphate buffered saline; EGF: epidermal growth factor; FBS: foetal bovine serum; HBSS: Hank's Buffered Salt Solution; ICF: immunocytofluorescence; ITS: insulin-transferrin-selenium; MIAMI: marrow isolated adult multilineage inducible; MSC: marrow stromal cell; NSC: neural stem cell; NT3: neurotrophin 3; Ntrk: neurotrophic tyrosine kinase receptor; RT-qPCR: real-time quantitative polymerase chain reaction; SD: standard deviation; SEM: scanning electron microscopy; WB: western blot.

I N T R O D U C T I O N

Stem cells hold a great promise in regenerative medicine due to their capacity to self-replicate and to produce various differentiated cell types. Nevertheless, the contribution of each stem cell type has to be clearly evaluated in the context of a specific pathology. Among the different sources of stem cells, adult multipotent mesenchymal stromal cells (MSCs) are interesting for cell therapy studies due to their large differentiation potential and their immunoregulatory properties [START_REF] Delcroix | Adult cell therapy for brain neuronal damages and the role of tissue engineering[END_REF][START_REF] Blanc | HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells[END_REF][START_REF] Blanc | Immunomodulatory effects of fetal and adult mesenchymal stem cells[END_REF][START_REF] Maitra | Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation[END_REF][START_REF] Nasef | Immunosuppressive effects of mesenchymal stem cells: involvement of HLA-G[END_REF][START_REF] Gotherstrom | Immunologic properties of human fetal mesenchymal stem cells[END_REF][START_REF] Horwitz | Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement[END_REF]. Moreover, their easy isolation from bone marrow and their self-renewal capacity may allow autologous grafts to be performed. A number of studies have shown that subpopulations of MSCs differentiate into neuron-like cells in vitro, either by genetic manipulation, with the help of co-culture systems or by using different inducer molecules and growth factors [START_REF] Hermann | Epigenetic conversion of human adult bone mesodermal stromal cells into neuroectodermal cell types for replacement therapy of neurodegenerative disorders[END_REF][START_REF] Ross | Evaluation of neural plasticity in adult stem cells[END_REF][START_REF] Song | Brain as the Sea of Marrow[END_REF]. In regard of the difficulties in obtaining neural stem cells (NSCs) from adults and the inherent ethical problems linked with their isolation from foetal tissue, the identification of alternate sources of neuron-like cells for cell therapy in the central nervous system is of great importance [START_REF] Delcroix | Adult cell therapy for brain neuronal damages and the role of tissue engineering[END_REF].

MSCs transplanted in adult rat brains may respond to microenvironmental cues and a fraction of them differentiate into neuron-like cells [START_REF] Kopen | Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains[END_REF][START_REF] Zhao | Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats[END_REF][START_REF] Jendelova | Magnetic resonance tracking of transplanted bone marrow and embryonic stem cells labeled by iron oxide nanoparticles in rat brain and spinal cord[END_REF]. In addition, MSCs, that can migrate towards sources of lesions in the brain [START_REF] Jendelova | Magnetic resonance tracking of transplanted bone marrow and embryonic stem cells labeled by iron oxide nanoparticles in rat brain and spinal cord[END_REF][START_REF] Delcroix | Mesenchymal and neural stem cells labeled with HEDP-coated SPIO nanoparticles: in vitro characterization and migration potential in rat brain[END_REF][START_REF] Mahmood | Intracerebral transplantation of marrow stromal cells cultured with neurotrophic factors promotes functional recovery in adult rats subjected to traumatic brain injury[END_REF][START_REF] Sykova | In vivo tracking of stem cells in brain and spinal cord injury[END_REF][START_REF] Hellmann | Increased survival and migration of engrafted mesenchymal bone marrow stem cells in 6-hydroxydopamine-lesioned rodents[END_REF], may also provide a functional improvement in animal models, either directly or indirectly by their ability to produce various growth factors [START_REF] Mahmood | Intracerebral transplantation of marrow stromal cells cultured with neurotrophic factors promotes functional recovery in adult rats subjected to traumatic brain injury[END_REF][START_REF] Zhang | Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice[END_REF][START_REF] Chen | Ischemic rat brain extracts induce human marrow stromal cell growth factor production[END_REF][START_REF] Li | Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery[END_REF]. Finally, their administration in the central nervous system is feasible and seems to be safe in human subjects [START_REF] Bang | Autologous mesenchymal stem cell transplantation in stroke patients[END_REF].

For all of these reasons, use of MSCs may become a clinically viable technology for cell therapy of the central nervous system that is not hindered by ethical and tissue rejection-related concerns. However, further investigations are still required, particularly with human MSCs, to clearly evaluate their neuronal differentiation potential.

In the present study, we have used a homogenous subpopulation of developmentally immature human MSCs termed MIAMI (Marrow Isolated Adult Multilineage Inducible) cells. They are small cells with a reduced cytoplasm, that express markers typical of embryonic stem cells and are capable of differentiating in vitro into cell lineages derived from all three germ layers, as previously described [START_REF] Ippolito | Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential[END_REF]. Moreover, MIAMI cells, which are isolated and expanded under niche-like conditions, including a low oxygen tension environment, express the major receptor for hepatocyte growth factor, c-met [START_REF] Ippolito | Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential[END_REF], as well as a number of chemokine and growth factor receptors, that may enhance their migratory potential leading to an improved regenerative potential [START_REF] Rosova | Hypoxic preconditioning results in increased motility and improved therapeutic potential of human mesenchymal stem cells[END_REF]. We recently reported a 3-step neurotrophin 3 (NT3)-dependent differentiation of these cells using a protocol that aims to mimic the differentiation program of NSCs at the molecular level. At the end of this process, 50-60 % of MIAMI cells became immature neuron-like cells with a fraction showing ionic channel activity but no action potential [START_REF] Tatard | Neurotrophin-directed differentiation of human adult marrow stromal cells to dopaminergic-like neurons[END_REF]. For convenience purpose, in this study, this process . Based on these results, it appears necessary to improve the neuronal differentiation potential of MIAMI cells, as well as to better understand their behavior and the molecular mechanisms that direct it, in order to optimize their future use in cell therapy studies.

