Valérie Schreiber

Madame Le Professeur

Julie Kerr-Conte

Julien Thevenet

Nathalie Dellaleau

Gianni Pasquetti

Anaïs Coddeville

Valéry Gmyr

Matthias Huyghe

A Madame le Docteur

Table of contents

Résumé

Introduction : L'utilisation des îlots humains de Langerhans est la référence pour la recherche, tant physiologique que pour le développement de nouvelles molécules thérapeutiques pour le traitement du diabète de type 2. La demande d'îlots de Langerhans humains pour des projets de recherche est en constante augmentation, cependant, la disponibilité est limitée et les différentes préparations d'îlots de Langerhans révèlent une grande variabilité entre elles.

Objectifs : L'objectif principal de cette thèse était de proposer une alternative aux îlots de Langerhans humains natifs qui permettrait d'obtenir des îlots pancréatiques homogènes et en quantité abondante pour les projets de recherche.

Pour ce faire, nous avions deux objectifs principaux : 1) la production de pseudo-îlots de diamètre contrôlé à partir de pancréas humain, et l'évaluation de leur fonction in vitro et in vivo par rapport à leurs équivalents îlots natifs ; 2) l'optimisation de la production de cellules endocrines pancréatiques à partir de différentes lignées de cellules souches pluripotentes et l'évaluation des effets de la progérine sur la différenciation et la maturation des cellules produites. Les cellules souches pluripotentes utilisées provenaient de donneurs sains (H1, WiCell) et de patients atteints de Progeria (HGPS, iStem).

Matériel et méthodes :

Les pseudo-îlots ont été formés dans un milieu d'îlots clinique (CMRL 1066 albumine humaine, insuline) pendant 7 jours en utilisant les Sphericalplate 5D (Kulgelmeiers) et comparés aux îlots natifs J1 (jour 1) et J7 (jour 7) du même donneur.

La différenciation des cellules souches pluripotentes (cellules iPS DF19.9, H1 et iPS HGPS) a été optimisée par différents protocoles : le protocole Rezania, le SD Kit (StemCell Technologies) et le protocole Nostro. L'expression des gènes de maturation in vitro entre différentes lignées cellulaires a été évaluée par qPCR. L'expression des protéines a été évaluée par immunofluorescence et par cytométrie en flux (plateforme EGID).

Pour la maturation in vivo, après la transplantation sous la capsule rénale de souris immunodéficientes, des mesures de glycémie et de c-peptide humain ont été effectuées, ainsi que des tests métaboliques comme l'ipGTT.

Résultats : Les pseudo-îlots (n=4) générés ont sécrété significativement moins d'insuline in vitro que les îlots natifs à J1 mais sans différence significative avec les îlots natifs à J7. Dans les deux groupes à J7, on a observé une diminution significative de l'insuline intracellulaire comparativement aux îlots natifs à J1. In vivo, les îlots natifs à J1 sécrètent significativement plus de c-peptide humain que les îlots natifs à J7, alors que la différence n'est pas significative entre les îlots natifs à J1 et les pseudo-îlots à J7. De plus, l'analyse morphométrique des greffons a révélé que les pseudo-îlots ont tendance à avoir plus de cellules glucagon-positives que les deux autres groupes.

L'optimisation de la différenciation des cellules souches pluripotentes a permis d'obtenir plus de 95% d'endoderme pour les cellules H1 et 80% pour les cellules iPS HGPS. Pour les deux lignées, nous avons généré 95 % de progéniteurs pancréatiques. La comparaison des gènes de maturation a révélé que la progérine conduisait à une légère augmentation de la maturation cellulaire dans le groupe iPS HGPS par rapport aux cellules H1. Des marqueurs liés à l'âge (53BP1, IGF1r et yH2AX) ont été validés dans un pancréas provenant d'un donneur âgé et un insulinome. Cependant, aucune différence de la fonctionnalité in vivo n'a été observée. Six mois post transplantation, nous avons identifié yH2AX dans des cellules endocrines et non endocrine des greffons H1 alors que dans les greffons HGPS, nous l'avons observé dans une plus vaste proportion de cellules présentant différentes formes de noyaux.

Conclusion :

Ces travaux ont donné des résultats positifs : pseudo-îlots fonctionnels et de cellules souches dérivées de cellules endocrines pancréatiques. Toutefois, ces travaux demeurent préliminaires et d'autres études doivent être menées pour fournir des alternatives réalistes aux îlots de Langerhans humains natifs à des fins de recherche. showing nuclei deformation with high level of progerin (left) compared to low level of progerin (right) ..... The World Health Organization defines diabetes as a chronic disease that occurs when the pancreas does not secrete enough insulin or when the body is unable to effectively use the produced insulin.

Abbreviation list

This leads to chronic fasting hyperglycemia ( 7mM) resulting in severe microvascular (retinopathy, neuropathy, nephropathy) and macrovascular complications (cardiovascular disorders). The management of diabetes and its complications represents therefore a considerable socioeconomic burden for society. Indeed, the International Diabetes federation has identified 425 million diabetics worldwide and estimates that there will be 629 million by 2045, representing a 48% increase in diabetes prevalence [START_REF] Cho | IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045[END_REF]).

Blood glucose is tightly regulated (Figure 1) by the release of hormones by the pancreatic islets of Langerhans in a precise range of normal values (3,0 to 7,8 mM) throughout the day. of insulin secretion during the day in order to maintain blood glucose levels in physiological values [START_REF] Polonsky | Abnormal patterns of insulin secretion in non-insulin-dependent diabetes mellitus[END_REF]). Development of short-and long-acting analogues enable most of the patients to achieve this goal [START_REF] Mathieu | Insulin analogues in type 1 diabetes mellitus: getting better all the time[END_REF].

The US funded Diabetes Control and Complications Trials (DCCT) sought to determine if achieving near normal glucose levels would ameliorate the long-term complications (eye, kidneys, nerve disease) of diabetes. T1D patients (n=1441) were divided in two groups, 'conventional' (1 or 2 insulin injections per day) or 'intensive' (3 or more insulin injections per day) insulin regimen where patients achieved respectively a mean of 7.2% and 9.1% HbA1c which reflect glycemic equilibrium over 3 months. After 6.5 years follow-up, the risk of developing retinopathy, nephropathy, neuropathy and macrovascular complications was reduced by 76%, 34%, 69% and 34% respectively in the intensive group [START_REF] Control | The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus[END_REF]. In a subsequent This long-term effect on diabetes complications is attributed to the "metabolic memory" theory [START_REF] Nathan | Intensive diabetes therapy and carotid intima-media thickness in type 1 diabetes mellitus[END_REF][START_REF] Nathan | Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes[END_REF][START_REF] Holman | 10-year follow-up of intensive glucose control in type 2 diabetes[END_REF]. However, there are several side effects associated with the use of insulin, the most severe of which are severe hypoglycemia episodes, which require third party intervention and are life-threatening. In the DCCT study, severe hypoglycemia episodes were 3 times more frequent in the 'intensive' insulin regimen group compared to 'conventional' therapy group [START_REF] Control | The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus[END_REF]. Treatments achieving improved glycemic equilibrium like islet transplantation may have a long-term effect on diabetes complications.

'Closed-loop system' or 'artificial pancreas' are devices coupling insulin pump to a continuous glucose monitoring system (CGMS) in order to deliver insulin in response to measured glucose variations. Such devices improve glucose control as shown in Figure 3. Next generation 'artificial pancreas' systems under evaluation are bi-hormonal (insulin and glucagon) systems, which also prevents hypoglycemic episodes by secreting glucagon in response

to low blood glucose level. Beside its attractive treatment potential for T1D patients this technology has some limitations. Indeed, CGMS measures glucose from the interstitial fluid and not directly from the blood, there is, therefore, a lag time for the interstitial fluid glucose levels to reflect blood glucose levels. Lag time can vary from 5 to 10 minutes [START_REF] Basu | Time lag of glucose from intravascular to interstitial compartment in humans[END_REF][START_REF] Allen | Current Diabetes Technology: Striving for the Artificial Pancreas[END_REF].

Furthermore, such devices cost around 10 000 USD and some patients refuse to wear a device at all times.

Despite evident progress, about 1% of all T1D patients have difficulty in equilibrating their glycemia, even with modern devices, and suffer from severe debilitating hypoglycemic unawareness. In those cases, the benefit/risk ratio of transplantation and lifetime immunosuppressive treatment is in favor of  cell replacement therapy in the form of a pancreas transplantation, islet or  cell transplantation.

Pancreas transplantation is often done simultaneously (SPK) or after kidney (PAK) transplantation although it can be transplanted alone (PTA). Study at 10 years post-transplantation, reported more than 80% (SPK) and 60% (PTA) insulin independence at 10 years [START_REF] Gruessner | Pancreas transplant outcomes for United States and non United States cases as reported to the United Network for Organ Sharing and the International Pancreas Transplant Registry as of December 2011[END_REF][START_REF] Lombardo | Transplantation of the Pancreas [Internet[END_REF].

Islet transplantation is a less invasive validated treatment for severe forms of T1D. Islet transplantation can be performed alone or after kidney graft in T1D patients with severe hypoglycemic unawareness (Vantyghem et al. 2019b). The Collaborative Islet Transplant Registry (CITR) reported 1 086 T1D recipients between 1999-2015 (10 th annual CITR report 2017). Islet isolation (Figure 4) starts with the removal pancreas harvested from brain-deceased donors in France, or from cardiorespiratory-deceased donors in other countries. The pancreas is digested using an enzyme (collagenase) and a Ricordi digestion chamber. The endocrine and exocrine tissues are then separated and the islets are then purified (Figure 5). If the obtained islets meet the release criteria (number of islet equivalent (IEQ) > 200 000), they are then injected intraportally to the recipient via a mesenteric vein. The same T1D patient will need 2-3 islet preparation in order to reach the goal of 10000 IEQ/kg recipient bodyweight (Vantyghem et al. 2019b). and it is currently participating to its 5th clinical trial. The clinical results of the U1190 laboratory at 10 years after islet transplantation show that 28% of the patients are insulin independent (Vantyghem et al. 2019b(Vantyghem et al. , 2019a)). The current islet isolation protocol requires improvement. Indeed, we estimate the loss of  cell mass at approximately 50% after islet isolation (Figure 7, red square) and only 40% of pancreases reach the 200 000 IEQ release criteria. When islets cannot be used for clinical purposes and the family has granted consent for scientific use, they are released for research use. Table 1 shows the numbers of islet isolation, IEQ used for internal research and IEQ used for external research per year from 2012 to 2018. Our laboratory uses human islets for research in the context of the physiology of human islets of Langerhans and glucose transporters [START_REF] Bonner | Inhibition of the glucose transporter SGLT2 with dapagliflozin in pancreatic alpha cells triggers glucagon secretion[END_REF][START_REF] Saponaro | The GLP1R Agonist Liraglutide Reduces Hyperglucagonemia Induced by the SGLT2 Inhibitor Dapagliflozin via Somatostatin Release[END_REF], and their adaptive capacities to obesogenic environment [START_REF] Gargani | Adaptive changes of human islets to an obesogenic environment in the mouse[END_REF]. The U1190 laboratory, with its 25 years of expertise, sends human islets for research to many scientific collaborators within Europe and throughout the world. Most of the basic research published on diabetes is based on rodent models. The possibility of using human islets of Langerhans is a considerable asset to confirm or refute these results and this is increasingly requested by editors and peer review committees [START_REF] Kulkarni | Summary of the Keystone islet workshop (April 2014): the increasing demand for human islet availability in diabetes research[END_REF][START_REF] Nano | Human islet distribution programme for basic research: activity over the last 5 years[END_REF]. The heterogeneity and quality of human islets for research has been recently underlined [START_REF] Hart | Use of human islets to understand islet biology and diabetes: progress, challenges and suggestions[END_REF][START_REF] Nano | Islets for Research: Nothing Is Perfect, but We Can Do Better[END_REF]) and an obligatory checklist has been adopted to report specific characteristics of human islet preparations, harvesting data including cold ischemia time, culture duration, viability, and functional glucose stimulated insulin secretion tests (Annexe 1).

The goal is to ensure the transparency of the data in terms of donor information but also about quality and functionality of the human islets used for research.

Limitation of the use of human islets for research

The use of human islets of Langerhans is the gold standard for research, both for physiological research and for the development of new therapeutic molecules. However, human pancreatic islets are very heterogeneous. Within the same preparation of human islets, islets are highly variable in size, and insulin secretion from islets of Langerhans in response to glucose or pharmacological compounds is variable between different donors [START_REF] Henquin | Influence of organ donor attributes and preparation characteristics on the dynamics of insulin secretion in isolated human islets[END_REF][START_REF] Nasteska | The role of beta cell heterogeneity in islet function and insulin release[END_REF][START_REF] Dominguez-Gutierrez | Heterogeneity of human pancreatic β-cells[END_REF][START_REF] Dybala | Heterogeneity of the Human Pancreatic Islet[END_REF]. In the U1190 laboratory, more than 1000 human pancreases have been isolated. Each human islet preparation is assessed for insulin secretion in response to glucose whereby 400 IEQ (standardized unit of Islets EQuivalent to 150 microns in diameter) are placed in each perifusion chamber. The dynamics of insulin secretion of ten consecutive human islet preparations, assessed by perifusion, shows the typical physiological insulin secretion profiles (Figure 8): basal secretion at 3mM glucose, then a peak of secretion (first phase) followed by a plateau (second phase) at 15mM and, finally, a return to basal secretion when glucose is switched back to 3mM glucose. However, secretion is highly heterogeneous between donors. One of the reason for the observed islet heterogeneity has been attributed to phenotypic and clinical characteristics of donors such as sex, BMI, HbA1c, cause of death or number of days spent in intensive care unit [START_REF] Henquin | Influence of organ donor attributes and preparation characteristics on the dynamics of insulin secretion in isolated human islets[END_REF]. In addition to functional differences, the expression of key transcription factors (PDX1, NKX6.1, MAFA) in human islets that have been isolated and cultured ex vivo [START_REF] Teo | Single-cell analyses of human islet cells reveal de-differentiation signatures[END_REF]) varied considerably from 0 to 47% of the human islet cells.

Despite their heterogeneity, human pancreatic islets remained the 'gold standard' and there is, therefore, a considerable demand of human islets for research [START_REF] Nano | Human islet distribution programme for basic research: activity over the last 5 years[END_REF]. However, no evident increase in their availability have been observed. As a consequence, the scientific community is seeking alternatives to "native" human islets [START_REF] Nano | Islets for Research: Nothing Is Perfect, but We Can Do Better[END_REF]).

Pseudo-islets

Pseudo-islets were first described in the dog model by D.W. Scharp in the 80's [START_REF] Britt | Neonatal pig pseudo-islets. A product of selective aggregation[END_REF] as a new approach in the struggle to mass isolate the elusive islet. After complete digestion of the [START_REF] Britt | Neonatal pig pseudo-islets. A product of selective aggregation[END_REF][START_REF] Ju | Proliferation and functional assessment of pseudoislets with the use of pancreatic endocrine cells[END_REF][START_REF] Yu | Bioengineered human pseudoislets form efficiently from donated tissue, compare favourably with native islets in vitro and restore normoglycaemia in mice[END_REF], spontaneous reaggregation [START_REF] Schröder | Characterization of pseudo-islets formed from pancreatic islet cell suspensions of neonatal rats[END_REF][START_REF] Hopcroft | Structure-function relationships in pancreatic islets: support for intraislet modulation of insulin secretion[END_REF][START_REF] Halban | Spontaneous reassociation of dispersed adult rat pancreatic islet cells into aggregates with three-dimensional architecture typical of native islets[END_REF], hanging drops [START_REF] Cavallari | Rat pancreatic islet size standardization by the "hanging drop" technique[END_REF][START_REF] Pathak | Engineered islet cell clusters transplanted into subcutaneous space are superior to pancreatic islets in diabetes[END_REF][START_REF] Zuellig | Improved physiological properties of gravity-enforced reassembled rat and human pancreatic pseudo-islets[END_REF], micromolds or microwells [START_REF] Ramachandran | Assessment of re-aggregated human pancreatic islets for secondary drug screening[END_REF][START_REF] Hilderink | Controlled aggregation of primary human pancreatic islet cells leads to glucose-responsive pseudoislets comparable to native islets[END_REF].

Numerous commercial plates are now available (5D Sphericalplate, Ezsphere, PrimeSurface 3D).

The number of cells seeded per well determines the diameter of the pseudo-islets. Pseudo-islet formation requires 3 to 8 days (Table 2). The company InSphero provides pseudo-islets for research to academia and industry formed as described in Figure 9. Although the method is not new, pseudo-islet technology is generating renewed interest from the islet transplant community in particular due to a claim from the team in Zürich that islet size influence graft islet outcome. Immediately after transplantation and before revascularization, islets are hypoxic in the portal blood and get oxygen mainly by diffusion [START_REF] Lehmann | Superiority of small islets in human islet transplantation[END_REF].

Logically, smaller islets should outperform larger ones in this setting. Indeed, small islets showed improved survival rates under normoxic and hypoxic conditions compared to larger diameter islets, and superior insulin secretion capacity (Figure 10 A-D) and content (Figure 10 E). Pseudo-islets show similar endocrine cell distribution as native islets both in the pancreas and after isolation (Figure 11) with some reports of slightly increased glucagon in pseudo-islets. Likewise, pseudo-islets of small diameter (< 100 m) also show higher viability [START_REF] Ramachandran | Assessment of re-aggregated human pancreatic islets for secondary drug screening[END_REF], improved glucose-stimulated insulin secretion, increased tolerance to hypoxia and improved function and vascularization after transplantation in rodents [START_REF] Yu | Bioengineered human pseudoislets form efficiently from donated tissue, compare favourably with native islets in vitro and restore normoglycaemia in mice[END_REF].

E)

Human native islets Human pseudo-islets A B

Altogether, pseudo-islets technology allows the production of islets of a constant diameter in order to reduce heterogeneity, improve function, resistance to hypoxia and enhance vascularization post transplantation [START_REF] Yu | Bioengineered human pseudoislets form efficiently from donated tissue, compare favourably with native islets in vitro and restore normoglycaemia in mice[END_REF]. For these above mentioned reasons pseudo-islets are gearing up for clinical transplantation in North America [START_REF] Yu | Bioengineered human pseudoislets form efficiently from donated tissue, compare favourably with native islets in vitro and restore normoglycaemia in mice[END_REF]) and in Europe [START_REF] Lebreton | Insulin-producing organoids engineered from islet and amniotic epithelial cells to treat diabetes[END_REF]).

However, cell therapy with pseudo-islets requires native human islets as starting material or human pancreases and is, therefore, like islet transplantation affected by the scarcity of pancreatic tissue available (2000 organs/year in France). In 2011, Dr. P Ravassard and Dr. R Scharfmann published a long awaited functional human pancreatic  cell line, EndoC-H1 cells which expressed -cell specific markers, resembled native -cells in their expression of specific and epigenetic markers and secreted insulin in response to a glucose stimulation [START_REF] Ravassard | A genetically engineered human pancreatic β cell line exhibiting glucose-inducible insulin secretion[END_REF][START_REF] Lawlor | Multiomic Profiling Identifies cis-Regulatory Networks Underlying Human Pancreatic β Cell Identity and Function[END_REF].

This human  cell line has the advantage of being readily available and has been used worldwide since 2011 as a surrogate to native human -cells for research purpose. Nevertheless, EndoC-H1 cannot be used for cell therapy neither to study -cell development. A recent review (Table 3) highlights the advantages and limitations of the the EndoC-H1 cells compared to native human islets and pluripotent stem cell-derived  cells.

Embryogenesis and early pancreas development

Considerable efforts have been made to develop protocols for the differentiation of pluripotent stem cells to mature endocrine cells. To do this, it's important to understand the mechanisms of embryogenesis and identify the factors that are involved in the pancreas organogenesis, in order to mimic these pathways in vitro.

The oocyte begins its first segmentation to form the morula, which is composed of about thirty cells. The cells tighten together and then flatten to form an internal cell mass, giving place to a cavity, which is the blastocyst stage. Then, the cells of the internal cell mass shape themselves to form the ectoderm and mesendoderm, which during gastrulation will form the mesoderm and endoderm [START_REF] Lu | From fertilization to gastrulation: axis formation in the mouse embryo[END_REF][START_REF] Shook | Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development[END_REF]. It has been shown that Nodal, a member of the TGFb family signaling pathway, is necessary and sufficient to induce the formation of the endoderm and mesoderm [START_REF] Mccracken | Molecular pathways controlling pancreas induction[END_REF]. Nodal allows the gastrulation and formation of the mesendoderm, as well as the separation of the endoderm (high Nodal) and the mesoderm (low Nodal).

Subsequently, the cells of the endoderm will differentiate into the respiratory and digestive tract, the thyroid, thymus, lungs, liver and pancreas. The cells of the mesoderm will give rise to muscle, bone and blood vessels, while those of the ectoderm will form the epidermis and nervous system.

The endoderm acquires an antero-posterior pattern; several pathways activated by signaling factors such as wingless-related integration site (Wnt), fibroblast growth factor (FGF), Bone morphogeneic protein (BMP) and retinoic acid (RA) have a posteriorizing effect [START_REF] Tiso | BMP signalling regulates anteroposterior endoderm patterning in zebrafish[END_REF][START_REF] Dessimoz | FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo[END_REF][START_REF] Mclin | Repression of Wnt/beta-catenin signaling in the anterior endoderm is essential for liver and pancreas development[END_REF][START_REF] Bayha | Retinoic acid signaling organizes endodermal organ specification along the entire antero-posterior axis[END_REF]). This antero-posteriori identity is associated with cell morphogenesis events to form the primitive gut tube and induce pancreatic organogenesis [START_REF] Lawson | Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer[END_REF][START_REF] Tremblay | Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues[END_REF]. Dorsal and ventral buds fuse together to form the pancreas [START_REF] Slack | Developmental biology of the pancreas[END_REF][START_REF] Polak | Early pattern of differentiation in the human pancreas[END_REF][START_REF] Jennings | Development of the human pancreas from foregut to endocrine commitment[END_REF][START_REF] Jennings | Laser Capture and Deep Sequencing Reveals the Transcriptomic Programmes Regulating the Onset of Pancreas and Liver Differentiation in Human Embryos[END_REF]. The cells of the posterior foregut will give rise to PDX1 positive multipotent progenitor cells [START_REF] Wells | Early mouse endoderm is patterned by soluble factors from adjacent germ layers[END_REF] which will be able to differentiate into endocrine or exocrine cells [START_REF] Gu | Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors[END_REF].

Under high or low Notch signaling, multipotent progenitors will differentiate into trunk (bipotent cells) or tip (unipotent) cells [START_REF] Zhou | A multipotent progenitor domain guides pancreatic organogenesis[END_REF], respectively. Tip cells will give rise to acinus while trunk cells will give rise, to duct or endocrine cells, under high or low Notch signaling respectively.

Trunk cells express neurogenin 3 (NGN3), which is a pro-endocrine gene [START_REF] Gu | Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors[END_REF][START_REF] Salisbury | The window period of NEUROGENIN3 during human gestation[END_REF] whose expression is detected in two waves during mouse embryogenesis [START_REF] Villasenor | Biphasic Ngn3 expression in the developing pancreas[END_REF]). In the first wave, known as first transition, mainly  cells are generated at high NGN3 levels while during the second transition, NGN3 allows the differentiation into all endocrine cells including  cells (Figure 12) [START_REF] Johansson | Temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types[END_REF]. After cell differentiation, endocrine cells migrate to the surrounding mesenchyme and form the islets of Langerhans, composed of , , ,  and PP cells which produce glucagon, insulin, somatostatin, ghrelin and pancreatic polypeptides respectively.

