Method of expanding pancreatic progenitor cells and pancreatic progenitor cells produced by said method and uses thereof

Abstract

The present invention relates to a method of expansion of pancreas progenitors under GMP-compliant conditions. The present invention relates to a method of expansion of pancreas progenitors under GMP-compliant conditions, cells produced by said method (e.g., expanded PP (ePP) cells) as well as uses of said cells in various applications (e.g., clinical applications).

Description

METHOD OF EXPANDING PANCREATIC PROGENITOR CELLS AND PANCREATIC PROGENITOR CELLS PRODUCED BY SAID METHOD AND USES THEREOFSEQUENCE LISTING

[0001] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.FIELD OF THE INVENTION

[0002] The present invention relates to a method of expansion of pancreas progenitors under GMP-compliant conditions (e.g., serum-free and / or animal product free and / or does not comprise the use of a feeder layer), cells produced by said method (e.g., expanded PP (ePP) cells) as well as uses of said cells in various applications (e.g., clinical applications, e.g., for the diabetes treatment). In particular, the present invention relates to an in vitro or ex vivo method for expanding pluripotent stem (PS) cells-derived pancreatic progenitors (PP), said method comprising: (i) providing PS cells-derived pancreatic progenitors (PP); (a) activating MAPK signaling pathways in said cells by the means of adding one or more of the MAPK activators comprising: (i') one or more FGF molecules (e.g., FGF18); (ii') a combination of one or more of FGF (e.g., FGF18) and EGF molecules and / or (iii') a combination of one or more of FGF18, EGF and FGF2 molecules; (b) inhibiting and / or disabling and / or modulating retinoic acid signaling in said cells, preferably by the means of using a serum-free and / or animal product free supplement without Vitamin A for cell cultivation; (c) inhibiting and / or disabling and / or modulating TGF-p signaling pathway in said cells by the means of adding one or more inhibitor / s that block TGF-p1, -|32, and / or -p3 ligands and adding one or more inhibitor / s that block BMP-2, BMP-4 and / or BMP-7 signaling and (d) inhibiting Wnt signaling pathway in said cells, preferably by the means of adding one or more Wnt / |3-Catenin inhibitors; (ii) expanding said cells, preferably said expanding is carried out using a recombinant truncated vitronectin (VTN-N) coating, further preferably said expanding is carried out using fibronectin coating.1RECTIFIED SHEET (RULE 91) ISA / EPBACKGROUND OF THE INVENTION

[0003] Diabetes is a global epidemic affecting > 9% of the global population and its two main forms result from the immune destruction (type 1) or malfunction (type 2) of insulinproducing |3 cells residing in the pancreatic islets of Langerhans. In severe cases of diabetes, whole pancreas transplantation or pancreatic islet transplantation can restore excellent metabolic control and insulin independence but both approaches are critically limited by the scarcity of tissue donors. Moreover, such transplantations require the use of immunosuppressive drugs that hinder the already limited [3-cell self-renewal and are associated with severe side effects and morbidity.

[0004] The remarkable progress over the last decade in the differentiation of human pluripotent stem (hPS) cells into pancreatic islets (SC-islets) suggests that this approach could provide an unlimited source of [3-cells for transplantations and personalized medicine. This success was achieved through the application of developmental mechanisms elucidated using primarily the mouse as a model organism. The underlying strategy recapitulates the stepwise differentiation of PS cells in the early embryo initially to definitive endoderm and then to pancreatic endoderm, pancreatic endocrine progenitors and finally to pancreatic islet cells. Clinical trials that employed either GMP-grade PP cells in an non-immunoprotective macro-encapsulation device or GMP-grade SC-islet cells in an immunoprotective macroencapsulation device suggest that diabetes cell therapies may become reality.

[0005] However, there are still numerous obstacles to be overcome including the limited maturation of the resulting p-cells, incomplete conversion of hPS cells into endocrine cells and, particularly for clinical uses, the large number, at least 1 billion, of cells required for a single transplantation. Elucidating the mechanisms that maintain the self-renewal of pure pancreatic progenitors while inhibiting their differentiation would allow the establishment of expandable populations of pancreatic progenitors that would address the need for large numbers of pure pancreatic endocrine cells. A necessary requirement for cell therapeutic applicability would be the employment of GMP-compliant conditions.

[0006] Pancreatic tri-potent progenitors arise around embryonic day 8 (E8) in the mouse and during week 4 of development (WD4) in humans. Several signals intersect to induce the pancreatic anlage at the posterior foregut. Repression of posteriorly derived Wnt signaling is initially essential to define the foregut region. Then, the combination of RA signaling, which is derived from the somitic mesoderm, and endothelial signals, which are derived from the2RECTIFIED SHEET (RULE 91) ISA / EPdorsal aorta and repress shh signaling in the endoderm, induces the formation of pancreatic progenitors. Pancreas and liver develop in close proximity sharing a common progenitor. FGF10 and BMPs are important pro-hepatic signals secreted by the cardiac mesoderm and, therefore, specification of pancreatic progenitors is restricted to a region of the foregut away from this tissue. The emerging pancreatic buds contain mesenchymal cells as well as epithelial pancreatic progenitors that are characterized by the combined expression of several transcription factors, most notably Pdx1, Nkx6.1 and Sox9, which are essential for progenitor self-renewal and subsequent endocrine differentiation. Pdx1 expression is induced at the boundary between the Sox2-expressing anterior endoderm and the Cdx2-expressing posterior endoderm and it is necessary for the maintenance of the pancreatic identity. Its function is reinforced by Sox9 which cooperates with Pdx1 to repress Cdx2 expression in the pancreatic anlage. Loss-of-function experiments of Pdx1 and Sox9 result in pancreatic agenesis, despite initial induction of the pancreatic progenitors, further confirming their key role in maintaining pancreatic identity. Sox9 is subsequently essential for the induction of Neurog3, the transcription factor which is necessary and sufficient for the induction of the pancreatic endocrine lineage. Forced expression of Pdx1 in endocrine progenitors resulted in an a- to |3-cell conversion suggesting that Pdx1 may also act later on as a [3-cell lineage determinant. Nkx6.1 also contributes to the maintenance and self-renewal of pancreatic progenitors and it is necessary, in a subsequent step, for the segregation of duct / endocrine bipotent progenitors from acinar progenitors. It is also required for p-cell specification since ectopic Nkx6.1 expression directed endocrine precursors into [3-cells, whereas [3-cell specific ablation of Nkx6.1 diverted these cells into the other endocrine lineages.

[0007] Thus, defining conditions to expand PDX1 + / SOX9+ / NKX6.1 + pancreatic progenitor (PP) cells derived from hPS cells could provide a stepping stone for reproducible, efficient differentiation into SC-islets. These cells exist in vivo and undergo self-renewal for a limited amount of time. During development and under the influence of multiple signaling pathways, feed-forward loops that steer cells towards differentiation operate in parallel with maintenance and self-renewal mechanisms. Maintaining PP cells in vitro for unlimited expansion will require disentangling differentiation signals from proliferation / maintenance signals. Several pathways have been implicated in these processes. Notch signaling plays a key role both in mediating progenitor self-renewal as well as in lineage segregation. Downregulation of Notch signaling is necessary for the specification of the endocrine lineage3RECTIFIED SHEET (RULE 91) ISA / EPin bipotent progenitors. In turn, Notch signaling is regulated by the extracellular signal sphingosine-1 -phosphate (S1p). Wnt signaling has also been implicated in progenitor maintenance since epithelial overexpression of Wnt7b resulted in both acinar and endocrine differentiation defects. Low levels of endogenous RA signaling are involved in subsequent differentiation steps of both endocrine and exocrine lineages. Expression of TG Fp ligands and receptors is also widespread during pancreas development and it has been suggested that the TGFp pathway activity regulates the relative differentiation levels to exocrine and endocrine cells.

[0008] Elucidating the mechanistic requirements for PP expansion is necessary for reliably expanding hPS derived PP cells under GMP conditions.

[0009] To derive a mechanistic understanding of the signaling requirements for PP expansion, we undertook longitudinal transcriptome analyses and comparisons of nonexpanding and, occasionally expanding PP cells using a medium very similar to that reported previously. In these analyses we identified regulated signaling pathways and we reasoned that upregulated pathways in expanding cells would be promoting expansion whereas downregulated pathways would be blocking expansion and / or favoring differentiation. We leveraged these findings and employed a hypothesis-driven iterative process to define conditions that allowed robust, unlimited expansion, of hPS cell-derived PP cells. We found that the combined stimulation of specific mitogenic pathways, suppression of retinoic acid signaling and inhibition of selected branches of the TGFfJ and Wnt signaling pathways enabled the 2000-fold expansion of PP cells over ten passages and 40-45 days. Expansion conditions are GMP-compliant and enable the robust, reproducible expansion, as well as cryo-preservation of PP cells derived from diverse hPS cell lines with essentially identical growth kinetics. These conditions significantly enrich the numbers of PDX1 + / SOX9+ / NKX6.1 + PP cells up to 90%, suggesting that they will be advantageous for hPS cell lines that may differentiate less efficiently into PP cells. Expanded PP cells differentiated essentially identical efficiency to non-expanded cells into SC-islet clusters that contained functional p-cells as shown by glucose stimulated insulin secretion (GSIS) assays.

[0010] These findings will allow the establishment of large banks of PP cells derived under GMP conditions from diverse hPS cell lines. This will streamline the generation of SC-islet clusters for further development of the differentiation procedure, diabetes research, personalized medicine and cell therapies.4RECTIFIED SHEET (RULE 91) ISA / EP

[0011] The in vitro unlimited expansion of human progenitor cells could unlock many possibilities but remains an important challenge for regenerative medicine because it requires the decoupling of the mechanisms supporting progenitor self-renewal and expansion from feed-forward mechanisms that promote their differentiation. The expansion of hPS cell derived pancreatic progenitors (PP) will accelerate the development of novel therapies for diabetes.

[0012] Accordingly, the technical problem underlying the present application is thus to comply with this need. The technical problem is solved by providing the embodiments reflected in the claims, described in the description and illustrated in the examples and figures that follow.SUMMARY OF THE INVENTION

[0013] In the course of the present invention mechanistic insights were obtained into the expansion requirements of Pancreatic Progenitors (PP) cells and leveraged them to conduct a hypothesis-driven iterative search to identify conditions for the robust and unlimited expansion of human Pluripotent Stem (hPS) cell-derived PP cells under GMP-compliant conditions. The combined stimulation of specific mitogenic pathways, suppression of retinoic acid signaling and inhibition of selected branches of the TGFp and Wnt signaling pathways, enabled the 2000-fold expansion of PP cells over ten passages and 40-45 days and their enrichment in up to 90% PDX1 + / SOX9+ / NKX6.1 + cells. During expansion, pancreatic progenitor identity was enhanced and endocrine differentiation was repressed suggesting that we succeeded in decoupling pancreatic progenitor proliferation from differentiation. Using these conditions, PP cells derived from different hPS cell lines, expanded with very similar kinetics and efficiency. Expanded and non-expanded PP cells were differentiated in micro-patterned wells into homogeneous islet-like clusters (SC-islets) with identical efficiency. These clusters contained abundant p-cells of very similar functionality as assessed by glucose stimulated insulin secretion assays.

[0014] These findings established the signaling conditions allowing the expansion of hPS cell derived PP cells while suppressing differentiation. They will enable the establishment of large banks of PP cells derived under GMP conditions from diverse hPS cell lines. This will also streamline the generation of SC-islet clusters for further development of the differentiation process, diabetes research, personalized medicine and cell therapies.5RECTIFIED SHEET (RULE 91) ISA / EP

[0015] Accordingly, in one aspect the present invention relates to an in vitro or ex vivo method for expanding pluripotent stem (PS) cells-derived (e.g., induced pluripotent cells (iPSs)-derived) pancreatic progenitors (PP), preferably human PPs (e.g., expressing PDX1+ / SOX9+ / NKX6.1+, e.g., having corresponding UniProt Accession Numbers:P52945 / P48436 / P78426), e.g., H1-PP, H9-PP or CRTD1-PP cells): said method comprising: (i) providing PS cells-derived pancreatic progenitors (PP), e.g., providing H1 -PP (e.g., H1 cells- derived PPs, e.g., H1 cells having accession number: WA01 (hPSCReg-ID: WAe001 -A)), H9-PP (e.g., H9 cells derived PPs, e.g., H9 cells having accession number: WA09 (hPSCReg-ID: WAe009-A) or CRTD1-PP cells (e.g., CRTD1 cells-derived PPs, e.g., CRTD1 cells having accession number: CRTDiOO4-A (hPSCreg-ID: CRTDi004-A); (a) activating MAPK (Mitogen- activated protein kinase) signaling pathways in said cells by the means of adding one or more of the MAPK activators comprising: (i') one or more FGF (Fibroblast growth factor) molecu les, preferably comprising one or more FGF18 molecules, e.g., having UniProt Accession Number: 076093); (ii') a combination of one or more FGF molecules (preferably FGF18, e.g., having UniProt Accession Number: 076093) and one or more EGF (Epidermal growth factor) (e.g., having UniProt Accession Number: P01133) molecules and / or (iii') a combination of one or more FGF18 molecules (e.g., having UniProt Accession Number: 076093), one or more EGF molecules (e.g., having UniProt Accession Number: P01133) and one or more FGF2 (e.g., having UniProt Accession Number: P09038) molecules; (b) inhibiting and / or disabling and / or modulating retinoic acid signaling in said cells, preferably by the means of using a serum-free and / or animal product free (e.g., GMP-compliant) supplement without Vitamin A for cell cultivation; (c) inhibiting and / or disabling and / or modulating TGF-|3 (Transforming growth factor beta) signaling pathway in said cells, e.g., by the means of adding one or more inhibitor / s that block TGF-(31, -f>2, and / or -[33 ligands (e.g., StemMACS RepSox inhibitor) and adding one or more inhibitor / s that block BMP-2 (Bone Morphogenetic Protein-2, e.g., having UniProt Accession Number: P12643), BMP-4 (Bone Morphogenetic Protein-4, e.g., having UniProt Accession Number: P12644) and / or BMP-7 (Bone Morphogenetic Protein-7, e.g., having UniProt Accession Number: P18075) signaling (e.g., K02288 inhibitor) and (d) inhibiting Wnt signaling pathway (e.g., canonical Wnt signaling) in said cells, preferably by the means of adding one or more Wnt / p-Catenin inhibitors (e.g., Endo-IWR-1); (ii) expanding said cells, preferably said expanding is carried out using a recombinant truncated vitronectin (VTN-N) coating, further preferably said expanding is carried out using fibronectin coating.6RECTIFIED SHEET (RULE 91) ISA / EP

[0016] In one aspect the present invention relates to the method of the present invention, wherein said PPs are human PPs expressing PDX1+ / SOX9+ / NKX6.1+(e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426), preferably said PPs are derived from H1, H9 or CRTD1 cell lines: said method comprising: (ii) providing hPS cells derived pancreatic progenitors (PP), preferably H1 -PP, H9-PP or CRTD1 -PP cells; (a) activating MAPK signaling pathways in said cells by the means of adding one or more of the MAPK activators, preferably EGF and / or FGF18 and / or FGF2 molecules (e.g., EGF and FGF18 and FGF2); (b) inhibiting retinoic acid signaling in said cells, preferably by the means of using a serum-free supplement without Vitamin A for cell cultivation; (c) inhibiting TGFfJ signaling pathway in said cells, preferably by the means of adding one or more inhibitor / s that block TGF-ftl ligands (e.g., StemMACS RepSox inhibitor) and / or one or more BMP-2 inhibitors (e.g., K02288 inhibitor) and (d) inhibiting Wnt signaling pathway in said cells, preferably by the means of adding one or more Wnt / p-Catenin inhibitors (e.g., Endo-IWR-1); (ii) expanding said cells, preferably said expanding is carried out using a recombinant truncated vitronectin (VTN-N) coating.

[0017] In one aspect the present invention relates to the method of the present invention, wherein said method is capable of: (a) upregulating the expression of a PP marker GP2 (Pancreatic secretory granule membrane major glycoprotein GP2, e.g., having UniProt Accession Number: P55259) in said cells, preferably said upregulating is detectable on the RNA level (e.g., up to 140-fold induction) and / or on the protein level (e.g., from about 5% to about 70% upregulation); and / or (b) producing (e.g., produces) PDX17SOX97NKX6.17GP2+(e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426 / P55259) PP cells.