Epidermal growth factor and basic fibroblast growth factor (EGF and bFGF) are powerful mitogens and cell fate drivers for NSCs and neural precursor cells present in the developing and adult central nervous system, as well as for ES cell-derived neural precursors [START_REF] Bithell | Fibroblast growth factor 2 maintains the neurogenic capacity of embryonic neural progenitor cells in vitro but changes their neuronal subtype specification[END_REF][START_REF] Tsai | Fibroblast growth factor 2 negatively regulates the induction of neuronal progenitors from neural stem cells[END_REF][START_REF] Temple | The development of neural stem cells[END_REF][START_REF] Caldwell | Heparin stabilizes FGF-2 and modulates striatal precursor cell behavior in response to EGF[END_REF][START_REF] Guan | Embryonic stem cell-derived neurogenesis. Retinoic acid induction and lineage selection of neuronal cells[END_REF][START_REF] Reynolds | A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes[END_REF]. Interestingly, these factors may also permit the long-term proliferation of human mesencephalic precursors expanded under hypoxia [START_REF] Schwarz | Developmental perspectives on human midbrain-derived neural stem cells[END_REF], while bFGF may contribute to maintain the neurogenic niche in vivo [START_REF] Mudo | The FGF-2/FGFRs neurotrophic system promotes neurogenesis in the adult brain[END_REF]. It has been shown that MSCs, including MIAMI cells, may already express certain neural-related genes [START_REF] Tondreau | Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation[END_REF][START_REF] Woodbury | Adult bone marrow stromal stem cells express germline, ectodermal, endodermal, and mesodermal genes prior to neurogenesis[END_REF], and it was therefore assumed that an EGF-bFGF exposure may further improve their in vitro neuronal differentiation. In MSCs, the expression of Nestin, a marker expressed by NSCs [START_REF] Gilyarov | Nestin in central nervous system cells[END_REF], was up-regulated after EGF and bFGF exposure under adherent [START_REF] Song | Comparison of Neuron-Like Cells Derived from Bone Marrow Stem Cells to Those Differentiated from Adult Brain Neural Stem Cells[END_REF] or non-adherent conditions prior to in vitro neurogenesis [START_REF] Hermann | Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells[END_REF][START_REF] Kim | Neural differentiation potential of peripheral blood-and bone-marrow-derived precursor cells[END_REF][START_REF] Yang | A simple and efficient method for deriving neurospheres from bone marrow stromal cells[END_REF]. Furthermore, recent studies described the differentiation of MSCs, after a firststep treatment with EGF-bFGF, into dopamine producing cells leading to a functional improvement in a rat model of [START_REF] Barzilay | Induction of human mesenchymal stem cells into dopamine-producing cells with different differentiation protocols[END_REF][START_REF] Levy | Regenerative effect of neural-induced human mesenchymal stromal cells in rat models of Parkinson's disease[END_REF]. However, a precise characterization of the impact of an EGF-bFGF pre-treatment, prior to MSC neuronal differentiation, by comparing to non pre-treated cells and extensively analyzing several neuronal molecules is still lacking [START_REF] Song | Comparison of Neuron-Like Cells Derived from Bone Marrow Stem Cells to Those Differentiated from Adult Brain Neural Stem Cells[END_REF][START_REF] Hermann | Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells[END_REF][START_REF] Kim | Neural differentiation potential of peripheral blood-and bone-marrow-derived precursor cells[END_REF][START_REF] Yang | A simple and efficient method for deriving neurospheres from bone marrow stromal cells[END_REF][START_REF] Long | Neural cell differentiation in vitro from adult human bone marrow mesenchymal stem cells[END_REF]. It is therefore important to thoroughly assess the benefits of using EGF-bFGF pre-treatment prior to the induction of MSCs toward a neuronal lineage.

In this study, we first sought to assess the potential benefits of an EGF-bFGF pre-treatment in terms of neuronal lineage specification of MIAMI cells to further improve their neuronal differentiation in vitro. In addition, the effects of the EGF-bFGF pre-treatment was characterized and compared on MIAMI cells harvested from vertebral bodies or from iliac crest of living donors. As NT3 is essential for MIAMI cells neuronal-like differentiation, we also studied the effect of EGF-bFGF on the response of MIAMI cells to NT3, by evaluating the expression of the NT3 receptors and Erk phosphorylation involved in this transduction pathway. Neuronal differentiation involves the reduction of cell proliferation leading to post-mitotic cells, a process accompanied with a loss of stem cell molecules together with the acquisition of a neuronal morphology and the expression of neural and neuronal molecules. Therefore, we further analyzed the behavior of MIAMI cells, with or w/o pre-treatment, along the differentiation protocol in terms of neuronal morphology, proliferation, apoptosis and molecular expression pattern.

M A T E R I A LS A N D M E T H O DS

Bone marrow harvesting, selection & expansion of M I A M I cells. Whole bone marrow was obtained from vertebral bodies (T1 L5) of a 3 year old male cadaveric donor who died of fatal traumatic injury (MIAMI #519) [START_REF] Ippolito | Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential[END_REF] or from iliac crest of a 20 year old male living donor (Lonza Walkersville, Maryland; MIAMI #3515), following guidelines for informed consent set by the University of Miami School of Medicine Committee on the Use of Human Subjects in Research. As previously described [START_REF] Ippolito | Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential[END_REF], isolated whole bone marrow cells were plated at a constant density of 10 Medicine) and cultured as previously described [START_REF] Tatard | Neurotrophin-directed differentiation of human adult marrow stromal cells to dopaminergic-like neurons[END_REF].

E G F -b F G F pre-treatment. A 10 day EGF & bFGF pre-treatment was performed using either a low dose (5 ng/mL) or a high dose (20-50 ng/mL) of both EGF and bFGF (R&D Systems Europe, Lille, France) supplemented with 5 µg/mL heparin (Sigma) and a lipid mixture (working concentration of presented results were mainly obtained with 20 ng/mL of EGF-bFGF. To investigate the NT3 response of pre-treated cells, we checked the neurotrophin receptor expression in response to 0, 50 and a higher dose (100 ng/mL) of EGF-bFGF. As we already observed an effect at the intermediary dose (50 ng/mL), we continued with this dose for the detection of Erk/pErk. Briefly, cells were treated with NT3 (30 ng/mL) for 5, 10 and 15 minutes following a serum deprivation phase of 24 hours prior processing for western blotting.

M I A M I cells neuronal-like differentiation. This 3-step differentiation process was described in greater detail elsewhere [START_REF] Tatard | Neurotrophin-directed differentiation of human adult marrow stromal cells to dopaminergic-like neurons[END_REF]. Non pre-treated or EGF-bFGF pre-treated MIAMI cells passages 5 to 9 were seeded at 3000 4000 cells/cm 2 on a 2 µg/cm fibronectin-coated surface in DMEM F12 (GIBCO) supplemented with 20 % FBS, 10 ng/mL bFGF (for the non pre-treated), antibiotics and cultured for 24 h (Neural specification, step 1). At the end of the neural specification treatment, cells were washed and neuronal commitment (step 2) was induced by exposing the cells to 30 ng/ml NT3 (R&D Systems) in the presence of 1 mM -mercaptoethanol (Sigma), for 2 days. Neuronal differentiation (step 3) was induced by rinsing and then exposing the cells to 100 hydroxyanisole, 25 mM KCl, 2 and eluted in 50 µL RNAse free water (Gibco). Five microliters of cDNA (1:20) were mixed with iQ SYBR Green Supermix (Biorad) and primer mix (0.2 µM) in a final volume of 15 µL. Amplification was carried on a Chromo4 thermocycler (Biorad) with a first denaturation step at 95°C for 3 min and 40 cycles of 95°C for 10 s, 55°C for 15 s and 72°C for 15 s. After amplification, a melting curve of the products determined the specificity of the primers for the targeted genes. A mean cycle threshold value (Ct) was obtained from 2 measurements for each cDNA. Several housekeeping genes, Glyceraldehyde-3-phosphate dehydrogenase (Gapdh, NM_002046), Hypoxanthine phosphoribosyltransferase 1 (Hprt1, NM_000194), Beta actin (Actb, NM_001101), 30S ribosomal protein S18 (Rps18, NM_001093779)

and Heat shock 90 kD protein 1 beta (Hspcb, NM_007355) were tested for normalization. The www.primerdesign.co.uk/geNorm.asp) was used to determine that Gapdh, Hprt1 & Hspcb were the three most stable housekeeping genes. The relative transcript quantity (Q) was determined by the delta cT method Q=E (Ct min in all the samples tested-Ct of the sample) 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. [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF]. Q normalized = Q/(geometric mean of the 3 most stable housekeeping genes Q). In facilities, normalization was performed in a similar manner with two housekeeping genes, Eukaryotic translational elongation factor 1 alpha (Ef1a, NM_001402) and Ribosomal protein L13a (RpL13a, NM_01242). On figures, results are expressed as a percentage of change in the expression of the mRNA relative to the expression in expanding MIAMI cells ± average deviation (% change mRNA X=

[(Q x -Q expanding MIAMI cells )/Q expanding MIAMI cells ] x 100).
Western blotting. Cell protein extracts were separated into Triton X-100 soluble and insoluble lysates.