Pancreatic islet neogenesis from ducts occurs in human fetal pancreas (Figure 13). Further differentiation and rearrangement of the endocrine cells in the islets will occur until adulthood [START_REF] Jeon | Endocrine cell clustering during human pancreas development[END_REF]. Most of the knowledge on embryogenesis and pancreatic organogenesis is from murine models due to scarcity of human fetal samples [START_REF] Jennings | Development of the human pancreas from foregut to endocrine commitment[END_REF][START_REF] Nair | Islet formation in mice and men: lessons for the generation of functional insulinproducing β-cells from human pluripotent stem cells[END_REF]. Recapitulating murine embryogenesis in human pluripotent stem cell lines may not be optimal since some signaling pathways are well conserved between the mouse and humans but others diverge. This knowledge is used as a starting point for the development of new protocols for the differentiation of human pluripotent stem cells to endocrine  cells in vitro. Figure 14 highlights the similarities and differences between human and mouse pancreas morphogenesis [START_REF] Nair | Islet formation in mice and men: lessons for the generation of functional insulinproducing β-cells from human pluripotent stem cells[END_REF].

Stem cells & pluripotent stem cells

First described by Auguste Weissman in 1883, in contrast to terminally differentiated somatic cells, stem cells harbor a high grade of plasticity in order to differentiate in various cell types (pluripotency) and display self-renewal properties.

Pluripotency is the ability of a cell to differentiate into the three embryonic germ layers Pluripotency is a temporal state observed during embryogenesis and can be artificially maintained during in vitro culture by specific culture media. Indeed, components in culture medium allow to promote self-renewal while inhibiting differentiation [START_REF] Li | Ground rules of the pluripotency gene regulatory network[END_REF][START_REF] Smith | Formative pluripotency: the executive phase in a developmental continuum[END_REF][START_REF] Akberdin | Pluripotency gene network dynamics: System views from parametric analysis[END_REF].

In contrast to most in vitro cultured somatic cells that have a finite number of cell division (Hayflick limit <80), stem cells are capable of extensive proliferation (self-renewal) (> 160) without evidence of oncogenic transformation or senescence [START_REF] Zeng | Human embryonic stem cells: mechanisms to escape replicative senescence?[END_REF]. Self-renewal is achieved by symmetrical cell division, one stem cell to divide into two daughter cells, at least one of which remains undifferentiated. Thus, stem cells are constantly renewed which allows access to an unlimited cell reservoir [START_REF] Zeng | Human embryonic stem cells: long term stability, absence of senescence and a potential cell source for neural replacement[END_REF].

Stem cells have the ability to differentiate into different cell types (plasticity). While differentiating, the cells become more and more specialized. The mature cells will be able to function as a tissue or organ. Stem cells may be able to generate several types of differentiated cells. Once the progenitor is engaged in a path of differentiation, it loses the ability to self-renew.

Pluripotency can be demonstrated in vivo by the formation of teratoma composed of cells from the three embryonic germ layers after transplantation in immunodeficient mice [START_REF] Thomson | Embryonic stem cell lines derived from human blastocysts[END_REF].

Stem cells are characterized by a high nucleo-cytoplasmic ratio and form colonies when cultured in vitro. There are different sources of pluripotent stem cells, embryonic stem cells from the inner cell mass of the blastocyst or induced-pluripotent stem cell "iPS" generated from somatic cells (Figure 15).

1) Embryonic stem cell (ESC)

The development of a new organism begins with the zygote resulting from the fusion of a male and a female gamete. The zygote is composed of totipotent cells that have the potential for self-renewal and the ability to differentiate into all cells of the organism. During differentiation, cells gradually specialize into pluripotent, multipotent and unipotent cells to result in a fully differentiated cell state. Totipotent stem cells are cells at the morula stage. They are able to differentiate in all cell types of an organism as well as embryonic appendages [START_REF] Jaenisch | Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming[END_REF]. Pluripotent stem cells can differentiate into one of the three embryonic layers (endoderm, mesoderm, ectoderm) and lead to all the cell types of an organism. Contrary to totipotent stem cells, they do not differentiate and produce embryonic appendages. There are different types of pluripotent stem cells: embryonic stem cells and induced pluripotent stem cells. Multipotent stem cells are restricted in their differentiation potential within a tissue or lineage [START_REF] Young | Adult reserve stem cells and their potential for tissue engineering[END_REF]. They maintain tissue homeostasis by differentiating into the different cell types of this tissue, such as hematopoietic stem cells that have the potential to reconstitute hematopoiesis. Unipotent stem cells are only able to differentiate into a single cell type such as endothelial progenitor cells Embryonic stem (Figure 16) cells are generated from pluripotent stem cells present in the blastocyst stage within the inner cell mass. They were first characterized in mice by Evans andMartin in 1981 (Evans and[START_REF] Evans | Establishment in culture of pluripotential cells from mouse embryos[END_REF][START_REF] Martin | Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[END_REF]) and then in humans by [START_REF] Thomson | Embryonic stem cell lines derived from human blastocysts[END_REF][START_REF] Thomson | Embryonic stem cell lines derived from human blastocysts[END_REF]. Evans et al. showed that inner cellular mass cells (ECM) could be cultured in vitro while maintaining their pluripotency capacity for years in culture [START_REF] Rosler | Long-term culture of human embryonic stem cells in feeder-free conditions[END_REF].

Derivation methods:

There are different methods of embryonic stem cells derivation: isolation from the inner cell mass of the blastocyst, nuclear transfer and cell fusion.

Most of the embryonic stem cell lines used today are obtained by isolating, dissociating and culturing cells from the blastocyst's inner cell mass as described by Thomson. However, this requires the use of supernumerary human embryos initially produced as part of medical assisted procreation and obtained by in vitro fertilization, then donated to research with the informed consent of parents. In France, embryonic stem cells are subject to a strict regulatory framework (Law 2013-715). Research using embryonic stem cells must be authorized by a nominative derogation (Annexe 2) issued by the French Biomedical Agency (ABM). Major progress was made in 2013 where a non-destructive single cell biopsy of the inner cell mass at the blastocyste stage was performed (DPMAregister DDE 10 2004 062 184.5) [START_REF] Dittrich | Non-embryo-destructive Extraction of Pluripotent Embryonic Stem Cells: Implications for Regenerative Medicine and Reproductive Medicine[END_REF].

Nuclear transfer consists of removing the nucleus from a somatic cell and introducing it into the cytoplasm of an enucleated oocyte. This technique resulted in the birth of the Dolly sheep in 1997.

In 2013, the nuclear transfer was carried out from human cells, thus making it possible to generate human blastocysts called NT-ESC (Nuclear Transfert-Embryonic Stem Cells) [START_REF] Tachibana | Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer[END_REF]. This technique makes it possible to obtain embryonic stem cells specific to a patient and to model some genetic disease. Although nuclear transfer does not require the destruction of embryos, the use of human oocytes also presents ethical problems.

Cell fusion is the fusion of a somatic cell with an embryonic stem cell resulting in the formation of a tetraploid hybrid stem cell. This technique has made it possible to highlight the ability of embryonic stem cells to reprogram the nucleus of a somatic cell. Since then, many cell types have been reprogrammed. Starting cells that can be obtained easily and without invasive procedures and cells sensitive to reprogramming are favored. Most of the reprogrammed cells are cells from mesodermal origin: fibroblasts [START_REF] Takahashi | Induction of pluripotent stem cells from fibroblast cultures[END_REF]), hematopoietic lineage [START_REF] Giorgetti | Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2[END_REF][START_REF] Loh | Generation of induced pluripotent stem cells from human blood[END_REF][START_REF] Kunisato | Direct generation of induced pluripotent stem cells from human nonmobilized blood[END_REF] or adipose cells [START_REF] Qu | Induced Pluripotent Stem Cells Generated from Human Adipose-Derived Stem Cells Using a Non-Viral Polycistronic Plasmid in Feeder-Free Conditions[END_REF]. iPSCs have also been generated from cells derived from ectoderm: keratinocytes [START_REF] Aasen | Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes[END_REF], neural progenitors (Kim et al. 2009b), or melanocytes [START_REF] Utikal | Sox2 is dispensable for the reprogramming of melanocytes and melanoma cells into induced pluripotent stem cells[END_REF]) and from cells derived from endoderm: hepatocytes [START_REF] Liu | Generation of endoderm-derived human induced pluripotent stem cells from primary hepatocytes[END_REF] or pancreatic  cells (Bar-Nur et al. 2011).

The 4 stemness transcription factors also known as Yamanaka cocktail are Oct 3/4 (Octamerbinding transcription factor 3/4, also known as POU5F1), Sox2 (SRY-box 2), Klf4 (Krüppel-like transcription factor 4), and c-Myc. Oct3/4 is exclusively expressed in embryonic stem cells and primordial germ cell and is crucial for in vitro and in vivo pluripotency [START_REF] Schöler | A family of octamer-specific proteins 200 present during mouse embryogenesis: evidence for germline-specific expression of an Oct factor[END_REF][START_REF] Nichols | Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4[END_REF]). Sox2 expression is required for maintenance of pluripotency of epiblast cells and regulate OCT3/4 in mouse ESC [START_REF] Avilion | Multipotent cell lineages in early mouse development depend on SOX2 function[END_REF][START_REF] Masui | Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells[END_REF]). Oct3/4 and Sox2 have the ability to complex and stimulate the expression of the factor Nanog, which has a role in the acquisition of inner cell mass pluripotency [START_REF] Chambers | Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells[END_REF][START_REF] Chambers | Nanog safeguards pluripotency and mediates germline development[END_REF][START_REF] Mitsui | The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[END_REF][START_REF] Silva | Nanog is the gateway to the pluripotent ground state[END_REF]). Klf4 is an important factor for the self-renewal of embryonic stem cells, it is also involved in embryonic development, proliferation, differentiation and cellular apoptosis [START_REF] Dang | The biology of the mammalian Krüppel-like family of transcription factors[END_REF]. Klf4 interacts with the Oct3/4-Sox2 complex to regulate the expression of other factors involved in the self-renewal and differentiation of embryonic stem cells such as Lefty1 [START_REF] Nakatake | Klf4 cooperates with Oct3/4 and Sox2 to activate the Lefty1 core promoter in embryonic stem cells[END_REF]. C-myc is a proto-oncogene involved in cell cycle regulation, proliferation and apoptosis [START_REF] Bretones | Myc and cell cycle control[END_REF]. Oct3/4, Sox 2 and Nanog are the core factors in pluripotency gene network necessary for induction, maintenance and loss of pluripotency during cell reprogramming [START_REF] Chen | Mechanisms of pluripotency maintenance in mouse embryonic stem cells[END_REF][START_REF] Li | Ground rules of the pluripotency gene regulatory network[END_REF]. They act together as an interconnected network to regulate pluripotency gene expression [START_REF] Boyer | Core transcriptional regulatory circuitry in human embryonic stem cells[END_REF]. Nanog is not necessary to initiate the reprogramming process but is essential to maintain the pluripotency of the reprogrammed cells [START_REF] Mitsui | The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[END_REF][START_REF] Chambers | Nanog safeguards pluripotency and mediates germline development[END_REF]) and is activated by the Oct3/4-Sox2 complex. Klf4

promote reprogramming via a direct interaction Oct3/4-Sox2 complex [START_REF] Wei | Klf4 interacts directly with Oct4 and Sox2 to promote reprogramming[END_REF]).

Alternatives to retroviruses have been developed in order to decrease the risk of insertional mutagenesis as they randomly integrate into the host genome and the risk of reactivation of the reprogramming factor. Reprogramming can be done using an integrative or a non-integrative method, their advantages and disadvantages are described in table 4. The reprogramming efficiency of the Yamanaka's cocktail was quite low. In the last year the reprogramming outcome was significantly improved by alternatives at the original reprogramming cocktail (Table 5). 3) Pluripotent stem cell applications Pluripotent stem cells are a promising tool for therapeutic applications and also for the study of embryonic development, to model human diseases and for drug discovery (Figure 17). preserved between species, recent studies have shown that there may also be differences. For instance, in human, GATA 6 is essential for pancreatic organogenesis [START_REF] Fisher | GATA6 is essential for endoderm formation from human pluripotent stem cells[END_REF]Shi et al. 2017b;[START_REF] Tiyaboonchai | GATA6 Plays an Important Role in the Induction of Human Definitive Endoderm, Development of the Pancreas, and Functionality of Pancreatic β Cells[END_REF], while both GATA 4 and GATA6 control mouse pancreatic organogenesis [START_REF] Carrasco | GATA4 and GATA6 control mouse pancreas organogenesis[END_REF][START_REF] Xuan | Pancreas-specific deletion of mouse Gata4 and Gata6 causes pancreatic agenesis[END_REF]. A recent study, used human iPSC technology to model human hepato-biliary-pancreatic organogenesis in order to observe interactions between neighboring components [START_REF] Koike | Modelling human hepatobiliary-pancreatic organogenesis from the foregut-midgut boundary[END_REF]. Using single-cell expression analysis on -like cells differentiated from human ESC, a study uncovered distinct subpopulations over the endocrine differentiation process and an early linage bifurcation toward polyhormonal cells or monohormonal -like cells [START_REF] Petersen | Single-Cell Gene Expression Analysis of a Human ESC Model of Pancreatic Endocrine Development Reveals Different Paths to β-Cell Differentiation[END_REF]. Another single-cell transcriptome analysis of pancreatic progenitors and endocrine cells derived from mice and human ESC cells, provided comparative data on biological development in mouse and human available on https://lynnlab.shinyapps.io/embryonic_pancreas [START_REF] Krentz | Single-Cell Transcriptome Profiling of Mouse and hESC-Derived Pancreatic Progenitors[END_REF]. These data will provide a better understanding of the underlying molecular mechanisms of embryonic development, and also to improve differentiation protocols.

Disease modeling / drug screening application

The development of differentiation protocols allows the use of the patient derived cells to identify the underlying pathological mechanisms of a disease. To this end, iPS cells have many advantages, in particular that of providing an unlimited differentiated-cell source from patients suffering from diseases as T1D, T2D, MODY (Maturity Onset Diabetes of the young) or pancreatic agenesis [START_REF] Park | Disease-specific induced pluripotent stem cells[END_REF][START_REF] Maehr | Generation of pluripotent stem cells from patients with type 1 diabetes[END_REF][START_REF] Thatava | Intrapatient variations in type 1 diabetes-specific iPS cell differentiation into insulin-producing cells[END_REF][START_REF] Chan | Three-Dimensional Vascular Network Assembly From Diabetic Patient-Derived Induced Pluripotent Stem Cells[END_REF][START_REF] Khatib | Tumor-Free Transplantation of Patient-Derived Induced Pluripotent Stem Cell Progeny for Customized Islet Regeneration[END_REF][START_REF] Millman | Generation of stem cellderived β-cells from patients with type 1 diabetes[END_REF][START_REF] Griscelli | Generation of an induced pluripotent stem cell (iPSC) line from a patient with maturity-onset diabetes of the young type 13 (MODY13) with a the potassium inwardly-rectifying channel, subfamily J, member 11 (KCNJ11) mutation[END_REF][START_REF] Griscelli | Generation of an induced pluripotent stem cell (iPSC) line from a patient with maturity-onset diabetes of the young type 3 (MODY3) carrying a hepatocyte nuclear factor 1-alpha (HNF1A) mutation[END_REF][START_REF] Rajaei | Pancreatic Endoderm-Derived From Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells[END_REF][START_REF] Kondo | iPSC technology-based regenerative therapy for diabetes[END_REF].

Although pluripotent stem cell-based modeling has shown its tremendous value to model early onset diseases such as Progeria [START_REF] Cicero | Pathological modelling of pigmentation disorders associated with Hutchinson-Gilford Progeria Syndrome (HGPS) revealed an impaired melanogenesis pathway in iPS-derived melanocytes[END_REF] or long QT syndrome [START_REF] Moretti | Patient-specific induced pluripotent stem-cell models for long-QT syndrome[END_REF][START_REF] Bellin | Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome[END_REF], most cells derived from pluripotent stem cells show poor maturity and functionality making it more challenging to model late-onset diseases such as type 2 diabetes, or neurodegenerative diseases like Alzheimers disease or Parkinsons.

CRISPR/Cas9 technology can be used to induce disease-mutations in wild type iPS and to correct mutations in patient specific iPS to create isogenic controls. CRISPR/Cas 9 technology has been used to mutate specific genes, previously identified by GWAS involved in T2D, to study their effect on  cell function [START_REF] Zeng | An Isogenic Human ESC Platform for Functional Evaluation of Genome-wide-Association-Study-Identified Diabetes Genes and Drug 206 Discovery[END_REF].

Pluripotent stem cell technology can also be used for efficacy and toxicity drug screening. The first large-scale drug screening identified 1 compound among 6912 to improve aberrant splicing responsible for dysautonomia, on neural crest precursors derived from family dysautonomia by the iPSC [START_REF] Lee | Large-scale screening using familial dysautonomia induced pluripotent stem cells identifies compounds that rescue IKBKAP expression[END_REF]. Such technology was also used for drug screening for Progeria disease [START_REF] Blondel | Drug screening on Hutchinson Gilford progeria pluripotent stem cells reveals aminopyrimidines as new modulators of farnesylation[END_REF][START_REF] Cicero | A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells[END_REF].

Clinical application of pluripotent stem cells

The discovery of iPSC has provided a great interest for regenerative medicine to replace the patient's damaged cells or regenerate them. Clinical trials with embryonic stem cell-derived cells are currently underway for the treatment of various diseases (Table 6) however, ethical concerns limit the utilization of these cells. On the other hand, iPSC cells are independent of ethical concerns and might be used for both autologous and allogeneic applications (Table 7) [START_REF] Trounson | Pluripotent stem cells progressing to the clinic[END_REF]. Autologous transplants are assumed to be safer to avoid immune rejection. Nevertheless, currently, this strategy does not appear feasible as a general treatment option due to the time required to generate patient derived iPS cell line and excessive cost.

An accepted strategy currently under evaluation is to produce differentiate cells from HLA (Human Leukocyte Antigen) homozygous donor iPS cells. Indeed, using HLA compatible cells will a reduce the risk of rejection and the time needed to provide HLA-matched iPSC products to significant numbers of patients. Although, the benefit of HLA matching to minimize rejection has been demonstrated, an immunosuppressive treatment may be required [START_REF] Turner | Toward the development of a global induced pluripotent stem cell library[END_REF][START_REF] Wilmut | Development of a global network of induced pluripotent stem cell haplobanks[END_REF][START_REF] Lee | Repurposing the Cord Blood Bank for Haplobanking of HLA-Homozygous iPSCs and Their Usefulness to Multiple Populations[END_REF]. For this purpose, HLA haplobanks has been envisaged [START_REF] Okita | A more efficient method to generate integration-free human iPS cells[END_REF][START_REF] Gourraud | The role of human leukocyte antigen matching in the development of multiethnic "haplobank" of induced pluripotent stem cell lines[END_REF][START_REF] Taylor | Generating an iPSC bank for HLAmatched tissue transplantation based on known donor and recipient HLA types[END_REF][START_REF] Jacquet | Strategy for the creation of clinical grade hESC line banks that HLA-match a target population[END_REF]) and a Global Alliance for iPSC Therapies (GAiT) developed to achieve a consensus on clinical-grade pluripotent stem cells. A major step forward was the efficient differentiation of hESC to definitive endoderm [START_REF] Kubo | Development of definitive endoderm from embryonic stem cells in culture[END_REF][START_REF] D'amour | Efficient differentiation of human embryonic stem cells to definitive endoderm[END_REF] et al. 2006;[START_REF] Kroon | Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo[END_REF]. This protocol was further optimized [START_REF] Schulz | A scalable system for production of functional pancreatic progenitors from human embryonic stem cells[END_REF] and it is currently used in clinical trials in USA and Canada by ViaCyte as a regenerative approach for the treatment of T1D (NCT02239354, NCT03163511, NCT03162926).

These publications were obtained with specific embryonic stem cell lines (CyT49 and CyT203) not readily available. Since then, many modified and improved protocols have been published using several cell lines (Table 8) [START_REF] Chen | Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer[END_REF][START_REF] Ameri | FGF2 specifies hESC-derived definitive endoderm into foregut/midgut cell lineages in a concentration-dependent manner[END_REF][START_REF] Mfopou | Noggin, retinoids, and fibroblast growth factor regulate hepatic or pancreatic fate of human embryonic stem cells[END_REF][START_REF] Nostro | Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells[END_REF][START_REF] Nostro | Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines[END_REF][START_REF] Xu | Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells[END_REF][START_REF] Rezania | Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice[END_REF][START_REF] Rezania | Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo[END_REF]. Although there has been no direct comparisons between the different protocols, differentiated cells were often polyhormonal (figure 18), poorly functional (not responsive to glucose) and resemble fetal cells [START_REF] Hrvatin | Differentiated human stem cells resemble fetal, not adult, β cells[END_REF].

While polyhormonal cells are rare in human adult pancreas (1.34%+ 0.6%), they appear not to be an artifact of in vitro differentiation as they are frequent in human fetal pancreas (11-13 week-old:  cell differentiation is a complex process that is orchestrated by the expression of key transcription factors (Figure 19 and 20). To study  cell commitment in vitro, differentiation protocols were prolonged to stage 5 including NGN3+ endocrine progenitors and stage 6 insulin+ -like cells (BLCs) [START_REF] Pagliuca | Generation of functional human pancreatic β cells in vitro[END_REF][START_REF] Rezania | Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells[END_REF][START_REF] Russ | Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro[END_REF]. BLCs generated in vitro at stage 6 (or in some groups stage 7) were 50% insulin positive; these cells respond to static glucosestimulated insulin secretion and restore normal glycemia in 8-12 weeks when transplanted in immunodeficient diabetic mice [START_REF] Rezania | Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells[END_REF]). However, those in vitro generated cells were still immature as they had major differences to native  cells, such as gene expression profiles (particularly high NGN3 and proinsulin and low urocortin 3 (UCN3) expression levels), calcium signaling and lack of response in dynamic GSIS [START_REF] Pagliuca | Generation of functional human pancreatic β cells in vitro[END_REF] to obtain BLC with an enhanced functional maturation as they display both first and second phase response similar to the response of native human islets in a perifusion experiment. In addition, differentiated cells had an increase in their insulin expression and content. Both studies, show in vivo function of transplanted cells rapidly after grafting (10 days). These two studies represent a major technical advance in the differentiation of human pluripotent cells, although a difference in UCN3 and MAFA mRNA expression persists between the cells produced and human islets. Table 8 summarizes the characteristics of the key published protocols. The finding that hPSC derived cells are functionally immature has been reported for pancreatic  cells, hepatocytes, neurons, and hematopoietic cells [START_REF] Miller | Human iPSC-based modeling of late-onset disease via progerin-induced aging[END_REF]). This is a major MAFA is up-regulated by glucose and activates the insulin gene [START_REF] Kataoka | MafA is a glucose-regulated and pancreatic beta-cell-specific transcriptional activator for the insulin gene[END_REF][START_REF] Olbrot | Identification of beta-cell-specific insulin gene transcription factor RIPE3b1 as mammalian MafA[END_REF][START_REF] Matsuoka | The MafA transcription factor appears to be responsible for tissue-specific expression of insulin[END_REF]), during rat development, MAFA gradually increases and shifts its cytoplasmic localization to a nuclear localization [START_REF] Matsuoka | The MafA transcription factor appears to be responsible for tissue-specific expression of insulin[END_REF][START_REF] Artner | MafA and MafB regulate genes critical to beta-cells in a unique temporal manner[END_REF][START_REF] Aguayo-Mazzucato | Mafa expression enhances glucose-responsive insulin secretion in neonatal rat beta cells[END_REF]) and leads to a positive GSIS response. Increases of MAFA, p16 and improved GSIS have been induced by tri-iodothyronine hormone (T3) (Aguayo-Mazzucato et al. 2013, 2015, 2018). ERRy expression increased during pancreas development and is required for post-natal  cell maturation as well as metabolic maturation of -like cells derived from human iPSC [START_REF] Yoshihara | ERRγ Is Required for the Metabolic Maturation of Therapeutically Functional Glucose-Responsive β Cells[END_REF]).