[0018] In one aspect the present invention relates to the method of the present invention, wherein said method is characterized by one or more of the following: (a) said method produces and / or expands (e.g., selectively expands) and / or enriches (e.g., selectively enriches) PDX17SOX97NKX6.1+PP cells; (b) said method is capable of enriching (e.g., increasing the amount / concentration) of the PDX17SOX97NKX6.1+PP cells in pancreatic progenitors (PP) cell culture; (c) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are pancreatic progenitors (PPs) capable of proliferation (e.g., their proliferation ability is active / un-inhibited) while inhibiting differentiation and / or inhibiting endocrine differentiation of said PPs; (d) said progenitor cells are human pluripotent stem 7RECTIFIED SHEET (RULE 91) ISA / EP(hPS), preferably iPSs, cells derived pancreatic progenitors (PP), wherein said method is capable of expanding said hPS cell derived PP cells while suppressing their differentiation; (e) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into islet-like clusters (SC-islets), preferably insulin producing SC-islets (e.g., said SC-islets are capable of producing the essentially same amount of insulin as in vitro derived non-expanded PPs as defined in claim 1 (i) and / or having the essentially same amount of differentiated endocrine cells in said SC-islets; (f) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into insulin-producing cells, preferably said cells are capable of secreting insulin in the presence of glucose; (g) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into endocrine cells, preferably pancreatic endocrine cells, including insulinproducing beta cells; (h) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of cryopreservation (e.g., at least about - 195°C); (i) said method does not comprise the use of a feeder layer (e.g., GMP-compliant); (j) said method comprises the use of PP grown in a monolayer.

[0019] In one aspect the present invention relates to the method of the present invention, wherein said progenitor cells are human pluripotent stem (hPS) cell derived pancreatic progenitors (PP).

[0020] In one aspect the present invention relates to the method of the present invention, wherein said method is one or more of the following: said method is not limited by a number of cell divisions of said progenitor cells; said method is a method for unlimited expansion of said progenitor cells; said method is capable of producing (e.g., produces) progenitor cells that are capable of avoiding replicative senescence and / or replicative exhaustion, preferably continuously avoiding replicative senescence and / or replicative exhaustion; said method is capable of producing (e.g., produces) progenitor cells that are capable of avoiding arrest of cell division, preferably continuously avoiding arrest of cell division; said method is capable of producing (e.g., produces) progenitor cells that are capable of proliferation, preferably continuous proliferation; said method is capable of producing (e.g., produces) progenitor cells that are capable of self-renewal, preferably continuous self-renewal; said method is capable of producing (e.g., produces) progenitor cells that are capable of indefinitely renewing itself; said method is capable of producing (e.g., produces) progenitor cells that are 8RECTIFIED SHEET (RULE 91) ISA / EPcapable of expansion, preferably continuous expansion; said method is capable of producing (e.g., produces) progenitor cells that are capable of differentiation (e.g., into pancreatic endocrine cells), while suppressing their differentiation; said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into islet-like clusters (SC-islets); said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are pancreatic progenitors capable of proliferation (e.g., their proliferation ability is active / un-inhibited), wherein said method is simultaneously capable of inhibiting the endocrine, hepatic and / or gut differentiation in said cells; said method is capable of producing (e.g., produces) progenitor cells that are capable of maintaining their differential potential, preferably continuously maintaining their differential potential; said method does not comprise the use of artificial modification (e.g., siRNA) of the genome of said progenitor cells.

[0021] In one aspect the present invention relates to the method of the present invention, wherein said method is capable of producing (e.g., produces) at least 100-fold (e.g., 500, 700, 800, 900, 1000-fold, or 2000-fold) expansion of progenitor cells (e.g., PP cells), preferably said expansion is carried out during about at least 5 passages (e.g., 10 passages) and / or in a period from about 20 (e.g. 40) to about 25 (e.g., 45) days) and / or said progenitor cells comprising at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%) of PDX1 + / SOX9+ / NKX6.1 + cells (e.g., PDX1 + / SOX9+ / NKX6.1 + / GP2+).

[0022] In one aspect the present invention relates to an isolated PP cell or an isolated population of PP cells, wherein said cell / s are PDX17SOX97NKX6.1+cell / s (e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426), wherein said cells are capable of indefinitely renewing themselves, wherein said cell / s further having one or more of the following characteristics: said cell / s expressing PP marker GP2 (Pancreatic secretory granule membrane major glycoprotein GP2, e.g., having UniProt Accession Number: P55259); said cells are capable of proliferation while having their differentiation inhibited / repressed; said cells are capable of unlimited expansion; optionally, said cells are obtainable (e.g., obtained) from pluripotent stem cells (e.g., iPSs), preferably providing human pluripotent stem (hPS) cells, further preferably providing hPS cells derived pancreatic progenitors (PP), e.g., H1-PP, H9-PP or CRTD1-PP cell lines.9RECTIFIED SHEET (RULE 91) ISA / EP

[0023] In one aspect the present invention relates to a population of progenitor cells or a progenitor cell produced and / or expanded and / or modified by method of the present invention, preferably said cells are PDX1+ / SOX97NKX6.1+cells (e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426).

[0024] In one aspect the present invention relates to the population of progenitor cells of the present invention, wherein population comprising at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%) of PDX1+ / SOX97NKX6.1+cells, preferably said method of the present invention comprising activating MAPK signaling pathways in said cells by the means of adding MAPK activators EGF, FGF18 and FGF2.

[0025] In one aspect the present invention relates to a composition, preparation or kit comprising the population of progenitor cells (e.g., isolated) or the progenitor cell (e.g., isolated) of the present invention.

[0026] In one aspect the present invention relates to the composition, preparation or kit of the present invention, wherein said composition, preparation or kit is a pharmaceutical and / or diagnostic composition, preparation or kit.

[0027] In one aspect the present invention relates to the population of progenitor cells (e.g., isolated), a progenitor cell (e.g., isolated), composition, preparation or kit of the present invention, for use as a medicament and / or in therapy.

[0028] In one aspect the present invention relates to the population of progenitor cells (e.g., isolated), a progenitor cell (e.g., isolated), composition, preparation or kit of the present invention for use in one or more of the following methods: method of treatment, amelioration, prophylaxis and / or diagnostics of an endocrine disease (e.g., Diabetes, Type 1 Diabetes, Type 2 Diabetes, Gestational Diabetes, Mature Onset Diabetes of the Young (MODY)); method of treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; method for monitoring development and / or assessing the efficacy of prophylaxis and / or therapy of Diabetes, preferably Type 1 Diabetes; method for screening a candidate compound for use in method of treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; method of regenerative treatment of a cell, tissue, organ and / or body; method of transplantation (e.g., beta-cell transplantation); method of cell therapy (e.g., beta-cell therapy); disease modelling (e.g., diabetes modelling); method of insulin production and / or insulin supplementation; method10RECTIFIED SHEET (RULE 91) ISA / EPaccording to any one of the preceding claims; any combination of methods as above; method as above, wherein said method is an in vitro, ex vivo or in vivo method.

[0029] In one aspect the present invention relates to a use of the population of progenitor cells (e.g., isolated), a progenitor cell (e.g., isolated), composition, preparation or kit of the present invention for one or more of the following: for treatment, amelioration, prophylaxis and / or diagnostics of an endocrine disease (e.g., Diabetes, Type 1 Diabetes, Type 2 Diabetes, Gestational Diabetes, Mature Onset Diabetes of the Young (MODY)); for treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; for monitoring development and / or assessing the efficacy of prophylaxis and / or therapy of Diabetes, preferably Type 1 Diabetes; for screening a candidate compound for use in method of treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; for regenerative treatment of a cell, tissue, organ and / or body; for transplantation (e.g., beta-cell transplantation); for cell therapy (e.g., beta-cell therapy); for insulin production and / or insulin supplementation; disease modelling (e.g., diabetes modelling); use as above; any combination of methods as above; for any use as above, wherein said use is an in vitro, ex vivo or in vivo use.OVERVIEW OF THE SEQUENCE LISTING

[0030] SEQ ID NO: 1 is a DNA Adaptor-Oligo 1 having ACA CTC TTT CCC TAG ACG ACG CTC TTC CGA TCT sequence.

[0031] SEQ ID NO: 2 is a DNA Adaptor-Oligo 2 having GAT CGG AAG AGC ACA CGT CTGAAC TCC AGT CAC sequence.

[0032] SEQ ID NO: 3 is a DNA Primer 1 having AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T sequence.

[0033] SEQ ID NO: 4 is a DNA Primer 2 having GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T sequence.

[0034] SEQ ID NO: 5 is a DNA Primer 3 having CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN GTG ACT GGA GTT sequence.

[0035] SEQ ID NO: 6 is an antigen amino acid sequence depicted in Fig. 13 (H).

[0036] SEQ ID NO: 7 is a human NKX6.1 sequence fragment depicted in Fig. 13 (H).11RECTIFIED SHEET (RULE 91) ISA / EP

[0037] SEQ ID NO: 8 is a human NKX6.2 sequence fragment depicted in Fig. 13 (H).

[0038] SEQ ID NOs: 9-44 are exemplary DNA forward and reverse primers for qPCR.BRIEF DESCRIPTION OF THE FIGURES

[0039] Figure 1: Initial expansion and differentiation of H1-PP cells. (A) Regression lines of two samples showing exponential expansion of PP cells for 32 d. (B, C) Immunofluorescence staining of H1 -derived PPs showed that expression of PP markers at P0 (B) was retained for at least five passages (p5) of the expansion under the initial expansion condition (C). (D) qPCR analyses indicated transcript levels of pancreatic progenitor markers were retained at P5 and P10. Values at p5 and pl 0 were normalized against the value of the corresponding expansion at pO to give the calculated fold change. Horizontal lines represent the mean ± standard deviation (SD). (E) Stained cryo-sections of expanded PP-derived endocrine cell clusters showing expression of endocrine markers. Scale bar corresponds to 50 pm (B, C, E).

[0040] Figure 2: Regulated genes and signaling pathways during PP expansion under initial conditions. (A) Schematic showing the number of genes significantly up- or down- regulated after filtering the RNA Seq data for 1.6 < fold change < 0.6, normalized counts > 100 and padj < 0.1. (B) MDS plot representing the Euclidian distance of the samples pO (n=4), p5 (n=3) and pl 0 (n=3). (C-G) Average transcript levels in normalized RNA-Seq counts of pO (n=4), p5 (n = 3) and pl 0 (n=3) cell populations for genes encoding additional ligands and receptors of TGF-B (C), FGF (D) and PDGF (E) signaling, the RA producing enzyme ALDH1A1 as well as ligands and receptors of NOTCH (G) signaling. Horizontal lines represent the mean ± SD. Statistical tests were one-way ANOVA using pO as the control condition for the comparison with p < 0.033 (*), p < 0.002 (**), < 0.0002 (***) and < 0.0001

[0041] Figure 3: Reproducible expansion of PP cells in condition 5. (A) Growth curves and regression analysis for PP cells expanded in CO, C1 and C5 for at least 10 passages. The doubling time (Td) of C5-expanded cells (n = 7) was 2.3 days with a 95% confidence interval (Cl) of 2.13-2.51 days. This was clearly increased compared to CO- (n = 2, Td = 3.92 days, 95% Cl = 3.22-4.98 days) and C1-expanded cells (n=2, Td=3.55 days, 95% Cl = 2.88-4.62 days). The translucent shading represents the 95% Cl of the growth rate of the12RECTIFIED SHEET (RULE 91) ISA / EPdifferent conditions. (B-D) Gene expression profile of CO-, C1- and C5-expanded cells as shown by qPCR for expression of the key pancreas progenitor markers PDX1 (B), NKX6.1 (C) and S0X9 (D) during the expansion. Expression is normalized against expression of each marker at pO. (E-G) Representative images of immunofluorescent staining of pO PR cells (E) as well as C5-expanded cells at p5 (F) and pl 0 (G) for the PP transcription factors PDX1, NKX6.1 and SOX9. (H-J) Flow cytometry analysis of pO PP cells (H), as C5-expanded cells at p5 (I) and P10 (J) for PDX1, NKX6.1 and SOX9. (K) Cumulative results of the flow cytometry analyses for PDX1 + / SOX9+ and PDX1 + / SOX9+ / NKX6.1 + C5-expanded cells at pO, p5 and p10. Horizontal lines represent the mean ± SD. Statistical tests were two-way ANOVA with Tukey's test, using pO as the control condition for the comparison with p < 0.033 (*), p < 0.002 (**), <0.0002 (***) and < 0.0001 (****). Scale bar corresponds to 50 pm.

[0042] Figure 4: PP expansion conditions promote primarily proliferation rather than survival of PP cells. (A, B) Histogram plots showing the % of cells that had incorporated EdU-Alexa488 in the CINIexpanded PP cells (A) in comparison to the C5-expanded PP cells (B). (C) Summary of flow cytometry data comparing proliferation using EdU-Alexa488 incorporation (n=4) as well as cell death by Annexin V / 7-AAD staining in INI- and C5- expanded PP cells (n=6). Horizontal lines represent mean ± SD. Means were compared with multiple t-tests and significance is p < 0.033 (*), p < 0.0021 (**), p < 0.0002 (***) or p < 0.0001****)

[0043] Figure 5: Reproducible expansion in condition 6 promotes PP identity. (A)Growth curves and regression analysis for PP cells expanded in C5- and C6 for ten passages. The regression line for C5 showed a doubling time of 2.3 d (n=7) compared to C6 with 2.5 d (n= 10). (B-D) Representative images of immunofluorescent staining of pO PP cells (B) as well as C6-expanded cells at p5 (C) and p 10 (D) for the PP transcription factors PDX1, NKX6.1 and SOX9. (E-G) Flow cytometry analysis of non-expanded pO PP cells (E) and C6-expanded cells at p5 (F) and plO for PDX1 + / SOX9+ / NKX6.1 + cells (G). (H, I) Cumulative results of the flow cytometry analyses for PDX1 + / SOX9+ and PDX1 + / SOX9+ / NKX6.1 + C6-expanded cells at pO, p5 and plO (H) and comparison of the % of C5- and C6- expanded PDX1 + / SOX9+ / NKX6.1 + cells at p5 and pl 0 (I). (J) Karyotyping of C6-expanded PP cells after sixteen passages showed no chromosomal abnormalities. Horizontal lines represent the mean ± SD. Statistical tests were two-way ANOVA with Tukey's test, using pO as the control condition for the comparison13RECTIFIED SHEET (RULE 91) ISA / EPwith p < 0.033 (*), p < 0.002 (**), < 0.0002 (***) and < 0.0001 (’ ). Scale bar corresponds to50 pm.

[0044] Figure 6: Expansion stabilizes PP cell identity by repressing endocrine differentiation. (A, B) Correlation analyses of the transcriptome profiles of non-expanded (pO) and plO expanded PP cells (A) and the transcriptome profiles of p5 and p10 expanded cells (B). The numbers of upregulated and downregulated genes (normalized counts > 200 and 0.5 > FC > 2) are shown in red and blue, respectively and r is the correlation coefficient. (C) Most affected biological processes between pO and p10. (D) PCA of feeder expanded cells and corresponding pO cells (shades of green), fibronectin (FN) expanded cells and corresponding pO cells (shades of blue) as well as vitronectin-N (VTN-N) expanded cells and corresponding pO cells (shades of red). Darker shades correspond to earlier passages. (E) Expression levels of GP2 in normalized RNA-Seq counts. (F) GP2 immunofluorescence of p12 expanded PP cells. (G) Expression levels of progenitor and endocrine markers in normalized RNA-Seq counts.

[0045] Figure 7: Expansion of H9-derived and CRTD1 -derived PP cells and their differentiation containing functional p-cells. (A, B) Growth curve and regression analysis of the expansion of H9-derived PP cells (A) and CRTD1 -derived PPs cells (B). (C, D) Flow cytometry analysis for PDX1 + / SOX9+ and PDX1 + / SOX9+ / NKX6.1 + cells during the expansion in C6 of H9-derived PP cells (C) and CRTD1 -derived PP cells (D) at pO and during their expansion in C6 at p5 and pl 0. (E, F) Immunofluorescence analysis of SC-islets derived from pO PP cells (dPP) or expanded PP cells for at least ten passages (ePP) for INS and GCG expression (E) or INS and SST expression (F). (G) Percentages of INS+, INS+ / GCG+ as well as GCG+ cells in SC-islets derived from dPP or ePP cells as determined by flow cytometry. (H) Expression levels of NKX6.1, PDX1 and SLC30A8 in relation to expression levels in hPS cells (fold induction) as determined by qPCR. (I) Secretion of C-peptide following sequential stimulation by 16.7 mM glucose and 16.7 mM glucose / 30 mM KCI after exposure in basal conditions with 2.8 mM glucose. Stimulation index is determined by the ratio of secretion under these conditions to secretion in basal conditions. Horizontal lines represent the mean ± SD. Statistical tests were two-way ANOVA with Tukey's test, using pO as the control condition for the comparison with p < 0.033 (*), p < 0.002 (**), < 0.0002 (***) and< 0.0001 (****). Scale bar corresponds to 50 um. Scale bar corresponds to 100 pm (E, F).14RECTIFIED SHEET (RULE 91) ISA / EP

[0046] Figure 8: Initial expansion and differentiation of H1 -PP cells. (A) Differentiation scheme to PP cells and differentiation of expanded cells using the ALI approach.