Protease inhibitor cocktail (Sigma) was added to both lysis buffers directly prior to use. After rinsing, the cells were incubated with the secondary biotinylated anti-mouse antibody ( after EGF-bFGF treatment (fig. 2A). However, EGF-bFGF treatment did not further up-regulate Ntrk3 expression in iliac-crest isolated MIAMI cells, which express relatively higher basal levels of this receptor. In these cells, NT3 stimulation (30 ng/mL) induced Erk1/2 phosphorylation (p42/p44), which peaked after 5 min, decreased after 10 min and was not detectable after 15 min of stimulation. EGF-bFGF treatment resulted in a more sustained phosphorylation of pErk1/2 (p42 and p44: p ), still present at 10 min of NT3 stimulation compared to non-treated cells (fig. 2B,C). This result shows a differential response to NT3 stimulation after EGF-bFGF treatment, suggesting that the pre-treatment may permit the cells to better respond to NT3 during the neuronal differentiation.

E ffects of E G F -b F G F pre-treatment during neuronal differentiation. To better understand the effects of the EGF-bFGF treatment, we describe in this second section its effects on the neuronal differentiation potential of MIAMI cells.

Decreased proliferation during neuronal differentiation. The antiproliferative gene p21 was upregulated during EGF-bFGF treatment (10.2 ± 0.7 fold increase) and Trypan blue counting suggested that cell doubling rate was reduced after exposure to EGF-bFGF. This reduced cell doubling rate was also observed during differentiation when cells were pre-treated with 5 ng/mL EGF-bFGF, while cell doubling was almost abolished with 20 ng/mL EGF-bFGF pre-treatement (fig. 3A). Notch1 expression, which was already down-regulated after EGF-bFGF treatment, further decreased during neuronal differentiation, both for non pre-treated and EGF-bFGF pre-treated cells (fig. 3B). Moreover, expression of the cell cycle markers p21 & Cyclin D1 at the RNA level confirmed the decreased proliferation observed during differentiation after pre-treatment (fig. 3C,D). Indeed, p21 was low in non pre-treated cells, it increased during differentiation in 5 ng/mL pre-treated cells and it was already elevated after 20 ng/mL pre-treatment, prior to the beginning of the differentiation. Conversely, Cyclin D1 expression was high in non pre-treated cells and increased during differentiation. In 5 ng/mL EGF-bFGF pre-treated cells, Cyclin D1 decreased during differentiation whereas it was always low in cells pre-treated with a higher dose of EGF-bFGF. Importantly, dead cells were never detected during neuronal differentiation, with or w/o pre-treatment. To further confirm that there was no contribution from apoptosis to the reduced proliferation rate observed, a Caspase-3 apoptosis assay was performed.

No Caspase-3 activity was detected, strongly suggesting that MIAMI cells, pre-treated or not with EGF-bFGF, did not undergo detectable apoptosis. In addition, it is interesting to note that at the end of differentiation, non pre-treated and EGF-bFGF pre-treated cells were sensitive to an apoptotic stress induced by 1 µg/mL staurosporine (fig. 4A). When exposed to staurosporine, the cells exhibited an increased Caspase 3 activity as well as a retracted morphology and nucleus fragmentation (fig. 4, B & C). However, under expansion conditions, both non pre-treated and pre-treated MIAMI cells seemed to be resistant to the proapoptotic inducer staurosporine as Caspase-3 activity was never detected (fig. 4A).

Homogeneous cell morphology and induced neurite extensions. Under low density expansion conditions, MIAMI cells are very small, slightly bipolar cells with a reduced cytoplasm compared to traditional MSCs. When treated with a combination of EGF-bFGF, MIAMI cells exhibited an even more homogeneous morphology with a refractile body, compared to non treated cells (fig. 5A,B). In addition, EGF-bFGF pre-treatment resulted in important morphological changes reminiscent of neurons at the end of the differentiation protocol (step 3). During the six days of neuronal differentiation, the small MIAMI cells gradually extended, thereby acquiring a neuron-like morphology with long neuritelike extensions (fig 5C ,D). The total cell length could achieve more that 400 µm at the end of the differentiation when the EGF-bFGF pre-treatment was applied (table 2). EGF-bFGF pre-treated cells were also thinner than non pre-treated cells (fig. 5C,D & table 2) and numerous connexions could be observed by SEM at the end of differentiation (fig. 5E,F). Similar morphological changes were observed during neuronal differentiation when pre-treating iliac crest-isolated MIAMI cells with the EGF-bFGF pre-treatment.

Improved neuronal expression pattern during neuronal differentiation. Nestin expression was upregulated during pre-treatment and its expression decreased during differentiation as observed by ICF at the end of step 3 and even more drastically after 12 days of step 3 (data not shown). The pattern of expression of the early neuronal marker -Tubulin followed the neuronal developmental program only if cells were pre-treated with EGF-bFGF. -Tubulin mRNA increased during neuronal differentiation before decreasing at the end of step 3 (fig. 6A). Moreover, its expression was already high after pretreatment compared to non pre-treated cells. Using ICF, -Tubulin expression at the end of step 2 and 3 was always significantly higher for EGF-bFGF pre-treated cells compared to non pre-treated cells, both in terms of the percentage of -Tubulin expressing cells and marker intensity (fig. 6B,E). In addition, assessment of -Tubulin expression at the end of step 2 by ICF confirmed that removal of NT3 lowered the -Tubulin-positive cells at the end of step 2 (data not shown) as previously described [START_REF] Tatard | Neurotrophin-directed differentiation of human adult marrow stromal cells to dopaminergic-like neurons[END_REF], further supporting the role of NT3 on the neuronal differentiation of MIAMI cells.

The mRNA expression of the mature neuronal marker Neurofilament medium (N F M) underwent a strong decrease at the end of step 3 for non pre-treated cells. In contrast, expression of N F M increased throughout the differentiation protocol when the pre-treatment was used (fig. 6C). -Tubulin, NFM protein expression was higher for pre-treated cells at the end of step 3 (fig. 6D,E).

Neurofilament light (N F L), N F M and Neurofilament heavy (N F H) mRNAs were all up-regulated at the end of step 2 when iliac crest-isolated MIAMI cells were pre-treated with EGF-bFGF compared to non pre-treated cells (54.3 ± 9.3, 6.8 ± 0.2 & 2.0 ± 0.3 fold increase for N F L, N F M and N F H, respectively).

Additionally, the unphosphorylated microtubule associated protein Tau, characteristic of immature neurons, was readily detectable throughout the neuronal differentiation procedure by Western blot analysis while we did not detect its phosphorylated form (data not shown). The major changes observed with and without EGF-bFGF pre-treatment before and during neuronal differentiation are summarized in table 3.