UCN3 is highly expressed in adult human  cells and increases during in vivo maturation of insulin expressing cells derived from hPSC [START_REF] Blum | Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3[END_REF][START_REF] Van Der Meulen | Urocortin 3 marks mature human primary and embryonic stem cell-derived pancreatic alpha and beta cells[END_REF]). However, UCN3 is a passive maturation marker as UCN3 treatment does not promote maturation of stem cell-derived endocrine cells [START_REF] Blum | Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3[END_REF].

In terms of functionality, it has been shown that adult murine  cells show changes in methylation patterns which repress proliferation and activate genes involved in insulin secretion resulting in higher insulin secretion in  cells from older mice than  cells from young mice [START_REF] Avrahami | Aging-Dependent Demethylation of Regulatory Elements Correlates with Chromatin State and Improved β Cell Function[END_REF]. However, this finding is controversial as others found no intrinsic functional difference between old and young islets and rather point to an aging extracellular matrix and vessels [START_REF] Almaça | Young capillary vessels rejuvenate aged pancreatic islets[END_REF]. Similarly, insulin secretion in human islets from adult donors (>28 years old) was superior to human islets from young donors (<9 year old). Additionally, p16 ink4a (p16), which is expressed during normal aging of  cells and, transgenic mice in which p16 ink4a is activated both show increased insulin secretion [START_REF] Helman | p16(Ink4a)-induced senescence of pancreatic beta cells enhances insulin secretion[END_REF]).

Different methods have been tested to age in vitro hPSC-derived lineages, one of which includes overexpressing progerin.

Directing aging using progerin

Although human pluripotent stem cell differentiation is a useful tool to deciphering embryogenesis, physiopathology, model disease and also for cell replacement, lack of maturation and fetal-like properties of most of the PSC-differentiated cells types can be a challenge, particularly to model late-onset diseases. Indeed, from a development perspective there is a continuity between cell specification, maturation and aging. Cellular specification is characterized by the development of a specific transcriptional program and the expression of markers, maturation through the acquisition of a function and aging through the progressive decline of this function [START_REF] Studer | Programming and Reprogramming Cellular Age in the Era of Induced Pluripotency[END_REF].

Indeed, iPS cells derived from old donors (as young donors) show rejuvenation patterns during reprogramming in particular an increase of telomer size [START_REF] Marion | Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells[END_REF]) and loss of senescence markers [START_REF] Prigione | The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells[END_REF][START_REF] Suhr | Mitochondrial rejuvenation after induced pluripotency[END_REF], and months of post-differentiation maturation are necessary for the acquisition of a functionality [START_REF] Saha | Technical challenges in using human induced pluripotent stem cells to model disease[END_REF]Liu et al. 2012a).

Several strategies have been developed in order to induce maturation or aging in differentiated iPSC cells, such as cellular stressors which target mitochondrial function or protein degradation [START_REF] Nguyen | LRRK2 mutant iPSCderived DA neurons demonstrate increased susceptibility to oxidative stress[END_REF][START_REF] Cooper | Pharmacological rescue of mitochondrial deficits in iPSC-derived neural cells from patients with familial Parkinson's disease[END_REF]Liu et al. 2012b;Shi et al. 2017a), overexpressing progerin [START_REF] Miller | Human iPSC-based modeling of late-onset disease via progerin-induced aging[END_REF]), 3D co-culture which enhance cell-cell interaction [START_REF] Giacomelli | Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells[END_REF]Shi et al. 2017a;[START_REF] Correia | 3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes[END_REF][START_REF] Doss | Current Challenges of iPSC-Based Disease Modeling and Therapeutic Implications[END_REF][START_REF] Yan | Cell population balance of cardiovascular spheroids derived from human induced pluripotent stem cells[END_REF], or the use of telomerase inhibitors [START_REF] Vera | Generating Late-Onset Human iPSC-Based Disease Models by Inducing Neuronal Age-Related Phenotypes through Telomerase Manipulation[END_REF]) (Figure 21). In 2011, Izpisua Belmonte group showed that iPS-derived from progeria patients did not express aging markers and progerin, while after differentiation, the smooth muscle cells they expressed signs of premature senescence phenotypes [START_REF] Liu | Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome[END_REF] The onset of Progeria is due to a point mutation of the LMNA gene. There are more than 400 mutations associated with this disease reported to date (http://www.umd.be/LMNA/) but the most frequently observed is the replacement of a cytosine by a thymine in position 1824 (c.1824>T) (De [START_REF] Sandre-Giovannoli | Lamin a truncation in Hutchinson-Gilford progeria[END_REF][START_REF] Eriksson | Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome[END_REF][START_REF] Verstraeten | Compound heterozygosity for mutations in LMNA causes a progeria syndrome without prelamin A accumulation[END_REF][START_REF] Moulson | Increased progerin expression associated with unusual LMNA mutations causes severe progeroid syndromes[END_REF].

This mutation activates a cryptic splicing site, responsible for the deletion of 150 base pairs in the lamin A transcript. Therefore, the protein produced, lamin A50 also called progerin, is truncated of 50 amino acids on its C-terminal region. The produced protein undergoes the same posttranslational modifications as lamin A except for the last cleavage. Indeed, the deletion is responsible for the absence of the ZMPSTE24 (Zinc Metallopeptidase STE24) enzyme recognition site which allows the last proteolysis. Therefore, the protein produced (progerin) remains farnesylated and carboxymethylated.

Lamin A is one of the intermediate filaments of type V constituting the nuclear lamina. Nuclear lamina is the structural support of the nuclear envelope, and is also involved in many cellular mechanisms such as gene regulation and transcription, cell differentiation, DNA replication and repair, and nuclear envelope assembly [START_REF] Goldman | Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome[END_REF][START_REF] Capell | Human laminopathies: nuclei gone genetically awry[END_REF][START_REF] Frock | Lamin A/C and emerin are critical for skeletal muscle satellite cell differentiation[END_REF][START_REF] Shumaker | Mutant nuclear lamin A leads to progressive alterations of epigenetic control in premature aging[END_REF][START_REF] Dechat | Alterations in mitosis and cell cycle progression caused by a mutant lamin A known to accelerate human aging[END_REF][START_REF] Young | Targeting protein prenylation in progeria[END_REF].

The mutation of the lamin A responsible for HGPS alters the structure and function of the nuclear lamin. As result, the nuclei of the cells have an abnormal shape (blebbing) (Figure 24). Children suffering from progeria show no evidence of the disease at birth, the first clinical signs appear in the early years of life: growth retardation, characteristic facial features (prominent forehead and scalp veins, pinched nose, thin lips and abnormal dentition), alopecia, loss of subcutaneous adipose tissue, skeletal dysplasia, diminished bone mineral density, skeletal muscle wasting, severe cardiovascular decline with progressive coronary atherosclerosis [START_REF] Hennekam | Hutchinson-Gilford progeria syndrome: review of the phenotype[END_REF][START_REF] Merideth | Phenotype and course of Hutchinson-Gilford progeria syndrome[END_REF]. HGPS is a "segmental ageing disease", the main tissues affected are of mesodermal origin, in particular blood vessels, bones, heart, muscles, skin and dander, whereas organs such as liver, kidneys, lungs, gastrointestinal tract and brain are not affected [START_REF] Gordon | Disease progression in Hutchinson-Gilford progeria syndrome: impact on growth and development[END_REF][START_REF] Ullrich | Hutchinson-Gilford progeria syndrome[END_REF]. A team using genome-scale expression profiling found that in patients with progeria, the most affected gene was MEOX2/GAX (Mesenchyme Homeobox 2/ Growth Arrest-specific Homeobox) which is a transcription factor that is a negative regulator of mesodermal tissue proliferation [START_REF] Csoka | Genome-scale expression profiling of Hutchinson-Gilford progeria syndrome reveals widespread transcriptional misregulation leading to mesodermal/mesenchymal defects and accelerated atherosclerosis[END_REF]). Indeed, abnormal oral glucose-tolerance test (OGTT) have been observed in only 2 out of 13 patients and an abnormal fasting glycemia over 100mg/dl was observed in only 1 out of 13 HGPS patients [START_REF] Merideth | Phenotype and course of Hutchinson-Gilford progeria syndrome[END_REF]).

Premature-aging features observed in HGPS are shared with normal aging although HGPS patients do not express all physiological aging features such as osteoporosis, cancer or dementia (Figure 25). The sparing of the cognitive functions may be attributed to the upregulation of miR-9. Indeed, it has been shown that miR9 microRNA expression in neural cells allows their preservation [START_REF] Nissan | Unique preservation of neural cells in Hutchinson-Gilford progeria syndrome is due to the expression of the neural-specific miR-9 microRNA[END_REF].The similitude between HGPS and physiological aging can be explained by the implication of lamin A defects in normal aging. Indeed, fibroblasts from healthy aged individuals presented nuclear abnormalities comparable to HGPS fibroblasts as well as changes in histone modifications and increased DNA damage [START_REF] Scaffidi | Lamin A-dependent nuclear defects in human aging[END_REF]. This discovery has been further confirmed by the detection of progerin in fibroblasts of healthy patients responsible for mitotic defects associated with HGPS (Cao et al. 2007b;[START_REF] Mcclintock | The mutant form of lamin A that causes Hutchinson-Gilford progeria is a biomarker of cellular aging in human skin[END_REF]). In addition, progerin accumulation has been detected in the adventitial layer of arteries from healthy aged donors [START_REF] Olive | Cardiovascular pathology in Hutchinson-Gilford progeria: correlation with the vascular pathology of aging[END_REF].

Objectives

Our first objective (Chapter 3) was to test the feasibility and reproducibility of methods already published in the literature to produce pseudo-islets from human pancreas and to adapt them to clinical transplantation constraints (animal derived component free). Moreover, the objective was to compare the functionality of pseudo-islets with the native human islets, in vitro and in vivo, and to evaluate if the use of pseudo-islets could be a valid strategy to reduce the donor derived heterogeneity observed in native human islets. An important functional heterogeneity is observed between the different human islet preparations [START_REF] Nasteska | The role of beta cell heterogeneity in islet function and insulin release[END_REF][START_REF] Dybala | Heterogeneity of the Human Pancreatic Islet[END_REF] and there are several explanations for this heterogeneity [START_REF] Kayton | Human islet preparations distributed for research exhibit a variety of insulin-secretory profiles[END_REF][START_REF] Lyon | Research-Focused Isolation of Human Islets From Donors With and Without Diabetes at the Alberta Diabetes Institute IsletCore[END_REF][START_REF] Westacott | Age-Dependent Decline in the Coordinated [Ca2+] and Insulin Secretory Dynamics in Human Pancreatic Islets[END_REF][START_REF] Henquin | Influence of organ donor attributes and preparation characteristics on the dynamics of insulin secretion in isolated human islets[END_REF].

Therefore, our laboratory was interested in testing the feasibility and reproducibility of this technology. By producing 80-100m pseudo-islets and comparing their function in vitro and in vivo with their matched native human islet counterparts to evaluate whether inter-donor heterogeneity (donor's signature) persists or is abolished.

Our second objective (Chapter 4) was to produce endocrine cells from pluripotent stem cells and subsequently test if progerin could accelerate the maturation of the pancreatic endocrine cells of endodermal origin. To achieve this, we used several types of stem cells: the human embryonic stem cell H1 and induced pluripotent stem cells (iPS) DF19.9 from healthy individuals, and iPS HGPS 1972 from patients with progeria. We first aimed to optimize the differentiation protocol for each specific cell lines mainly stage 1 definitive endoderm and then S4 pancreatic progenitors, and then, to determine if progerin was expressed during the differentiation steps and to investigate if it had an effect on endocrine differentiation and maturation both in vitro and in vivo.

The production of functional cells from pluripotent stem cells remains an important issue. The majority of cells generated in vitro from human pluripotent stem cells are immature post differentiation which is, for instance, substantiated by the expression in iPS-generated hepatocytes of  fetoprotein and low albumin levels [START_REF] Baxter | Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes[END_REF]. Beta cells generated from pluripotent stem cell technology are immature and do not typically respond to glucose [START_REF] Hrvatin | Differentiated human stem cells resemble fetal, not adult, β cells[END_REF]).

Studer's laboratory recently presented a strategy to artificially induce aging in vitro, which is particularly interesting to obtain model of late-onset diseases as Parkinson or Alzheimer, and perhaps type 2 diabetes mellitus. In 2013, Studer's group exploited progerin, the protein involved 67 in Hutchinson-Gilford Progeria Syndrome. They demonstrated that after the differentiation of iPS cells into fibroblasts and neurons, progerin overexpression accelerated in vitro aging [START_REF] Miller | Human iPSC-based modeling of late-onset disease via progerin-induced aging[END_REF]). The over-expression of progerin was sufficient to induce aging markers similar to elderly donor cells. Although progerin is a pathological protein, low levels of progerin expression have been found in healthy individuals [START_REF] Scaffidi | Reversal of the cellular phenotype in the premature aging disease Hutchinson-Gilford progeria syndrome[END_REF]. Progerin has been implicated in the aging of skin induced by UV light [START_REF] Takeuchi | Longwave UV light induces the aging-associated progerin[END_REF].

Therefore, our third objective (Chapter 5) was to study the influence of the overexpression of progerin on pancreatic endocrine cell differentiation. To do this we used iPS cells derived from patients with the premature aging disease HGPS or Progeria. We compared these cells to H1 cells and in order to confirm the specific role of progerin, we planned to overexpress progerin in H1 cells during differentiation.

1. Materials 1) Human pluripotent cell lines iPS DF19-9-7T (referred to herein as iPS DF19.9) were purchased from WiCell ® . They were reprogrammed from foreskin fibroblasts from a male donor with no reported disease and using a non-integrating method Sendai virus (OCT4, SOX2, NANOG, LIN28, L-MYC, KLF4, SV40LT) by Dr. J. Thomson at University of Wisconsin (UW, USA) [START_REF] Yu | Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences[END_REF]). WA01 cells (referred to herein as H1) were purchased from WiCell ® . Dr. Julie Kerr-Conte is authorized to import, conserve and do research on H1 cell line by the ABM (Annexe 2). This embryonic stem cell line was derived from a human blastocyt by Dr. J.

Thomson (UW, USA) [START_REF] Thomson | Embryonic stem cell lines derived from human blastocysts[END_REF]. iPS HGPS AG01972 cells (referred to herein as iPS HGPS) were kindly provided by Dr. X. Nissan and Dr. M. Peschanski (I-STEM, France). iPS HGPS AG01872 cells were reprogrammed from HGPS patient fibroblasts (Fibro HGPS Ag001972) using Yamanaka's method with OCT4, KLF4, SOX2, c-MYC [START_REF] Takahashi | Induction of pluripotent stem cells from fibroblast cultures[END_REF]).

2) Human islets

Human islets are isolated from pancreata obtained from brain dead donors by Prof. F.

Pattou's team (Inserm U1190) using the Edmonton protocol [START_REF] Shapiro | Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen[END_REF]. Islet isolation and evaluation was assessed by trained employees of the biotherapy platform.

To gauge the differentiation state of the in vitro stem cell-derived endocrine cells we used a pool of 10 human islets coming from 10 different donors for qPCR experiments. For "previous results", qPCR results were compared to a single donor.

The pseudo-islets generated from each donor were compared to native islets at Day 1 and to native islets at D8 from the same donor. Islet donor characteristics are described in the table 9.

Cell culture conditions

All cells were cultured at 37°C and 5% CO2.

Thawing and freezing

Vials with cryopreserved cells were thawed and transferred to a 15mL falcon tube to which DMEM media was added dropwise. Cell suspension was centrifuged at 200g during 5 minutes. Supernatant was removed and the cell pellet was resuspended in specific media for each cell line as detailed in materialscell culture section.

For freezing of pluripotent stem cells, EDTA (Versene ™) treatment during 3 to 5 minutes was used to remove pluripotent stem cells. Cells were detached using 2mL of DMEM media 3 times; cell suspension was centrifuged at 300g for 5 minutes. Supernatant was removed and the cell pellet was resuspended in mFreSR™ media. Cells were stored in Mr. Frosty ™ Freezing Containers (Thermo Fisher) with isopropylic alcohol during 24 hours at -80°C before storage in liquid nitrogen.

Cell maintenance

Pluripotent stem cells were grown in 6-well plates coated with Matrigel™ diluted to 1:60. After 3 to 5 days cells reached approximatively 70-80% confluence and their passage was done by adding 1mL EDTA per well for 3 to 5 min. Cells were detached using 2mL of culture media with 10uM of rock inhibitor, three times. Cell suspension was then centrifuged at 200g for 5 minutes.

Fibroblasts were cultivated in T75 flasks. Media was changed every other day.

Human islets and human pseudo-islets were cultivated in T150 flasks. Media was changed every other day. For native human islets and pseudo islets groups, cells were kept for up to 8 days postisolation.

Cell differentiation

Cell differentiation was done without antibiotics, on Matrigel ™ and according to:

-Odorico protocol (confidential agreement, adaptated from [START_REF] Xu | Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells[END_REF])

-

Pseudo-islet formation:

One day after isolation, human islets were dissociated using Accutase (1mL/1000IEQ).

Dissociation was done alternately by 1 minute cycles of pipetting (P1000) up and down and water bath incubation at 37°C; the number of cycles was islet preparation dependent. The enzymatic reaction was stopped by adding 2 volumes of culture media [START_REF] Lukowiak | Identification and purification of functional human beta-cells by a new specific zinc-fluorescent probe[END_REF].

Single cells were counted and 187 500 cells were seeded per well of a 24-well sphericalplate (Kugelmeiers, Switzerland) making that 250 cells per microwell. Media was changed every other day.

Perifusion of human islets

As previously described, 300 IEQ were placed in the perifusion chamber [START_REF] Henquin | In vivo and in vitro glucose-induced biphasic insulin secretion in the mouse: pattern and role of cytoplasmic Ca2+ and amplification signals in betacells[END_REF].

Equilibration in Krebs solution with BSA (1mg/mL) at low glucose (3mM) for 50 min was done prior to starting. The islets were then perfused 3 min at 3mM glucose, 40 min at 15mM glucose and 20 min at 3mM glucose. During the experiments, samples were collected every 2 min. Outflow, pressure, temperature and oxygen were kept stable during the process. After perifusion, islets and pseudo-islets were lysed in ethanol acid and collected for intracellular insulin measurements (Dxl Access Immunoassay System, Beckman Coulter) and insulin secretion measurements.

The reading range is between 0.3 and 300 IUU/ml. The samples are supplemented with albumin at 1mg/ml to promote insulin detection. Intracellular insulin was extracted from the cells after utrasonication in ethanol-acid (1.5% concentrated HCl; 70% pure ethanol; 28.5% H2O) on ice. Each intracellular insulin sample is diluted in 3 different concentrations (1/2114; 1/3844; 1/5761).

Molecular biology

RNA isolation

Cells were removed from culture and lysed in RLT buffer (Qiagen) complemented with 2mercaptoethanol (10L/mL). RNeasy mini Kit (Qiagen) was used according to the manufacturer's recommendations to extract RNA. RNA quality was controlled using a Biorad Experion chip, RQI > 7 were considered of sufficient quality to perform retrotranscription. RNA dosage was done using Nanodrop (Thermofisher).

Retrotranscription

RNA was reversely transcribed using the iScript cDNA Synthesis Kit (Bio-Rad) as follows:

iScript reaction mix 4 L iScript Reverse Transcriptase 1 L

RNA qs 300ng

Nuclease free water qs 20 L

The cDNA synthethis was performed in a 3-step cycler program on the Veriti (Applied Biosystem) using the following protocol: 5 min at 25°C, 20 min at 46°C and 1 min at 95°C.

The cDNA was stored at -80°C.

Quantitative Real Time PCR (qRT-PCR)

To normalize our results, we used as the house-keeping genes RPL27 for SYBR Green Q-PCRs and GAPDH for Taqman experiments. Experiments were done in duplicate and the 2 -Ct method was used for calculations in accordance with the MIQE guidelines [START_REF] Bustin | The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[END_REF]). Reactions were conducted on the CFX Connect RT-PCR Detection System (Bio-Rad) as follows:

For SYBR Green experiments:

SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) 5L

Forward primer (100M) 0,25L PFA and embedded in paraffin. Sections of 10 m thick sections were cut. Samples were deparaffinized in xylene, followed by a decreasing ethanol row of 100%, 90%, 70%, 50% and rehydrated in water and PBS. Antigen retrieval was performed in citrate buffer (pH6), 9 minutes at 750 watts in the microwave. When needed, sections were permeabilized in 0,1% Triton-X100.

Protein block (Dako) was applied for 15 min to avoid unspecific binding. Primary antibody was incubated overnight at 4°C. The next day, slices were washed 3 times in PBS under agitation. Then, secondary antibody was incubated for 1 hour at room temperature in the dark. Finally, slices were then washed 3 times in PBS and DAPI was added before mounting.

Flow Cytometry and Flurorescence Activated Cell Separation

One well during differentiation experiments was taken for flow cytometry. TrypLE was used to obtain single cells. The cells were collected with FACS Buffer (PBS (w/o Ca 2+ and Mg 2+ ) + 10%FBS) and put in wells of a 96-well round bottom plate and centrifuged at 1400 rpm during 5 minutes.

For surface antigen:

One wash with FACS Buffer was done before adding antibodies and isotypes. After 20 min at room temperature, two washes were done before resuspension in FACS Buffer and prior to the flow cytometry run.

For intracellular antigen:

Cells were resuspended in CytoFix Solution (BD Biosciences) for 20-30 min at room temperature in the dark and washed twice with Wash/Perm Solution (BD Biosciences) at 2000 rpm during 2 minutes. Cells were incubated with antibody or isotypes overnight at 4°C. The following day, cells were washed once with Wash/Perm Solution prior to adding secondary antibodies for 30 min in the dark and at room temperature. After incubation, cells were washed twice before resuspension in FACS buffer for flow cytometry run.

Gating strategy:

First, we gated our population using forward and side scatters for size and granularity, debris were excluded. From the previous population, we used height (H) and width (W) for FSC and SSC scatters in order to gate single cells. From that single population, we gated the population of interest on isotypes sample (Figure 26).