[0047] Figure 9: Regulated genes and signaling pathways during PP expansion under initial conditions. (A-C) Enrichment plots for regulated genes (pO vs P10) of the TGF-B signaling pathway (A) E2F target genes (B) and DNA replication (C) showing a negative correlation of the TGF-B pathway with the expansion but a positive correlation of E2F target genes and DNA replication with the expansion. (D-F) Average transcript levels in normalized RNA-Seq counts of pO (n=4), p5 (n=3) and p 10 (n =3) PP cells for genes encoding ligands and receptors of the TGF-B (D), PDGF (E), RA and NOTCH signaling pathways (F). (G, H) Average transcript levels in normalized RNA-Seq counts of the liver marker AFP (G) and gut marker CDX2 (H). Horizontal lines represent the mean ± SD. Statistical tests were one-way ANOVA using pO as the control condition for the comparison with p < 0.033 (*), p < 0.002 (**), < 0.0002 (***) and < 0.0001 (****).

[0048] Figure 10: Reproducible expansion of PP cells in condition 5. (A-D) Gene expression profile of C0-, C1 - and C5-expanded cells as shown by qPCR for the PP markers PTF1A (A) and FOXA2 (B) as well as the liver marker AFP (C) and the gut marker CDX2 (D). Expression is normalized against expression of each marker at pO. (E-J) Representative images of immunofluorescent staining of non-expanded pO PP cells and C5-expanded cells at p5 and p10 for FOXA2 (E-G) and AFP / CDX2 expression (H-J).(K-M) Flow cytometry analysis of non-expanded pO PP cells and C5-expanded cells at p5 and plO for PDX1 + / SOX9+ cells. (N) Flow cytometry analysis of PP cells incubated with only secondary antibodies. Horizontal lines represent the mean ± SD. Statistical tests were two- way ANOVA with Tukey's test, using pO as the control condition for the comparison with p < 0.033 (*), p < 0.002 (**), < 0.0002 (***) and < 0.0001 (****). Scale bar corresponds to 50 pm.

[0049] Figure 11: PP expansion conditions promote primarily proliferation rather than survival of PP cells. (A) Cells with no EdU incorporation but processed as the sample were used to set the threshold for the assay. (B, C) Dot plot diagrams of the cell death flow cytometry assays showed dead cells staining for both Annexin V-FITC and 7-AAD in the upper right quadrant using CINI- (B) and C5- (C) expanded PP cells.15RECTIFIED SHEET (RULE 91) ISA / EP

[0050] Figure 12: Reproducible expansion in condition 6 promotes PP identity. (A) Gene expression profile of C6-expanded cells relative to C5 at p5 (green) and p10 (red) as shown by qPCR. (B) The expression of the hepatic markers AFP, TTR and HHEX, as determined by qPCR, were significantly lower in C6 than in C5 by plO. Fold induction was calculated with reference to expression levels at the PS cell stage. (C-H) Representative images of immunofluorescent staining of non-expanded pO PP cells and C6-expanded cells at p5 and pl 0 for FOXA2 (C-E) and AFP / CDX2 expression (F-H). (I-K) Flow cytometry analysis of non-expanded pO PP cells and C6-expanded cells at p5 and p10 for PDX1 + / SOX9+ cells. (I-K) Flow cytometry analysis of non-expanded pO PP cells (I) and C6-expanded cells at p5 (J) and p10 (K) for PDX1 + / SOX9+ cells. (L) Flow cytometry analysis of PP cells incubated with only secondary antibodies. Horizontal lines represent the mean ± SD. Statistical tests were two-way ANOVA with Tukey's test, using pO as the control condition for the comparison with p < 0.033 (*), p < 0.002 (**), < 0.0002 (***) and < 0.0001 (****). Scale bar corresponds to 50 pm.

[0051] Figure 13: Expansion stabilizes PP cell identity by repressing endocrine differentiation. (A) PCA analysis of our pO as well as p5 and pl O expanded PP cells. (B) Correlation analyses of the transcriptome profile of pO non-expanded cells and p5 expanded PP cells. The numbers of upregulated and downregulated genes (normalized counts > 200 and 0.5 > FC > 2) are shown in red and blue, respectively and r is the correlation coefficient. (C) Most affected molecular functions, cellular compartments and KEGG pathways between pO and p10. (D) PCA of feeder expanded cells and corresponding pO cells (shades of green), fibronectin (FN) expanded cells and corresponding pO cells (shades of blue), vitronectin-N (VTN-N) expanded cells and corresponding pO cells (shades of red) as well as FACS isolated subpopulations of human fetal pancreas progenitor cells (shades of grey). Darker shades correspond to earlier cells. (E) Heat map of the genes found to drive the difference between our expanded PP cells and other, in vitro derived PP cells, using the variance stabilizing transformation. Darker shades of red correspond to stronger upregulation. (F) Most affected molecular functions, cellular compartments, biological processes and KEGG pathways in genes that separate our expanded PP cells from other in vitro derived PP cells. (G) Heat map of the expression levels of duct progenitor and differentiated cells based on RNASeq analyses. (H) Alignment of the mouse Nkx6.1 peptide, used to generate the NKX6.1, with the human NKX6.1 and NKX6.2 sequences (e.g., mouse16RECTIFIED SHEET (RULE 91) ISA / EPNkx6.1 sequence can be derived from mouse Nkx6.1 having Uniprot accession number: Q99MA9, human NKX6.1 sequence can be derived from human NKX6.1 having Uniprot accession number P78426; human NKX6.2 sequence can be derived from human NKX6.2 having Uniprot accession number: A6NCS4). (I) Expression levels of progenitor and endocrine markers in normalized RNA-Seq counts.

[0052] Figure 14: Expansion of H9-derived and CRTD1 -derived PP cells and their differentiation containing functional p-cells. (A-G) Gene expression profile of PP cells derived from H1, H9 and the CRTD1 hPS cells at pO, p5 and plO as shown by qPCR for the progenitor markers PDX1 (A), NKX6. 1 (B), SOX9 (C) FOXA2 (D) and PTF1A (E) as well as for the liver marker AFP () and the gut marker CDX2 (G). Expression levels are normalized against the expression levels of H1-PP cells at the corresponding passages (pO, p5 and p10). (H, I) Karyotyping of C6-expanded H9-PP cells after twelve passages (H) and C6-expanded CRTD1 -PP cells after thirteen passages (I) showed no chromosomal abnormalities. (J, K) Immunofluorescence analysis of SC-islets for INS / NKX6.1 expression (J) or INS / MAFA expression (K) derived from pO PP cells (dPP) or expanded PP cells for at least ten passages (ePP). (L, M) Representative flow cytometry analysis of SC-islet cells incubated with only secondary antibodies (L) or with both C-PEP and GCG primary and corresponding secondary antibodies (M). (N) Expression levels of INS, GCG, MAFA, and SST in relation to expression levels in hPS cells (fold induction) as determined by qPCR. Horizontal lines represent the mean ± SD. Statistical tests were two-way ANOVA with Tukey's test, using pO as the control condition for the comparison with p < 0.033 (*), p < 0.002 (**), < 0.0002 (***) and < 0.0001 (****). Scale bar corresponds to 100 pm (J, K).

[0053] Figure 15: Titration of the effects of the BMP receptor inhibitor K02288 in expanding PP cells. PP cells derived from the differentiation of H1 hPS cells were expanded for five passages as described in the SOP in the absence or presence of different amounts of K02288. At the end of that period expression of the PP markers PDX1, NKX6.1 and SOX9 as well as of the liver marker AFP and the gut marker CDX2 was assessed by qRT-PCR. It was determined that K02288 at 0.25 M is effective in repressing expression of the liver and gut markers without affecting the expression of the PP markers, p < 0.05 (*), p < 0.005 (**), p < 0.0005 (***), p < 0.0001 (****).17RECTIFIED SHEET (RULE 91) ISA / EP

[0054] Figure 16: Titration of the effects of the BMP receptor inhibitor K02288 in expanding PP cells. PP cells derived from the differentiation of H1 hPS cells were expanded for five passages as described in the SOP in the absence or presence of different amounts of K02288. At the end of that period expression of the PP markers PDX1, NKX6.1 and SOX9 as well as of the liver marker AFP and the gut marker CDX2 was assessed by qRT-PCR. It was determined that K02288 at 0.25 M effectively represses the expression of the liver and gut markers without affecting the expression of the PP markers, p < 0.05 (*), p < 0.005 (**), p < 0.0005 (***), p < 0.0001 (****).DETAILED DESCRIPTION OF THE INVENTION

[0055] As described herein references can be made to UniProtKB Accession Numbers(http: / / www.uniprot.org / , e.g., as available in UniProt release 2023_02 (published May 03 2023).

[0056] The following detailed description refers to the accompanying Examples and Figures that show, by way of illustration, specific details and embodiments, in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilized such that structural, logical, and eclectic changes may be made without departing from the scope of the invention. Various aspects of the present invention described herein are not necessarily mutually exclusive, as aspects of the present invention can be combined with one or more other aspects to form new embodiments of the present invention.

[0057] As used herein, the term "pluripotent stem cells" (or "PSCs") may refer to cells capable of differentiating into cells of any type of tissue, e.g., which could be obtained by means other than the destruction of an embryo (e.g., human embryo) (e.g., Yu et al., 2007, Science. 2007 Dec 21;318(5858): 1917-20). Any suitable pluripotent stem cells can be used according to the invention (see herein for examples). Exemplary pluripotent stem cells include, but are not limited to: H1 cells having accession number: WA01 (hPSCReg-ID: WAe001 -A), H9 cells having accession number: WA09 (hPSCReg-ID: WAe009-A), CRTD1 cells having accession number: CRTDi004-A (hPSCreg-ID: CRTDi004-A), HUES4 cells (HUES-4; HUES4; HuES4; HVRDeOO4-A) or STBCi322-A (RRID:CVCL_ZX55) etc.18RECTIFIED SHEET (RULE 91) ISA / EP

[0058] As used herein, the term "embryonic stem cells" (or "ESCs") may refer to embryonic pluripotent stem cells that may be produced without destroying an embryo (e.g., human embryo) (e.g., Chung et al. Cell Stem Cell, February 2008, Vol. 2, pages 113-117).

[0059] As used herein, the term "induced pluripotent stem cells" (or "iPSCs") may refer to pluripotent stem cells that can be generated directly from adult cells (e.g., Yu et al., 2007), i.e., they are obtainable without destruction of embryos, e.g., they are obtainable from somatic cells (e.g., fibroblasts).

[0060] As used herein, the term "pancreatic progenitors" (or "PP") may refer to multipotent stem cells, e.g., originating from the developing fore-gut endoderm or cell lines, which have the ability to differentiate into the lineage specific progenitors responsible for the developing pancreas. They give rise to both the endocrine and exocrine cells.

[0061] As used herein, the term "expanding" may refer to an in vitro or ex vivo process of growing and proliferating cells derived from tissues or cell lines.

[0062] As used herein, the term "PDX1" may refer to Pancreas / duodenum homeobox protein1, e.g., having UniProt Accession Number: P52945.

[0063] As used herein, the term "SOX9" may refer to Transcription factor SOX-9, e.g., having UniProt Accession Number: P48436.

[0064] As used herein, the term "NKX6.1" may refer to Homeobox protein Nkx-6.1, e.g., having UniProt Accession Number: P78426.

[0065] As used herein, the term "GP2" may refer to Pancreatic secretory granule membrane major glycoprotein GP2, e.g., having UniProt Accession Number: P55259.

[0066] The term "polypeptide" is equally used herein with the term "protein". Proteins (including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids) comprise one or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids). The term "polypeptide" as used herein describes a group of molecules, which, for example, consist of more than 30 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. An example for a heteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical19RECTIFIED SHEET (RULE 91) ISA / EPheavy polypeptide chains. The terms "polypeptide" and "protein" also refer to naturally modified polypeptides / proteins wherein the modification is effected e.g. by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.

[0067] The term "patient" or "subject" as used herein may refer to a human or non-human animal, generally a mammal. Particularly envisaged is a mammal, such as a rabbit, a mouse, a rat, a Guinea pig, a hamster, a dog, a cat, a pig, a cow, a goat, a sheep, a horse, a monkey, an ape or preferably a human. Thus, the methods, uses and compounds described in this document are in general applicable to both human and veterinary disease.

[0068] The term "treatment" in all its grammatical forms includes therapeutic or prophylactic treatment. A "therapeutic or prophylactic treatment" comprises prophylactic treatments aimed at the complete prevention of clinical and / or pathological manifestations or therapeutic treatment aimed at amelioration or remission of clinical and / or pathological manifestations of the diseases. The term "treatment" thus also includes the amelioration or prevention of diabetes.

[0069] Dose: Preferably, a therapeutically effective amount of the product of the present invention (e.g., isolated PP cell (e.g., expanded (e.g., in vitro or ex vivo expanded) PP (ePP) cells) or an isolated population of PP cells (e.g., population of expanded (e.g., in vitro or ex vivo expanded) PP (ePP) cells, e.g., preferred product of the present invention is / are differentiated cells (e.g., SC-islets) that are derived from the expanded PP cells, e.g., both the ePP cells as well as their SC-islets derived from them can be micro-encapsulated or macroencapsulated before administration) as described herein is administered, e.g., expanded PP cells of the present invention can be used to treat diabetes, e.g., said cells are capable of differentiating into islet cells including beta cells after transplantation, and / or said cells can also be differentiated in vitro into SC-islets, e.g., which can be subsequently transplanted. By "therapeutically effective amount" is meant an amount of the product as described herein that elicits a therapeutic effect. The exact dose of cell / s, composition, preparation of the present invention will depend on the purpose of the treatment (e.g. remission maintenance vs. treatment of acute flare of the disease), and will be ascertainable by one skilled in the art using known techniques. Adjustments for route of administration, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition20RECTIFIED SHEET (RULE 91) ISA / EPmay be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

[0070] Administration: a variety of routes are applicable for administration of the product according to the present invention (e.g., isolated PP cell (e.g., expanded (e.g., in vitro or ex vivo expanded) PP (ePP) cells) or an isolated population of PP cells (e.g., population of expanded (e.g., in vitro or ex vivo expanded) PP (ePP) cells, e.g., preferred product of the present invention is / are differentiated cells (e.g., SC-islets) that are derived from the expanded PP cells, e.g., both the ePP cells as well as their SC-islets derived from them can be micro-encapsulated or macro-encapsulated before administration), including, but not limited to, orally, topically, transdermally, subcutaneously, intravenously, intraperitoneally, intramuscularly or intraocularly. However, any other route may readily be chosen by the person skilled in the art if desired.

[0071] Kit: a kit is also provided herein. The kit may be a kit of two or more parts, and comprises the cell / s of the present invention, preferably in a therapeutically effective amount and in a pharmaceutically acceptable form. The components of the kit may be contained in a container or vials. The kit is envisaged to comprise additional agents useful in treating diabetes, as described elsewhere herein (e.g., expanded PP cells of the present invention can be used to treat diabetes, e.g., said cells are capable of differentiating into islet cells including beta cells after transplantation, and / or said cells can also be differentiated in vitro into SC- islets, e.g., which can be subsequently transplanted). Exemplary additional agents include, without limitation, metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin and insulin derivatives (insulin glulisine, insulin lispro, insulin aspart, insulin glargine, insulin detemir, insulin isophane), and combinations thereof.