Neuronal neurotransmitter phenotypes during neuronal differentiation. Acetylcholinesterase Glial phenotypes during neuronal differentiation. Galactosylceramidase (GalC), characteristic of oligodendrocytes, was slightly expressed by MIAMI cells and further increased during neuronal differentiation for pre-treated cells, whereas its expression does not significantly vary in non pre-treated cells (fig. 7A). In opposition, Oligodendrocyte lineage transcription factor 2 (Olig2), another early marker of oligodendrocytes, was never detected in MIAMI cells. Glial fibrillary acidic protein (G F AP), specific of astrocytes, was not detected at the end of the differentiation. Previous results suggested that an insulin, transferin, selenium and lipids complement (ITS+3) improved cell morphology and membrane potential when used in step 3 (data not shown). However, when cultured in this lipid rich medium all along the differentiation protocol, numerous perinuclear lipid vesicles appeared at the end of step 2 and remained until the end of step 3, in a more significant manner for pretreated cells, as observed with oil red staining (fig. 7C,D). The lipid nature of these vesicles was confirmed using back-scattered electron SEM imaging (fig. 7B), with visualization of osmium on double bonds-molecules rich sites.

Mesodermal phenotypes during neuronal differentiation. We analysed the expression of several genes related to mesodermal phenotypes, the major differentiation pathways of MSCs. Sox9, specific to chondrogenic progenitors, was slightly up-regulated during neuronal differentiation of non pre-treated cells (1.5 ± 0.1 fold increase); whereas it was down-regulated when cells were pre-treated with EGF-bFGF (2.5 ± 0.6 fold decrease), thereby underlining the importance of the pre-treatment for neuronal differentiation purposes. The osteo-chondro marker Runt-related transcription factor 2 (Runx2) was always detected at very low levels, while the osteogenic marker Osterix (Sp7) was never detected.

Peroxisome proliferator-activated receptor gamma (PPAR gamma), commonly related to the adipogenic/neurogenic lineage, was expressed by MIAMI cells. It was further up-regulated during differentiation, without significant changes with or w/o pre-treatment (11.4 ± 4.0 fold increase) whereas the solely adipogenic Lipoprotein lipase (LpL) was never detected. Noteworthy, exposure to ITS+3 during neuronal differentiation, described in the previous section, did not affect PPAR gamma expression and LpL remained undetected.

D ISC USSI O N

The differentiation of MSC subpopulations into neuron-like cells, a tightly controlled process involving a series of stimuli, has been recently reviewed [START_REF] Ross | Evaluation of neural plasticity in adult stem cells[END_REF]. In order to be able to use these cells for regenerative medicine studies in the brain, it is important to have a deeper understanding of the molecular events mediating this process. We have demonstrated that an EGF-bFGF treatment of human MIAMI cells, a subpopulation of MSCs, initiated their cell cycle exit and directed their gene expression pattern towards a neural/neuronal lineage. In addition, we reported in a previous study that NT3 was essential for the differentiation of MIAMI cells to cells with morphological, biochemical and electrophysiological characteristics of immature neurons [START_REF] Tatard | Neurotrophin-directed differentiation of human adult marrow stromal cells to dopaminergic-like neurons[END_REF]. In the present study, we observed that Ntrk1 and Ntrk3 receptors, both mediators of NT3 responses [START_REF] Reichardt | Neurotrophin-regulated signalling pathways[END_REF][START_REF] Ivanisevic | TrkA receptor "hot spots" for binding of NT-3 as a heterologous ligand[END_REF], were up-regulated after EGF-bFGF treatment, a process accompanied with a more sustained phosphorylation of Erk in response to NT3. In this way, EGF-bFGF treatment enables the cells to better respond to the neuronal differentiation stimuli. We effectively observed that, during the induction protocol, EGF-bFGF pretreated MIAMI-derived neuron-like cells stopped proliferating, presented longer neurite extensions and acquired an expression pattern more consistent with a neuronal differentiation program. These results shed light on the molecular events induced by an EGF-bFGF pre-treatment during the neuronal differentiation of MSC subpopulations. Thus, this pre-treatment may increase the benefit of using MSCs for regenerative medicine approaches of the nervous system.

MIAMI cells have a broad differentiation potential, express embryonic markers, including Rex1

and SSEA synthase [START_REF] Ippolito | Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential[END_REF][START_REF] Tatard | Neurotrophin-directed differentiation of human adult marrow stromal cells to dopaminergic-like neurons[END_REF], as well as Oct4A, described as essential for the pluripotency and selfrenewal properties of stem cells [START_REF] Masui | Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells[END_REF][START_REF] Boyer | Core transcriptional regulatory circuitry in human embryonic stem cells[END_REF][START_REF] Liedtke | Oct4 expression revisited: potential pitfalls for data misinterpretation in stem cell research[END_REF]. The latter was down-regulated over EGF-bFGF exposure, suggesting a certain commitment of the cells towards a differentiation program as previously suggested [START_REF] Hermann | Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells[END_REF]. In this regard, a recent study demonstrates that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency [START_REF] Kim | Oct4-induced pluripotency in adult neural stem cells[END_REF]. Nestin expression by pluripotent stem cells is considered to be a prerequisite for the commitment of the cells toward the neural lineage [START_REF] Shiota | Isolation and characterization of bone marrow-derived mesenchymal progenitor cells with myogenic and neuronal properties[END_REF][START_REF] Wislet-Gendebien | Regulation of neural markers nestin and GFAP expression by cultivated bone marrow stromal cells[END_REF] and this intermediate filament protein is commonly used to identify NSCs and neural precursors [START_REF] Gilyarov | Nestin in central nervous system cells[END_REF], even if its expression may be found in other cell types. Using flow cytometry, all MIAMI cells were found to express Nestin, as reported in other studies although results vary depending on the detection method, cell species and expansion method used [START_REF] Tatard | Neurotrophin-directed differentiation of human adult marrow stromal cells to dopaminergic-like neurons[END_REF][START_REF] Tondreau | Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation[END_REF][START_REF] Song | Comparison of Neuron-Like Cells Derived from Bone Marrow Stem Cells to Those Differentiated from Adult Brain Neural Stem Cells[END_REF][START_REF] Hermann | Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells[END_REF][START_REF] Wislet-Gendebien | Regulation of neural markers nestin and GFAP expression by cultivated bone marrow stromal cells[END_REF]. EGF-bFGF pre-treatment induced Nestin over-expression by MIAMI cells, as previously described for adherent or floating MSCs [START_REF] Hermann | Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells[END_REF][START_REF] Long | Neural cell differentiation in vitro from adult human bone marrow mesenchymal stem cells[END_REF][START_REF] Shiota | Isolation and characterization of bone marrow-derived mesenchymal progenitor cells with myogenic and neuronal properties[END_REF]; a mechanism that may be mediated through Erk pathway [START_REF] Huang | Thrombin induces nestin expression via the transactivation of EGFR signalings in rat vascular smooth muscle cells[END_REF].

Notch1, which is involved in proliferation and maintenance of an undifferentiated state by NSCs [START_REF] Kageyama | Roles of bHLH genes in neural stem cell differentiation[END_REF], was down-regulated in response to EGF-bFGF treatment. Notch1 down-regulation could have led to the observed decreased expression of HES5 after EGF-bFGF pre-treatment [START_REF] Kageyama | Roles of bHLH genes in neural stem cell differentiation[END_REF].