Single cells Single cells Autofluorescence Isotypes

Cell population

Mice

All animal experimentations were approved by our local animal ethical committee (CEEA75 -Lille). Under anesthesia and local analgesia, left kidney was exposed to proceed to the transplantation under the kidney capsule as previously described [START_REF] Caiazzo | Quantitative in vivo islet potency assay in normoglycemic nude mice correlates with primary graft function after clinical transplantation[END_REF].

Mice were housed in a SOPF facility with an ad libitum access to water and food, on a 12 hour dark/light cycle. Each animal was followed each 2 weeks for body weight, glycemia and blood sampling for c-peptide measurements.

Preliminary studies in our laboratory using human islets transplanted in normoglycemic nude mice (n=4) compared glucose stimulated insulin secretion (3g/Kg) with an oral glucose tolerance test (OGTT) vs intraperitoneal tolerance test (IPGTT) showing the highest secretion at 30 minutes with ipGTT (Figure 27). 91

The objective here was first to test the feasibility and reproducibility of pseudo-islet technology in clinically approved culture media and to compare their function in vitro and in vivo with their matched native human islet counterparts at D1 (day of dissociation) and D7 (day required for reaggregation). Secondly, we attempted to evaluate whether donor heterogeneity or the donor's signature in human intact islets persisted in pseudo-islets or was abolished.

Figure 28 gives an overview of the pseudo-islet experiments. The viability before dissociation of all four human islet preparations used in this experiment which was assessed by trypan blue staining and it was always above 90%. One day after isolation, the native D1 islets (red) were compared to donor matched pseudo-islets formed from dissociated single cells of native islets that were reaggregated for one week (pseudo-islets D7, blue) and to the native islets cultured for one week (Native islets D7, grey) (Figure 28). The islet functionality of the three groups was evaluated in vitro by a dynamic perifusion technique to study the insulin secretion capacity of 300 islet equivalents (IEQ) in response to glucose [START_REF] Henquin | In vivo and in vitro glucose-induced biphasic insulin secretion in the mouse: pattern and role of cytoplasmic Ca2+ and amplification signals in betacells[END_REF]). The secreted insulin was normalized to the intracellular insulin content. For the in vivo evaluation, 500 IEQs were grafted under the renal capsule of non-diabetic immunodeficient mice and, over a month, glycemia and fasting human c-peptide secretion were measured [START_REF] Caiazzo | Quantitative in vivo islet potency assay in normoglycemic nude mice correlates with primary graft function after clinical transplantation[END_REF]. Afterwards, the animals were sacrificed and the insulin and glucagon positive area within the grafts was morphometrically evaluated.

Figure 28: Overview of pseudo-islets experiments. Donor matched pseudo-islets (blue) were generated from dissociated single cells obtained from human islet donors one day after isolation and compared to intact native islets at day 1 (red) or day 7 (grey). Islet functionality was evaluated in vitro and in vivo (n=3-4 donors). 1. In vitro results

In vitro Evaluation

The islet donors had very diverse characteristics and medical histories (age, BMI, sex, alcohol, tobacco, cardiorespiratory arrest or time spent in intensive care unit). The phenotypic and clinical characteristics of human islet donors used in the present study are reported in Table 9 (see Material and Method). None of the islet preparations used were from diabetic donors. Pseudo-islets were successfully formed from 4/5 human islet preparation tested. For the in vitro evaluation of pseudoislets vs. islets at D1 or D7, 300IEQ of each group were perifused with basal glucose (3mM), followed by high glucose (15mM) and finally again with basal levels (Figure 29). As expected, in all groups the insulin secretion profiles showed a negligible release of insulin at basal glucose (G3), and a secretion peak (first phase) at 15mM glucose (G15) followed by a stable plateau (second phase) until the glucose concentration was decreased to basal glucose (Figure 29). While the insulin secretion pattern of native human islets at D1 was rather heterogenous between donors (Figure 29A), the insulin release of native islets at D7 (Figure 29B) and pseudo-islets at D7 (Figure 29C)

showed less variability. However, the total amount of secreted insulin was significantly higher for native islets at D1 compared to native islets at D7 (**p<0,01) and pseudo-islets at D7

(****p<0,0001) (Figure 29D). Insulin content was drastically reduced (75%) upon culture in our clinical grade culture medium (CMRL 1066 with 0,0625% human serum albumin and recombinant insulin), both in pseudo-islets and intact native islets at D7 groups compared to native islets D1 insulin content (Figure 30). For this reason, insulin secretion levels between D1, D7 native islets and pseudo-islets were not expressed in percent of insulin content.

In vivo results

To evaluate the functionality of the three different groups in vivo, we performed grafts in an immunodeficient mouse model [START_REF] Caiazzo | Quantitative in vivo islet potency assay in normoglycemic nude mice correlates with primary graft function after clinical transplantation[END_REF]. Islets were manually counted prior to transplantation and 500 IEQ (IEQ = islet at 150 m in diameter) were transplanted under the kidney capsule of mice. The in vivo function of pseudo-islets was evaluated as human c-peptide secretion in response to glucose (c-peptide/glycemia) and it was compared to native D1 human islets from the same donor and to native human islets cultured 7 days. The kinetic results over one month showed a tendency for native human islets at D1 to secrete higher levels of human c-peptide than pseudo-islets D7 (Figure 31A, pseudo-islets vs. native islets D1 p=0,2327) and native islets cultured 7 days, albeit only the later was statistically significant (Figure 31A, native islets D7 vs. native islets D1 p=0,0098). Mean of the c-peptide/glycemia ratio over one month was calculated and confirmed the superiority of intact native islets D1 over D7 islets and pseudo-islets (Figure 31B p=0,0038 and p=0,0294 respectively). Next, we quantified by morphometry, the distribution of endocrine cells in the 3 different groups, one month after transplantation under the kidney capsule of nondiabetic immunodeficient mice.

The surface of insulin or glucagon positive cells was similar between D1 and D7 native islets, but a trend towards an increase in the glucagon surface area was observed in the pseudo-islet group, compared to the other two groups (Figure 32). Finally, preliminary results on 4 islet preparations suggest that the donor heterogeneity persisted after pseudo-islet formation in vitro (Figure 33) both in the first and second phase of insulin secretion. As controls, both undifferentiated H1 cells (white bar) and human islets isolated from one donor (red bar) were used and compared to differentiated H1 cells (grey bars). As expected, these key pancreatic genes were highly and progressively induced in human islets compared to undifferentiated H1 cells, to which the expression was normalized. Although non-significant, a gradual increase in CHGA, a pan-endocrine marker [START_REF] Travis | Survival analysis of 200 pulmonary neuroendocrine tumors with clarification of criteria for atypical carcinoid and its separation from typical carcinoid[END_REF]) was observed (Figure 36A). This coincided with a slight increase in CK19 mRNA levels, a known ductal marker [START_REF] Kerr-Conte | Ductal cyst formation in collagen-embedded adult human islet preparations. A means to the reproduction of nesidioblastosis in vitro[END_REF]. Of note, two key transcription factors crucial for  cell specification [START_REF] Nelson | The transcription factors Nkx6.1 and Nkx6.2 possess equivalent activities in promoting beta-cell fate specification in Pdx1+ pancreatic progenitor cells[END_REF]), PDX1 and NKX6.1, the pancreatic homeodomain transcription factor and a homeobox transcription factor, were also upregulated in stage 4 differentiated cells compared to undifferentiated cells (Figure 36A). Stage 4 cells expressed the same PDX1 levels as human islets, however, NKX6.1 expression remained one log apart from human islet expression. We then verified if the differentiated cells expressed INS and GCG. In comparison with human islets from one donor, the differentiated cells expressed chromogranin A, CK19 and PDX1 at the same magnitude, but one log less for NKX6.1 and INS and 1,5 log less for GCG.

We then verified protein expression for Chromo A (pan-endocrine marker), CK19, Insulin and Glucagon by immunofluorescence techniques. Figure 36B (experiment 4) shows chromogranin A and CK19 positive cells in the 3D structure "budding" of. In addition, double positive cells for insulin and glucagon were observed (Figure 36B, yellow square) as previously reported [START_REF] Hrvatin | Differentiated human stem cells resemble fetal, not adult, β cells[END_REF]. After in vitro differentiation, S4 cells were grafted under the kidney capsule of nondiabetic immunodeficient mice for in vivo maturation [START_REF] Caiazzo | Quantitative in vivo islet potency assay in normoglycemic nude mice correlates with primary graft function after clinical transplantation[END_REF]). All mice were followed over a period of 3 months. In 9 mice out of a total of 29, fasting c-peptide secretion was detectable with the ultrasensitive human c-peptide kit (Mercodia, Sweden). The maximum secretion did not exceed 200 ng/L in non-fasted animals, while the average range of fasting c-peptide of 500 human islet equivalents (IEQ) was 500 -1000 ng/L (represented by the red dot in Figure 37A). However, with time the amount of detected human c-peptide was decreased in all grafted mice (Figure 37A).

Immunofluorescent staining from a graft of cells from experiment 9, at 12 weeks after transplantation, showed positive cells for chromogranin A or CK19 (Figure 37B). In addition, insulin or glucagon positive cells were also identified by immunofluorescent staining (Figure 37B).

Furthermore, identical to pancreatic islet neogenesis in the adult and fetal human islet a budding of chromogranin A positive endocrine cells was detected next to a Cytokeratin 19 positive duct, what resembles pancreatic islet neogenesis human islet (Figure 37C) [START_REF] Swartz | An islet of Langerhans located within the epithelium of a human pancreatic duct[END_REF][START_REF] Kerr-Conte | Ductal cyst formation in collagen-embedded adult human islet preparations. A means to the reproduction of nesidioblastosis in vitro[END_REF]. A typical adverse event of stem cell transplantation in animals is the formation of teratomas, it occurs when undifferentiated cells persist in the transplanted cells. Therefore, we analyzed the animals and identified 13% (4 mice from a total of 29) teratoma formation (Figure 37D). The frequency of observed teratomas in transplanted animals is in line with observations from other groups [START_REF] Kroon | Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo[END_REF]. In conclusion, by using H1 cells differentiated according to the Odorico 2012 protocol and subsequently transplanted in immunodeficient mice, it was possible to obtain endocrine and ductal cells in vivo. Although these results were very encouraging, polyhormonal cells were observed as well as important teratoma formation in vivo suggesting that both, the differentiation efficiency toward endoderm and pancreatic progenitor were insufficient. Therefore, more emphasis was put on optimizing the efficiency of definitive endoderm (stage 1) and "second generation" protocols which generate large number of stage 4 PDX1+/NKX6.1+ cells with low expression of NGN3, thus optimizing S1 and S4 differentiation and lowering the teratoma formation.

2. Future strategies for the generation of pancreatic progenitors with iPS DF19.9 cells using second generation protocols following cancellation of H1 authorization.

As mentioned above, the most favorable results were obtained with H1 cells, however due to the loss of our authorization to work with embryonic stem cells we focused on the optimization of the differentiation of iPS DF19.9 cells, since this was the cell line used in Prof. Odorico's laboratory in 2012 [START_REF] Xu | Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells[END_REF]. In general, differentiation protocols were less efficient with the DF19.9 cell line than with H1 cells.

Subsequent studies focused on iPS DF19.9 cell differentiation using "second generation" protocols, either the SD Kit (STEMDiff Pancreatic Progenitor Kit) from stage 1 to 4 and Rezania 2014 for stage 4 to 7, or Rezania 2014 alone for stage 1 to 7 (adaptation of the protocol: no single cells at stage 4 and no transition to the air/liquid interface for stages 5 to 7 as described by Rezania) (Figure 38). We evaluated the differentiated cells in vitro at stage 1, stage 4 and stage 7. In some experiments we grafted cells at stage 4 and in others at stage 7, all animals were assessed for glycemia and c-peptide secretion. et al. 2005). Gene expression analysis at stage 1 (black) using the Rezania protocol (3 days) showed an increase in CXCR4 and FOXA2 mRNA expression in comparison to undifferentiated iPS DF19.9 cells (Figure 39A white bar). Then, quantification of their expression by immunofluorescent staining was performed. For each experiment, 100 cells were identified by DAPI, and both CXCR4 and FOXA2 positive cells were counted (Figure 39B). In total, 90% double positive cells were detected (Figure 39B). Because this method is time consuming and not accurate, a quantification method by flow cytometry was performed (Figure 39C) which was recommended in my 2 nd PhD progress meeting by the jury members. For endoderm cytometry In vitro evaluation

SD

In vivo evaluation

Fasting C-peptide Glycemia Immunofluorescence on explanted graft iPS DF19.9

109 analysis, we quantified endoderm formation by CXCR4 and c-Kit (tyrosine-protein kinase kit, also known as CD117) [START_REF] Korytnikov | Generation of polyhormonal and multipotent pancreatic progenitor lineages from human pluripotent stem cells[END_REF]. First, dead cells and debris were excluded. In a second step, the isotype controls were used to define the gates for each marker and channel (Figure 26). With the iPS DF19.9 we compared 2 protocols, either the Rezania 2014 protocol or the kit commercialized by StemCell Technologies (SD Kit). First CXCR4 expression reached in some experiments 79-91% of the cells however, only 21-25% of cells were double positive for CXCR4

and C-Kit with the Rezania protocol while with the SD Kit more than 34-58% of all differentiated cells were double positive (Figure 39C-D). It should be mentioned that for these experiments working banks were not yet made so the passage number between runs was not always constant. Immunofluorescent quantification of endoderm differentiation at stage 1 using CXCR4 and FOXA2 staining showing more than 90% endoderm formation (Expt 28). Scale bar = 30m. (C) Representative image of FACS plots illustrating protein expression of CXCR4 and C-Kit in populations of iPS DF19.9-derived stage 1 cells (Expt 49) (D) FACS quantification of endoderm formation -Table shows percentage of stage 1 cells expressing CXCR4 and C-Kit with Rezania protocol and SD Kit. SD Kit: STEMdiff Kit.

2) Evaluation of stage 4 pancreatic progenitors using the Rezania 2014 protocol and STEMDiff Pancreatic Progenitor Kit (stage 4 cells)

For gene expression comparison, a pool of 10 human islet donors was systematically used in order to reduce heterogeneity and ensure consistency. Also, contrary to our previous results, endocrine marker expression was reported as a fold change relative to human islets, since our goal was to differentiate towards pancreatic endocrine cells. Gene expression levels of stage 4 cells generated with the Rezania 2014 protocol or SD Kit were compared to human islets (red). We observed, for all genes, that cells produced with the SD Kit had a higher mRNA expression than those obtained with the Rezania protocol (Figure 40). After in vitro differentiation, 5 million cells were grafted in mice for in vivo maturation over 3 months. In 33% of grafted animals (3 out of 9) we were able to detect human fasting c-peptide (Figure 41). As for previous results, the c-peptide secretion was below 200 ng/mL while normal secretion range for 500 IEQ is between 500 and 1000 ng/L (Figure 41 red dot). Recently, several groups published -like cell differentiation protocols in which the generated cells resemble a rather mature phenotype and in vivo maturation time to acquire functional properties is significantly reduced [START_REF] Pagliuca | Generation of functional human pancreatic β cells in vitro[END_REF][START_REF] Rezania | Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells[END_REF][START_REF] Russ | Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro[END_REF].

For this thesis, either the Rezania protocol was used to generate stage 1 to stage 7 cells or the STEMDiff Pancreatic Progenitor Kit (SD Kit) for the production of stage 1 to stage 4 (S4) followed by the Rezania protocol to complete stages 5 to 7 (Figure 38).

Gene expression of the differentiation markers chromogranin A, CK19, PDX1 and NKX6.1 was increased between stage 4 and stage 7 in cells differentiated with the Rezania protocol but not in cells differentiated with the SD Kit + Rezania protocol (Figure 26). This increase allows the S7 cells of the Rezania protocol to achieve the gene expression of the S4 cells produced with the SD Kit. Despite this increase, insulin and glucagon expression remained 4 log apart from the expression levels in a pool of human islets from 10 donors (Figure 42). Gene expression of PDX1, NKX6.1, Chromogranin A, CK19, Insulin and Glucagon from iPS DF19.9derived cells at differentiation stage 4 and 7. RPL27 was used as house-keeping gene and results are expressed as a fold change relative to a pool of 10 human islet donors. Gene expression is shown on a logarithmic scale. SD Kit: STEMdiff Kit.

After more than 2 months of in vivo maturation, we observed a significant increase in fasting human c-peptide, almost reaching the reference level of c-peptide secreted by 500 human islets. This finding indicates that "second generation" protocol generated cells showed advanced -like cell maturation. In 3 mice out of 5 (1 mouse grafted with cells from the Rezania protocol and 2 mice

Glucagon

SD Kit S1-S4 Rezania S5-S7 Rezania S1-S7 Islets S4 S7 S4 S7 10 -2 10 -1 10 0 10 1 NKX6.1 SD Kit S1-S4 Rezania S5-S7 Rezania S1-S7
Relative to human islets 3. New authorization for H1 granted -December 2017.

Embryonic stem cells remain the gold standard for most published differentiation protocols. By the end of December 2017, we obtained a new authorization for embryonic stem cell research from the French ABM. Two months were required to re-establish the MTA and to import the cells again and finally in February 2018, we were able to start differentiation experiments using H1 cells. Just as for the iPS DF19.9 cell line, we first focused on endoderm differentiation optimization and then, on pancreatic progenitor differentiation. Visiting the laboratory of Dr. Nostro was a game changer in our goal of optimizing pancreatic differentiation as we learned to generate high rates of pancreatic progenitor cells for several cell lines. For the next differentiation experiments, we compared the newly learned protocol from Dr. Nostro laboratory to the STEMDiff Kit (Figure 44).

For definitive endoderm quantification, the flow cytometry-based method analysis used in Toronto was adopted, standardized and used as a new gold standard of evaluating differentiation efficiencies.

In vitro evaluation

In vivo evaluation

Fasting C-peptide Glycemia Immunofluorescence on explanted graft 118 1) Optimization of definitive endoderm differentiation after Toronto visit H1 cells were differentiated into stage 1 cells using the SD Kit. In experiment 53, the generation of stage 1 cells was optimized and lasted either 3 vs 4 days where 87,5% or 95,6% double positive cells for CXCR4 and C-Kit were obtained, respectively (Figure 45A). For additional experiments in Lille, the 4 days differentiation protocol was applied and resulted in 93,13% double positive cells on an average for CXCR4/C-Kit (Figure 45A). The evaluation of the 6 experiments included in this thesis revealed a similar dot plot pattern (as reported in Figure 45B), representing all achieved flow cytometry results for stage 4 determination.

2) Optimization of pancreatic progenitor differentiation

Using SD Kit:

Differentiation into pancreatic progenitors using the SD Kit was heterogeneous and not efficient enough. The best percentage of NKX6.1/PDX1 double positive cells was less than 25% (Figure 46A). Hence, as an attempt to improve differentiation efficiency before evaluation, we decided to 120 FACS sort the cells for GP2 (Glycoprotein 2), which is a specific cell surface maker for pancreatic progenitor cells [START_REF] Ameri | Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2+ Human Pancreatic Progenitors[END_REF][START_REF] Cogger | Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors[END_REF]. After FACS purification for GP2, we were able to obtain 48,30% of NKX6.1 positive cells. However, only 0,40 % of pre-sort cells were positive for GP2 (Figure 46B-C), indicating that the differentiation protocol was highly inefficient. Using the Nostro protocol:

After applying Dr Nostro's differentiation protocol to H1 cells, we obtained a reproducible number of more than 95% of double positive cells for NKX6.1 and PDX1 (Figure 47A). In addition, more than 88% of cells were positive for GP2. In figure 47B we reported the flow cytometry plots for the isotype controls and PDX1 / NKX6.1 for stage 4 cells. Gene expression analysis at stage 4 for the differentiation markers chromogranin A, CK19, PDX1, NKX6.1, insulin and glucagon compared to a pool of 10 human islets donors (red bar) revealed that cells produced with SD Kit + Nostro's protocol had higher mRNA expression of chromogranin A, CK19 and PDX1 than the pool of 10 human islet donors. By contrast, they had lower expression of insulin and glucagon (2,5 and 1,5 log difference respectively) and identical NKX6.1 expression (Figure 48). Finally, we considered that the differentiation of H1 cells into pancreatic progenitor cells was sufficiently optimized when using the Endoderm Kit for stage 1 followed by the Nostro protocol to obtain S4 pancreatic progenitors. Mean endoderm efficiency was 93%. Subsequently, at the end of the differentiation process, we obtain more than 95% double positive cells for NKX6.1 and PDX1.

To assess final maturation of pancreatic progenitor cells in vivo, 5 million double positive PDX+/NKX6.1 cells were grafted under the kidney capsule of immunodeficient mice. We obtained reproducibly, a fasting c-peptide secretion close to 100ng/L (the lower limit of the ultra-sensitive human c-peptide assay is 7,6ng/L). The red dot indicates human c-peptide secretion levels from 500-1000 IEQ (Figure 49). After 6 months in vivo, two kidneys transplanted with 5 million H1 derived S4 cells were harvested and the graft was submitted to perifusion, a dynamic GSIS assay (low glucose, high glucose, high glucose + arginine (10mM) and low glucose). The pluripotent stem cells derived grafted S4 cells were compared to a 2000 IEQ human islets graft (Figure 50). While the human islet transplant (Figure 50, red dot) depicted a 1 st phase response to both high glucose and high glucose + arginine stimulation, the H1 graft (figure 50, black square) only responded to high glucose + arginine albeit 4. iPS Progeria.

As an approach to accelerate the maturation and aging of differentiated cells, an iPS cell line from patients with Progeria was first tested for its capacity to differentiate into definitive endoderm and then pancreatic progenitors. Indeed, it has been shown that overexpression of progerin is sufficient to induce markers of aging in fetal-like neurons differentiated from pluripotent stem cells [START_REF] Miller | Human iPSC-based modeling of late-onset disease via progerin-induced aging[END_REF]. The optimization of iPS cell line differentiation into pancreatic progenitor cells was the prerequisite to study the maturation and aging of differentiated cells (Figure 51). To improve endoderm differentiation efficiency of the iPS HGPS, we sorted the cells by FACS for CXCR4 and C-Kit. The percentage of double positive cells after sorting was 94%, demonstrating the feasibility of the strategy (Figure 53). This strategy also allowed us to obtain comparable S1 efficiencies to subsequently compare the H1 cells directly to CXCR4/c-kit FAC purified iPS-HGPS cells. However, the cells were contaminated 1 day after culturing the sorted cell. Trouble shooting in Dr. Nostro's laboratory: importance of confluency.

For all cell lines and protocols the confluence status the day prior to differentiation is an important factor for differentiation efficiency [START_REF] Korytnikov | Generation of polyhormonal and multipotent pancreatic progenitor lineages from human pluripotent stem cells[END_REF]. In the laboratory of Dr. Nostro in Toronto, we optimized endoderm differentiation using the SD Kit for the H1 and iPS cell lines.

We tried differentiation with cells that were confluent at 50% or 70%. The lowest confluence gave the best results at day 4 (data not shown). Back in Lille we reproduced the same conditions for the stage 1 differentiation experiments with the SD kit, as well as trying again on D3 and D5 (Figure 129 54A). The mean of CXCR4/C-Kit double positive cells was 80,35% at D4. Representative FACS plots of experiment 71 are shown in Figure 55B.