[0072] Accordingly, in one aspect the present invention relates to an in vitro or ex vivo method for expanding pluripotent stem (PS) cells-derived (e.g., induced pluripotent cells (iPSs)-derived) pancreatic progenitors (PP), preferably human PPs (e.g., expressing PDX1+ / SOX9+ / NKX6.1+, e.g., having corresponding UniProt Accession Numbers:P52945 / P48436 / P78426), e.g., H1-PP, H9-PP or CRTD1-PP cells): said method comprising: (i) providing PS cells-derived pancreatic progenitors (PP), e.g., providing H1 -PP (e.g., H1 cells- derived PPs, e.g., H1 cells having accession number: WA01 (hPSCReg-ID: WAe001 -A)), H9-PP (e.g., H9 cells derived PPs, e.g., H9 cells having accession number: WA09 (hPSCReg-ID:21RECTIFIED SHEET (RULE 91) ISA / EPWAe009-A) or CRTD1-PP cells (e.g., CRTD1 cells-derived PPs, e.g., CRTD1 cells having accession number: CRTDiOO4-A (hPSCreg-ID: CRTDi004-A); (a) activating MAPK (Mitogen- activated protein kinase) signaling pathways in said cells by the means of adding one or more of the MAPK activators comprising: (i') one or more FGF (Fibroblast growth factor) molecules, preferably comprising one or more FGF18 molecules, e.g., having UniProt Accession Number: 076093); (ii') a combination of one or more FGF molecules (preferably FGF18, e.g., having UniProt Accession Number: 076093) and one or more EGF (Epidermal growth factor) (e.g., having UniProt Accession Number: P01133) molecules and / or (iii') a combination of one or more FGF18 molecules (e.g., having UniProt Accession Number: 076093), one or more EGF molecules (e.g., having UniProt Accession Number: P01133) and one or more FGF2 (e.g., having UniProt Accession Number: P09038) molecules; (b) inhibiting and / or disabling and / or modulating retinoic acid signaling in said cells, preferably by the means of using a serum-free and / or animal product free (e.g., GMP-compliant) supplement without Vitamin A for cell cultivation; (c) inhibiting and / or disabling and / or modulating TGF-f (Transforming growth factor beta) signaling pathway in said cells, e.g., by the means of adding one or more inhibitor / s that block TGF-pi, -f>2, and / or -|33 ligands (e.g., StemMACS RepSox inhibitor) and adding one or more inhibitor / s that block BMP-2 (Bone Morphogenetic Protein-2, e.g., having UniProt Accession Number: P12643), BMP-4 (Bone Morphogenetic Protein-4, e.g., having UniProt Accession Number: P12644) and / or BMP-7 (Bone Morphogenetic Protein-7, e.g., having UniProt Accession Number: P18075) signaling (e.g., K02288 inhibitor) and (d) inhibiting Wnt signaling pathway (e.g., canonical Wnt signaling) in said cells, preferably by the means of adding one or more Wnt / |3-Catenin inhibitors (e.g., Endo-IWR-1); (ii) expanding said cells, preferably said expanding is carried out using a recombinant truncated vitronectin (VTN-N) coating, further preferably said expanding is carried out using fibronectin coating.

[0073] In one aspect the present invention relates to the method of the present invention, wherein said PPs are human PPs expressing PDX1+ / SOX9+ / NKX6.1+(e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426), preferably said PPs are derived from H1, H9 or CRTD1 cell lines: said method comprising: (ii) providing hPS cells derived pancreatic progenitors (PP), preferably H1 -PP, H9-PP or CRTD1 -PP cells; (a) activating MAPK signaling pathways in said cells by the means of adding one or more of the MAPK activators, preferably EGF and / or FGF18 and / or FGF2 molecules (e.g., EGF and FGF18 and FGF2); (b) inhibiting retinoic acid signaling in said cells, preferably by the means of using a22RECTIFIED SHEET (RULE 91) ISA / EPserum-free supplement without Vitamin A for cell cultivation; (c) inhibiting TGF[3 signaling pathway in said cells, preferably by the means of adding one or more inhibitor / s that block TGF-ftl ligands (e.g., StemMACS RepSox inhibitor) and / or one or more BMP-2 inhibitors (e.g., K02288 inhibitor) and (d) inhibiting Wnt signaling pathway in said cells, preferably by the means of adding one or more Wnt / p-Catenin inhibitors (e.g., Endo-IWR-1); (ii) expanding said cells, preferably said expanding is carried out using a recombinant truncated vitronectin (VTN-N) coating.

[0074] In one aspect the present invention relates to the method of the present invention, wherein said method is capable of: (a) upregulating the expression of a PP marker GP2 (Pancreatic secretory granule membrane major glycoprotein GP2, e.g., having UniProt Accession Number: P55259) in said cells, preferably said upregulating is detectable on the RNA level (e.g., up to 140-fold induction) and / or on the protein level (e.g., from about 5% to about 70% upregulation); and / or (b) producing (e.g., produces) PDX17SOX97NKX6.1+ / GP2+(e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426 / P55259) PP cells.

[0075] In one aspect the present invention relates to the method of the present invention, wherein said method is characterized by one or more of the following: (a) said method produces and / or expands (e.g., selectively expands) and / or enriches (e.g., selectively enriches) PDX1+ / SOX9+ / NKX6.1+PP cells; (b) said method is capable of enriching (e.g., increasing the amount / concentration) of the PDX1+ / SOX9+ / NKX6.1+PP cells in pancreatic progenitors (PP) cell culture; (c) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are pancreatic progenitors (PPs) capable of proliferation (e.g., their proliferation ability is active / un-inhibited) while inhibiting differentiation and / or inhibiting endocrine differentiation of said PPs; (d) said progenitor cells are human pluripotent stem (hPS), preferably iPSs, cells derived pancreatic progenitors (PP), wherein said method is capable of expanding said hPS cell derived PP cells while suppressing their differentiation; (e) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into islet-like clusters (SC-islets), preferably insulin producing SC-islets (e.g., said SC-islets are capable of producing the essentially same amount of insulin as in vitro derived non-expanded PPs as defined in claim 1 (i) and / or having the essentially same amount of differentiated endocrine cells in said SC-islets; (f) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor 23RECTIFIED SHEET (RULE 91) ISA / EPcells are capable of differentiating into insulin-producing cells, preferably said cells are capable of secreting insulin in the presence of glucose; (g) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into endocrine cells, preferably pancreatic endocrine cells, including insulinproducing beta cells; (h) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of cryopreservation (e.g., at least about - 195°C); (i) said method does not comprise the use of a feeder layer (e.g., GMP-compliant); (j) said method comprises the use of PP grown in a monolayer.

[0076] In one aspect the present invention relates to the method of the present invention, wherein said progenitor cells are human pluripotent stem (hPS) cell derived pancreatic progenitors (PP).

[0077] In one aspect the present invention relates to the method of the present invention, wherein said method is one or more of the following: said method is not limited by a number of cell divisions of said progenitor cells; said method is a method for unlimited expansion of said progenitor cells; said method is capable of producing (e.g., produces) progenitor cells that are capable of avoiding replicative senescence and / or replicative exhaustion, preferably continuously avoiding replicative senescence and / or replicative exhaustion; said method is capable of producing (e.g., produces) progenitor cells that are capable of avoiding arrest of cell division, preferably continuously avoiding arrest of cell division; said method is capable of producing (e.g., produces) progenitor cells that are capable of proliferation, preferably continuous proliferation; said method is capable of producing (e.g., produces) progenitor cells that are capable of self-renewal, preferably continuous self-renewal; said method is capable of producing (e.g., produces) progenitor cells that are capable of indefinitely renewing itself; said method is capable of producing (e.g., produces) progenitor cells that are capable of expansion, preferably continuous expansion; said method is capable of producing (e.g., produces) progenitor cells that are capable of differentiation (e.g., into pancreatic endocrine cells), while suppressing their differentiation; said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into islet-like clusters (SC-islets); said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are pancreatic progenitors capable of proliferation (e.g., their proliferation ability is active / un-inhibited), wherein said method is simultaneously capable of inhibiting the endocrine, hepatic and / or gut differentiation in said 24RECTIFIED SHEET (RULE 91) ISA / EPcells; said method is capable of producing (e.g., produces) progenitor cells that are capable of maintaining their differential potential, preferably continuously maintaining their differential potential; said method does not comprise the use of artificial modification (e.g., siRNA) of the genome of said progenitor cells.

[0078] In one aspect the present invention relates to the method of the present invention, wherein said method is capable of producing (e.g., produces) at least 100-fold (e.g., 500, 700, 800, 900, 1000-fold, or 2000-fold) expansion of progenitor cells (e.g., PP cells), preferably said expansion is carried out during about at least 5 passages (e.g., 10 passages) and / or in a period from about 20 (e.g. 40) to about 25 (e.g., 45) days) and / or said progenitor cells comprising at least about 50% (e.g., at least about 60%, at least about 70%, at least about80%, at least about 90%) of PDX1 + / SOX9+ / NKX6.1 + cells (e.g., PDX1 + / SOX9+ / NKX6.1 + / GP2+).

[0079] In one aspect the present invention relates to an isolated PP cell or an isolated population of PP cells, wherein said cell / s are PDX1+ / SOX9+ / NKX6.1+cell / s (e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426), wherein said cells are capable of indefinitely renewing themselves, wherein said cell / s further having one or more of the following characteristics: said cell / s expressing PP marker GP2 (Pancreatic secretory granule membrane major glycoprotein GP2, e.g., having UniProt Accession Number: P55259); said cells are capable of proliferation while having their differentiation inhibited / repressed; said cells are capable of unlimited expansion; optionally, said cells are obtainable (e.g., obtained) from pluripotent stem cells (e.g., iPSs), preferably providing human pluripotent stem (hPS) cells, further preferably providing hPS cells derived pancreatic progenitors (PP), e.g., H1-PP, H9-PP or CRTD1-PP cell lines.

[0080] In one aspect the present invention relates to a population of progenitor cells or a progenitor cell produced and / or expanded and / or modified by method of the present invention, preferably said cells are PDX1+ / SOX9+ / NKX6.1+cells (e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426).

[0081] In one aspect the present invention relates to the population of progenitor cells of the present invention, wherein population comprising at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%) of PDX1+ / SOX9+ / NKX6.1+25RECTIFIED SHEET (RULE 91) ISA / EPcells, preferably said method of the present invention comprising activating MAPK signaling pathways in said cells by the means of adding MAPK activators EGF, FGF18 and FGF2.

[0082] In one aspect the present invention relates to a composition, preparation or kit comprising the population of progenitor cells (e.g., isolated) or the progenitor cell (e.g., isolated) of the present invention.

[0083] In one aspect the present invention relates to the composition, preparation or kit of the present invention, wherein said composition, preparation or kit is a pharmaceutical and / or diagnostic composition, preparation or kit.

[0084] In one aspect the present invention relates to the population of progenitor cells (e.g., isolated), a progenitor cell (e.g., isolated), composition, preparation or kit of the present invention, for use as a medicament and / or in therapy.

[0085] In one aspect the present invention relates to the population of progenitor cells (e.g., isolated), a progenitor cell (e.g., isolated), composition, preparation or kit of the present invention for use in one or more of the following methods: method of treatment, amelioration, prophylaxis and / or diagnostics of an endocrine disease (e.g., Diabetes, Type 1 Diabetes, Type 2 Diabetes, Gestational Diabetes, Mature Onset Diabetes of the Young (MODY)); method of treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; method for monitoring development and / or assessing the efficacy of prophylaxis and / or therapy of Diabetes, preferably Type 1 Diabetes; method for screening a candidate compound for use in method of treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; method of regenerative treatment of a cell, tissue, organ and / or body; method of transplantation (e.g., beta-cell transplantation); method of cell therapy (e.g., beta-cell therapy); disease modelling (e.g., diabetes modelling); method of insulin production and / or insulin supplementation; method according to any one of the preceding claims; any combination of methods as above; method as above, wherein said method is an in vitro, ex vivo or in vivo method.

[0086] In one aspect the present invention relates to a use of the population of progenitor cells (e.g., isolated), a progenitor cell (e.g., isolated), e.g., isolated PP cell (e.g., expanded (e.g., in vitro or ex vivo expanded) PP (ePP) cells) or an isolated population of PP cells (e.g., population of expanded (e.g., in vitro or ex vivo expanded) PP (ePP) cells, e.g., preferred product of the present invention is / are differentiated cells (e.g., SC-islets) that are derived 26RECTIFIED SHEET (RULE 91) ISA / EPfrom the expanded PR cells, e.g., both the ePP cells as well as their SC-islets derived from them can be micro-encapsulated or macro-encapsulated before administration), composition, preparation or kit of the present invention for one or more of the following: for treatment, amelioration, prophylaxis and / or diagnostics of an endocrine disease (e.g., Diabetes, Type 1 Diabetes, Type 2 Diabetes, Gestational Diabetes, Mature Onset Diabetes of the Young (MODY)); for treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; for monitoring development and / or assessing the efficacy of prophylaxis and / or therapy of Diabetes, preferably Type 1 Diabetes; for screening a candidate compound for use in method of treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; for regenerative treatment of a cell, tissue, organ and / or body; for transplantation (e.g., beta-cell transplantation); for cell therapy (e.g., betacell therapy); for insulin production and / or insulin supplementation; disease modelling (e.g., diabetes modelling); e.g., expanded PP cells of the present invention can be used to treat diabetes, e.g., said cells are capable of differentiating into islet cells including beta cells after transplantation, and / or said cells can also be differentiated in vitro into SC-islets, e.g., which can be subsequently transplanted; use as above; any combination of methods as above; for any use as above, wherein said use is an in vitro, ex vivo or in vivo use.

[0087] The unlimited expansion of progenitor cells can offer many therapeutic advantages but remains an important challenge for regenerative medicine. Progenitor cells are dynamic entities and the key objective in these efforts is to uncouple survival and proliferation from widely employed feed-forward mechanisms that promote their differentiation. The latter are not exclusively regulated from extrinsic signals but rely to a large extent on internal regulators as well as autocrine signals. Thus, the hallmarks of efficient progenitor expansion would be the maintenance of key progenitor features, the efficient suppression of differentiation programs and alternative lineages and the capacity to efficiently differentiate under appropriate conditions. To be suitable for therapeutic applications, such expansion should be efficient, reproducible, applicable across different cell lines and compatible with chemically defined culture media.

[0088] Regarding pancreatic development in particular, several feed-forward networks have been documented. In the early pancreatic progenitors, a Sox9 / Fgf feed-forward loop is essential to escape liver fate and promote pancreas identity and expansion. FgflO was identified as a possible extrinsic signal but it was shown that it also promotes liver identity. At 1RECTIFIED SHEET (RULE 91) ISA / EPa later stage, Ptf 1 a initially forms heterodimers with Rbpj to promote expansion of pancreatic progenitors and activate transcription of Rbpjl which eventually replaces Rbpj in its complexes with Rtfla. The Ptfla. Rbpjl complexes then promote the specification of acinar progenitors. Si p signaling in pancreas progenitors promotes progenitor survival but also their differentiation into the acinar and endocrine lineages through the attenuation of Notch signaling. Subsequently, the transcription factors Myt1 and Neurog3 form a feed-forward loop to promote the final commitment into the endocrine lineage.

[0089] However, the identity of signaling pathways that promote progenitor proliferation and of those initiating differentiation is unclear precluding a rational approach to expanding PP cells in vitro while blocking differentiation. To address this, we first undertook longitudinal transcriptome analyses and comparisons of non-expanding and, occasionally expanding PP cells. The analyses focused on components of signaling and several candidates were identified providing a rational, mechanistic approach to address the expansion of PP cells. We followed a hypothesis-driven, iterative, approach to identify conditions enabling the robust unlimited expansion of hPS cell derived PP cells under chemically defined conditions that are applicable for different hPS cell lines. We found that a combination of EGF, FGF2 and FGF18 promoted robust expansion of hPS cell derived PP cells. EGF promotes NKX6.1 activation but on its own it could not support consistent PP cell expansion (CINI, Figure 8) and thus we looked for additional mitogens. FGF2 is a widely used mitogen and whereas it enhanced PP proliferation (Cl) it was less efficient than FGF18 (C5, Figure 8). There was a clear synergy of the two mitogens in promoting expansion and possibly the enrichment in NKX6.1 + cells (C6, Figure 8). NKX6.1 regulates multiple cell cycle genes and thus some of the effects of these mitogens might be indirectly reinforced through NKX6.1. Pancreas progenitor cells express the enzymes necessary to convert vitamin A into retinoic acid (RA) (Figure 10) which promotes differentiation of the pancreas progenitors. Vitamin A is one of the B-27 components in its most common formulation and to suppress the differentiation drive of PP cells we used, in all conditions tested apart from CINI, the vitamin A free variant of B-27 (Figure 8). The TGFfJ signaling pathway plays a complex role in the induction, maintenance and endocrine differentiation of pancreas progenitors and the diverging gene expression kinetics of receptors and ligands during expansion in CINI reflected this complexity.Accordingly, our experiments suggested that the highly specific ALK5i II inhibitor (C5, C6) was more efficient than the widely specific A83-01 inhibitor (CINI) in promoting PP expansion.28RECTIFIED SHEET (RULE 91) ISA / EPFurther inhibition of the pathway with the addition of LDN193198 which targets ALK3 more efficiently than ALK5i II dramatically reduced PP expansion (C7, Figure 10). In line with the effects of these interventions affecting primarily the proliferation of PP cells, we documented that preferential expansion of progenitors rather than cell death was the main mechanism. Finally, the addition of the canonical Wnt inhibitor IWR-1 significantly reduced AFP expression and promoted a large enrichment in NKX6.1 + cells without affecting proliferation, presumably by reducing divergence of expanding cells towards the hepatic fate.