Characteristic of decreased HES5 expression is the up-regulation of the early neuronal marker Ngn2 [START_REF] Kageyama | Roles of bHLH genes in neural stem cell differentiation[END_REF], which we also observed after EGF-bFGF treatment. Noteworthy, this response has previously been described in Nestin expressing cells after treatment with bFGF [START_REF] Vergano-Vera | FGF-2 increases the expression of neurogenic genes and promotes the migration and differentiation of neurons derived from transplanted neural stem/progenitor cells[END_REF]. Pax6, known to promote neurogenesis in neural stem cells [START_REF] Kallur | Pax6 promotes neurogenesis in human neural stem cells[END_REF] was also up-regulated after pre-treatment. These results suggest a potential progression of MIAMI cells towards a neuronal phenotype.

Cell cycle gene analysis and cell counting confirmed the propensity of EGF-bFGF pre-treated cells to decrease their proliferation rate during neuronal differentiation. Indeed, non pre-treated cells exhibited a high level of Cyclin D1, the key cell-cycle regulatory protein governing progression from the G1 to S phase, and low level of the antiproliferative gene p21, a Cyclin-dependent kinase and Proliferating cell nuclear antigen (PCNA) inhibitor [START_REF] Luo | Cell-cycle inhibition by independent CDK and PCNA binding domains in p21Cip1[END_REF]. We can note that such a gene expression pattern is specific for integrin-mediated adhesion to fibronectin [START_REF] Danen | Fibronectin, integrins, and growth control[END_REF], the substrate used in this study.

Conversely, p21 was up-regulated in pre-treated cells, and remained at high level during neuronal differentiation, while Cyclin D1 was down-regulated. These events, which take place during the exit from the cell cycle, have already been described in the context of terminal differentiation [START_REF] Pacary | Synergistic effects of CoCl(2) and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells[END_REF][START_REF] Ekholm | Regulation of G(1) cyclin-dependent kinases in the mammalian cell cycle[END_REF]. In addition to its anti-proliferative role, p21 may rescue human MSCs from apoptosis induced by low density culture [START_REF] Van Den Bos | p21(cip1) rescues human mesenchymal stem cells from apoptosis induced by low-density culture[END_REF], with p21 loss increasing apoptosis [START_REF] Seoane | Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage[END_REF]. In our study, we did not observe apoptotic cells, neither in low density expansion nor in differentiation. However, differentiated MIAMI cells became sensitive to the pro-apoptotic molecule used as a positive control, confirming a previous report made with MSCs [START_REF] Pelletier | Caspase-3 can be pseudo-activated by a Ca2+dependent proteolysis at a non-canonical site[END_REF]. To explain this phenomenon, we may hypothesize that inhibition of Caspase 3, or of another related molecule, occurs in expansion and that this inhibition is abolished during differentiation. For example, the Heat shock protein Hsp20, highly expressed by MIAMI cells (data not shown), has recently been described to inhibit Caspase 3 via an interaction with the pro-apoptotic protein Bax [START_REF] Fan | Hsp20 and its cardioprotection[END_REF][START_REF] Fan | Novel cardioprotective role of a small heat-shock protein, Hsp20, against ischemia/reperfusion injury[END_REF].

Altogether, these results demonstrate that EGF-bFGF treatment induces the specification of MIAMI cells toward the neural lineage. During neuronal differentiation, cells exhibited a strong propensity to exit from the cell cycle, which suggests that they are on their way to ultimately become post-mitotic. Moreover, the neuronal gene expression pattern closely mimicked the neuronal developmental program. In vivo, Nestin is down-regulated during the switch from neural precursors to differentiated post-mitotic neurons [START_REF] Gilyarov | Nestin in central nervous system cells[END_REF][START_REF] Zimmerman | Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors[END_REF], similarly to what we observed during differentiation of MIAMI cells by ICF. During the neuronal differentiation of pre--Tubulin expression pattern was also similar to that of neuronal precursors, with a first increase followed by a terminal decrease at the end of the neuronal differentiation protocol [START_REF] Ginzburg | Regulation of three betatubulin mRNAs during rat brain development[END_REF]. NFL, NFM and NFH expression increased during neuronal differentiation, therefore following the expression pattern observed during normal brain development [START_REF] Moskowitz | Transcriptional and post-transcriptional mechanisms regulating neurofilament and tubulin gene expression during normal development of the rat brain[END_REF], whereas NFM decreased strongly at the end of step 3 for non pre-treated cells. In support of these results, we observed by ICF that EGF-bFGF pre-treatment resulted in a stronger expression of NFM -Tubulin at the end of step 3. Pretreated cells exhibited narrower and longer neurite-like extensions consistent with the filamentous protein expression. Therefore, EGF-bFGF pre-treatment enhanced the neuronal differentiation ability of MIAMI cells, although a fully mature phenotype was still not obtained as the neuronal marker Tau was not detected in its phosphorylated form. Without specific phenotype inducers, EGF-bFGF pretreatment did not direct the neuronal differentiation toward a default neuronal pathway as mature neurotransmitter markers were only slightly detected. However, we previously demonstrated that MIAMI cells were able to differentiate toward the dopaminergic phenotype in response to the correct instructive cues, i.e. sonic hedgehog, fibroblast growth factor 8 and retinoic acid [START_REF] Tatard | Neurotrophin-directed differentiation of human adult marrow stromal cells to dopaminergic-like neurons[END_REF].

For central nervous system cell therapy purposes, it is important to ascertain that MIAMI cells not only acquire a neuronal phenotype but also loose their mesodermal differentiation potential. In this study, differentiated MIAMI cells did not express osteogenic and adipogenic specific genes while the EGF-bFGF pre-treatment induced a decreased expression of Sox9, present in chondrogenic progenitors [START_REF] Marshall | Molecular mechanisms of SOX9 action[END_REF]. Only PPAR gamma was up-regulated during differentiation, a marker commonly related to the adipogenic but also neurogenic lineages [START_REF] Shiota | Isolation and characterization of bone marrow-derived mesenchymal progenitor cells with myogenic and neuronal properties[END_REF][START_REF] Casteilla | PPARs and Adipose Cell Plasticity[END_REF][START_REF] Cimini | Emerging roles of peroxisome proliferator-activated receptors (PPARs) in the regulation of neural stem cells proliferation and differentiation[END_REF][START_REF] Wada | Peroxisome proliferator-activated receptor gamma-mediated regulation of neural stem cell proliferation and differentiation[END_REF], thereby playing a role in proliferation and differentiation of NSCs [START_REF] Cimini | Emerging roles of peroxisome proliferator-activated receptors (PPARs) in the regulation of neural stem cells proliferation and differentiation[END_REF][START_REF] Wada | Peroxisome proliferator-activated receptor gamma-mediated regulation of neural stem cell proliferation and differentiation[END_REF]. Moreover, lipid droplets were never observed inside the cells, unless if differentiated in a lipid rich medium supplemented with insulin, transferin and selenium, commonly used for neuronal differentiation purposes [START_REF] Song | Comparison of Neuron-Like Cells Derived from Bone Marrow Stem Cells to Those Differentiated from Adult Brain Neural Stem Cells[END_REF][START_REF] Okabe | Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro[END_REF][START_REF] Sanchez-Ramos | Adult bone marrow stromal cells differentiate into neural cells in vitro[END_REF][START_REF] Tao | Cytokine-induced stable neuronal differentiation of human bone marrow mesenchymal stem cells in a serum/feeder cell-free condition[END_REF][START_REF] Levy | Induction of neuron-specific enolase promoter and neuronal markers in differentiated mouse bone marrow stromal cells[END_REF]. PPAR gamma expression and the ability to uptake lipids are also observed in oligodendrocyte cell lines [START_REF] Roth | PPAR gamma activators induce growth arrest and process extension in B12 oligodendrocyte-like cells and terminal differentiation of cultured oligodendrocytes[END_REF]. In this regard, the increased ability of differentiated EGF-bFGF pre-treated cells to produce lipid vesicles may be related to the observed upregulation of the oligodendrocyte specific gene GalC even if Olig 2, mainly involved in the development of oligodendrocyte lineage [START_REF] Jakovcevski | Olig transcription factors are expressed in oligodendrocyte and neuronal cells in human fetal CNS[END_REF], was never detected. In addition, Notch1 down-regulation renders possible the oligodendrocytic differentiation of NSCs [START_REF] Wang | Notch receptor activation inhibits oligodendrocyte differentiation[END_REF], suggesting that MIAMI cells may commit toward a neuronal and preoligodendrocytic lineage after EGF-bFGF pre-treatment. In this respect, it has been proposed that some neurons and oligodendrocytes may share common progenitors during central nervous system development [START_REF] Rowitch | An 'oligarchy' rules neural development[END_REF].