2) Optimization of pancreatic progenitor differentiation Using the Nostro protocol, we obtained a mean of 95% of double positive cells for NKX6.1 and PDX1 (Figure 55A). Flow cytometry plots from the experiment performed in Toronto are shown in Figure 55B. chromogranin A, CK19 and PDX1 compared to human islets, a similar NKX6.1 expression and lower expression of insulin and glucagon (3 log difference) (Figure 56). Glucagon from iPS HGPS cells at differentiation stage 4. RPL27 is used as house-keeping gene and results are expressed as a fold change relative to a pool of 10 human islet donors (red bar). Gene expression is on logarithmic scale. SD Kit: STEMdiff Kit.

After trying several differentiation techniques, we finally succeeded with the help of Dr Nostro's group in obtaining more than 80% of definitive endoderm at stage 1 and more than 90% of PDX/NKX6.1 double positive cells at the end of differentiation with iPS-HGPS cell line.

Stage 4 pancreatic progenitor cells were then grafted under the kidney capsule of non-diabetic immunodeficient mice for in vivo maturation. Human fasting c-peptide was around 100ng/L for all mice (Figure 57). The objective here was to study the influence of the expression of progerin on pancreatic endocrine cell differentiation in vitro and in vivo using the previous optimized experiments (Table 11 and Figure 58). 59).

Role of Progerin on inducing maturation

To assess the influence of progerin on the endocrine maturation of the produced cells, we aimed to determine if there was a difference in the expression of the 4 maturation genes (MafA, MafB, NeuroD1 and Urocortin 3), in cells produced with H1 cells compared to cells produced with the iPS HGPS cells. MafA (Musculoaponeurotic fibrosarcoma oncogene family A) and MafB are required for pancreatic  cell development. It has been previously reported that they control  cell function in rodents [START_REF] Artner | MafB is required for islet beta cell maturation[END_REF][START_REF] Artner | MafA and MafB regulate genes critical to beta-cells in a unique temporal manner[END_REF][START_REF] Aguayo-Mazzucato | Mafa expression enhances glucose-responsive insulin secretion in neonatal rat beta cells[END_REF]) and more recently that MafA and Mafb have an age-dependent expression in human [START_REF] Arda | Age-Dependent Pancreatic Gene Regulation Reveals Mechanisms Governing Human β Cell Function[END_REF]). In addition, others have shown that NeuroD1 (Neuronal Differentiation 1) is necessary for the maturation of  cells as well as the acquisition of their glucose response [START_REF] Gu | Pancreatic beta cells require NeuroD to achieve and maintain functional maturity[END_REF]). Furthermore, it has been shown that cells differentiated from stem cells that exhibit glucose responsiveness after in vivo maturation also display an increase of Urocortin 3 gene expression [START_REF] Blum | Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3[END_REF].

We compared the expression of these 4 genes in human pancreatic islets and H1 or iPS-HGPS derived cells at stage 4. While for MafB and NeuroD1 the gene expression levels were lower than in human islets, for MafA and Urocortin 3 the expressions were comparable in all groups. For all analyzed genes, a trend for higher expression in the HGPS group was observed compared to H1 differentiated cells (Figure 60). As described above, 5 million double-positive PDX1/NKX6.1 cells generated from H1 or HGPS cells were grafted under the kidney capsule of immunodeficient mice. The functionality of the grafts was assessed by measuring glycemia and human fasting c-peptide. At 4 months, an intraperitoneal glucose tolerance test (ipGTT) was performed to evaluate cells responsiveness to glucose.

Mice grafted with cells differentiated from H1 cells (n=5) secrete the same levels of fasting human c-peptide as mice grafted with HGPS cells (n=8) except for the timepoint of 4 weeks (Figure 60A). Of note, these values are above the detection limit of the Mercodia ultra-sensitive kit. When these values are expressed as an average, the values of the HGPS group were slightly increased compared to H1 group (Figure 60B). When the value of fasting c-peptide was normalized to blood glucose levels, similar secretion profiles over time were observed in both groups (Figure 60C). The average of the normalized values were about 20 arbitrary units higher in the HGPS group but no significance was reached (Figure 60D). After 4 months of in vivo maturation, in both groups, we were unable to detect any glucose stimulated human c-peptide secretion in response to an ipGTT (Figure 60E), when expressed as c-peptide divided by glycemia ratio (Figure 60F). Out of the 7 grafts (Table 11) fixed for histology and immunohistochemistry, 2 showed few or no persistant grafted cells and a large infiltrate. Previous kinetic studies in the U1190 laboratory with human islets showed that ipGTT was superior to OGTT in our immunodeficient nondiabetic model where islets are grafted under the kidney capsule (see Material and Methods Figure 27).

Progerin inducing aging

Before analyzing the impact of progerin on differentiated cell transplants, it was necessary to verify if progerin was expressed in grafts and more importantly in pancreatic endocrine cells.

Interestingly, some patients with progeria may develop insulin resistance over time without the development of type 2 diabetes [START_REF] Coppedè | The epidemiology of premature aging and associated comorbidities[END_REF]). To our knowledge progerin has never been described in human pancreatic islets. In order to verify if progerin was expressed in islets, we performed costainings of progerin and chromogranin A in an old human pancreas (H1066) and on an insulinoma. An insulinoma is a benign tumor of the pancreas composed of immature cells.

Progerin was detected in the cytoplasm of a subpopulation of endocrine cells from the old donor but not in the insulinoma cells (Figure 65A). As progerin is a mutated form of lamin A, we also stained the same samples for lamin A/C. Lamin A/C expression was detected in both the old pancreas and the insulinoma, although the lamin A/C signal was weak in endocrine islets cells from the old donor but positive in the neighboring exocrine pancreas (Figure 65A). The figure 65B depicts progerin immunofluorescent staining and nuclear deformation in human HGPS fibroblasts (positive control). 148

During the early experiments with iPS HGPS cell line, both endoderm and pancreatic progenitor stages were not optimized and thus, few cells were positive for pancreatic progenitor markers after transplantation in mice and large teratomas were present. However, progerin was detected only in grafts of differentiated iPS HGPS cells and not in grafts from H1 cells (Figure 66). Endoderm and pancreatic progenitor stages have since been optimized. Similarly, progerin was detected in HGPS grafts but not in H1 grafts. All human nuclei were not progerin positive in iPS HGPS grafts (Figure 66). Endocrine positive cells were not progerin positive. However, progerin positive cells were detected in teratoma like structure in HGPS graft (Figure 67). To further confirm if the above-mentioned markers are also expressed on protein levels, immunofluorescent stainings were performed on paraffin sections of pancreas from a 70-year-old donor (H1066) and a human insulinoma (Figure 68). All markers were co-stained for chromogranin A to identify endocrine cells. Positive staining for 53BP1 was observed in the nuclei of positive chromogranin A cells of the elderly pancreas donor while no signal was detected in the chromogranin A positive cells of the insulinoma. For yH2AX a positive nuclear labelling in the pancreas H1066 was observed while the endocrine cells of the insulinoma were negative. Of note, the yH2AX staining in the pancreas of the old donor was stronger in non-endocrine cells than in chromogranin A positive endocrine cells. IGF1r labelling was detected in the exocrine pancreas of the elderly donors but was absent in pancreatic islets whereas in the insulinoma no staining was detected. Previous results in the laboratory showed p16 is negative in young islets (5 year old donor pancreas) but positive in islets of a 20 and 60 year old donor (not shown). Results on grafs are in progress. yH2AX appeared to be one of the most sensitive markers since the expression was positive in the 70 year old pancreas but not in the more immature insulinoma. Immunofluorescent labeling of yH2AX did not show foci in endocrine cells of human fetal pancreas or young donor (<10 year old pancreas) but showed visible foci in an adult pancreas 20 year old pancreas (not shown). Figure 69 shows yH2AX after 6 months transplantation of H1-grafts in endocrine and non-endocrine cells, while the expression in iPS HGPS-grafts appeared in the majority of cells, which had various shape of nuclei. Furthermore, 53BP1 or IGF1r positive cells were not identify in both type of grafts. To convincingly prove the role of progerin in the maturation of pluripotent pancreatic endocrine derived cells, we planned to over express progerin using PMO in pancreatic progenitors derived from the H1 cell line. In preparation for this study we optimized both the concentration, and the duration of the transfection of adult human fibroblasts derived from the human islet fraction. As others have previously shown [START_REF] Egesipe | Metformin decreases progerin expression and alleviates pathological defects of Hutchinson-Gilford progeria syndrome cells[END_REF] we could increase the expression of progerin determined by Taqman probes after 48h at 10 with PMOs to levels at 25% or 80% of expression levels detected in HGPS-patient derived fibroblasts respectively from HGPS AG1972 or HGPS Coriell. In our hands, western blots of patient derived fibroblasts required immunoprecipitation with a progerin specific antibody prior to western blotting (Annexe 3).

RS2278

Chromo A Chromo A / yH2AX In preparation for this clinical trial, we explored the hypothesis that the standardization of human islets into pseudo-islets of a controlled diameter may be superior both in vitro and in vivo to native human islets. This work is based on the well described capacity of dissociated pancreatic epithelial cells to self-assemble into 3D structures that conserve the architecture of native islets [START_REF] Halban | Spontaneous reassociation of dispersed adult rat pancreatic islet cells into aggregates with three-dimensional architecture typical of native islets[END_REF]. In preparation for clinical application of pseudo-islets, we purposely reaggregated pancreatic cells in culture media that was authorized in several past and ongoing clinical trials in Lille, even though most published experimental studies used media supplemented with fetal calf serum. Our clinical culture media consists of CMRL 1066 culture media containing human serum albumin (0.0626%), human recombinant insulin and antibiotics (penicillin / streptomycin).

In our experience, the formation of pseudo-islets was a reproducible technique in 80% of preparations (4/5) and the pseudo-islets were formed after 7 days. The failed islet preparation had a high percentage of dead cells after culture in both native islets and pseudo-islets groups potentially due to the long digestion of the pancreas during islet isolation (more than 20 minutes).

Aggregates of 80 m diameter were formed where the majority of cells were endocrine but exocrine cells were visible in the aggregates in contrast to reports by InSphero (https://insphero.com/products/islet/). InSphero reports that remaining exocrine cells rarely integrate the newly formed pseudoislet (InSphero whitepaper) and the nonincorporated exocrine cells eventually die. For their Insphero 3D InSight™ Islet Microtissues, they developed refined post isolation procedures to optimize the formation of uniform 3D pseudo-islets from "qualified donor islet preparations" received from different centers. They use a mild optimized dissociation and patented recovery and preconditioning technology that is able to eliminate the contaminating exocrine fraction and improve recovery followed by their patented GravityPlus plate technology (US2011306122) and media supplements that enhance reaggregation. However, no information was found on the latter technology. Evaluation by in vitro perifusion revealed that native islets at D1 were superior than both groups at D7 which can be attributed to the insulin content drop observed in both D7 groups since glucose stimulated insulin secretion is expressed as percent of content. This decrease has also been observed in 5-6 days pseudo-islets cultured in fetal calf serum supplemented media [START_REF] Halban | Spontaneous reassociation of dispersed adult rat pancreatic islet cells into aggregates with three-dimensional architecture typical of native islets[END_REF] Other improvements planned in Lille include testing new molds to reduce the time required to form pseudo-islets (collaboration with Dr. Aart van Apeldoorn, University of Maastricht) in particular as this may then prevent this cell therapy procedure from being regulated by the EMA as an ATMP (Advanced Therapy Medicinal Product -Regulation 1394), which requires GMP production of cells.

Although preliminary (4 preparations), the donor derived heterogeneity of islet preparations including the in vitro GSIS was not abolished by the pseudo-islet formation technique. This is in contrast to InSphero's claims albeit they do show that pseudo-islets show persistent "donor dependent GLP-1 responsiveness" (InSphero whitepaper). Lisa Stehno-Bittel's group showed that there was no statistically significant difference in the stimulation index in reaggregated donors.

Secondly, she consistently showed that re-aggregated human pseudo-islets respond better than native islets to drugs but that assay variability was decreased with re-aggregated pseudo-islets in 23 human donors [START_REF] Ramachandran | Assessment of re-aggregated human pancreatic islets for secondary drug screening[END_REF].

In Lille, we were unable to confirm if smaller diameter islets have improved function in vitro and in vivo as claimed by Lehmann and colleagues [START_REF] Lehmann | Superiority of small islets in human islet transplantation[END_REF]. Concerning Oxford's finding that islet number correlates with clinical function and not islet size [START_REF] Hughes | Does Islet Size Really Influence Graft Function After Clinical Islet Transplantation?[END_REF], we must first mention that in Lille, islet count before culture does not correlate with graft function in our series of islet transplant patients (Mikael Chetboun, U1190, personal communication). However, we will verify if post culture counts correlate with graft function. It should be pointed out that Lehmann's original observation was based on human islet preparations that were immediately transplanted without culture. Oxford could not confirm Lehmann's findings but they used cultured islets and they hypothesized that if the large islets survive culture, then they will function well in vivo post transplantation and this was responsible for their finding that islet number correlated with clinical islet function.

Morphological comparisons between native human islets and donor matched pseudo-islets allowed us to confirm an increase of the glucagon positive surface area in pseudo-islets in the graft compared to grafted native islets, which has been confirmed in humans by Zuellig et al. and in rat by Ichihara et al. (Ichihara et al. 2016;[START_REF] Zuellig | Improved physiological properties of gravity-enforced reassembled rat and human pancreatic pseudo-islets[END_REF] Several foreseeable limitations exist to current pseudo-islet technology. First pseudo-islet technology should allow formation and transplantation within 72 hours, in agreement with current islet transplantation phase 3 clinical trials authorized by national regulatory authorities. The technique used herein requires a 7 day culture with the Kugelmeier plates which may not be accepted by the French FDA. The Edmonton group is currently upscaling their 3 day centrifugalforced-aggregation pseudo-islet technology in bioreactors [START_REF] Yu | Bioengineered human pseudoislets form efficiently from donated tissue, compare favourably with native islets in vitro and restore normoglycaemia in mice[END_REF]) for clinical transplantation. In Europe the status and thus regulations of human pseudo-islet production for clinical allografts must be submitted to the CAT (Committee of Advanced Technologies) at the EMA. Extended culture time (more than 72h) and major modifications may result in an ATMP (and not a cell therapy product) status which will require that islet centers have GMP compliance.

Another foreseeable limitation of scaling up of pseudo-islet technology for the clinics is the inefficiency of current human islet isolation techniques. The multiple steps before islet transplantation result in a sequential loss of functional  cell mass relative to the number of cells initially present in the pancreas. On average, less than 85% of the pancreas is digested in our center, where 1080 human pancreases have been processed, and subsequent purification steps with density gradients recover only 50-60% of isolated human islets. The current technology to form pseudoislets involves two enzymatic digestions in two separate steps: enzymatic isolation of pancreatic islets and enzymatic dissociation of islets into isolated cells after several days of culture and before the production of pseudo-islets. We plan to substantially increase human endocrine cell yields for pseudo-islet technology with a single enzymatic digestion step and standardize procedures capable

Pluripotent Stem cells and the role of progerin in pancreatic cell differentiation and maturation:

The goal of this section was to investigate if the expression of a truncated form of lamin A progerin, associated with cellular aging and exploited to accelerate aging in neurons, could improve islet cell maturation. First of all, the differentiation of the H1 human embryonic stem cell line was compared to the differentiation both in vitro and in vivo of an iPS cell line derived from patients with progeria (cell line iPS HGPS 1972 obtained from iSTEM). Although our laboratory had some experience with the differentiation of H1 and the iPS DF19.9 cell line, the huge amount (20 %) of teratomas, the low human c-peptide level secretion post maturation in vivo and the low number of neuroendocrine and hormone positive cells post differentiation suggested that the efficiency of the definitive endoderm (Stage 1) and subsequent pancreatic progenitors (S4) was insufficient. By reading different publications we attempted to improve the differentiation of the cell lines. We tried several differentiation protocols (Odorico, Rezania, SD Kit, Nostro), conditions (2D and 3D) with two healthy (iPS DF19.9 and H1) and one HGPS (iPS HGPS AG1972) stem cell lines in order to obtain endocrine pancreatic cells. Our attempt with the iPS DF19.9 will not be further commented here as we discontinued use with it. Since our early differentiation efficiencies were variable between the 2 cell lines that we wanted to compare, we envisaged to standardize S1 cell purity by FAC purifying with CXCR4 and c-kit and then further enriching S4 pancreatic progenitors by FACs purifying for GP2 [START_REF] Cogger | Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors[END_REF]. After a PhD progress meeting with Dr Nostro in 2018 it appeared that this may be a useful approach but that the efficiencies of both S1 and S4 were too low and needed to be significantly improved. Indeed, each stem cell line has an intrinsic propensity for differentiation in certain lineages [START_REF] Osafune | Marked differences in differentiation propensity among human embryonic stem cell lines[END_REF][START_REF] Bock | Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines[END_REF][START_REF] Singec | Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling[END_REF][START_REF] Southard | Generation and selection of pluripotent stem cells for robust differentiation to insulin-secreting cells capable of reversing diabetes in rodents[END_REF]. Most of the protocols have been published with a limited number of cell lines. Furthermore, there has never been a direct comparison of the different protocols, using the same cell line under the same conditions (on feeder, feeder-free or 3D).

Optimization was done for each cell line during a visit to Dr Nostro's lab in Toronto. The Nostro protocol was the best to obtain sufficient levels of definitive endoderm (>90% CXCR4+/c-kit+) and pancreatic progenitor (>97% CXCR4+/c-kit+) cells for both H1 and iPS HGPS cells which was what was previously published [START_REF] Nostro | Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines[END_REF]). This appears to be the first report of in vitro differentiation in definitive endoderm from an iPS HGPS cell line, which is not the major afflicted lineage in the progeria disease. Pancreatic progenitor efficiency was high (>90% NKX6.1+/PDX-1+) for both cell lines. We learned during this PhD thesis that flow cytometry, which was used in the final years of the thesis is a more stringent and informative technique than following cell differentiation with Q-PCR, which was used in the first 2 years.

When comparing pancreatic progenitors generated from iPS HGPS AG1972 compared to H1 by Q-PCR, it did not allow us to document any differences in PDX1, NKX6.1, INS, GCG, ChromoA, CK10 mRNA expression. However, MAFA and UCN3 expression were slightly increased in iPS HGPS generated S4 cells compared to H1 generated S4 cells. Indeed, MAFA and UCN3 expression have been shown to be expressed in mature  and  cells (van der [START_REF] Van Der Meulen | Urocortin 3 marks mature human primary and embryonic stem cell-derived pancreatic alpha and beta cells[END_REF]) and linked to an improvement in static GSIS function [START_REF] Ghazizadeh | ROCKII inhibition promotes the maturation of human pancreatic beta-like cells[END_REF]). However, they appear nonessential for insulin glucose response as published recently [START_REF] Nair | Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived β cells[END_REF][START_REF] Velazco-Cruz | Acquisition of Dynamic Function in Human Stem Cell-Derived β Cells[END_REF]). We did not perform in vitro glucose stimulation before grafting since S4 cells have been notoriously shown to be insensitive to glucose and we were expecting increased in vivo function.

However, in the 6 runs performed here with optimized S1 and S4 efficiencies, we were not able to observe any significant difference in vivo in human c-peptide secretion between the two groups and mostly the grafts were poorly responsive to glucose stimulation. One explanation may be that grafts were rejected by the mice. Indeed, we were not able to find most of the graft after sacrifice.

For these studies, we used SCID beige mice as published (Viacyte) while other groups used NSG mice [START_REF] Pagliuca | Generation of functional human pancreatic β cells in vitro[END_REF][START_REF] Rezania | Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells[END_REF][START_REF] Nostro | Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines[END_REF][START_REF] Nair | Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived β cells[END_REF][START_REF] Velazco-Cruz | Acquisition of Dynamic Function in Human Stem Cell-Derived β Cells[END_REF]. SCID beige mice have dendritic, macrophage and NK activity which may have contributed to the graft loss. Infiltration of several grafts was observed suggesting rejection, or contamination of cells post transplantation. We did not formally determine the cell type at this time but in future studies, we will take a sample of culture supernatant for microbiological assessment, to validate the sterility of the preparation, just as it is routinely performed prior to clinical islet transplantation.

Hebrok's group showed with elegant imaging studies that more than 50% of differentiated human pluripotent stem cell derived insulin producing cells die shortly after transplantation in mice [START_REF] Faleo | Mitigating Ischemic Injury of Stem Cell-Derived Insulin-Producing Cells after Transplant[END_REF]. Furthermore, they verified that cell death was attributed to nutrient deprivation and hypoxia. Amino acid supplementation to prevent nutrient deprivation together with hypoxia preconditioning improved post transplantation survival. This was optimized to successfully culture and graft human islets under the kidney capsule and further supplementation was not done.

Identical to the problematic for pseudo-islets [START_REF] Furuyama | Diabetes relief in mice by glucose-sensing insulin-secreting human α-cells[END_REF], stem cell engraftment was improved by adding mesenchymal stem cells, endothelial cells [START_REF] Takahashi | Self-Condensation Culture Enables Vascularization of Tissue Fragments for Efficient Therapeutic Transplantation[END_REF], or amniotic epithelial cells [START_REF] Lebreton | Insulin-producing organoids engineered from islet and amniotic epithelial cells to treat diabetes[END_REF]) to the graft. This could be easy to test to improve the survival of the highly enriched pancreatic progenitors (>95%).

By using Taqman probes we were able to detect that progerin was expressed during maturation of iPS HGPS AG1972 cell line but not the H1 line, where we expected to see progerin positive nuclei.

After transplantation progerin positive nuclei were readily detectable in iPS grafts but again were not seen in the H1 derived grafts. It was unexpected was that not all human nuclei in the iPS grafts expressed progerin and notably the endocrine cells were indeed human in origin (positive for the human nuclear antigen antibody) but negative for progerin (further slides will be process to definitively confirm this finding). We could infer from this that perhaps since the major cell lineage afflicted in progeria patients is the mesoderm, that the endoderm lineage was not affected.

Identification of mesoderm teratoma tissue with progerin positive human nuclei could confirm this hypothesis. We did not explore the expression of progerin in the ectoderm lineage.

No major differences were observed in the function of pancreatic progenitors between the H1 and iPS HGPS AG1972 cell lines, both measured for fasting human c-peptide, a quantitative measure of human islet mass. Levels reached 200 ng/L (0.2ng/ml) of human c-peptide but with time the levels dropped down to about 100 ng/ml or below the level of detection. Furthermore, early studies from Novocell describe proinsulin producing pancreatic progenitors [START_REF] Kroon | Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo[END_REF]. Therefore,

for future experiments, we aim to dose human proinsulin in serum from the different grafts.

Perifusion of explanted grafts showed that H1-derived cells after 6 months in vivo were insensitive to glucose but displayed increased insulin secretion in response to high glucose and arginine. We hypothesize that arginine could elevate cAMP levels what is a known mechanism that increases the secretion capacity of both insulin and glucagon.

We then turned to quality differences between H1 grafts and iPS HGPS AG1972 grafts (3-6 months post transplantation) with aging markers previously published in the literature. First, we showed differential expression in insulinoma (immature cells) and human pancreas from an old donor. For example, for the yH2AX marker, we planned to enumerate the number of foci per nuclei was calculated to gauge cell aging, however although nuclei were positive for this marker no foci where visible. In our hands the autophagy marker ATG 12 did not appear to be informative as the differences in expression between different ages of islets was not discriminatory.