[0090] We report here that this is a robust procedure resulting in 2000-fold expansion of up to 90% PDX1 + / SOX9+ / NKX6.1 + PP cells after ten passages over 40-45 days. High levels of NKX6-1 expression suggested that the expanded cells may resemble more bipotent progenitors as in mice and it has also been proposed that high levels of NKX6-1 are necessary to produce |3 cells in humans. The same procedure applied to H9- and CRTD1 -PP cells gave essentially identical results, even though the initial differentiation of these hPS cells into PP cells appeared less efficient. This suggested that this procedure is advantageous also for hPS cell lines with reduced initial capacity to differentiate into PP cells. Of note, for all cell lines a high initial density was necessary in achieving robust early expansion.

[0091] Culture conditions to expand hPS cell-derived PP cells have recently been reported. The first relied on feeder layers of transformed cells and a BET bromodomain inhibitor to expand an, up to 90% PDX1 + / SOX9+ / NKX6.1 +, population of PP cells and the second relied on a simple expansion medium and fibronectin as a cell culture substrate. While the first is limited by the use of feeder layers, which preclude its use in a clinical setting, the second did not document reproducibility of the expansion procedure and the percentage of NKX6.1 + cells reached only 40% with large variability among different cell lines. Nevertheless, we compared their transcriptome profile with that of our ePP cells. Our procedure is unique in effecting a strong upregulation of GP2, a unique marker of human fetal pancreas progenitors with a high preference for endocrine differentiation and strong upregulation of NKX6.2, another marker of PP cells which complements NKX6.1 function but is not retained in differentiated endocrine cells. Expression of other progenitor markers such as FOXA2 and RBPJ were retained at similar but variable levels. Levels of TFs that are engaged in the duct program appeared higher in the FN expansion procedure whereas expression of acinar TFs was very low in all three procedures. As expected, the expression of TFs driving the endocrine program is strongly reduced in all three procedures albeit our procedure is more efficient in29RECTIFIED SHEET (RULE 91) ISA / EPthis respect, particularly with regard to RFX3 and RFX6 expression. Expression of AFP and HHEX, markers of the liver lineage, were significantly higher in the FN-expansion procedure whereas all three procedures resulted in substantial upregulation of CDX2, a gut marker.

[0092] I mportantly, ePP cells from H1, H9 and CRTD1 hPS cells all differentiated with similar efficiency to dPP cells into SC-islets containing similar numbers of B-cells of comparable functionality. This may appear surprising because ePP cells contained a much higher percentage of PDX1 + / SOX9+ / NKX6.1 + cells. It should be noted, however, that the liver marker AFP and, particularly, the gut marker CDX2 were upregulated, whereas expression of PTF1A, recently shown to promote endocrine differentiation of hPS cells, was essentially lost. The same pattern, and even higher upregulation of gut and liver markers, was also seen in the other expansion procedures. Additionally, the strong upregulation of NKX6.2 in our procedure suggested that our ePP cells may have retracted to an earlier PP stage. Thus, means to suppress AFP and CDX2 expression during expansion and restore PTF1A expression during the resumption of the differentiation are expected to result in a very high percentage of endocrine cells in the SC-islets derived from expanded cells.

[0093] The unlimited expansion of PP cells reported here is applicable to different hPS cell lines and presents several advantages in the efforts to scale-up the generation of islet cells, including p-cells, for the cell therapy of diabetes. It reduces the number of differentiation procedures to be carried out starting at the hPS cell stage, thus eliminating a source of variability. It even allows the selection of the most optimally differentiated PP cell population for subsequent expansion and storage. Since it is currently acknowledged that current differentiation procedures do not produce fully functional p-cells, these ePP cells will provide a convenient springboard to refine downstream differentiation procedures. Suitable surface markers for the selection of endocrine cells at the end of the differentiation procedure have been reported but such procedures may prove to expensive in a clinical setting. Alternatively, expanded PP cells could be directly used for transplantations as it has been shown that they can mature in vivo. Whether PP cells or terminally differentiated SC-islet cells would be the best approach in future tansplantations is still discussed but, in any of these cases, the availability of a highly pure GMP-grade, hPS cell derived PP cell population has several advantages as discussed above. Therefore, expansion of PP cells will facilitate the generation of unlimited number of endocrine cells, initially for studying diabetes, drug screening for personalized medicine and eventually cell therapies. Thus, the chemically defined expansion30RECTIFIED SHEET (RULE 91) ISA / EPprocedure we report here will be an important step toward generating large numbers of human pancreatic endocrine cells that are of great interest for biomedical research and regenerative medicine.

[0094] ITEMS OF THE INVENTION

[0095] The present invention may also be summarized by the following items:1. An in vitro or ex vivo method for expanding pluripotent stem cells (PSCs)-derived (e.g., induced pluripotent cells (iPSCs)- or embryonic stem cells (ESCs)-derived) pancreatic progenitors (PP), preferably human PPs (e.g., expressing PDX1+ / SOX9+ / NKX6.1+genes / proteins, e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426), e.g., H1-PP, H9-PP or CRTD1-PP cells): said method comprising:(i) providing PS cells-derived pancreatic progenitors (PP) (e.g., isolated PPs), e.g., providing H1 -PP (e.g., H1 cells-derived PPs, e.g., H 1 cells having accession number: WA01 (hPSCReg-ID: WAe001 -A)), H9-PP (e.g., H9 cells derived PPs, e.g., H9 cells having accession number: WA09 (hPSCReg-ID: WAe009-A) or CRTD1 -PP cells (e.g., CRTD1 cells- derived PPs, e.g., CRTD1 cells having accession number: CRTDi004-A (hPSCreg-ID: CRTDiOO4-A) or other suitable PS cells-derived pancreatic progenitors (PP); a) activating MAPK (Mitogen-activated protein kinase) signaling pathways in said cells, e.g., by the means of adding one or more of the MAPK activators, e.g., comprising: (i') one or more FGF (Fibroblast growth factor) molecules, preferably comprising one or more FGF 18 molecules (e.g., FGF18 at a concentration from about 5 to about 20 ng / pl), e.g., having UniProt Accession Number: 076093); (ii') a combination of one or more FGF molecules (preferably FGF18, e.g., having UniProt Accession Number: 076093, e.g., FGF18 at a concentration from about 5 to about 20 ng / pl) and one or more EGF (Epidermal growth factor) (e.g., having UniProt Accession Number: P01133) molecules and / or (iii') a combination of one or more FGF1831RECTIFIED SHEET (RULE 91) ISA / EPmolecules (e.g., having UniProt Accession Number: 076093) e.g., FGF18 at a concentration from about 5 to about 20 ng / pl, one or more EGF molecules (e.g., having UniProt Accession Number: P01133) and one or more FGF2 (e.g., having UniProt Accession Number: P09038) molecules; b) inhibiting and / or disabling and / or modulating retinoic acid signaling in said cells, preferably by the means of using a serum-free and / or animal product free (e.g., GMP-compliant) supplement without Vitamin A for cell cultivation; c) inhibiting and / or disabling and / or modulating TGF-p (Transforming growth factor beta) signaling pathway in said cells, e.g., by the means of adding one or more inhibitor / s that block TGF- / 31, -f>2, and / or -p3 ligands (e.g., StemMACS RepSox inhibitor) and / or adding one or more inhibitor / s that block BMP-2 (Bone Morphogenetic Protein-2, e.g., having UniProt Accession Number: P12643), BMP-4 (Bone Morphogenetic Protein-4, e.g., having UniProt Accession Number: P12644) and / or BMP-7 (Bone Morphogenetic Protein-7, e.g., having UniProt Accession Number: P18075) signaling (e.g., K02288 inhibitor) and d) inhibiting Wnt signaling pathway (e.g., canonical Wnt signaling, e.g., (e.g., G0:0016055; e.g., Wnt / |3-catenin signaling pathway is the series of molecular signals initiated by binding of a Wnt protein to a frizzled family receptor on the surface of the target cell and ending with a change in cell state (Huelsken J, Birchmeier W. New aspects of Wnt signalling pathways in higher vertebrates. Current Opinion in Genetics & Development. 2001 Oct;11 (5):547-553.)) in said cells, preferably by the means of adding one or more Wnt / |3-Catenin inhibitors (e.g., Endo-IWR- D;RECTIFIED SHEET (RULE 91) ISA / EPii) expanding said cells, preferably said expanding is carried out using a recombinant truncated vitronectin (VTN-N) coating, further preferably said expanding is carried out using fibronectin coating.2. The method of any one of the preceding items, wherein said method is carried out under GMP-conditions (Good manufacturing practice, e.g., as set by FDA and / or EMA or other competent national or international authority, e.g., serum-free and / or animal product free and / or does not comprise the use of a feeder layer), preferably said method additionally comprises growing said PPs in a monolayer. 3. The method of any one of the preceding items, wherein said PPs are human PPs expressing PDX1+ / SOX9+ / NKX6.1+(e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426), preferably said PPs are derived from H1, H9 or CRTD1 cell lines: said method comprising:(i) providing hPS cells derived pancreatic progenitors (PP), preferably HI PP, H9-PP or CRTD1-PP cells; a) activating MAPK signaling pathways in said cells by the means of adding one or more of the MAPK activators, preferably EGF and / or FGF18 and / or FGF2 (e.g., EGF and FGF18 and FGF2); b) inhibiting retinoic acid signaling in said cells, preferably by the means of using a serum-free supplement without Vitamin A for cell cultivation; c) inhibiting TGFp signaling pathway in said cells, preferably by the means of adding one or more inhibitor / s that block TGF-ftl ligands (e.g., StemMACS RepSox inhibitor) and / or one or more BMP-2 inhibitors (e.g., K02288 inhibitor) and d) inhibiting Wnt signaling pathway in said cells, preferably by the means of adding one or more Wnt / |3-Catenin inhibitors (e.g., Endo-IWR-1); ii) expanding said cells, preferably said expanding is carried out using a recombinant truncated vitronectin (VTN-N) coating.4. The method of any one of the preceding items, wherein said method is capable of:33RECTIFIED SHEET (RULE 91) ISA / EPa) upregulating the expression of a PR marker GP2 (Pancreatic secretory granule membrane major glycoprotein GP2, e.g., having UniProt Accession Number: P55259) in said cells, preferably said upregulating is detectable on the RNA level (e.g., up to 140-fold induction) and / or on the protein level (e.g., from about 5% to about 70% upregulation); and / or b) producing (e.g., produces) PDX17SOX9 NKX6.17GP2+(e.g., having corresponding UniProt Accession Numbers:P52945 / P48436 / P78426 / P55259) PP cells. 5. The method of any one of the preceding items, wherein said method is characterized by one or more of the following: a) said method produces and / or expands (e.g., selectively expands) and / or enriches (e.g., selectively enriches) PDX17SOX97NKX6.1+PP cells; b) said method is capable of enriching (e.g., increasing the amount / concentration) of the PDX17SOX97NKX6.1+PP cells in pancreatic progenitors (PP) cell culture; c) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are pancreatic progenitors (PPs) capable of proliferation (e.g., their proliferation ability is active / un-inhibited) while inhibiting differentiation and / or inhibiting endocrine differentiation of said PPs; d) said progenitor cells are human pluripotent stem (hPS), preferably iPSs, cells derived pancreatic progenitors (PP), wherein said method is capable of expanding said hPS cell derived PP cells while suppressing their differentiation; e) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into islet-like clusters (SC-islets), preferably insulin producing SC-islets (e.g., said SC- islets are capable of producing the essentially same amount of insulin as in vitro derived non-expanded PPs as defined in claim 1 (i) and / or having the essentially same amount of differentiated endocrine cells in said SC- islets;34RECTIFIED SHEET (RULE 91) ISA / EPf) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into insulinproducing cells, preferably said cells are capable of secreting insulin in the presence of glucose; g) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into endocrine cells, preferably pancreatic endocrine cells, including insulinproducing beta cells; h) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of cryopreservation (e.g., at least about -195°C); i) said method does not comprise the use of a feeder layer (e.g., GMP- compliant); j) said method comprises the use of PP grown in a monolayer; k) said method is capable of producing (e.g., produces) PP cells that do not produce insulin and / or capable of differentiating into islet cells (e.g., including beta cells, e.g., after transplantation). l) said method is capable of producing (e.g., produces) PP cells capable of differentiating in vitro into SC-islets, (e.g., which can be subsequently transplanted). The method of any one of the preceding items, wherein said progenitor cells are human pluripotent stem (hPS) cell derived pancreatic progenitors (PP).The method of any one of the preceding items, wherein said method is one or more of the following: a) said method is not limited by a number of cell divisions of said progenitor cells; b) said method is a method for unlimited expansion of said progenitor cells; c) said method is capable of producing (e.g., produces) progenitor cells that are capable of avoiding replicative senescence and / or replicative35RECTIFIED SHEET (RULE 91) ISA / EPexhaustion, preferably continuously avoiding replicative senescence and / or replicative exhaustion; d) said method is capable of producing (e.g., produces) progenitor cells that are capable of avoiding arrest of cell division, preferably continuously avoiding arrest of cell division; e) said method is capable of producing (e.g., produces) progenitor cells that are capable of proliferation, preferably continuous proliferation; f) said method is capable of producing (e.g., produces) progenitor cells that are capable of self-renewal, preferably continuous self-renewal; g) said method is capable of producing (e.g., produces) progenitor cells that are capable of indefinitely renewing itself; h) said method is capable of producing (e.g., produces) progenitor cells that are capable of expansion, preferably continuous expansion; i) said method is capable of producing (e.g., produces) progenitor cells that are capable of differentiation (e.g., into pancreatic endocrine cells), while suppressing their differentiation; j) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into islet-like clusters (SC-islets); k) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are pancreatic progenitors capable of proliferation (e.g., their proliferation ability is active / un-inhibited), wherein said method is simultaneously capable of inhibiting the endocrine, hepatic and / or gut differentiation in said cells; l) said method is capable of producing (e.g., produces) progenitor cells that are capable of maintaining their differential potential, preferably continuously maintaining their differential potential; m) said method does not comprise the use of artificial modification (e.g., siRNA) of the genome of said progenitor cells; n) said method is capable of producing (e.g., produces) PP cells that do not produce insulin and / or capable of differentiating into islet cells (e.g., including beta cells, e.g., after transplantation).RECTIFIED SHEET (RULE 91) ISA / EPo) said method is capable of producing (e.g., produces) PP cells capable of differentiating in vitro into SC-islets, (e.g., which can be subsequently transplanted). The method of any one of the preceding items, wherein said method is capable of producing (e.g., produces) at least 100-fold (e.g., 500, 700, 800, 900, 1000-fold, or 2000- fold) expansion of progenitor cells (e.g., PP cells), preferably said expansion is carried out during about at least 5 passages (e.g., 10 passages) and / or in a period from about 20 (e.g. 40) to about 25 (e.g., 45) days) and / or said progenitor cells comprising at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%) of PDX17SOX97NKX6.F cells (e.g., PDX17SOX97NKX6.17GP2+). The method of any one of the preceding items, wherein said method is carried out as depicted in example 1, example 2 and / or example 3 herein (e.g., as depicted in any one of the Figures 1-16). An isolated PP cell (e.g., expanded (e.g., in vitro or ex vivo expanded) PP (ePP) cells) or an isolated population of PP cells (e.g., population of expanded (e.g., in vitro or ex vivo expanded) PP (ePP) cells), wherein said cell / s are PDX17SOX97NKX6.1+cell / s (e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426), wherein said cells are capable of indefinitely renewing themselves, wherein said cell / s further having one or more of the following characteristics: a) said cell / s expressing PP marker GP2 (Pancreatic secretory granule membrane major glycoprotein GP2, e.g., having UniProt Accession Number: P55259); b) said cells are capable of proliferation while having their differentiation inhibited / repressed; c) said cells are capable of unlimited expansion; d) optionally, said cells are obtainable (e.g., obtained) from pluripotent stem cells (e.g., iPSs), preferably providing human pluripotent stem (hPS) cells, further preferably providing hPS cells derived pancreatic progenitors (PP), e.g., H1 -PP, H9-PP or CRTD1 -PP cell lines.37RECTIFIED SHEET (RULE 91) ISA / EPThe isolated PP cell (e.g., expanded (e.g., in vitro or ex vivo expanded) PP (ePP) cells) or an isolated population of PP cells (e.g., population of expanded (e.g., in vitro or ex vivo expanded) PP (ePP) cells), according to any one of the preceding items, wherein said cells: a) do not produce insulin and / or capable of differentiating into islet cells (e.g., including beta cells, e.g., after transplantation); b) capable of differentiating in vitro into SC-islets, (e.g., which can be subsequently transplanted). The isolated PP cell (e.g., expanded (e.g., in vitro or ex vivo expanded) PP (ePP) cells) or an isolated population of PP cells (e.g., population of expanded (e.g., in vitro or ex vivo expanded) PP (ePP) cells), according to any one of the preceding items, produced by the method according to any one of the preceding items. A population of progenitor cells or a progenitor cell produced and / or expanded and / or modified by method according to any one of the preceding items, preferably said cells are PDX17SOX97NKX6.1+cells (e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426). The population of progenitor cells according to any one of the preceding items, wherein population comprising at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%) of PDX17SOX9+ / NKX6.1+cells, preferably said method comprising activating MAPK signaling pathways in said cells by the means of adding MAPK activators EGF, FGF18 and FGF2. The population of progenitor cells according to any one of the preceding items, wherein said population of progenitor cells is characterized as depicted in example 1 and or example 2 herein (and / or derived as disclosed elsewhere herein). A composition, preparation or kit comprising the population of progenitor cells (e.g., isolated) or the progenitor cell (e.g., isolated) according to any one of preceding items.38RECTIFIED SHEET (RULE 91) ISA / EP17. The composition, preparation or kit according to any one of the preceding items, wherein said composition, preparation or kit is a pharmaceutical and / or diagnostic composition, preparation or kit.18. The population of progenitor cells (e.g., isolated), a progenitor cell (e.g., isolated), composition, preparation or kit according to any one of the preceding items, for use as a medicament and / or in therapy and / or for use in a manufacture of a medicament. 19. A method of treatments of a subject (e.g., patient) in need thereof, comprising administering a therapeutically effective amount of the population of progenitor cells (e.g., isolated), a progenitor cell (e.g., isolated), composition, preparation or kit according to any one of the preceding items to said subject.20. The population of progenitor cells (e.g., isolated), a progenitor cell (e.g., isolated), composition, preparation or kit according to any one of the preceding items, for use in one or more of the following methods: a) method of treatment, amelioration, prophylaxis and / or diagnostics of an endocrine disease (e.g., Diabetes, Type 1 Diabetes, Type 2 Diabetes, Gestational Diabetes, Mature Onset Diabetes of the Young (MODY)); b) method of treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; c) method for monitoring development and / or assessing the efficacy of prophylaxis and / or therapy of Diabetes, preferably Type 1 Diabetes; d) method for screening a candidate compound for use in method of treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; e) method of regenerative treatment of a cell, tissue, organ and / or body; f) method of transplantation (e.g., beta-cell transplantation); g) method of cell therapy (e.g., beta-cell therapy); h) disease modelling (e.g., diabetes modelling) i) method of insulin production and / or insulin supplementation;39RECTIFIED SHEET (RULE 91) ISA / EPj) method of producing PP cells that do not produce insulin and / or capable of differentiating into islet cells (e.g., including beta cells, e.g., after transplantation); k) method of producing PP cells capable of differentiating in vitro into SC- islets, (e.g., which can be subsequently transplanted); l) method according to any one of the preceding items; m) any combination of methods according (a)-(l); n) method of any of (a)-(m), wherein said method is an in vitro, ex vivo or in vivo method. Use of the population of progenitor cells (e.g., isolated), a progenitor cell (e.g., isolated), composition, preparation or kit according to any one of the preceding items for one or more of the following: a) for treatment, amelioration, prophylaxis and / or diagnostics of an endocrine disease (e.g., Diabetes, Type 1 Diabetes, Type 2 Diabetes, Gestational Diabetes, Mature Onset Diabetes of the Young (MODY)); b) for treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; c) for monitoring development and / or assessing the efficacy of prophylaxis and / or therapy of Diabetes, preferably Type 1 Diabetes; d) for screening a candidate compound for use in method of treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; e) for regenerative treatment of a cell, tissue, organ and / or body; f) for transplantation (e.g., beta-cell transplantation); g) for cell therapy (e.g., beta-cell therapy); h) for insulin production and / or insulin supplementation; i) for producing PP cells that do not produce insulin and / or capable of differentiating into islet cells (e.g., including beta cells, e.g., after transplantation); j) for producing PP cells capable of differentiating in vitro into SC-islets, (e.g., which can be subsequently transplanted);40RECTIFIED SHEET (RULE 91) ISA / EPk) disease modelling (e.g., diabetes modelling) l) use according to any one of the preceding items; m) any combination of methods according (a)-(l); n) for any of (a)-(m), wherein said use is an in vitro, ex vivo or in vivo use.22. The method, use, isolated PP cell (e.g., expanded (e.g., in vitro or ex vivo expanded) PP (ePP) cells) or an isolated population of PP cells, composition, preparation or kit, population of progenitor cells or a progenitor cell according to any one of the preceding items, produced or carried out as described in example 1, 2 and / or 3 herein (e.g., as shown in any one of Figures 1-16 herein).