C O N C L USI O NS

These results show that EGF-bFGF pre-treatment of MIAMI cells specified the cells toward the neural lineage and improved their neuronal differentiation program in vitro. In opposition to our method, others described culture of MSC spheroids in an EGF-bFGF containing medium prior to neuronal differentiation, but many of the cells died in the process of sphere formation [START_REF] Hermann | Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells[END_REF].

Importantly, MIAMI cells isolated from the iliac crest of living donors exhibited similar responses to EGF-bFGF pre-treatment than MIAMI cells isolated from vertebral bodies, therefore allowing a simplified harvesting for autologous transplantation in clinical protocols. To our knowledge, this study is the first to precisely characterize the effects of an EGF-bFGF priming on human MSCs prior to neuronal differentiation. The results underline the advantages of pre-treating human MSCs with EGF-bFGF for neuronal differentiation purposes. In the future, EGF-bFGF pre-treatment may become an attractive, and easy to implement, method for enhancing the benefits of nervous system cell therapy based on the use of MSCs. -T ubulin and N F M expression pattern during neuronal differentiation. EGF-bFGF pre-treated cells exhibited an increase and a final decrease -Tubulin mRNA expression during differentiation (A), as well as a constant increase in N F M mRNA (C), similarly to what is observed during neuronal development in vivo. At the protein -Tubulin and NFM expression were higher for pre-treated cells as observed by ICF in terms of marker intensity (B, D respectively). EGF-bFGF pre-treatment also resulted in an increased number of for B3-Tubulin and NFM-positive cells with a neuronal morphology (E). Presented ICF data were obtained from a representative experiment and differences were considered significant at P=0.05*.

  The cell pellet was resuspended in buffer A [500 mM Tris HCl pH 6.8, 50 mM EGTA, 1 M KCl, 10 % Triton X-100 (all from Sigma)], triturated, vortexed and centrifugated at 13000 g for 20 minutes to pellet the Triton X-100 insoluble pellet. Buffer A containing the supernatant Triton X-100 soluble fraction was stored at -80°C. The Triton X-100 insoluble pellet was washed twice with buffer B [500 mM TrisHCl pH6.8, 50 mM EGTA, 850 mM sucrose, 10 % Triton X-100 (all from Sigma)] then centrifugated at 13000 g for 20 minutes. Buffer B was aspirated from the Triton X-100 insoluble pellet and the pellet was reconstituted in Laemmli buffer [10 % SDS, 500 mM Tris HCl pH 6.8, 4 % glycerol, 1 % ß-ME, bromophenol blue (all from Sigma)], boiled for 5 minutes and stored at -80°C. The protein concentration of the Triton X-100 pellet was determined using the BCA protein assay (Pierce, Rockford, IL, USA). Primary antibodies included rabbit anti-Ntrk1 (1:2000, polyclonal, #sc-118, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-Ntrk3 (1:500, clone 75213, #MAB3731, R&D Systems), mouse anti-pErk p44/42 (1:2000, #9106S, Cell Signaling Technology, Danvers, MA, USA), mouse anti-Erk p44/42 (1:2000, #9107, Cell Signaling Technology), goat anti-pTau (Ser396) (1:500, polyclonal, #sc-12414, Santa Cruz Biotechnology) & mouse anti-Tau (1:500, monoclonal, #sc-32274, Santa Cruz Biotechnology). Secondary antibodies used were sheep anti-mouse IgG-horseradish peroxidase (HRP) (1:2000, GE Healthcare, USA), goat anti-rabbit IgG-HRP and bovine anti-goat IgG-HRP (1:1000, both from Santa Cruz Biotechnology) depending on the primary antibody. 50 µg protein extracts were electrophoresed on 6 % or 10 % gels and transferred onto Immobilon-P 0.2 µM membranes (Millipore). Membranes were blocked with 5 % BSA-DPBS for 1 hour then incubated with primary antibody in 5 % BSA-DPBS. Secondary antibodies were diluted in DPBS alone and incubated on the membrane for 45 minutes. Membranes were developed with ECL Plus Western Blot Detection System (GE Healthcare) and an AFP Imaging Mini-Medical Services Auto developer. Image analysis was completed using ImageJ (http://rsbweb.nih.gov/ij/). Coomasie blue staining of the blot was used to control the total amount of proteins loaded. Caspase 3 assay. MIAMI cells were plated in 1.9 cm fibronectin-coated wells and Caspase 3 activity was assessed following the Caspase 3 assay kit (Sigma) guidelines. Briefly, positive controls were induced by exposing the cells to Staurosporine (1 µg/mL, Sigma) for 4 hours at 37°C. After apoptotic induction of the positive controls, cell culture medium of every well was replaced by assay buffer and frozen at -80°C. After thawing, Caspase 3 substrate (Ac-DEVD-AMC) was added and the solutions transferred to black plates (Greiner bio-one, Courtaboeuf, France). Fluorescence was read in a kinetic mode for 1 hour at room temperature using a Fluoroskan Ascent FL microplate reader (Thermo Fisher scientific, Illkirch, France) with excitation/emission wavelengths of 355 nm-460 nm, respectively. Caspase 3 activity was read from a 7-amino-4-methylcoumarin standard curve. Caspase 3 specificity of the fluorescent signal was assessed with the Caspase 3 inhibitor (Ac-DEVD-CHO). Every condition was performed in triplicate. Immunocytofluorescence (I C F ). Uninduced and induced cells at the end of step 2 (neuronal commitment) and step 3 (neuronal differentiation) of neuronal induction were used for Nestin, 3-Tubulin and NFM ICF. After washing the slides 3 times with DPBS, cells were fixed with 4 % paraformaldehyde at 4°C for 15 min and then permeabilized with 0.2 % Triton X-100 (Sigma) for 5 min. Slides were blocked with DPBS, 10 % normal goat serum (Sigma), 4 % bovine serum albumin (BSA) (Sigma) at room temperature for 45 min. After washing, slides were incubated overnight at 4°C with anti-NFM (1:50, clone NN18, #N5264, Sigma), anti-3-Tubulin (1:1000, clone SDL.3D10, #T8660, Sigma) or anti-nestin (1:200, clone 10C2, #MAB5326, Chemicon, St Quentin en Yvelines, France) antibodies in DPBS, 4 % BSA, 0.2 % Triton X-100. Isotypic controls were made with IgG1k (clone MOPC-31C, #557273, BD Biosciences) and IgG2bk (clone 27-35, #555740, BD Biosciences).