This work is still in progress and will be important in confirming the positive role of progerin in accelerating the maturation of pancreatic endocrine cells. Another elegant proof would have been to correct, using CRISPR-Cas9 technology, the mutation in the iPS HGPS AG1972 cell line and then to compare the mutated line and its corrected isogenic control as it is routinely done. This was not in the scope of this thesis but may be required for publication of our results.

In summary, this work allowed us to generate endocrine positive cells from H1 and iPS HGPS AG1972 cell lines that were detectable after in vivo transplantation (3-6 months) in immunodeficient mice. Prior to transplantation the iPS HGPS derived S4 pancreatic progenitors expressing higher levels of maturation markers MafA and urocortin 3 but this did not translate into high human c-peptide levels nor higher GSIS in vivo. In grafts 6-7 months post transplantation, the lack of progerin in endocrine cells derived from H1 was logical, but the lack in iPS HGPS AG1972derived endocrine cells in immunofluorescence in contrast to a strong expression in neighboring human cells in teratoma-like structures was a surprise in particular since a high percentage of pancreatic progenitors cells was observed by flowcytometry at S4. We can only infer from these results that the endoderm may not be sufficiently affected in the time frame explored in this study.

More fine aging markers like p16 may allow us to better characterize the cells to determine if there are any differences between the H1 and iPS HGPS derived grafts. We also must admit that these results are based on only one HGPS cell line derived from AG1972 fibroblasts and that multiple iPS cell lines are available.

Perspective studies include overexpression of progerin with PMO technology in S4 pancreatic progenitors, since the expression is irreversible or at later stages, and improving early graft outcome by using pre-vascularized sites or by adding feeder cells like human amniotic epithelial cells with regenerative and angiogenic properties to increase the mass of pancreatic progenitors that survive initial grafting.

We were intrigued by the finding [START_REF] Nissan | Unique preservation of neural cells in Hutchinson-Gilford progeria syndrome is due to the expression of the neural-specific miR-9 microRNA[END_REF]) that the unaltered cognitive capacity of patients with Progeria syndrome may be explained by the protection of neural cells in progeria syndrome patients from progerin-induced damage (premature senescence, loss of proliferation, blebbing) by Mir-9, which decreases lamin A and progerin expression in these cells to provide protection . Terminally differentiated neurons including motor neurons, and neural stem cells appear to be negative for lamin A/C but express high levels of lamin B1. Overexpression of Mir9 decreased progerin and lamin A expression and damage (nuclear blebbing).

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Figure 1 :

 1 Figures list

Figure 2 :

 2 Figure 2: Glycated hemoglobin (percent) between patients treated by conventional or intensive insulin therapy .............................................................................................................................................................

Figure 3 :

 3 Figure 3: Continuous glucose monitoring for 5 consecutive days of a child with T1D before and after treatment by 'artificial pancreas' .....................................................................................................................

Figure 4 :

 4 Figure 4: Graphical overview showing the all relevant steps from islet isolation from the donor until the islet transplantation into the recipient .....................................................................................................................

Figure 5 :

 5 Figure 5: Representative image of a human pancreatic islets from one donor stained with dithizone to assess the purity of the preparation ............................................................................................................................

Figure 6 :

 6 Figure 6: Continuous glucose monitoring of a T1D patient before and after islet transplantation .................

Figure 7 :

 7 Figure 7: Graph depicts the loss of  cell mass from donor islet isolation until the islets are grafted into the recipient ...........................................................................................................................................................

Figure 8 :

 8 Figure 8: A dynamic perifusion system was used to determine insulin secretion of human islet preparations in response to changing glucose concentrations (3mM, 15mM, 3mM) ..........................................................

Figure 9 :

 9 Figure 9: Schematic representation of the 3D Select ™ process to generate standardized islet microtissues Figure 10: Glucose-stimulated insulin secretion (GSIS) profile and insulin content of intact native islets compared to pseudo-islets. ..............................................................................................................................

Figure 11 :

 11 Figure 11: Islets and pseudo-islets architecture and cellular composition. .....................................................

Figure 12 :

 12 Figure 12: Pancreatic cell lineage determination during pancreas organogenesis ..........................................

Figure 13 :

 13 Figure 13: Immunocytochemical staining of the endocrine marker synaptophysin (in black) on a fetal pancreas at 16 weeks of gestation shows single cell or small endocrine cell aggregates in direct contact with pancreatic duct (D) ..........................................................................................................................................

Figure 14 :

 14 Figure 14: Similarities and differences during pancreas morphogenesis in mouse (top) and Human (bottom).

Figure 15 :Figure 16 :

 1516 Figure 15: Sources of pluripotent stem cells: pluripotent stem cells are hallmarked by self-renewal capacity and differentiation in cells of all the three germ layers (ectoderm, mesoderm, endoderm) Figure 16: Representative image of an embryonic stem cell (H1) cultured in m-TeSR. ................................

Figure 17 :

 17 Figure 17: Possible application for pluripotent stem cells in research and for clinical treatment strategy .....

Figure 18 :

 18 Figure 18: (A) Insulin and glucagon polyhormonal cells, pluripotent stem cell derived insulin and glucagon positive cells are ARX positive (B) and NKX6.1 negative (C) .......................................................................

Figure 19 :

 19 Figure 19: Overview of lineage segregation in human pancreatic development .............................................

Figure 20 :

 20 Figure 20: Overview of human pluripotent stem cells (hPSC) step-wise differentiation towards  cells. Different differentiation stages from pluripotent stem cell towards stage 7. ..................................................

Figure 21 :

 21 Figure 21: Inducing cellular aging in hPSC-derived lineages .........................................................................

Figure 22 :

 22 Figure 22: Age-associated changes induced by progerin overexpression in iPSC-mDA Neurons derived from young and old donors .............................................................................................................................

Figure 23 :

 23 Figure 23: Schematic overview of the biogenesis of lamin A and progerin ...................................................

Figure 24 :

 24 Figure 24: Representative immunofluorescent image of HGPS cells: progerin (red) and human DNA (blue)

Figure 25 :

 25 Figure 25: Similarities and differences between HGPS (left) and physiological aging ..................................

Figure 26 :

 26 Figure 26: Strategy gating flowchart. ..............................................................................................................

Figure 27 :

 27 Figure 27: Glycemia, human c-peptide and human c-peptide divided by glycemia during glucose challenge (3g/kg) in normoglycemic nude mice with a human islet transplant under the kidney capsule. .....................

Figure 28 :

 28 Figure 28: Overview of pseudo-islets experiments. ........................................................................................

Figure 29 :

 29 Figure 29: Dynamic insulin secretion by perifusion in response to glucose variations. .................................

Figure 30 :

 30 Figure 30: Insulin content of native human islets at D1 and D7 and pseudo-islets at D7 ...............................

Figure 31 :

 31 Figure 31: In vivo function of D1 native human islets, D7 native islets and pseudo-islets.

Figure 32 :

 32 Figure 32: Morphometric analysis of grafted native human islets D1, D7 and pseudo-islets .........................

Figure 33 :

 33 Figure 33: Correlation of glucose-stimulated insulin secretion between native human islets and pseudo-islets Figure 34: Timeline of stem cell differentiation at U1190, Lille………………………………………….

Figure 35 :

 35 Figure 35: Overview of applied differentiation protocols from 2012 to 2015. ...............................................

Figure 36 :

 36 Figure 36: Phenotype of H1 cells stage 1-4 during differentiation with the Odorico protocol…………..

Figure 37 :

 37 Figure 37: In vivo evaluation of engrafted stage 4-H1 cells differentiated according to the Odorico protocol………………………………………………………………………………………………….

Figure 38 :

 38 Figure 38: Overview of differentiation protocol applied with iPS DF19.9 cells. ............................................

Figure 39 :

 39 Figure 39: CXCR4 and FOXA2 gene and protein expression from iPS DF19.9-derived cells at differentiation stage 1 with SD Kit compared to the Rezania protocol. ..........................................................

Figure 40 :

 40 Figure 40: Stage 4 cells generated using the Rezania protocol or SD Kit with iPS DF19.9 cellsin vitro evaluation. .......................................................................................................................................................

Figure 41 :

 41 Figure 41: Stage 4 cells generated with iPS DF19.9 cells using SD Kit -In vivo evaluation .........................

Figure 42 :

 42 Figure 42: iPS DF19.9 cells at stage 7 using SD Kit + Rezania or Rezania alone -in vitro evaluation……………………………………………………………………………………….

Figure 43 :

 43 Figure 43: Stage 7 cells generated using the Rezania protocol or SD Kit with iPS DF19.9 cells engrafted under the kidney capsulein vivo evaluation.. ..............................................................................................

Figure 44 :

 44 Figure 44: Overview of applied differentiation protocols with H1 cells.. .......................................................

Figure 45 :

 45 Figure 45: Stage 1 cells generated using SD Kit with H1 cells optimized in Toronto………… ………..

Figure 46 :

 46 Figure 46: Stage 4 cells generated using SD Kit with H1 cells -FACS quantification of pancreatic progenitors.. .....................................................................................................................................................

Figure 47 :

 47 Figure 47: Stage 4 cells generated using the Nostro protocol with H1 cells -FACS quantification of endoderm formation.. ......................................................................................................................................

Figure 48 :

 48 Figure 48: Stage 4 cells generated by using the Nostro protocol with H1 cellsin vitro evaluation………………………………………………………………………………………………....

Figure 49 :

 49 Figure 49: In vivo evaluation of stage 4 cells generated with H1 cells using SD Kit and the Nostro protocol over 4 months (n=8 mice). ..............................................................................................................................

Figure 50 :

 50 Figure 50: Dynamic GSIS by perifusion of explanted H1-derived S4 cells grafts, 6 months after transplant (black) and an explant human islet graft (control, red)…………………………………………………..

Figure 52 :

 52 Figure 52: Stage 1 cells generated using SD Kit or the Rezania protocol with patient specific iPS HGPS cells -Definitive endoderm quantification. ....................................................................................................

Figure 53 :

 53 Figure 53: Stage 1 cells generated using the Rezania protocol with patient specific iPS HGPS cells -FACS sorting on CXCR4/C-Kit………………………………………………………………………………….

Figure 54 :

 54 Figure 54: Stage 1 cells generated using a SD Kit (Nostro adaptation) with patient specific iPS HGPS AG1972-Definitive endoderm quantification……………………………………..…………………….

Figure 55 :

 55 Figure 55: Stage 4 cells generated using the Nostro protocol with patient specific iPS HGPS AG1972 cells -FACS quantification of pancreatic progenitor………………………………………………………….

Figure 56 :

 56 Figure 56: Stage 4 cells generated using the Nostro protocol with patient specific iPS HGPS AG1972-in vitro evaluation…………………………………………………………………………………………….

Figure 57 :

 57 Figure 57: Fasting human c-peptide levels (ng/L) measured over 4 months (n=8 mice). …………..

Figure 58 :

 58 Figure 58: Overview of the objectives of using progeria to induce aging in cells derived from pluripotent cells. .................................................................................................................................................................

Figure 59 :

 59 Figure 59: Gene expression profile of Progerin, Lamin A and Lamin C was determined in fibroblasts from patients with HGPS-Progeria (Fibro HGPS -AG1972), undifferentiated iPS HGPS cells (dedifferentiated from Fibro HGPS -AG1972), S4 cells generated from iPS HGPS or H1 cells and human islets by Taqman qPCR. ..............................................................................................................................................................

Figure 60 :

 60 Figure 60: MAFa, MAFb, NeuroD1 and Urocortin 3 expression in iPS HGPS (AG1972) and H1-derived cells at stage 4 and human islets………………………………………………………………………….

Figure 61 :

 61 Figure 61: Human c-peptide secretion of differentiated H1 or iPS HGPS cells 4 months after transplantation……………………………………………..……………………………………………..

Figure 62 :

 62 Figure 62: Representative image of a trichrome staining showing duct (top) and endocrine like structure (bottom) in human pancreas (left panel) and iPS HGPS graft 5 months post-transplantation (right panel). ..

Figure 63 :Figure 65 :

 6365 Figure 63: Immunofluorescent staining of graft of iPS HGPS-derived stage 4 cells, harvested 6 months after transplantation (Expt 68) for chromogranin A endocrine (Chromo A, red) and CK19 epithelium (green) ....Figure 64: Immunofluorescent staining of graft of H1-derived stage 4 cells, harvested 6 months after transplantation (Expt 73) for chromogranin A (Chromo A, red) and DAPI (blue) .........................................

Figure 66 :

 66 Figure 66: Progerin (red), human nuclei (green) and DAPI (blue) staining in iPS HGPS graft versus H1 graft. ................................................................................................................................................................

Figure 67 :

 67 Figure 67: Representative immunofluorescent images for progerin in iPS HGPS-derived stage 4 cells graft, in a teratoma like structure harvested 6 months after transplantation (Expt 68) .............................................

Figure 68 :

 68 Figure 68: Validation of aging markers on 70-year-old pancreas (H1066) and an insulinoma. 53BP1, yH2AX, IGF1r (green) were co-stained with pan-endocrine marker chromogranin A (red) in the pancreas of a 70-year-old donor (H1066) and in an insulinoma (benign tumor composed of immature cells) .............

Figure 69 :

 69 Figure 69: Immunofluorescent staining of graft of H1-derived stage 4 cells, harvested 6 months after transplantation (Expt 73) for chromogranin A (Chromo A, red) and yH2AX (green). ...................................

Figure 70 :

 70 Figure 70: Immunofluorescent staining of graft of iPS HGPS-derived stage 4 cells, harvested 6 months after transplantation (Expt 68) for chromogranin A (Chromo A, red) and yH2AX (green) ....................................

Figure 1 :

 1 Figure 1: Blood glucose (mg/dL and mM) and insulin levels (UI/mL and pM) during 24 hours from 14 healthy adult individuals. Lines represent the means and shading the SEM (Latres et al. 2019).

  study the Epidemiology and Diabetes Interventions and Complications (EDIC) study 93% of patients in the DCCT study were followed over 30 years after training the patients in the conventional group how to better control their glycemia. Although both groups subsequently had similar (Figure 2) HbA1c levels of 8%, patients who originally had 'intensive' insulin therapy had a >30% reduced risk of complications(Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) Study Research Group 2016).

Figure 2 :

 2 Figure 2: Glycated hemoglobin (percent) between patients treated by conventional or intensive insulin therapy (Nathan 2014).

Figure 3 :

 3 Figure 3: Continuous glucose monitoring for 5 consecutive days of a child with T1D before and after treatment by 'artificial pancreas' (Latres et al. 2019).

Figure 4 :

 4 Figure 4: Graphical overview showing the all relevant steps from islet isolation from the donor until the islet transplantation into the recipient (Merani and Shapiro 2006).

Figure 5 :

 5 Figure 5: Representative image of a human pancreatic islets from one donor stained with dithizone to assess the purity of the preparation. Scale bar =100m

Figure 6 :

 6 Figure 6: Continuous glucose monitoring of a T1D patient before and after islet transplantation (Latres et al. 2019).

Figure 7 :

 7 Figure 7: Graph depicts the loss of  cell mass from donor islet isolation until the islets are grafted into the recipient. Tx: Transplantation (F. Pattou unpublished).

Figure 8 :

 8 Figure8: A dynamic perifusion system was used to determine insulin secretion of human islet preparations in response to changing glucose concentrations (3mM, 15mM, 3mM). Islets were incubated in 3mM glucose for 1 hour prior to experimentation to equilibrate basal secretion. The secretion pattern of each donor is represented with a different color. Insulin secretion was normalized to the percentage of insulin content (U1190 unpublished data).

Figure 9 :

 9 Figure 9: Schematic representation of the 3D Select ™ process to generate standardized islet microtissues. A heterogeneous fraction of isolated human islets is dissociated to obtain a single cell suspension. Pancreatic cells are reaggregate to achieve pseudo-islets with uniform size and cellular composition. Each preparation is assessed for viability and glucose-stimulated insulin secretion. Exocrine contaminant cells are not incorporated into nascent islet microtissues and decay (from http://www.insphero.com).

Figure 10 :

 10 Figure 10: Glucose-stimulated insulin secretion (GSIS) profile and insulin content of intact native islets compared to pseudo-islets. Insulin secretion profiles from perifusion assays of intact native human islets (A), pseudo-islets after 7 and 14 days in culture composed of 250 cells (B), 750 cells (C) and 1500 cells (D). Data were normalized to total islet volume and per condition n=100 islets were used. (E) The total insulin content was expressed per cell from intact native human islets of different sizes or pseudo-islets (250, 750 and 1500 cells). Data are expressed as mean  SEM (Zuellig et al. 2017).

Figure 11 :

 11 Figure 11: Islets and pseudo-islets architecture and cellular composition. (A) Immunofluorescent staining of insulin (green), glucagon (red) and somatostatin (blue), scale bar = 50m. (B) Relative abundance of hormone-expressing cells. Data are expressed as mean  SD (Zuellig et al. 2017).

Figure 12 :

 12 Figure 12: Pancreatic cell lineage determination during pancreas organogenesis (Larsen and Grapin-Botton 2017).

Figure 13 :

 13 Figure 13: Immunocytochemical staining of the endocrine marker synaptophysin (in black) on a fetal pancreas at 16 weeks of gestation shows single cell or small endocrine cell aggregates in direct contact with pancreatic duct (D) (Rooman et al. 1997).

Figure 14 :

 14 Figure 14: Similarities and differences during pancreas morphogenesis in mouse (top) and Human (bottom). e: embryonic day, dpc: day post conception, wpc: week post conception (Nair and Hebrok 2015).

Figure 15 :

 15 Figure 15: Sources of pluripotent stem cells: pluripotent stem cells are hallmarked by self-renewal capacity and differentiation in cells of all the three germ layers (ectoderm, mesoderm, endoderm) (Grandy et al. 2019).

Figure 16 :

 16 Figure 16: Representative image of an embryonic stem cell (H1) cultured in m-TeSR.

  by Prof. Yamanaka and awarded the Nobel Prize for Medicine and Physiology in 2012, induced Puripotent Stem cells (iPSC) are reprogrammed mature adult cells back to an pluripotent state using reprogramming factors. The generated cells are genetically identical to the donor's cells, and highly similar to embryonic stem cells in terms of morphology, proliferation and self-renewal capacity and cell differentiation capacity in cells from the three embryonic layers. Yamanaka et al. identify 4 reprogramming factors by screening 24 factors, previously observed in mouse embryonic fibroblast, enabling the reprogramming of mouse fibroblasts[START_REF] Takahashi | Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[END_REF]) and human fibroblasts[START_REF] Takahashi | Induction of pluripotent stem cells from fibroblast cultures[END_REF]) using a retroviral transduction.

Figure 17 :

 17 Figure 17: Possible application for pluripotent stem cells in research and for clinical treatment strategy (Kiskinis and Eggan 2010). In vitro model of development Most embryonic development studies are based on work done in rodents hence the in vitro differentiation of human pluripotent stem cells allows the study of the molecular mechanisms involved during human embryonic development. Although most molecular mechanisms are well

  (stage 1), a prerequisite for subsequent differentiation to pancreatic progenitors. From 2006-2009 protocols were published to guide definitive endoderm cells to primitive gut tube (stage 2) by vitamin C and FGF7 activation and further to posterior foregut (stage 3) by inhibiting BMP and SHH while maintaining FGF7 signaling pathway. The next stage (stage 4) was a breakthrough in mimicking pancreas organogenesis, which leads to the generation of multipotent pancreatic progenitors (PP). The cells can be generated with high efficiency by inhibition of BMP signaling pathway and tankyrases and the activation of EGF. Once transplanted in vivo, the multipotent PPs differentiate into all pancreatic cell types including the insulin producing  cells (D'Amour

  28.79%+5.6%) (Riedel Kieffer Diabetologia 2012). It has been previously shown that polyhormonal insulin/glucagon positive cells have been shown to be NKX6.1 negative/ PDX-1 negative and ARX positive (Riedel Kieffer Diabetologia 2012). With time these cells become glucagon positive  cells (Figure18).

Figure 18 :Figure 19 :

 1819 Figure 18: (A) Insulin and glucagon polyhormonal cells, pluripotent stem cell derived insulin and glucagon positive cells are ARX positive (B) and NKX6.1 negative (C). Proportion of insulin positive, glucagon positive and double positive cells in pre-transplanted clusters and in grafts at 1, 3 and 8 months post-transplantation showing that transplanted cells mainly convert into  cells. (Rezania et al. 2012).Strategies to obtain monohormonal insulin positive cells became a priority. Cell progenitor cells positive for NKX6.1 have been shown to rather develop into insulin positive cells associated with improved in vivo function[START_REF] Rezania | Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo[END_REF]. Another study identified key signaling pathways to generate NKX6.1 positive cells from various human pluripotent stem cell (hPSC) lines and demonstrated that NKX6.1 positive progenitors lead to mono-hormonal positive cells[START_REF] Nostro | Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines[END_REF]. The NKX6.1 positive pancreatic progenitor cell should be c-peptide negative at S4. Russ et al. also demonstrated that NKX6.1+/PDX1+ progenitor cells generate mono-hormonal insulin positive cells[START_REF] Russ | Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro[END_REF].

Figure 20 :

 20 Figure 20: Overview of human pluripotent stem cells (hPSC) step-wise differentiation towards  cells. Different differentiation stages from pluripotent stem cell towards stage 7 (maturing  cell). Characteristic stage specific markers are shown below the corresponding stages (Petersen et al. 2018).

  inconvenience for clinical applications, basic research, and drug screening. The production of fully functional cells with characteristics of mature cells is an essential goal in stem cell biology. Most of the in vitro differentiated cell types reach maturation only after prolonged culture or in vivo transplantation for long periods of time. Many research groups that use pluripotent stem cells and patient derived stem cells to model late onset neurodegenerative diseases such as Parkinson's disease and Alzheimer disease, or metabolic diseases associated with aging and obesity such as T2D, face with a particular challenge since pathological features may only arise upon aging.The prolonged gestational period in humans compared to rodents may be in part responsible for the "fetal state" of differentiated hPSC. Indeed, Scharfmann et al. compared the endocrine development of early murine and human embryonic pancreas in the muscle of immunodeficient mice that[START_REF] Castaing | Blood glucose normalization upon transplantation of human embryonic pancreas into beta-cell-deficient SCID mice[END_REF]) and showed that the endocrine differentiation of early human embryonic pancreas occurs over 60 weeks (> 1 year) compared to only 2 weeks with murine embryonic E12.5 pancreas.7. Factors associated with the maturation of pancreatic  cellsAdult endocrine cells are long-lasting cells like post-mitotic cortical neurons[START_REF] Cnop | The long lifespan and low turnover of human islet beta cells estimated by mathematical modelling of lipofuscin accumulation[END_REF][START_REF] Arrojo E Drigo | Age Mosaicism across Multiple Scales in Adult Tissues[END_REF]. Pancreatic islet cells undergo dramatic changes during life in terms of functionality and in terms of their proliferation capacity. Neonatal islet cells are highly proliferative and insulin secretion is at low levels and normally increases during development and with time. In contrast, islets from elderly people display low cell replication capacities[START_REF] Hellerström | Functional maturation and proliferation of fetal pancreatic beta-cells[END_REF][START_REF] Bonner-Weir | Dynamic development of the pancreas from birth to adulthood[END_REF]. Furthermore, few proteins have been linked to  cell maturation, among which MAFA, ERRy (Estrogen-related receptor y), NeuroD and UCN3 (Urocortin 3).