[0096] Unless otherwise stated, the following terms used in this document, including the description and items, have the definitions given below.

[0097] Those skilled in the art will recognize, or be able to ascertain, using not more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0098] It is to be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0099] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0100] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".41RECTIFIED SHEET (RULE 91) ISA / EP

[0101] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes, however, also the concrete number, e.g., about 20 includes 20.

[0102] Throughout this specification and the items which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having".

[0103] When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0104] In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

[0105] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the items.

[0106] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.EXAMPLES

[0107] The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the items.42RECTIFIED SHEET (RULE 91) ISA / EP

[0108] Example 1: Regulation of multiple signaling pathways promotes exponential expansion of human pancreas progenitors in chemically defined conditions

[0109] MATERIALS AND METHODS

[0110] Derivation of the CRTD1 human iPS cell line

[0111] The CRTD1 human iPS cell line (hPSCreg: CRTDI004-A) was generated from previously published foreskin fibroblasts (termed Theo) of a consenting healthy donor (Wolf et al., 2016). Isolation of cells and reprogramming to hiPS cells was approved by the ethics council of TU Dresden (EK169052010 und EK386102017). Theo fibroblasts were reprogrammed at the CRTD Stem Cell Engineering Facility at the Technical University of Dresden using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fischer Scientific A16517) according to the supplier's recommendations for transduction. Following transduction with the Sendai virus, cells were cultured on irradiated CF1 Mouse Embryonic Fibroblasts (Thermo Fisher Scientific 15943412) in KOSR-based medium containing 80% DMEM / F12 (Thermo Fisher Scientific 31330095), 20% KnockOut Serum Replacement (Thermo Fisher Scientific 10828028), 2 mM L-glutamine (Thermo Fisher Scientific 25030024), 1% Nonessential Amino Acids (Thermo Fisher Scientific 11140035) and 0,1 mM 2- mercaptoethanol (Thermo Fisher Scientific 21985023) supplemented with 10 ng / ml human FGF2 (Stem Cell Technologies 78003). Individual iPSC colonies were mechanically picked, expanded as clonal lines and adapted to Matrigel (Corning 354277), mTeSRI (Stem Cell Technologies 85850) and ReLeSR (Stem Cell Technologies 05873) conditions after several passages. Master and working hiPS cell banks were established from clones with the best morphology.

[0112] To characterize the newly generated CRTD1 hiPS cell line, several tests were performed, accessible at https: / / hpscreg.eu / cell-line / CRTDi004-A. Pluripotency was analyzed by Alexa Fluor 488 conjugated anti-Oct3 / 4 (BD Pharmingen 560253), PE conjugated anti-Sox2 (BD Pharmingen 560291), V450 conjugated anti-SSEA4 (BD Pharmingen 561156), and Alexa Fluor 647 conjugated anti-Tra-1 -60 (BD Pharmingen 560122) used according to the manufacturer's recommendations and analyzed on a BD LSRII Flow Cytometer. Three germ layer differentiation was performed as described previously (Cheung et al., 2011) and resulting cells were stained using the 3-germ layer Immunocytochemistry Kit (Thermo Fisher Scientific A25538) according to the instruction manual. For endoderm differentiation, a43RECTIFIED SHEET (RULE 91) ISA / EPS0X17 primary antibody (Abeam ab84990) followed by Alexa Fluor 488 goat anti-mouse IgG (Thermo Fisher Scientific A11001) was used. Quantitative RT-PCR for pluripotency and trilineage spontaneous differentiation was performed according to the instruction manual of the human ES cell Primer Array (Takara Clontech).

[0113] Cells were analyzed for chromosomal abnormalities using standard G banding karyotyping. Cells were treated with 100 ng / ml KaryoMAX Colcemid solution (Thermo Fisher Scientific 15212012) for 4 h at 37°C, harvested and enlarged with 0.075 M KCI solution (Thermo Fisher Scientific 10575090) for 20 min at 37°C. After fixing with 3:1 methanol (VWR 20846.326) : glacial acetic acid (VWR 20102.292), cells were spread onto glass slides and stained with Giemsa at the Institute of Human Genetics, Jena University, Germany. G- bandings of at least 20 metaphases were analyzed.

[0114] Isolation of human pancreatic islets

[0115] Human islets were obtained through the TU Dresden Islet Transplantation Program approved by the TU Dresden Institutional Review Board (EK 255062022) with written informed consent obtained from each islet donor participant. Islets were isolated and purified from resected pancreas tissue according to a modified Ricordi method. Briefly, Collagenase, neutral protease (Serva Electrophoresis, Heidelberg, Germany), and Pulmozyme (Roche, Grenzach, Germany,) were infused into the main pancreatic duct. Islets were separated from exocrine tissue by centrifugation on a continuous Biocoll gradient (Biochrom AG, Berlin, Germany) in a COBE 2991 cell processor (Lakewood, CO, USA).

[0116] Maintenance and karyotyping of human pluripotent stem cell lines

[0117] The H1 and H9 hES cell lines were purchased from WiCell (Wisconsin, USA). H1 and H9 hES cells as well as CRTD1 iPS cells were maintained on cell culture dishes coated with hES cell qualified Corning Matrigel (BD Bioscience, 354277) diluted 1:50 with DMEM / F- 12 (Gibco, 21331 -020) and daily changes of mTeSRI medium (STEMCELL Technologies, 85850) supplemented with 1x penicillin / streptomycin (Gibco, 15140-122). The cells were passaged at around 70% confluency, approximately every 4 days at a ratio of 1 :6 to 1:9, as small aggregates using ReLeSR (STEMCELL Technologies, 05872). Karyotyping for H1 and H9 cells was as described above for the CRTD1 iPS cell line and cells were routinely tested for mycoplasma contamination by PCR as published previously (Young et al., 2010).44RECTIFIED SHEET (RULE 91) ISA / EP

[0118] Differentiation of hPS cells to PP cells

[0119] Initially, the H1 ES cell line was differentiated to PP cells using the STEMdiff™ Pancreatic Progenitor Kit (STEMCELL Technologies, 05120) according to the manufacturer's instructions. In short, the hES cell colonies were dissociated into single cells using TrypLE Express (Gibco, 12604-013) and seeded on Matrigel coated plates as described above at a concentration of 95,000 cells / cm2in mTeSR supplemented with 20 pM ROCKi. The medium was replaced the next day by mTeSR and differentiation was initiated by replacing it with the SI di differentiation medium when cells were 60-70% confluent, typically two days after the initial seeding. Daily washes with DPBS (Gibco, 14190250) and media changes were done until S4d5 when the cells reached the end of PP stage. The monolayer of PP cells was then dissociated using Accumax™ (STEMCELL Technologies, 07921) and cells were used for expansion under INI conditions.

[0120] Later, H1, H9 and CRTD1 iPS cells were differentiated into PP cells using an adaptation of published procedures (Mahaddalkar et al., 2020; Rezania et al., 2014; Shi et al., 2017) with changes as described (corresponding Table herein, Figure 11). The monolayer of PP cells was then dissociated using TrypLE Express (Gibco, 12604-013) for 2 minutes and cells were used for expansion under CO to C8 conditions.

[0121] Expansion and cryopreservation of PP cells

[0122] The monolayer of PP cells was dissociated using TrypLE Express (Gibco, 12604- 013) and cells were used for expansion. Expansion cultures were maintained on polystyrene cell culture plates (Corning, CLS3516) coated with Matrigel (Corning, 354277) or Cultrex (R&D systems, 3434-005-02), diluted 1 :50 in DMEM / F-12 or with 20 ug / mL recombinant truncated vitronectin (VTN-N) (Thermo Fischer Scientific, A31804) diluted in DMEM / F-12 (Gibco, 21331 - 020) for 1 hr at room temperature. PP cells were resuspended in PP expansion media CINI-C8 (corresponding Table herein, Figure 8) and seeded at a density of 2.1 to 3.5 x 105 / cm2. Expansion media were supplemented, during the first day, with 10 pM ROCKi. The expansion medium was then changed daily, and cells were typically passaged every 4thday using TrypLE Express dissociation into single cells. Karyotyping for expanded PP cells and mycoplasma testing was as described above for the hPS cell lines.

[0123] Expanding PP cells were routinely frozen at later passages using mFreSR (StemCell technologies, 05854), supplemented with 20 pM ROCKi, at a density of 10 million 45RECTIFIED SHEET (RULE 91) ISA / EPcells / ml. To ensure proper controlled freezing (- 1 °C / min), cryotubes were placed in Mr. Frosty™ Freezing Container (Thermo Fischer Scientific, 5100-0001) at - 80 °C. After 24 hours, cryotubes were transferred to a liquid nitrogen chamber. For thawing, frozen cells were placed at 37°C and then transferred to 6ml DMEM / F-12 at room temperature for centrifugation. After spinning down the cells at 600xg, the pellet was resuspended using PP expansion media and cells were counted using the Countess II Automated Cell Counter (Thermo Fischer Scientific) and Trypan blue. Typical recovery rates were above 85%. Cells were then seeded as described above for expansion.

[0124] Differentiation of PP cells expanded in CINI into pancreatic endocrine cells using ALI culture

[0125] PP cells generated using the STEMdiff™ Pancreatic Progenitor Kit and expanded under CINI conditions were dissociated into single cells using Accutase™ (STEMCELL Technologies, 07920) at 37 °C and then resuspended in PEP medium (corresponding Table herein) supplemented with 10 M ROCKi at concentration of 50,000 cells / pl. The Falcon® Cell Culture Inserts (Corning, 353493) were placed in their companion plate wells (Corning, 353502) containing 1.5 ml of complete PEP media supplemented with 10 pM ROCKi. Ten droplets of 5 pl of the cell suspension were dropped on the insert to create ten 3D clusters per well, each containing 250,000 cells. Daily media changes were conducted according to corresponding Table herein (Table 2), with the following changes : (a) during S5, the XX Notch inhibitor (Millipore, 565789) was added at concentration of 10nM (b) final glucose concentration at S6 and S7 was 20 mM and (c) no sodium pyruvate was used at S7.

[0126] Differentiation of PP cells into SC-islets using micropatterned wells

[0127] PP cells were dissociated using TrypLE Express at 37°C for 2-3 mins and seeded in microwells of AggreWell 800 plates (STEMCELL Technologies, 34825), using the recommended procedure by the supplier. Directly differentiated and expanded PP cells were seeded at a density of 5000 or 2000 cells per microwell, respectively. Media were an adaptation of published procedures (Balboa et al., 2022; Mahaddalkar et al., 2020; Rezania et al., 2014; Shi et al., 2017) (corresponding Table herein). Expanded PP cells were kept in expansion media for 24 hours and, during the first day of S5, the medium was supplemented46RECTIFIED SHEET (RULE 91) ISA / EPwith one fourth of the concentration of C6 additional factors. Media of S5-S7 were as described in corresponding Table herein and the medium was changed daily.