R 6 (

 6 ESU L TS Direct effects of MI A M I cell exposure to E G F -b F G F . EGF and bFGF receptors, E G FR and F G FR1 respectively, were highly expressed when assessed by RT-qPCR, suggesting that MIAMI cells may respond to the stimuli induced by these factors. In the following section, we first focus on the direct effects induced in MIAMI cells by an EGF-bFGF exposure. E xpression of stem and early neural-related genes after E G F -b F G F treatment. RT-qPCR demonstrated that expanding MIAMI cells expressed the stemness marker Oct4A, which was downregulated after treatment with EGF-bFGF for 10 days (2.5 ± 0.5 fold decrease). MIAMI cells also expressed a panel of early neural-related genes, including Nestin, Microtubule-associated protein 1b (MAP1b), Musashi homolog 1 (Msi1), Notch1, Hairy and enhancer of split 5 (H ES5), Paired box Pax6) & Neurogenin 2 (Ngn2). The expression of Nestin was stimulated (2.9 ± 0.8 fold increase) by the EGF-bFGF treatment and this was confirmed by flow cytometry and ICF with 5 and 20 ng/mL EGF-bFGF (fig. 1 A, B), while a higher concentration (50 ng/mL) did not further increase Nestin expression. Moreover, Nestin was detected in almost all non-treated or treated cells, but its expression always remained lower than for NSCs used as a positive control (data not shown). Pax6 increased after 20 ng/mL EGF-bFGF treatment (1.8 ± 0.1 fold increase), whereas the expression of Notch1, which plays a role in the control of NSC proliferation, slightly decreased (1.4 ± 0.1 fold decrease). Expression of H ES5 decreased and became almost undetectable while Ngn2 increased (2.1 ± 0.1 fold), possibly as a consequence of Notch1 down-regulation. Importantly, expression of stem and early neural-related genes was similar for iliac crest-derived MIAMI cells isolated from an older individual, with equivalent changes induced in response to EGF-bFGF exposure. All these results illustrate an EGF-bFGF induced specification of the MIAMI cells towards a neural phenotype, independently from their anatomical site of origin, i.e. vertebral bodies or iliac crest. E ffects of E G F -b F G F on the N T3 transduction pathway. Analysis of the NT3 receptor, neurotrophin tyrosine receptor kinase 3 (Ntrk3), revealed that both the full length isoform (gp145, FL-Ntrk3) and the tyrosine kinase deficient isoform (gp100, Tkd-Ntrk3) of Ntrk3 were up-regulated along with Ntrk1,

(

  AChE), expressed by cholinergic and noradrenergic neurons, was detected at very low level by RT-qPCR during expansion and tended to increase during neuronal differentiation (10.4 ± 3.3 fold) when cells were pre-treated with EGF-bFGF. Glutamate decarboxylase (GAD 25/67), expressed by GABAergic neurons, Choline acetyltransferase (ChAT), another cholinergic phenotype enzyme and dopaminergic markers (Tyrosine hydroxylase (TH), Dopamine transporter (DAT) and Nurr1) were not detected or at very low levels.

F

  I G U R E L E G E N DS F ig. 1. Nestin up-regulation during E G F -b F G F treatment. Nestin expression by MIAMI cells increased after exposure to EGF-bFGF. After treatment, expression by flow cytometry increased (A) and was confirmed by ICF (B). Depicted results were obtained from a representative experiment and differences were considered significant at P=0.05*. F ig. 2. Representative Ntr k up-regulation and E r k sustained phosphorylation. During EGF-bFGF pre-treatment, expression by MIAMI cells of Ntrk1, Ntrk3 and Tkd-Ntrk3 receptors were strongly up-regulated (A). Total proteins were detected by Coomasie blue staining. In response to NT3 stimulation, Erk phosphorylation peaked after 5 minutes and decreased at 10 min for non pre-treated cells. After 50 ng/mL EGF-bFGF pre-treatment, pErk peaked at 5 minutes and remained at a high level at 10 minutes of NT3 stimulation, compared to non pre-treated cells. This sustained phosphorylation was similar for p42 and p44 (B, C). *Differences were considered significant at P=0.05. Abbreviations: Ntrk: neurotrophic tyrosine kinase receptor. F ig. 3. Reduced cell proliferation and cell cycle exit during neuronal differentiation. Trypan blue exclusion (A) demonstrated that the number of cells increased during differentiation while almost no increase was detected with 20 ng/mL EGF-bFGF pre-treated MIAMI cells. Analysis of Notch1 (B) and cell cycle genes (p21 & Cyclin D1, C & D respectively) expressions tended to confirm the reduced cell proliferation and cell cycle exit observed during differentiation after pretreatment. *Differences were considered significant at P=0.05.F ig. 4. C aspase 3 activity. MIAMI cells lack significant Caspase 3 activity (A), unless if treated with 1 µg/mL staurosporine (an apoptosis inducer). This significant increase was detected only in differentiated cells whereas proliferative cells were resistant to staurosporine treatment. The top right bar of the histogram was obtained with a caspase 3 solution as a positive control. Picture B depicts morphology of pre-treated MIAMI cells after differentiation compared to the same cells after staurosporine exposure (C). Abbreviations: Ssp: staurosporine. *Significantly different medians at P=0.05. Scale bars: 100 µm.F ig. 5. Induced neural/neuronal morphology after E G F-b F G F pre-treatment. Expanding MIAMI cells (A) were larger than EGF-bFGF treated cells, which were narrower and more refractile (B). At the end of neuronal differentiation, EGF-bFGF pre-treated cells exhibited longer neurites as observed after NFM ICF (D) compared to non pre-treated cells (C). Neurite-like extensions and inter-connexions of EGF-bFGF pre-treated cells are depicted on SEM images at the end of differentiation (E, F, respectively). Scale bars A-E: 100 µm; F: 5 µm.

F

  ig. 7. E G F-b F G F may prime M I A M I cells toward a pre-oligodendrocyte lineage. GalC expression by MIAMI cells slightly increased during differentiation after EGF-bFGF pre-treatment (A). Use of ITS+3 during the neuronal differentiation protocol led to the formation of lipid vesicles (B, D, white arrowheads), especially in EGF-bFGF pre-treated cells. In presence of ITS+3, MIAMI cells exhibited red vesicles (D) at the end of differentiation compared to control cells (C) as observed with oil red staining. The lipid nature of the vesicles observed in EGF-bFGF pre-treated cells was confirmed by SEM. Osmium specifically links to double bounds-molecules such as lipids and back-scattering imaging reveals the localization of the osmium inside the cells (B). Scale bars B: 20 µm ; C, D: 100 µm.