Figure 21 :

 21 Figure 21: Inducing cellular aging in hPSC-derived lineages (Cornacchia and Studer 2017).

Figure 22 :

 22 Figure 22: Age-associated changes induced by progerin overexpression in iPSC-mDA Neurons derived from young and old donors. yH2AX (DNA-damage, pink) and MAP2 (dendritic marker, red) (Miller et al. 2013).

Figure 23 :

 23 Figure 23: Schematic overview of the biogenesis of lamin A and progerin. Under physiological conditions, prelamin A is synthesized as precursor, and after 4 post-translational maturation steps, it becomes lamina A (left panel). In Progeria patients, prelamin A is deficient of 50 amino acids, which hinders the last stage of maturation. Progerin remains farnesylated and carboxymethylated in the C-terminal position (right panel) (Coutinho et al. 2009).

Figure 24 :

 24 Figure 24: Representative immunofluorescent image of HGPS cells: progerin (red) and human DNA (blue) showing nuclei deformation with high level of progerin (left) compared to low level of progerin (right). From K Djabali / TUM

Figure 25 :

 25 Figure 25: Similarities and differences between HGPS (left) and physiological aging (Strandgren et al. 2017).

Figure 26 :

 26 Figure 26: Strategy gating flowchart. FSC: Forward Scatter. SSC: Side Scatter. A: Area. W: Width. H: Height.

Figure 27 :

 27 Figure 27: Glycemia, human c-peptide and human c-peptide divided by glycemia during glucose challenge (3g/kg) in normoglycemic nude mice with a human islet transplant under the kidney capsule. OGTT: Oral Glucose Tolerance Test; IPGTT: Intra-Peritoneal Glucose Tolerance Test.

Figure 29 :

 29 Figure 29: Dynamic insulin secretion by perifusion in response to glucose variations. (A) native islets one day after isolation (n=4) (B) native islets one week after isolation (n=3) (C) pseudo-islets one week after isolation (n=4) (D) avarage of donors from each group. Data are expressed as uUI/mL. One-way Anova and Kruskal-Wallis multiple comparison test were used. G3: glucose 3mM. G15: glucose 15mM.

Figure 30 :

 30 Figure 30: Insulin content of native human islets at D1 and D7 and pseudo-islets at D7. Native islets and pseudo-islets were cultured in clinical grade islet medium (CMRL 1066 with 0,0625% human serum albumin).

Figure 31 :

 31 Figure 31: In vivo function of D1 native human islets, D7 native islets and pseudo-islets. Fasting human c-peptide / glycemia ratio measured from immunodeficient mice transplanted with D1 native human islets (red), D7 native islets (grey) and pseudo-islets (blue). (A) secretion profiles each 7 days and (B) mean of secretion over one month. AU: Arbitrary units. Data are expressed as mean  SEM.

Figure 32 :

 32 Figure 32: Morphometric analysis of grafted native human islets D1, D7 and pseudo-islets. (A) Representative images of the distribution of insulin (red) and glucagon (green) positive cells in grafts of native islets D1, D7 and pseudo-islets one month after transplantation. (B) Percentage of insulin and glucagon positive cells from graft of native islets D1, D7 and pseudo-islets in n=3 mice per group.

Figure 33 :

 33 Figure 33: Correlation of glucose-stimulated insulin secretion between native human islets and pseudo-islets. Insulin secretion in response to high glucose was assessed by perifusion and the correlation between the native human islets and pseudo-islets insulin secretory response during first (left panel) and second phase (right panel) of stimulation was calculated.

Figure 34 :

 34 Figure 34: Timeline of stem cell differentiation at U1190, Lille. Stars indicate the timeframe of approval for the use of embryonic stem cell research in our laboratory by the French Biomedical Agency (ABM). The blue diamonds indicate the time spent as visiting scientist/professor at both the University of Wisconsin (UW) and University of Toronto (U of T). The blue dotted line indicates in which chapter the corresponding results are shown.

Figure 35 :

 35 Figure35: Overview of applied differentiation protocols from 2012 to 2015. H1 or iPS DF19.9 cell lines were differentiated towards stage 4 pancreatic progenitor cells according to Rezania 2012 protocol[START_REF] Rezania | Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice[END_REF] or an adapted protocol from Prof. Odorico's laboratory[START_REF] Xu | Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells[END_REF]. Stage 4 cells were analyzed in vitro by qPCR and immunofluorescent staining prior to transplantation under the kidney capsule of immunodeficient mice. Mice were followed for 3 months to evaluate human fasting c-peptide and glycaemia in vivo. Grafts were analyzed by various immunofluorescent stainings. Hereafter, only the experiments with H1 cells and J. Odorico's protocol will be mentioned. Arrows represent timepoint of cell engraftment.

Figure 36 :

 36 Figure 36: Phenotype of H1 cells stage 1-4 during differentiation with the Odorico protocol. (A) Gene expression of differentiated H1 cells at stage 4 compared to undifferentiated H1 cells (n=2).

Figure 37 :

 37 Figure 37: In vivo evaluation of engrafted stage 4-H1 cells differentiated according to the Odorico protocol. (A) Human c-peptide levels (ng/L) were measured under random fed conditions over 3 months.

  Human c-peptide was detected in 9 mice out of a total of 29. Normal range of c-peptide secretion from 500 human islets is in red (500-100ng/L). (B) Immunofluorescent staining of graft of stage 4 cells, harvested 12 weeks after transplantation (Expt 9obtained with H1 cells) for chromogranin A (CHGA, panendocrine marker) in green, cytokeratin 19 (CK19, ductal marker) in red in the upper panels, and glucagon (GCG) in red and insulin (INS) in green in the lower panels. Scale bar = 100 m. (C) Human islet neogenesis from ducts in a human pancreatic section. (D) Hematoxylin and eosin staining of a teratoma (left image), mouse developing a teratoma at 4 weeks after transplantation (right image).

Figure 38 :

 38 Figure 38: Overview of differentiation protocol applied with iPS DF19.9 cells. iPS DF19.9 cell line was differentiated towards stage 4 pancreatic progenitor cells or stage 7 maturing  cell according to Rezania 2012 or STEMDiff Pancreatic Progenitor Kit (SD KIT). Stage 1 cells were analyzed in vitro by qPCR, immunofluorescent staining and flow cytometry. Stage 4 and 7 cells were analyzed by qPCR and immunofluorescent staining prior to transplantation under the kidney capsule of immunodeficient mice. Mice were followed up to 3 months to assess human fasting c-peptide and glycemia in vivo. Grafts were analyzed by immunofluorescent staining. Arrows represent timepoint of cell engraftment. SD Kit: STEMdiff Kit.

Figure 39 :

 39 Figure39: CXCR4 and FOXA2 gene and protein expression from iPS DF19.9-derived cells at differentiation stage 1 with SD Kit compared to the Rezania protocol. (A) Gene expression of CXCR4 and FOXA2 from iPS DF19.9-derived cells at differentiation stage 1 normalized to undifferentiated iPS DF19.9. RPL27 was used as house-keeping gene. Gene expression is shown on a logarithmic scale. (B)

Figure 40 :

 40 Figure40: Stage 4 cells generated using the Rezania protocol or SD Kit with iPS DF19.9 cellsin vitro evaluation. Gene expression of PDX1, NKX6.1, Chromogranin A, CK19, Insulin and Glucagon from iPS DF19.9-derived cells at differentiation stage 4. RPL27 was used as house-keeping gene; results are expressed as a fold change relative to a pool of 10 human islet donors (red). Gene expression is shown on a logarithmic scale. SD Kit: STEMdiff Kit.

Figure 41 :

 41 Figure41: Stage 4 cells generated with iPS DF19.9 cells using SD Kit -In vivo evaluation. Fasting human c-peptide (ng/L) levels were measured over 3 months. Normal range of c-peptide secretion from 500 human islets is 500-100ng/L (red dot). SD Kit: STEMdiff Kit.

Fig

  Fig X: iPS DF19.9 in vivo evaluation A. Immuno B. Human fasting c-peptide (ng/L) kinetic over 3 months. Best previous results (9 mice on a total of 29). Normal range of c-peptide secretion from 500 human islets is in red (500-1000 ng/L).

Figure 42 :

 42 Figure42: iPS DF19.9 cells at stage 7 using SD Kit + Rezania or Rezania alone -in vitro evaluation. Gene expression of PDX1, NKX6.1, Chromogranin A, CK19, Insulin and Glucagon from iPS DF19.9derived cells at differentiation stage 4 and 7. RPL27 was used as house-keeping gene and results are expressed as a fold change relative to a pool of 10 human islet donors. Gene expression is shown on a logarithmic scale. SD Kit: STEMdiff Kit.

Figure 44 :

 44 Figure 44: Overview of applied differentiation protocols with H1 cells. H1 cell line was differentiated towards stage 4 pancreatic progenitor cells according to the SD Kit from stage 1 to stage 4 or the SD Kit for stage 1 and the Nostro protocol for stages 2 to 4. Stage 1 cells were analyzed in vitro by flow cytometry. Stage 4 cells were analyzed in vitro by qPCR and flow cytometry prior to transplantation under the kidney capsule of immunodeficient mice. Mice were followed for 3 months to assess human fasting c-peptide and glycemia in vivo. Grafts were analyzed by immunofluorescent staining. Arrows represent timepoint of cell engraftment. SD Kit: STEMdiff Kit.

Figure 45 :

 45 Figure 45: Stage 1 cells generated using SD Kit with H1 cells optimized in Toronto. FACS quantification of endoderm formation. (A) Table of percentage of stage 1 cells expressing CXCR4 and C-Kit that were differentiated with SD Kit. (B) Representative FACS dot plots of experiment 70 illustrating protein expression of CXCR4 and C-Kit in populations of H1-derived stage 1 cells. SD Kit: STEMdiff Kit.

Figure 46 :

 46 Figure 46: Stage 4 cells generated using SD Kit with H1 cells -FACS quantification of pancreatic progenitors. (A) Table of percentage of stage 4 cells expressing PDX1 and NKX6.1 with SD Kit and GP2

Figure 47 :

 47 Figure 47: Stage 4 cells generated using the Nostro protocol with H1 cells -FACS quantification of endoderm formation. (A) Table of percentage of stage 4 cells expressing NKX6.1, PDX1 and GP2 with the Nostro protocol. (B) Representative FACS plots illustrating protein expression of NKX6.1 and PDX1 in populations of H1-derived stage 4 cells (Expt 73: 96,9% co-expression of NKX6.1 and PDX1). SD Kit: STEM diff Kit.

Figure 48 :

 48 Figure 48: Stage 4 cells generated by using the Nostro protocol with H1 cellsin vitro evaluation. Gene expression of PDX1, NKX6.1, Chromogranin A, CK19, Insulin and Glucagon from H1-derived cells at differentiation stage 4. RPL27 was used as house-keeping gene and results are expressed as a fold change

Figure 49 :

 49 Figure 49: In vivo evaluation of stage 4 cells generated with H1 cells using SD Kit and the Nostro protocol over 4 months (n=8 mice). Normal range of c-peptide secretion from 500 human islets (red dot) is 500-1000 ng/L. Stage 4 cells were obtained using SD Kit (S1) and the Nostro protocol (S2-S4) from H1 cells. SD Kit: STEMdiff Kit.

  level. When glucose levels were returned to basal levels, insulin secretion in the H1 graft decreased to pre-stimulation levels, demonstrating the specificity of the insulin secretion.

Figure 50 :

 50 Figure 50: Dynamic GSIS by perifusion of explanted H1-derived S4 cells grafts, 6 months after transplant (black) and an explant human islet graft (control, red). G3: low glucose, G15: high glucose, G15+Arg: high glucose + 10mM arginine. Results are expressed in UI/minutes/mL.

Figure 51 :

 51 Figure 51: Overview of the differentiation protocols applied with patient specific iPS HGPS cells. iPS HGPS AG1972 cell line was differentiated towards stage 1 and stage 4 pancreatic progenitor cells. Stage 1 differentiation was achieved with the Rezania protocol or SD Kit and stage 4 differentiation with the Rezania protocol, SD Kit or the Nostro protocol. Stage 1 cells were analyzed in vitro by flow cytometry. Stage 4 cells were analyzed in vitro by qPCR and flow cytometry prior to transplantation under the kidney capsule of immunodeficient mice. Mice were followed over 3 months to assess human fasting c-peptide and glycemia. Grafts were analyzed by immunofluorescent staining. Arrows represent timepoint of cell engraftment. SD Kit: STEMdiff Kit.

Figure 52 :

 52 Figure 52: Stage 1 cells generated using SD Kit or the Rezania protocol with patient specific iPS HGPS cells -Definitive endoderm quantification. (A) Table of percentage of stage 1 cells expressing CXCR4 and C-Kit with the Rezania protocol. (B) Table of percentage of stage 1 cells expressing CXCR4 and C-Kit with SD Kit. (C) Representative FACS plots illustrating protein expression of CXCR4 and C-Kit in populations of iPS HGPS-derived stage 1 cells (Expt 56: 61,9% co-expression of CXCR4 and C-Kit). SD Kit: STEMdiff Kit.

Figure 53 :

 53 Figure 53: Stage 1 cells generated using the Rezania protocol with patient specific iPS HGPS cells -FACS sorting on CXCR4/C-Kit. (A) Representative FACS plot illustrating CXCR4/C-Kit sort of experiment 63. (B) Table of percentage of stage 1 cells expressing CXCR4 and C-Kit before and after CXCR4/C-Kit sort.

Figure 54 :

 54 Figure 54: Stage 1 cells generated using a SD Kit (Nostro adaptation) with patient specific iPS HGPS AG1972-Definitive endoderm quantification. (A) Table of percentage of stage 1 cells expressing CXCR4 and C-Kit with a SD Kit. (B) Representative FACS plots illustrating protein expression of CXCR4 and C-Kit in populations of iPS HGPS-derived stage 1 cells (Expt 71: 82,2% co-expression of CXCR4 and C-Kit). SD Kit: STEMdiff Kit

Figure 55 :

 55 Figure 55: Stage 4 cells generated using the Nostro protocol with patient specific iPS HGPS AG1972 cells -FACS quantification of pancreatic progenitor. (A) Table of percentage of expression of NKX6.1, PDX1 and GP2 stage from at stage 4 of differentiation with the Nostro protocol. (B) Representative FACS plots illustrating protein expression of NKX6.1 and PDX1 in populations of iPS HGPS-derived stage 4 cells (Expt TORONTO: 97,8% co-expression of NKX6.1 and PDX1). SD Kit: STEMdiff kit.Gene expression analysis at stage 4 for the differentiation markers chromogranin A, CK19, PDX1, NKX6.1, insulin and glucagon compared to a pool of 10 human islets donors (red bar) revealed that, cells produced with SD Kit + Nostro's protocol had higher mRNA expression levels of

Figure 56 :

 56 Figure 56: Stage 4 cells generated using the Nostro protocol with patient specific iPS HGPS AG1972 cellsin vitro evaluation. Gene expression of PDX1, NKX6.1, Chromogranin A, CK19, Insulin and

Figure 57 :

 57 Figure57: Fasting human c-peptide levels (ng/L) measured over 4 months (n=8 mice). Normal range of c-peptide secretion from 500 human islets is 500-1000 ng/L (red dot). Stage 4 cells were obtained using the SD Kit (S1) and the Nostro protocol (S2-S4) from iPS-HGPS AG1972 cells. SD Kit: STEMdiff Kit.

Figure 59 :

 59 Figure 59: Gene expression profile of Progerin, Lamin A and Lamin C was determined in fibroblasts from patients with HGPS-Progeria (Fibro HGPS -AG1972), undifferentiated iPS HGPS cells (dedifferentiated from Fibro HGPS -AG1972), S4 cells generated from iPS HGPS or H1 cells and human islets by Taqman qPCR. GAPDH was used as house-keeping gene and results are expressed as fold change relative to Fibro HGPS AG1972.

Figure 60 :

 60 Figure 60: MAFa, MAFb, NeuroD1 and Urocortin 3 expression in iPS HGPS (AG1972) and H1derived cells at stage 4 and human islets. RPL27 was used as house-keeping gene and results are expressed as a fold change relative to a pool of 10 human islet donors (red).

Figure 61 :

 61 Figure 61: Human c-peptide secretion of differentiated H1 or iPS HGPS cells 4 months after transplantation. (A) Human c-peptide secretion measured each 2 weeks after transplantation. (B) Mean of human c-peptide secretion over 4 months. (C) Human c-peptide secretion/glycemia measured every 2 weeks after transplantation. (D) Mean of human c-peptide secretion/glycemia over 4 months. (E) Human c-peptide secretion after an overnight fast (t0) and after intraperitoneal injection of 3g/kg glucose (t30 min). (F) Human c-peptide secretion/glycemia after an overnight fast (t0) and after intraperitoneal injection of 3g/kg glucose (t30 min). For each group n=8 mice. Results are expressed as mean ±SEM.

  depicts ductal cells and endocrine like structures in iPS HGPS graft 5 months posttransplantation (right) which resemble structures in the human pancreas (left) after routine trichrome straining.

Figure 62 :

 62 Figure 62: Representative image of a trichrome staining showing duct (top) and endocrine like structure (bottom) in human pancreas (left panel) and iPS HGPS graft 5 months post-transplantation (right panel).Chromogranin A positive endocrine cells were intercalated in CK19 positive epithelium cells in iPS HGPS graft (Figure63). Although CK19 epithelium was observed in grafts in our early experiments with H1 differentiated cells (Figure37), in optimized experiments only chromogranin A positive cells were detected in H1 cells (Figure64). Cytokeratine 19 is a marker for ductal epithelium but we can not exclude the presence of other types of epithelium notably intestinal epithelium that contains neuroendocrine cells. Carbohydrate 19.9 (also known as sialyl-Lewis

Figure 63 :Figure 64 :

 6364 Figure 63: Immunofluorescent staining of graft of iPS HGPS-derived stage 4 cells, harvested 6 months after transplantation (Expt 68) for chromogranin A endocrine (Chromo A, red) and CK19 epithelium (green). Scale bar = 25 m.

Figure 65 :H1066

 65 Figure 65: Representative immunofluorescent image for progerin, lamin A and chromogranin A. (A) Co-staining of chromogranin A (red) with progerin and lamin A/C (green) on a pancreas section from a 70 year-old donor (H1066) and an insulinoma. (B) Staining of progerin on HGPS fibroblasts (AG1972). Nuclei was stained with DAPI (blue). Scale bar = 60m

Figure 66 :Figure 67 :

 6667 Figure 66: Progerin (red), human nuclei (green) and DAPI (blue) staining in iPS HGPS graft versus H1 graft. Scale bar = 40m.

Figure 68 :

 68 Figure 68: Validation of aging markers on 70-year-old pancreas (H1066) and an insulinoma. 53BP1, yH2AX, IGF1r (green) were co-stained with pan-endocrine marker chromogranin A (red) in the pancreas of a 70-year-old donor (H1066) and in an insulinoma (benign tumor composed of immature cells). Scale bar = 15m.

FigureFigure 70 :

 70 Figure 69: Immunofluorescent staining of graft of H1-derived stage 4 cells, harvested 6 months after transplantation (Expt 73) for chromogranin A (Chromo A, red) and yH2AX (green). Scale bar = 25 m

  technology has taken off both for drug testing and more recently for clinical transplantation. Human islet transplantation for severe forms of diabetes is under evaluation in France for reimbursement which should be approved in 2020. The clinical activity will increase access to human islets for research. Mastering pseudo-islet technology will allow our laboratory to more efficiently use human islets for research and reduce donor derived heterogeneity which is a major disadvantage when working with native human islets. Tweaking this approach to further improve each step will remain a priority of our laboratory. Our laboratory has excellent long-term clinical islet results and is well situated to test pseudo-islets in the clinics to confirm or disprove their advantage this will be done within a consortium. Partnering up for this clinical trial with InSphero who has years of experience and numerous patented technologies on the subject should be the direction to take.Human pluripotent stem cell derived islets will one day be the future for both research and clinical transplantation for diabetes. In particular type 2 diabetic iPS cell lines are available and they would provide an excellent and unlimited source of cells to do research for comparison between healthy and diabetic cell lines. The complexity of stem cells and their differentiation to pancreatic islets, especially in terms of aging, requires profound know-how and resources. During this thesis, we had frequent contact with external expert (Dr. Nostro) which helped and guided us throughout our stem cell experiments. As a trained pharmacist, this PhD thesis however allowed me to become an expert in human stem cell culture and aseptic work, flow cytometry, confocal microscopy, and immunofluorescence to cite only a few techniques. My future work project is to work in a rather clinically environment with the pharmacy and clinicians in bringing ATMP cell therapy to the clinics, and in particular CAR-T cells. This thesis prepared me well for my future professional project.

Table list Table 1 :

 list1 Number of islet isolation, number of IEQ used for internal and external research (U1190 unpublished data) ........................................................................................................................

Table 2 :

 2 Different methodology and timing to obtain pseudo-islets from pig, dog, rodent and human native islets. ....................................................................................................................................

Table 3 :

 3 Advantages and limitations of native human -cell, stem cell-derived -cell and EndoC-H-cell (Scharfmann et al. 2019). ..................................................................................................

Table 4 :

 4 Advantages and disadvantages of published delivery methods used for reprogramming[START_REF] Brouwer | Choices for Induction of Pluripotency: Recent Developments in Human Induced Pluripotent Stem Cell Reprogramming Strategies[END_REF][START_REF] Takahashi | A decade of transcription factor-mediated reprogramming to pluripotency[END_REF][START_REF] Karagiannis | Induced Pluripotent Stem Cells and Their Use in Human Models of Disease and Development[END_REF]. .........................

Table 5: Evaluation of reprogramming factors capable of reprogramming human cells. EMT: epithelial to mesenchymal transition. MET: mesenchymal to epithelial transition. (Brouwer et al.

2016; Omole and Fakoya 2018). .................................................................................................... Table 6: Clinical trials with embryonic stem cell-derived cells ..................................................... Table 7: Clinical trials with induced pluripotent stem cell-derived cells ....................................... Table 8: Characteristics of published pancreatic -like cell differentiation protocols................... Table 9: Islet donor characteristics for pseudo-islets experiments................................................. Table 10: Acronyms used in differentiation protocols ................................................................... Table 11: Summary of optimized experiments with H1 and IPS HGPS cells ............................. 25 1. Background

Table 1 : Number of islet isolation, number of IEQ used for internal and external research (U1190 - unpublished data)

 1

	Organ Donation	Alloimmunity Autoimmunity
	Islet isolation	Xenobiotics
		Glucotoxicity and islet exhanstion
	Culture
	Hypoxia
	Innate immunity	Revascularization

Table 2 : Different methodology and timing to obtain pseudo-islets from pig, dog, rodent and human native islets.