[0128] Immunofluorescence analyses

[0129] For immunofluorescence (IF), cells were cultured on 12 mm diameter Matrigel- coated coverslips (Carl Roth, P231.1) placed in 12-well wells (Corning, CLS3513) and differentiated or expanded as described above. Cells were then fixed in 4% paraformaldehyde (PFA) for 20 min at 4° C and washed with PBS. Cells were blocked and permeabilized for 1 h at RT using a 5% serum / 0.3% Triton X-100 PBS solution. Samples were then incubated at 4°C in 2.5% serum I 0.3% Triton X-100 in PBS containing the primary antibodies in the appropriate concentration (see corresponding Table herein). The following day, samples were incubated for 1 h at RT in 2.5% serum / 0.3% Triton X-100 in PBS containing the appropriate secondary antibodies, conjugated with either Alexa 488, 568 or 647, at a 1:500 dilution (corresponding Table herein). The coverslip with the stained cells was then placed on a microscope slide, covered with Prolong™ Gold Antifade mounting medium with DAPI (Invitrogen, P36931) and overlayed with a rectangular coverslip.

[0130] IF images were acquired using a Zeiss Axio Observer Z1 microscope coupled with the Apotome 2.0 imaging system and with consistent exposure times for the Alexa 488, 568 and 647 channels in between passages and conditions to allow for direct comparison of the signal intensities.

[0131] Fluorescence-activated cell sorting (FACS) of hPS cell derived PP and endocrine cells

[0132] Cells were first washed with 2% BSA in DMEM-F12 and then dissociated into single cells using TrypLE Express (Gibco, 12604-013). Following dissociation, the cells were counted using the Countess II Automated Cell Counter (Thermo Fischer Scientific) and Trypan blue. Then, cells were washed with PBS and fixed at a concentration of 4 million / ml in in 4% PFA for 10 min at 4°C. For staining with transcription factor antibodies, cells were washed with PBS and permeabilized using the Foxp3 Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00) for 1 h in dark at 4°C. Cells were then washed again using the 1x permeabilization buffer. Thereafter, 2x106cells / sample were blocked using 100 pl of 5% serum in 1x permeabilization buffer and then incubated with primary antibodies at the appropriate concentration (corresponding Table herein) in the same buffer overnight at 4°C.47RECTIFIED SHEET (RULE 91) ISA / EPThen, they were washed twice with 1x permeabilization buffer and incubated with secondary antibodies at the appropriate concentration (corresponding Table herein) in the same buffer at room temperature for 1 hour in dark. For cytoplasmic factors, 2x106cells / sample were blocked for 30 mins at RT in 100 pl PBS containing 2% serum and 0.3% Triton X-100. Then cells were washed with 0.1% Triton X-100 in PBS solution and incubated with primary antibodies at the appropriate concentrations (corresponding Table herein) in the same buffer overnight. Thereafter, cells were washed twice with 0.1% Triton X-100 in PBS solution and incubated with secondary antibodies at the appropriate concentration (corresponding Table herein) at room temperature in the dark for 1 hour. For conjugated antibodies, the appropriate number of cells was used, as directed by the manufacturer, for both the isotype control and staining sample. Samples were stained overnight with conjugated antibodies and then washed with 0.1% Triton X-100 in PBS solution. FACS data were acquired using BD FACSCanto™ II and analysed using the FlowJo software.

[0133] Proliferation and cell death assays

[0134] The Edll proliferation assay was performed with the Click-iT™ Plus Edll Alexa Flour™ 488 Flow Cytometry Assay Kit (Invitrogen, C10632), that contains all the necessary reagent except PBS and BSA, and according to the kit protocol. In short, on the day of passaging the expanding cells, the expansion media was supplemented with 10 pM Edll for 2 h and cells were then dissociated and washed in 3 ml of 1% BSA in PBS. A pellet of 1 million cells was resuspended in 100 pl of Click-iT™ fixative for 15 min and washed again with 3 ml of1% BSA in PBS. The pelleted cells were resuspended in 100 pl of permeabilization and wash reagent for 15 min. Following the incubation, 0.5 ml of the Click-iT™ Plus reaction cocktail containing 1X buffer additive, the Alexa Fluor™ 488 picolyl azide and a copper protectant in PBS, was added to the sample and incubated in the dark at RT for 30 min. The cells were then washed with 3 ml of 1X permeabilization and wash reagent. The pelleted cells were resuspended in 0.5 ml of permeabilization and wash reagent, passed through a 40 pm strainer (PluriSelect, 43-10040) and analysed on the BD FASCanto™ II.

[0135] The flow cytometry staining for necrosis and apoptosis was was performed with the FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend, 640922). Once the cells were incubated in 10 pM of EdU, they were detached and washed in 1% BSA in PBS and an aliquot of 250,000 cells was taken and pelleted in a 1.5 ml microcentrifugation tube. The48RECTIFIED SHEET (RULE 91) ISA / EPcell pellet was resuspended in 100 pl of Annexin V Binding Buffer containing 5 pl of FITC Annexin V and 5 pl of 7-AAD Viability Staining Solution and cells were incubated for 15 min at RT in the dark. After the incubation, an additional 400 pl of Annexin V Binding Buffer was added to the sample and passed through a 40 pm strainer (PluriSelect, 43-10040) before the live cells were analysed on the BD FACSCanto™ II.

[0136] Static glucose-stimulated insulin secretion (GSIS) assay

[0137] At the end of S7 (S7d10 - S7d14) 150 clusters were collected, washed with PBS and incubated for 1 hour in 700 pl of fresh Kreb's buffer (2.5mM CaCI2, 129mM NaCI, 4.8mM KCI, 1.2mM MgSO4, 1.2mM KH2PO4, 1 mM Na2HPO4, 5mM NaHCO3, 10mM HEPES, 0.1% BS, pH adjusted to 7.4 with 5M NaOH). After first incubation, clusters were incubated with Kreb's buffer containing low glucose (2.8mM) for 1 hr, then high glucose (16.7mM) for 1 hr and finally KCI / high glucose (30 mM / 16.7 mM) for 1 hr. Incubations were for exactly one hour and then supernatant was collected. Collected supernatants and pelleted cells were frozen at -80 °C until the analysis. C-peptide detection was performed using the human C-peptide ELISA kit (Mercodia, 10-1141-01), readings were taken using an ELISA plate reader and the standard curve was generated. Cell pellets were used to isolate genomic DNA with the DNeasy Blood & Tissue kit (Qiagen, 69504) and quantification was done using a Nanodrop spectrophotometer.

[0138] RNA isolation and qRT-PCR (qPCR)

[0139] Total RNA was prepared using the RNeasy kit with on-column genomic DNA digestion (Qiagen, 74004) following the manufacturer's instructions. First strand cDNA was prepared using the TAKARA PrimeScript RT Master Mix (TAKARA RR036A). Real-time PCR primers (corresponding Table herein, SEQ ID NOs: 9-44) were designed using the Primer 3 software (SimGene), their specificity was ensured by in silica PCR and they were further evaluated by inspection of the dissociation curve. Reactions were performed with the FastStart Essential DNA Green Master mix (Roche 06924204001) using the Roche LightCycler 480 and primary results were analyzed using the on-board software. Reactions were carried out in technical triplicates from at least three independent biological samples. Relative expression values were calculated using the AACt method by normalizing to H1 undifferentiated expression levels and the TBP housekeeping gene.

[0140] RNA sequencing and bioinformatics analysis49RECTIFIED SHEET (RULE 91) ISA / EP

[0141] Cells were differentiated and from hPS cells as described above. Three independent samples from distinct differentiations and independent expansions were used as biological replicates. Total RNA prepared as above with an integrity number of > 9 was used and subsequent steps were performed at the Biotec Sequencing Core of TU Dresden. mRNA was isolated from 1 ug of total RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturer's instructions. Final elution was done in 15ul 2x first strand cDNA synthesis buffer (N EBnext, NEB). After chemical fragmentation by incubating for 15 min at 94°C the sample was directly subjected to the workflow for strand specific RNA-Seq library preparation (Ultra Directional RNA Library Prep, NEB). For ligation custom adaptors were used 1 : (SEQ ID NO: 1 : Adaptor-Oligo 1 5’-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3’, SEQ ID NO: 2: Adaptor-Oligo 2: 5'-R-GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC-31). After ligation, adapters were depleted by an XP bead purification (Beckman Coulter) adding bead in a ratio of 1:1. Indexing was done during the following PCR enrichment (15 cycles) using custom amplification primers caring the index sequence indicated with 'NNNNNN'. (SEQ ID NO: 3: (Primer 1) Oligo_Seq AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T; SEQ ID NO: 4: (Primer 2) GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T; SEQ ID NO: 5: (Primer 3) CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN GTG ACT GGA GTT). After two more XP beads purifications (1 :1) libraries were quantified using Qubit dsDNA HS Assay Kit (Invitrogen). For Illumina flowcell production, samples were equimolarly pooled and distributed on all lanes used for 75bp single read sequencing on Illumina HiSeq 2500.

[0142] After sequencing, FastQC (http: / / www.bioinformatics.babraham.ac.uk / ) was used to perform a basic quality control of the resulting sequencing data. Fragments were aligned to the human reference genome hg38 with support of the Ensembl 104 splice sites using the aligner gsnap (v2020-12-16) (Wu and Nacu, 2010). Counts per gene and sample were obtained based on the overlap of the uniquely mapped reads with the same Ensembl annotation using featurecounts (v2.0.1) (Liao et al., 2014). Normalization of raw fragments based on library size and testing for differential expression between the different cell types / treatments was done with the DESeq2 R package (v1.30.1) (Love et al., 2014). Sample to sample Euclidean distance, Pearson’ and Spearman correlation coefficient (r) and PCA based upon the top 500 genes showing highest variance were computed to explore correlation between biological replicates and different libraries. To identify differential expressed genes,50RECTIFIED SHEET (RULE 91) ISA / EPcounts were fitted to the negative binomial distribution and genes were tested between conditions using the Wald test of DESeq2. Resulting p-values were corrected for multiple testing with the using Independent Hypothesis Weighting (v1.18.0) (Ignatiadis et al., 2016). Genes with a maximum of 5% false discovery rate (padj < 0.05), 0.5>fold regulation>2.0 and counts above 200 were considered as significantly differentially expressed.

[0143] To directly compare the transcriptome profile of our expanded PP cells with previously published data sets, raw sequencing data of the GEO archives GSE156712 (Ma et al., 2022) were downloaded. The array express, E-MTAB-9992 archive from EBI (Nakamura et al., 2022) and EGAS00001003127 from EGA (Ramond et al., 2018) offered bam files. Here, the fastq files were extracted with picard tools (v2.25.6). All these data sets underwent the same processing procedure. Fragments / Reads were aligned to the human reference genome hg38 with support of the Ensembl 104 splice sites using the aligner gsnap (v2020-12-16). Counts per gene and sample were obtained based on the overlap of the uniquely mapped reads with the same Ensembl annotation using featurecounts (v2.0.1) (Liao et al., 2014). The various strand-specificity of the several projectes was taken into account for the gene counting.

[0144] Normalization of raw fragments based on library size and scaling the count on a Iog2 scale was done with the DESeq2 R package (v1.30.1) (Love et al., 2014) and the Variance Stabilizing Transformation (vst) function. These values were used for plotting.

[0145] Original RNA Seq data have been deposited in GEO under the GSE216266 accession number and accessible with the token sfwbsqyslfirbcj (C6 ePP transcriptome) and under the GSE216179 accession number and accessible with the token enyjwwycfjsjdub (C6 ePP transcriptome). GO and KEGG analyses have been carried out using the Enrichr suite https: / / maayanlab.doud / Enrichr / (Kuleshov et al., 2016) and GSEA analyses using the UCSD Broad Institute suite (https: / / www.gsea-msigdb.org / gsea / index.jsp) (Kuleshov et al., 2016).

[0146] Expansion conditions and reagents51RECTIFIED SHEET (RULE 91) ISA / EP

[0147] Table 1. Formulation of all expansion media tested.RECTIFIED SHEET (RULE 91) ISA / EP53RECTIFIED SHEET (RULE 91) ISA / EPTable 2. Formulation of media used for the differentiation of PP cells (expanded and nonexpanded) (e.g., can be used for analysis of the pp cells).54RECTIFIED SHEET (RULE 91) ISA / EPRECTIFIED SHEET (RULE 91) ISA / EPTable 3. Primary Antibodies used for immunofluorescense (IF) and flow cytometry (FC).Table 4. Secondary Antibodies used for the analyses.56RECTIFIED SHEET (RULE 91) ISA / EPTable 5. Primers used for the qPCR analyses.57RECTIFIED SHEET (RULE 91) ISA / EP

[0148] RESULTS

[0149] Broad TGFp inhibition does not suffice to reproducibly expand hPS cell- derived PP cells

[0150] H1 hPS cells were differentiated into PP cells (Figure 8). PP cell expansion was first attempted using an adaptation of a medium (initial conditions - Cl N I) used to expand PP cells generated through human fibroblast reprogramming (Zhu et al., 2016). This is a simple medium using A38-01, a broad TGF|3 inhibitor of ALK4 / 5 / 7 (Tojo et al., 2005), and very similar to the medium used earlier where SB431542, a broad TGFp inhibitor of the same specificity (Inman et al., 2002), was employed (corresponding Table herein). PP cells were plated at high density, 200K to 350K cells / cm2, on Matrigel-coated plates and passaged every 4-6 days. During the first five passages, cell numbers remained either constant or decreased, eventually leading to growth arrest in most instances. However, in a minority of cases, PP cells kept expanding exponentially (Figure 1A). Expanding cells were maintained for up to ten passages and initial immunofluorescence experiments as well as qPCR analyses suggested that they maintained their PP identity since expression of PDX1, NKX6.1 and SOX9 remained stable at both the protein (compare Figure 1 B to 1 C) and transcript levels (Figure 1 D). These cells could differentiate into endocrine cells in air-to-liquid interface (ALI) differentiation cultures (Rezania et al., 2014) (Figure 1 E, 8).

[0151] Mechanistic insights into signaling pathways for the expansion of pancreas progenitors58RECTIFIED SHEET (RULE 91) ISA / EP

[0152] To identify signaling pathways that may promote or repress the expansion of PP cells we performed RNA Seq and compared cells directly after their differentiation into pancreatic progenitors (dPP) (pO) (n=4), and expanded PP expanded cells (ePP) at passage 5 (p5) (n = 3) and (p10) (n=3). Analyzed pO samples included one sample that was subsequently successfully expanded. Analysis for differentially expressed genes (DEG) identified four groups comprised of genes that were continuously up or down-regulated or comprised of genes that were up or down-regulated only within the first five passages (Figure 2A and Figure 9). Metric multidimensional scaling (MDS) plot of the Euclidian distances of the samples showed clustering of the samples strictly according to their passage number suggesting that expanding cells adapted to the culture conditions and that this adaptation was reproducible. This adaptation occurred to a large extent during the first five passages (Figure 2A). The single pO sample, which subsequently expanded successfully, clustered closely with the other pO samples suggesting that it was not fundamentally different from samples that failed to expand (Figure 2B). GSEA analysis showed that branches of the TGFp signaling pathway was negatively correlated with expansion whereas DNA replication and E2F targets were positively correlated (Figure 9A-C). E2F transcription factors are indirectly activated by growth signals to regulate multiple cell cycle genes and promote cell proliferation (Ertosun et al., 2016; Rubin et al., 2020) and this raised the possibility that expanding cells upregulate their own growth factors engaging themselves in an autocrine growth loop. GSEA and related analyses would miss the regulation of signaling molecules and their receptors if the specific pathway was not regulated as a whole. Thus, to gain a better mechanistic insight into additional, possibly involved, signaling pathways we focused on the kinetics of expression of individual ligands and receptors during expansion.