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Effects of EGF-bFGF pre-treatment during neuronal differentiation Filaments

  EGF-bFGF pre-treated cells exhibited a decreased cell area (i.e. a narrower cell body) and an increased cell length, due to the presence of long neurite-like extensions at the end of step 3. Data represent the average length of the 3 longer cells of 5 different images in a representative experiment. * Significantly different means at P=0.05.T able 3. Major effects of MIAMI cells exposure to EGF-bFGFExposure of MIAMI cells to EGF-bFGF resulted in the differential expression of a large panel of markers, as observed using RT-qPCR, western blot, flow cytometry and immunocytofluorescence, thereby suggesting a neural specification of the cells. When applicable, fold change calculated by RT-qPCR are mentioned. After EGF-bFGF pre-treatment and further exposure to the 3 steps neuronal induction media, an increased expression of various neuronal filamentous proteins was observed, compared to non pre-treated MIAMI cells. In addition, a reduced proliferation rate during neuronal differentiation was observed only with EGF-bFGF pre-treated MIAMI cells, in accordance with a high expression of P21 and a downregulation of Cyclin D1. Thus, these results show that EGF and bFGF pre-treatment enhance the response of MIAMI cells to neuronal commitment. Abbreviations: NA: not applicable.

	T A B L ES T able 2. Cell area and length at the end of differentiation
	T able 1. Human specific primer sequences Gene F ull name Non pre-treated cells N M accession number E G F-b F G F pre-treated cells AChE Acetylcholinesterase 015831-00665 C ell area (µm⇢) 4378 ± 665 2893 ± 499* -CCCTCTCGAAACTACACG-Sequences -GGTCCAGACTAACGTACTGC-C ell length (µm ± SD) 289 ± 43 421 ± 50*	A mplicon 163
	-Tubulin	Homo sapiens tubulin, beta 3		006086		-CCAGTATGAGGGAGATCG--CACGTACTTGTGAGAAGAGG-	185
	C CND1	Cyclin D1		053056		-CTCTCCAAAATGCCAGAG--GATGGACAGGAAGTTGTTGG-	186
	ChAT	Choline acetyltransferase	020549-020984-020985-020986	HS_CHAT_1_SG QUANTITECT PRIMER ASSAY, REF QT00029624, QIAGEN	Unknown
	DAT	Dopamine transporter		001044		-GAAGGTGGTATGGATCACAG--GTAGAAGTCAACGCTCAGGT-	121
	E G FR	E G F receptor		005228		-CAGCCACCCATATGTACC--TGGAGTCTGTAGGACTTGG-	184
	F G FR1 GAD 25/67	F G F receptor 1 Glutamate decarboxylase	015850-023105-023106-023110-023111 000817-013445 Down/up--GGTAACTCTATCGGACTCTCC--GTGGAAGTCACTCTTCTTGG--CCTGGAAGAGAAGAGTCG--CTCCTCACCGTTCTTAGC-Fold change Markers regulation (RT-qPCR)	198 166
	GalC	Galactosylceramidase		000153-001037525 Oct4a	-GTTGCCTTATGGGAGATG--AAGCCATCAGTCAGAGGCTAC-2.5 ± 0.5	183
	G F AP	Glial fibrillary acidic protein	002055	Notch1	-TTGAGAGGGACAATCTGG--CGACTCAATCTTCCTCTCC-1.4 ± 0.1	161
	H ES5* LpL* MAP1b Msi1 Nes EGF-bFGF treatment Hairy and enhancer of split 5 Trancription factors 001010926 Lipoprotein lipase 000237 Microtubule-associated protein 1b 005909 Homo sapiens musashi homolog 1 002442 Nestin 006617 Filaments Cell cycle	Ngn2 HES5 Pax6 Nes P21 CyclinD1	-AGCCCCAAAGAGAAAAACCGA-2.1 ± 0.1 -GCTGTGCTTCAGGTAGCTGAC--ACAAGAGAGAACCAGACTCCAA--AGGGTAGTTAAACTCCTCCTCC-Undetectable 1.8 ± 0.1 -CCTCGAGACGTGATGAGTGA--TTGGGCGTCAGAGAGAAGTT--AGGTGAAGGAGTGTCTGG--TTCTTCGTTCGAGTCACC--AGAAACAGGGCCTACAGAG--AAAGCTGAGGGAAGTCTTG-2.9 ± 0.8 10.2 ± 0.7 <> NA	183 149 437 196 170
	N F L* N F M	Neurofilament, light polypeptide Neurofilament, medium polypeptide Receptors 006158 005382	Ntrk1 Ntrk3 Tkd-Ntrk3	-ATGAGTTCCTTCAGCTACGAGC-NA -GGGCATCAACGATCCAGAGC-NA -GACCTCAGCAGCTACCAG--CTAGTCTCTTCACCCTCCAG-NA	198 170
	N F H*	Neurofilament, heavy polypeptide Proliferation rate 021076		-GCAGTCCGAGGAGTGGTTC--TAGCGTCTGTGTTCACCTTGG-NA	71
	Ngn2*	Neurogenin 2		024019	3-Tubulin	-CGCATCAAGAAGACCCGTAG--GTGAGTGCCCAGATGTAGTTGTG-1.4 ± 0.3	173
	Notch1 Nurr1 (NR4A2)* Oct4A Olig2*	Notch homolog 1, translocation-associated nuclear receptor subfamily 4, group A, member 2 Pou class 5 homeobox 1 0ligodendrocyte lineage transcription factor 2 Cell cycle 017617 006186 002701 005806	NFL NFM NFH P21 Cyclin D1	-GACCTCATCAACTCACACG--AGAAACAGGGGTGTCTCC-54.3 ± 9.3 -TTGCCAGATGCGCTTCGACG-6.8 ± 0.2 -CCAACAGCCAGGCACTTCTG-2.0 ± 0.3 -TGGAGAAGGAGAAGCTGGAGCAAAA--GGCAGATGGTCGTTTGGCTGAATA--GCTGCGACGACTATCTTCCC--GCCTCCTAGCTTGTCCCCA-<> NA 6.7 ± 2.6	198 414 186 244
	Pax6*	Paired box 6	000280-001604-001127612 Oligodendrocyte GalC	-AGGTATTACGAGACTGGCTCC--TCCCGCTTATACTGGGCTATTT-7.5 ± 4.6	104
	p21	Cyclin-dependent kinase inhibitor 1A Proliferation rate 000389-078467	-GCAGAGGAAGACCATGTG--CCTCTTGGAGAAGATCAGC-NA	171
	PPAR gamma	Peroxisome proliferator-activated receptor gamma	138712-015869-138711-005037	-AACAGATCCAGTGGTTGC--CTCCACAGACACGACATTC-	176
	Runx2*	Runt-related transcription factor 2	001024630-004348	-TCCTATGACCAGTCTTACCCCT--GGCTCTTCTTACTGAGAGTGGAA-	190
	Sox9*	Sex determining region Y-box 9	000346		-GCCAGGTGCTCAAAGGCTA--TCTCGTTCAGAAGTCTCCAGAG-	213
	Sp7 (Osterix)*	Sp7 transcription factor		152860		-CCCAGGCAACACTCCTACTC--GGCTGGATTAAGGGGAGCAAA-	175
	TH*	Tyrosine hydroxylase		199292-000360-199293	-CACCATCTAGAGACCCGGCC--GCAATCAGCTTCCTGCGCTG-	290
	Primers		from Origen, USA and experiments with these primers performed in	facilities.

A C K N O W L E D G M E N TS

We thank the SCIAM (Service Commun d'Imagerie et d'Analyse Microscopique) of Angers for Rodriguez for their precious technical assistance, as well as Dr. Micheline McCarthy (Dept. Of Neurology, Univ. Of Miami) for supplying foetal neuroepithelial progenitor cells. This work was , the and by grants from the Department of Veterans Affairs, USA. T H E R E IS N O D ISC L OSU R E O F I N T E R EST I N T H IS PU B L I C A T I O N R E F E R E N C ES