 2

	Source	Method	Time (days)	Reference
	Pig/Dog	Spontaneous reaggregation + rotation	7	(Britt et al. 1981)
		Spontaneous reaggregation	4-6	(Schröder et al. 1983)
		Spontaneous reaggregation	3-4	(Hopcroft et al. 1985)
		Spontaneous reaggregation	5-6	(Halban et al. 1987)
		Hanging Drop	5-8	(Cavallari et al. 2007)
	Rodent	Spontaneous reaggregation + shaking	1	(Ju et al. 2013)
		Agarose microwells	6-7	(Ramachandran et al. 2014)
		Agarose microwells	7	(Ichihara et al. 2016)
		Hanging Drop	5	(Pathak et al. 2017)
		Hanging Drop	4-6	(Zuellig et al. 2017)
		Microtissues	-	InSphero
		Agarose microwells + rotation	3-5	(Ramachandran et al. 2014)
	Human	Agarose microwells	7	(Hilderink et al. 2015)
		Hanging Drop	4-6	(Zuellig et al. 2017)
		Centrifugal Forced Aggregation	2-3	(Yu et al. 2018)

Table 3 : Advantages and limitations of native human -cell, stem cell-derived -cell and EndoC-H- cell (Scharfmann et al. 2019).

 3

Table 4 : Summary of advantages and disadvantages of published transcriptor factors delivery methods used for cellular reprogramming (Brouwer et al. 2016; Takahashi and Yamanaka 2016; Karagiannis et al. 2019).

 4

	triggers immune	Rossi 2013; Yoshioka
	response	et al. 2013)

Table 5 : Evaluation of reprogramming factors capable of reprogramming human cells

 5

	Reprogramming	Function	Affected pathway	Effect on	References
	factors			pluripotency
	Oct4	Maintenance of pluripotency and	Core transcriptional	+	(Takahashi et al. 2007; Okita et al.
		self-renewal	circuitry		2011)
	Sox2	Maintenance of pluripotency and	Core transcriptional	+	(Takahashi et al. 2007)
		self-renewal	circuitry
	Klf4	Maintenance of pluripotency and	Core transcriptional	+	(Jiang et al. 2008; Zhang et al.
		self-renewal	circuitry		2010)
	c-Myc	Maintenance of pluripotency and	Core transcriptional	+	(Takahashi et al. 2007)
		self-renewal	circuitry
	Lin28	Maintenance of pluripotency,	Core transcriptional	+	(Yu et al. 2007)
		translational enhancer, inhibits	circuitry
		let7

. EMT: epithelial to mesenchymal transition. MET: mesenchymal to epithelial transition.

[START_REF] Brouwer | Choices for Induction of Pluripotency: Recent Developments in Human Induced Pluripotent Stem Cell Reprogramming Strategies[END_REF][START_REF] Omole | Ten years of progress and promise of induced pluripotent stem cells: historical origins, characteristics, mechanisms, limitations, and potential applications[END_REF]

.

Table 6 : Clinical trials with embryonic stem cell-derived cells

 6

	Disease	Cell type		NCT	Investigator	References
	Ischemic Heart Disease	hESC-derived	NCT02057900	APHP-France	(Menasché et al.
		CD15+	Isl-1			2018)
		progenitors
	Dry Age-related Macular	hESC-derived	NCT01344993	Astellas	(Schwartz et al.
	Degeneration	retinal pigment			2015)
		epithelium
	Spinal Cord Injury	hESC-derived	NCT02302157	Asterias
		oligodendrocyte		Biotherapeutics
		progenitors
	Parkinson disease	hESC-derived	NCT03119636	Chinese Academy of	(Wang et al. 2018)
		neural precursor		Sciences
		cells
	Type 1 Diabetes	hESC-derived	NCT03163511	Viacyte
		pancreatic
		endoderm cells

Table 7 : Clinical trials with induced pluripotent stem cell-derived cells

 7 [START_REF] Assady | Insulin production by human embryonic stem cells[END_REF] reported for the first time, the generation of insulin producing cells generated by spontaneous differentiation of human embryonic stem cells (hESC).

	Disease	Cell type		Clinical Trial Number Investigator	References
	Exudative Age-related	Autologous iPSC-	UMIN000011929	Riken	(Mandai et al.
	Macular	derived	retinal			2017)
	Degenerescence	pigment epithelial
	Graft-versus-host-	Allogenic	iPSC-	NCT02923375	Cynata Therapeutics
	disease (GvHD)	derived
		mesenchymal stem
		cells

  . UCN3 is a neuropeptide expressed in mature  and  cells (van der[START_REF] Van Der Meulen | Urocortin 3 marks mature human primary and embryonic stem cell-derived pancreatic alpha and beta cells[END_REF]. It has been shown that only functional  cells express UCN3 and it is induced in hESC-derived  cells after in vivo maturation[START_REF] Blum | Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3[END_REF][START_REF] Blum | Reversal of β cell de-differentiation by a small molecule inhibitor of the TGFβ pathway[END_REF]. Furthermore, Ghazizadeh et al. demonstrated that increased in vitro UCN3 expression is associated with increased static GSIS response[START_REF] Ghazizadeh | ROCKII inhibition promotes the maturation of human pancreatic beta-like cells[END_REF]).More recently, two studies demonstrated the enhanced functional maturation of BLC in vitro by mimicking endogenous endocrine cell clustering comparable to human pancreatic islets[START_REF] Nair | Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived β cells[END_REF][START_REF] Velazco-Cruz | Acquisition of Dynamic Function in Human Stem Cell-Derived β Cells[END_REF]). Nair et al. used an insulin-GFP labelled hPSC line and enrichment

by FACS purification before reaggregating single cells into clusters generated 99% of chromogranin A positive cells. For the first time, generated cells showed, in dynamic GSIS (perifusion), a robust first phase response however insulin secretion was not sustained in the second phase. Velazco-cruz et al. published a differentiation protocol without selection or sorting allowing

Table 9 : Islet donor characteristics for pseudo-islets experiments.

 9

	Lot	1063	1044	1037	1035
	Age (years)	59	39	30	49
	BMI	32	22	21	21
	Sex	F	M	M	M
	HbA1c (%)	5,7	5,8	5,1	5,5
	Alcohol	no	no	yes	no
	Tobacco	yes	no	yes	no
	Cold ischemia time	6,4	5,8	5,1	5,5
	Cardiorespiratory arrest (min) 0	15	30	0
	Intensive Care Unit stay (days) 5	5	1	8
	3) Human fibroblasts
	Peschanski (I-STEM, France).
	4) Cell culture
	Cell culture reagents
	Reagent		Company		Catalog #
	1-Thioglycerol (MTG)		Sigma		M6145
	Accutase		PAA Laboratories		L11-007
	Agarose		Sigma		A9045
	Ascorbic Acid		Sigma		A4544
	B-27 (without vitamin A)		Gibco		12587010
	 -Mercaptoethanol		Sigma		M3148
	BSA		Sigma		A9647
	CHIR99021		SelleckChem		S2924
	CMRL 1066		Connaught Medical Research	PM-C1051
			Laboratories
	DMEM High Glucose		Gibco		41966-029
	DMEM/F-12		Gibco		31330-038
	DMSO		Sigma		D8418

Human fibroblasts were isolated in the laboratory from the human islet fraction or from Hutchinson Progeria patients, fibroblasts HGPS Coriell, HGPS Ag001972, HGPS 8243 (referred to herein as fibro HGPS) were kindly provided by Dr. X Nissan and Dr. M.

  [START_REF] Rezania | Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice[END_REF]

				Stage 1	Stage 2	Stage 3	Stage 4
			D efinitive Endoderm	Prim itive G ut Tube	Posterior Foregut	Pancreatic Endoderm
			1 day : MCDB131 +1,5g/L
		70-80% confluence	NaHCO3+ 1X glx + 10mM Glc + 0,5%BSA + 100ng/ml GDF8 +1uM CHIR	2 days : MCDB131 +	2 days : MCDB131 + 2,5g/L NaHCO3 + 1XGlx + 10mM Glc + 2%BSA+ 0,25mM ascorbic acid	3 days : MCDB131 + 2,5g/l NaHCO3 + 1xGlx +
		H1 & iPS 1:30 matrigel	1day : MCDB +0,5%BSA + 1,5g/L NaHCO3 +1x Glx +10mM Glc + 100ng/ml GDF8 + 0,1uM CHIR	1,5g/L NaHCO3+1XGlx + 10mM Glc+ 0,25%BSA + 0,25mM Ascorbic acid + 50ng/mL FGF7	+ 50ng/ml FGF7 + 0,25uM SANT1 + 1uM Retinoic acid+ 100nM LDN+1/200 ITS-X + 200nM TPB	10mM Glc + 2%BSA + 0,25mM ascorbic acid+ 2ng/ml FGF7 + 0,25uM SANT1 + 0,1uM retinoic acid + 200nM LDN +
			1day : MCDB +0,5%BSA + 1,5g/L NaHCO3 +1x Glx +10mM Glc + 100ng/ml		MCDB131 could be susbstitued for BLAR medium	1/200 ITS-X + 100nM TPB
	-(Rezania et al. 2014)	GDF8
				Stage 5	Stage 6	Stage 7
	Rezania 2014		Progenitors Pancreatic Endocrine	Im m ature Beta Cells	Maturing Beta Cells
					7 days : MCDB131 +	7 days : MCDB131 +
					1,5g/L NaHCO3 + 1X	1,5g/L NaHCO3 + 1X
					Glx + 20mM Glc + 2%	Glx + 20mM Glc + 2%
					BSA + 100nM LDN +	BSA + 1/200 ITS-X +
					1/200 ITS-X + 1uM T3	1uM T3 + 10uM ALK5i
		Air-Liquide Interface	Stage 1 D efinitive Endoderm MCDB131 +1,5g/L NaHCO3 + 1X Glx + 20mM Glc + 2%BSA + 0,25um SANT-1 +	Stage 2 Prim itive G ut Tube + 10uM ALK5i II + 10uM ZnSO4 + 10nM gamma secretase inhibitor XX	Stage 3 Posterior Foregut II + 10uM ZnSO4 + 1mM Nacetyl cysteine + 10uM trolox + 2uM R428	Stage 4 Pancreatic Endoderm & Endocrine Precursors
	Rezania 2012	60-70% confluence; H1 cells; 1:30 matrigel	1 day : RPMI1640 + 0,2% FBS + 100ng/ml Act A +20ng/ml Wnt3A 2 days : RPMI1640 + 0,5% FBS + 100ng/ml ActA 0,005uM retinoic acid + 100nM LDN + 1/200 ITS-X + 1uM T3 + 10uM ALK5i II + 10uM ZnSO4 + 10ug/mL heparin	3 days : DMEM-F12 + 2% FBS + 50ng/ml FGF7 7 days : MCDB131 + 1,5g/L NaHCO3 + 1X Glx + 20mM Glc + 2% BSA + 100nM LDN + 1/200 ITS-X + 1uM T3 + 10uM ALK5i II + 10uM ZnSO4 + 10nM	4 days : DMEM-HG + 0,25umol SANT-1 + 2umol/L retinoic acid + 7 days : MCDB131 + 1,5g/L NaHCO3 + 1X Glx + 20mM Glc + 2% BSA + 1/200 ITS-X + 1uM T3 + 10uM ALK5i 100ng/ml Noggin + II + 10uM ZnSO4 + 1%B27 1mM Nacetyl cysteine	3 to 4 days : DMEM-HG + 1umol ALK5i II + 100ng/ml Noggin + 50nmol/L TPB + 1%B27
					gamma secretase	+ 10uM trolox + 2uM
					inhibitor XX +	R428 + 10ug/ml
					10ug/mL heparin	heparin
				Stage 1	Stage 2	Stage 3	Stage 4
			D efinitive Endoderm	Foregut	Pancreatic Endoderm	Endocrine Progenitors
		50-60 %	1day : SD Kit (10uL/mL		2 days : DMEM + 1%
		confluence	supp CJ and 10uL/mL	3 days : RPMI +1% Gln	Gln + 1% B27 + 50	5 days : H1 + 1% Gln +
	SD Kit + Nostro 2016	for iPS		supp MR)	+ 1%B27(w/o vitA) +	ug/ml ascorbic acid +	1% B27 + 50ug/ml
		HGPS Single			50ng/mL hFGF10 +	50ng/mL hNoggin +	ascorbic acid + 50ng/mL
		Cells + Rock			0,75uM Dorsomorphin	50ng/mL hFGF10 +	noggin + 100ng/ml hEGF
		inh for H1	2-3 days : SD Kit	+ 3ng/mL mWnt3a	0,25uM SANT1 + 2uM	+ 10mM nicotinamide
		cells	(10uL/mL supp CJ)		ATRA

-

[START_REF] Korytnikov | Generation of polyhormonal and multipotent pancreatic progenitor lineages from human pluripotent stem cells[END_REF]

STEMdiff ™ Definitive endoderm kit and STEMdiff ™ pancreatic progenitor kit (StemCell Technologies, Canada) were used and mixed with other protocols.

Acronyms are described in Table

10

.

Table 10 : Acronyms used in differentiation protocols

 10

	Acronyms	Name
	Act A	Activine A
	ALK5i II	TGF-β RI Kinase Inhibitor II
	ATRA	All-trans-retinoic acid
	B27	Vitamin B27
	BSA	Bovin serum albumin
	CHIR	Aminopyrimidine derivative Inhibitor of GSK3
	DMEM-F12	Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12
	DMEM-HG	Dulbecco's Modified Eagle Medium high glucose
	Dorsomorphin	BMP and AMPK pathway inhibitor
	FBS	Fetal bovin serum
	hFGF10	Human fibroblast growth factor
	FGF7	Fibroblast growth factor 7
	ySec Inh XX	Gamma secretase inhibitor XX
	GDF8	Growth Differentiation Factor 8
	Glc	Glucose
	Gln / Glx	Glutamine / glutamate
	hEGF	Human Epidermal Growth Factor
	ITS-X	Insulin-Tranferrin-Selenium-Ethanolamine
	NaHCO3	Sodium bicarbonate
	Nicotamide	Amide form of vitamin B3
	hNoggin or Noggin	Human NOG protein
	R428	Bemcentinib
	Retinoic acid	Retinoic acid receptor (RAR) agonist
	RPMI1640	Roswell Park Memorial Institute 1640
	SD kit	Stem cell technology kit stage 1
	Supp CJ	Stem cell technology supplement kit stage 1
	Supp MR	Stem cell technology supplement kit stage 1
	T3	Triiodothyronine
	Wnt3A	Wnt3 protein
	ZnSO4	Zinc Sulphate

Table 11 : Summary of optimized experiments with H1 and IPS HGPS cells both in vitro and in vivo. Figure 58: Overview of the objectives of using progeria to induce aging in cells derived from pluripotent cells.

 11 In the undifferentiated state, the iPS HPGS cells derived from patients with progeria have been shown to no longer express the features of aging cells such as shortening of telomeres, mitochondrial fitness and loss of senescence markers(Agarwal 2010[START_REF] Marion | Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells[END_REF][START_REF] Prigione | The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells[END_REF][START_REF] Suhr | Mitochondrial rejuvenation after induced pluripotency[END_REF], Lapasset 2011). First, we confirmed this finding and we questioned if pancreatic progenitor cells expressed Progerin. For this purpose, we performed Taqman qPCR gene expression analysis for Progerin, Lamin A and Lamin C, which are all encoded by the same gene.We compared human HGPS fibroblasts AG1972 obtained in collaboration with Dr. Nissan (iSTEM, France), undifferentiated iPS HGPS cells (dedifferentiated from iPS HGPS AG1972), S4 cells generated with iPS HGPS and H1 cells and human pancreatic islets (Figure43). In mature cells, human islets and human HGPS fibroblasts expressed equivalent levels of Lamin A and Lamin C, however these two genes and Progerin were undetectable in undifferentiated iPS HGPS samples in accordance withConstantinescu et al. (Constantinescu Stem cells 2006). As expected, human HGPS fibroblasts expressed Progerin (Taqman q-PCR) while levels in human pancreatic islets were undetectable. We next asked the question what happens during differentiation of iPS HGPS cells. After differentiation, S4 cells expressed Lamin A and Lamin C at lower levels than mature cells, and differentiated iPS-HGPS expressed around 30% less of Progerin than their HGPS patient derived fibroblast counterpart (Fibroblast AG1972) (Figure

	Experiment Transplantation date	Cell line	Stage	Transplantation site	Number of mice transplanted	In vivo time maturation	S1 C-kit+) (CXCR4+/	S4 PDX-1+) (NKX6.1+/	Use
	Expt 68	29/03/2019	IPS HGPS	S4	Kidney capsule	2	6 or 7 months	95,7	95,1	IHC/IF, RNA
	Expt 69	02/04/2019	IPS HGPS	S4	Kidney capsule	2	6,5 months	89,4	97,8	Perifusion, IHC/IF, RNA
	Expt 70	05/04/2019	H1	S4	Kidney capsule	2	6 or 7 months	78,2	95	IHC/IF, RNA
	Expt 71	09/04/2019	IPS HGPS	S4	Subcutaneous	4	6 or 7 months	75,9	95,8	IHC/IF, RNA, protein
	Expt 73	16/05/2019	H1	S4	Kidney capsule	3	6,5 months	95	95	IHC/IF, perifusion, RNA
	Expt 74	29/05/2019	H1	S4	Kidney capsule	3	6,5 months	87,5	90,9	IHC/If, RNA, protein
				H1 cells	iPS HGPS
					Stage 1 : CXCR4/c-kit
					quantification
					Stage 4 : NKX6.1/PDX1
					quantification
						PM O
						Progerin
				M aturation and aging m arkers

  . The decrease in intracellular insulin content might be due to the long culture time or the use of human serum albumin since other groups use fetal calf or bovine serum. Indeed, Yu et al reported no change in insulin content between native islets and D3 pseudo-islets using fetal bovine serum supplemented media[START_REF] Yu | Bioengineered human pseudoislets form efficiently from donated tissue, compare favourably with native islets in vitro and restore normoglycaemia in mice[END_REF] while InSphero reported an increase of insulin content after pseudo-islet formation. We previously published that native human islets can be cultured up to 5 days in CMRL 1066 supplemented with nicotinamide, pyruvate, 5% human AB serum, and antioxidant N-acetyl cysteine media without any change of insulin content[START_REF] Kerr-Conte | Upgrading pretransplant human islet culture technology requires human serum combined with media renewal[END_REF]. We are currently repeating the pseudo-islet experiments with the human AB serum and antioxidants to confirm if this medium improves our results.Surprisingly if the raw insulin secretion profiles of pseudo-islets were compared to native islets the conclusion would have been different that if insulin secretion was expressed as percentage of content.

human islets : 500-1000 ng/L

human islets: 500-1000 ng/L

Remerciements

thanks to Farida Sarangi for everything you taught me, to Frankie

Plasma was obtained from blood sampling using heparinized micro tubes which were centrifugation (2000 rpm 5 min) and stored at -80°C until the assay. The detection limit is < 2,5 pmol/L (0,0076ug/L).

Statistics

All values are shown as mean ± SEM. Prism 7 (GraphPad, USA) software was used for statistical analysis. The non-parametric Kruskal Wallis test was used to analyze the statistical significance in the pseudo-islet part.

Chapter 3: Pseudo-islets Chapter 4: Human pluripotent stem cell differentiation Reproduction of published protocols and optimization of two cell lines grafted with SD Kit + Rezania protocol) we observed a human c-peptide secretion higher than 200ng/L (Figure 43A). In a graft with cells differentiated with the Rezania protocol we detected a significant number of insulin positive cells (Figure 43B). In conclusion, although the same levels of expression of the pancreatic progenitor genes (NKX6.1 and PDX1) were detected by q-PCR after differentiation were high and identical to levels expressed in human islets, efficiency of endoderm differentiation was low when taking into account double positive CXCR4/c-kit cells. In the following chapters, our objective was to obtain more than 80% endoderm evaluated by flow cytometry. of recovering more than 80% of human endocrine cells from the entire pancreas. This technology may also allow us to obtain uniform "standardized islets" regardless of the status of donors, including young donors, but also marginal donors including obese and aged donors. Indeed, islets isolated from young donors (under 40 years of age) appear ideal for transplantation but are notoriously difficult to purify, due to surrounding exocrine tissue that increases their density making their separation from the heavy exocrine layer a challenge (less than 40% recovery). In addition, in our center, 30% of human organs come from obese donors, which is liked with a high islet mass and a larger diameter of each islet. It is known that large islets survive poorly in culture (central necrosis). Lastly, the age of donors proposed for organ harvesting is increasing throughout the world. In Lille, 30% of donors are over 55 years of age (15% are over 60 years old). Islets of aging donors show signs of inflammation and fibrosis of the islet blood vessels, but glucose detection and insulin secretion are intact [START_REF] Almaça | Young capillary vessels rejuvenate aged pancreatic islets[END_REF]. Eliminating the aging vascularization of islets during single cell production could broaden the scope of donors to older donors.

Islet vascular network is necessary for the preservation of the transplanted islet mass [START_REF] Coronel | Engineering a local microenvironment for pancreatic islet replacement[END_REF][START_REF] Vlahos | Modular tissue engineering for the vascularization of subcutaneously transplanted pancreatic islets[END_REF][START_REF] Weaver | Vasculogenic hydrogel enhances islet survival, engraftment, and function in leading extrahepatic sites[END_REF][START_REF] Citro | Can We Re-Engineer the Endocrine Pancreas?[END_REF]. Recent ingenious integration of human amniotic epithelial cells with anti-inflammatory, angiogenic, and regenerative properties during pseudo-islet reaggregation appears to be a solution to improve pseudo-islet survival in culture, viability and resistance to hypoxia, grafted pseudo-islet vascularization, insulin content and function, as very recently demonstrated [START_REF] Lebreton | Insulin-producing organoids engineered from islet and amniotic epithelial cells to treat diabetes[END_REF]).

Lastly, one key advantage of pseudo-islets is the possibility of cryopreserving the single cells before reaggregation into pseudo-islets [START_REF] Rawal | Long-term cryopreservation of reaggregated pancreatic islets resulting in successful transplantation in rats[END_REF]. This may allow us to freeze cell preparations directly after single cell isolation from several pancreata and subsequently prepare pseudo-islets for a single transplantation. Current procedures involve 2 to 3 transplant procedures per islet transplant recipient. The ultimate long-term goal in the laboratory is to optimize and automate the technology for pancreatic endocrine cell culture and the renewal of culture media as well as digestion, purification and culture steps. The laboratory participates in a consortium (Zurich, Leiden, Geneva, Lille, Kugelmeier, AG Switzerland) to bring pseudo-islets to clinical transplantation.

Indeed, islets are neuroendocrine cells and the old APUD (Amine Precursor Uptake Decarboxylase) system erroneously attributed these cells to be of ectodermal origin. In the literature, indeed Mir-9 has been reported to be expressed at high levels during human pancreatic development [START_REF] Joglekar | Expression of islet-specific microRNAs during human pancreatic development[END_REF][START_REF] Rosero | MicroRNA signature of the human developing pancreas[END_REF]. More recent work documents Mir-9-5p and miR-9-3p upregulation during the differentiation of human iPS cells to insulin producing cells [START_REF] Sebastiani | MicroRNA expression profiles of human iPSCs differentiation into insulin-producing cells[END_REF]. Thus, neuroendocrine pancreatic islet cells may also be protected by Mir-9 expression. We will explore this pathway further. This could explain why progerin was only detected in a subpopulation of endocrine cells in the islet and was localized in the cytoplasm. Studer was able to accelerate aging in terminal dopaminergic neurons by overexpressing progerin and thus to better model late onset neurological diseases and thus perhaps this aging approach could work for human islets cells derived from pluripotent stem cells however Mir-9 expression must be taken into account in our system.