[0153] We first examined the expression of TGF|3 ligands and receptors. High expression of TGF|3 ligands as well as Type I and Type II receptors suggested that all three branches of this signaling pathway are active at pO (corresponding Table herein, Figure 10). The negative correlation of the TGF|3 signaling pathway with expansion justified in retrospect the initial use of the broad TGF|3 signal inhibitor A38-01 which inhibits the ALK4 / 5 / 7 receptors (Tojo et al., 2005). However, this pathway has several branches, which often carry out opposing functions also depending on the cellular context. Thus we examined the expression kinetics of all expressed liagnds and receptors. The most highly expressed TGFp ligands in PP cells were TGFB1 and BMP2 and their expression was repressed nearly four-fold59RECTIFIED SHEET (RULE 91) ISA / EPduring expansion (Figure 2C, S2D). TGFB1 and BMP2 act through the ALK1 / 5 and ALK3 / 6 / 2 receptors respectively (Brown and Schneyer, 2010) and, therefore, A38-01 would block TGFB1 but not BMP2 signaling. Additionally, ALK4 and its ligand, GDF11, were strongly expressed throughout expansion; GDF11 was even upregulated (Figure 2D, corresponding Table herein, Figure 10), suggesting a possible positive role of this TGFp signaling branch in the expansion of pancreas progenitors. This mechanistic insight suggested that the use of A38-01 or SB431542 may not be optimal because these molecules do not inhibit BMP2 signaling whereas they also block ALK4 signaling, which might promote PP expansion through GDF11.

[0154] To account for the positive correlation of the expression of E2F targets with expansion, we examined changes in the gene expression of growth factors during the expansion. We found a striking upregulation of FGF18 expression by nearly 30-fold (Figure 2D and corresponding Table herein, Figure 10) suggesting a strong requirement for activation of the MAPK pathway for successful expansion. It is noteworthy that FGF18 has a selective affinity for FGFR3 and 4 (Zhang et al., 2006), the two most highly expressed FGFRs in pO and expanding PP cells. Other FGFs were weakly expressed and not upregulated during expansion (corresponding Table herein, Figure 10). This provided a mechanistic rationale to use this specific ligand for the reproducible expansion of PP cells.

[0155] Another growth factor signaling pathway that appeared to be of interest was the PDGF signaling pathway. PDGF receptors A and B were stably and strongly expressed during expansion but the initially strong expression of two of the expressed ligands, PDGFA and B was downregulated by 30- and 15-fold, respectively (Figure 2E, S2E, corresponding Table herein, Figure 10) providing another signaling pathway as a rational target for modulation.

[0156] Retinoic acid promotes differentiation of pancreatic progenitors in developing mouse pancreas (Lorberbaum et al., 2020; Martin et al., 2005; Ostrom et al., 2008; Vinckier et al., 2020). This signaling pathway could be acting in an autocrine manner in the presence of vitamin A, since the genes for retinol dehydrogenase 11 (RDH11) and aldehyde dehydrogenase 1 a1 (ALDH1A1), encoding enzymes involved in the conversion of vitamin A into retinoic acid, as well as the RA nuclear receptors RARA, RARG, RXRA and RXRB were highly expressed in PP cells. Strikingly, there was a dramatic 25-fold downregulation during expansion, exclusively in the expression of the RA-producing enzyme ALDH1A1 (Figure 2F,60RECTIFIED SHEET (RULE 91) ISA / EPcorresponding Table herein, Figure 10). Thus, PP cells are poised to initiate RA-mediated differentiation in an autocrine feed-forward loop and the PP state might be stabilized by eliminating vitamin A in the medium.

[0157] The Notch signaling pathway is implicated in multiple aspects of pancreatic development including expansion of earlier pancreatic progenitors and lineage selection (Afelik et al., 2012; Apelqvist et al., 1999; Murtaugh et al., 2003; Qu et al., 2013; Seymour et al., 2020; Shih et al., 2012). The expression of Notch receptors and ligands was also affected. Expression of the most strongly expressed receptors, NOTCH2 and NOTCH3, and the JAG1 ligand was downregulated nearly 3-fold. On the other hand, expression of the ligands DLL3 and 4 were strongly upregulated, whereas expression of HES1, a key readout of active Notch signaling, was reduced 4-fold, to still substantial levels, during the expansion (Figure 2G, S2F, corresponding Table herein, Figure 10). Thus gene regulation of Notch ligands and receptors was complex suggesting an overall attenuation, but not silencing, of the pathway.

[0158] It is also worth noting that, in the ePP cells, expression of the liver marker AFP and gut marker CDX2 remained constant during expansion (Figure 9G, H). In summary, the findings suggested several mechanisms to stabilize the PP state. Inhibition of the TGFB pathway with an inhibitor of restricted specificity, attenuation of the Notch pathway, stimulation with FGF18, and suppression of RA and PDGF signaling, alone or in combination, could lead to the reproducible expansion of the PP cells.

[0159] Elimination of RA and selective TGF inhibition allow reproducible expansion of PP cells

[0160] Several expansion culture conditions (summarized in corresponding Table herein, Figure 8) were then assessed based on the mechanistic insights discussed above and using PP cells generated with an adaptation of published procedures (Balboa et al., 2022; Mahaddalkar et al., 2020; Rezania et al., 2014; Shi et al., 2017) (corresponding Table herein, Figure 11). Our approach was to carry out gain-of-function or loss-of-function of specific signalling pathways alone or in combination in order to achieve reproducible, robust PP expansion. In all new expansion conditions, B27 containing vitamin A, the precursor of retinoic acid, was substituted with B27 devoid of vitamin A. Additionally, we substituted A38- 01 with the ALK5 II inhibitor (ALK5i II) that targets primarily ALK5, and to a lesser extentALK3 / 6, but not ALK4 (Gellibert et al., 2004). This was called ground condition (condition 0,61RECTIFIED SHEET (RULE 91) ISA / EPCO). Additionally, we tested conditions where FGF2 was substituted with FGF18 (C1), CO medium supplemented with the Notch inhibitor XXI (C2) (Seiffert et al., 2000), or with the PDGFR inhibitor CP673451 (C3) (Roberts et al., 2005) or with both Notch and PDGFR inhibitors (C4). Since FGF2 and FGF18 belong to different FGF subfamilies and have overlapping but not identical FGFR specificities (Zhang et al., 2006) we also addressed a possible synergistic effect of these FGFs in pancreatic progenitor expansion by providing both in C5. Three of these conditions, CO, C1 and C5 resulted in the reproducible expansion of PP cells for at least 10 (CO, C1, n=3) or 11 (C5, n = 5) passages with doubling times (Td) of 3.9, 3.6 and 2.3 days, respectively (Figure 3A, corresponding Table herein, Figure 8).

[0161] Assessment of the expression of key PP genes by qPCR at p5 and p10 suggested that in all three conditions initial high levels of PDX1, NKX6.1 and SOX9 expression were maintained (NKX6.1, SOX9) or even transiently increased at p5 in C5 (PDX1) (Figure 3B- D). Expression of PTF1A was dramatically decreased in all three conditions suggesting a restriction to a bipotent endocrine / duct progenitor identity (Figure 10A). Expression of FOXA2, which helps maintain pancreatic progenitor cells and is important for the development of the endocrine lineage (Gao et al., 2010; Gao et al., 2008; Lee et al., 2005; Lee et al., 2019), was upregulated in C5 indicating that cells expanded in this condition might be more amenable to terminal endocrine differentiation (Figure 10B). On the other hand, there was a statistically significant increase in the expression of the liver and gut markers AFP and CDX2 in CO and C1 and a similar trend in C5 (Figure 10C, D). This was in contrast to CINI, where expression of AFP and CDX2 remained constant (Figure 9G, H) suggesting that these conditions (CO, C1, C5) allowed cells to express aspects of liver and gut programs. Since C5 had a significantly lower Td and a less pronounced increase in AFP and CDX2 expression, we concentrated on the analysis and further improvement of C5. Notably, C5 ePP cells could be frozen and recovered after long storage with high survival rates (>85%) and no apparent loss of proliferative capacity.

[0162] To first confirm the qPCR results at the protein level, we assessed the expression of several markers by immunofluorescence for C5 ePP cells. Immunofluorescence suggested that PDX1 and SOX9 were uniformly expressed at pO, p5 as well as pl 0 and that a large number of PP cells were NKX6.1 + at all three different time points although expansion appeared to reduce the number of NKX6.1 + cells. (Figure 3E-G). Similarly, FOXA2 also remained widely expressed at pO, p5 and plO (Figure 10E-G). Expression of both AFP and 62RECTIFIED SHEET (RULE 91) ISA / EPCDX2 increased transiently upon expansion, at p5 (Figure 1OH-J). We then quantitated the expression of the key PP markers PDX1, SOX9 and NKX6.1 by flow cytometry at pO, p5 and pl 0. These experiments confirmed the immunofluorescence experiments showing that nearly 90% of all cells or more were PDX1 + / SOX9+ at all passages examined (Figure 10K-N), whereas nearly 50% of the cells at pO and p5 and nearly 40% at p10 were PDX1 + / SOX9+ / NKX6.1 +. There was a small progressive drop in the number of PDX1 + / SOX9+ as well as the number of PDX1 + / SOX9+ / NKX6.1 + cells which, however, did not reach statistical significance (Figure 3H-K, S3N).

[0163] Expansion conditions promote primarily proliferation of PP cells

[0164] To understand the mechanism of the successful expansion, we asked whether it was due to enhanced survival, proliferation, or a combination of both. De novo generated PP cells were expanded in either C5 or CINI and assayed at p3 for Edll incorporation and apoptosis. The EdU incorporation analysis revealed that C5 cultures contained significantly more EdU+ expanding cells (10.4 ± 1.2%) than CINI (4.2 ± 0.6%) cultures (n=4) (Figure 4A-C, S4A). The apoptosis assays showed that even though the percentage of 7-AAD+ / Annexin V+ cells appeared higher in CINI-expanded cells (22.1 ± 5.0%) than in C5-expanded cells (17.2 ± 4.7%) this difference did not reach statistical significance (n = 6) (Figure 4C and Figure 11, Figure 11B, C). Therefore, C5, as compared to CINI, promotes proliferation rather than survival of PP cells.

[0165] Canonical Wnt inhibition restricts upregulation of hepatic fate and promotes PP identity

[0166] The upregulation of AFP and CDX2 in CO, C1 and C5 expanded cells suggested a drift towards hepatic and intestinal fates that would hinder the efficiency of differentiation into endocrine cells and subsequent maturation (Nair et al., 2019). During mouse development, non-canonical Wnt signaling specifies bipotent liver / pancreas progenitors to pancreatic fates whereas canonical Wnt signaling leads to liver specification and the emergence of gastrointestinal identity (Ober et al., 2006; So et al., 2013; Munoz-Bravo et al., 2016; Rodnguez-Seguel et al., 2013). PP cells, at pO as well as subsequent passages, strongly and stably expressed several WNT receptors, co-receptors as well as canonical and non- canonical signals (corresponding Table herein, Figure 10). To suppress the upregulation of63RECTIFIED SHEET (RULE 91) ISA / EPhepatic and / or intestinal fates, we supplemented C5 with the canonical Wnt inhibitor IWR-1 to selectively inactivate the canonical Wnt signaling (Chen et al., 2009) in condition 6 (C6).

[0167] The growth curve of C6 ePP cells, over ten passages, showed that IWR-1 supplementation did not significantly affect the growth rate since the Td was 2.5 days as opposed to 2.3 for cells expanded in C5 (Figure 5A). Expansions in C6 were also undertaken using vitronectin-N (VTN-N) as a substrate instead of Matrigel without any changes in expansion efficiency and promotion of the PP identity (n = 3). VTN-N is a defined peptide that can be produced under GMP-conditions and, since all other media are chemically defined or can be produced under similar conditions, this finding established that this approach is also GMP-compliant (Figure 5A).

[0168] These cells retained strong expression of PDX1, NKX6.1, SOX9, PTF1A and FOXA2 to similar levels as in C5 ePP cells and, similarly to C5, expression of PTF1A was strongly reduced upon expansion (FIGURE 12, Figure 12A). Importantly, the expression of the liver markers examined, AFP, HHEX and TTR were all significantly lower by p 10 in the C6 ePP cells, as compared to C5 ePP cells (FIGURE 12, Figure 12B) but expression of CDX2 was only marginally reduced (FIGURE 12, Figure 12A). The expression of PDX1, NKX6.1, SOX9 and FOXA2 was also examined at the protein level by immunofluorescence which suggested that their expression at the protein level was stable and persisted at high levels (Figure 5B-D, FIGURE 12C-E). Similarly to C5 ePP cells, expression of AFP and CDX2 in C6 ePP cells was detectable by immunofluorescence at p5 but significantly reduced at p10 (FIGURE 12, Figure 12F-H). We then quantitated and compared the expression of the key PP markers by flow cytometry at pO, p5 and p10 (Figure 5E-I, FIGURE 121-L). Strikingly, the number of PDX1 + / SOX9+ / NKX6.1 + C6 ePP cells increased significantly from 48%± 11% at pO to 90%± 10% at p5 and 95%±5% at p10 (Figure 5H). This was an additional significant improvement over C5 ePP cells at either p5 or plO (Figure 51). Similarly to C5 ePP cells, C6 ePP cells could be frozen and recovered after long storage with high survival rates (>85%) and no apparent loss of proliferative capacity. Chromosomal stability was assessed after 16 passages analyzing the G-banding of at least 20 metaphases and no alterations were found (Figure 5J).

[0169] To further repress liver and gut programs, we reconsidered BMP inhibition.ALK5i II is considered a relatively weak inhibitor of ALK3 (Gellibert et al., 2004). However,64RECTIFIED SHEET (RULE 91) ISA / EPBMP2, a ligand of ALK3, was significantly downregulated in the CINI ePP cells (Figure 9D, corresponding Table herein, Figure 10) and, thus, we supplemented ALK5i II with LDN- 193189, which inhibits ALK3 with higher potency (Sanvitale et al., 2013). LDN-193189 was used alone or in combination with IWR-1 in C7 and C8, respectively, but none of these conditions was efficient in PP cell expansion (corresponding Table herein, Figure 8).

[0170] In summary, inhibition of the canonical Wnt signaling by IWR-1 promoted a very strong enrichment of PDX1 + / SOX9+ / NKX6.1 + in the expanding cell population and mitigated the upregulation of liver markers seen in C5 at p5 and pl 0 at both the gene and protein expression levels. Equally important, C6 is a robust, highly reproducible, GMP- compliant procedure suitable for its application in cell therapies.

[0171] Expansion stabilizes PP cell identity by repressing differentiation and alternative cell fates

[0172] To understand the effects of the expansion procedure on the transcriptome of PP cells we performed RNA-Seq analyses on PP cells (pO) derived from independent differentiations and ePP cells at p5 and p10. Principal component analyses (PCA) showed that the main component, PC1, represented 81% of the variance among samples and clearly separated pO PP cells from expanded p5 and pl O PP cells. Expanded cells were clustered remarkably close together on the PC1 axis and their difference was only a minor part of PC2, which itself was just 10% (Figure 13A). This suggested that the transcriptome stabilized quickly and this was confirmed by correlation analyses of the transcriptome profiles showing that all major changes occurred between pO and p5 (Figure 6A, B, 13B). Results from GO and KEGG analyses of DEGs between pO and pl 0 were consistent with a cell adaptation to culture conditions, the signaling molecules used for the expansion and an effect on the differentiation process (Figure 6C, 13C). We then compared the transcriptome of our ePP cells with that of PP cells expanded on feeders (Ma et al., 2022) and on fibronectin (FN) (Nakamura et al., 2022) as well as with FACSorted pancreatic progenitors from human fetuses (Ramond et al., 2018) after homogenization and normalization of all data sets. Initial PCA analyses suggested that all in vitro derived PP cells clustered away from fetal cells (Figure 13D) and thus we restricted subsequent comparisons only among in vitro derived PP cells.

[0173] Comparative PCA analysis, with other in vitro derived ePP cells (Ma et al., 2022;Nakamura et al., 2022), clustered our ePP cells separately and showed a very high similarity65RECTIFIED SHEET (RULE 91) ISA / EPamong our p5 and p 10 PP cells (Figure 6D). This segregated clustering would be attributed to the complex medium targeting several signaling pathways. To better identify the molecular basis of this difference, we used a variance stabilizing transformation (vst) function (Love et al., 2014) and hierarchical clustering to identify genes that are differentially regulated in our ePP cells in comparison to feeder (Ma et al., 2022) or FN ePP cells (Nakamura et al., 2022) (Figure 13E). Interestingly, these consisted only of upregulated genes and GO analyses of these genes showed that affected GO categories referred to epithelial maintenance and differentiation, cellular signaling response and metabolic processes (GO BP), membrane components (GO CC) and metabolism (GO MF and KEGG) (Figure 13F). One of the individual genes that stood out was GP2, a gene encoding a zymogen granule membrane glycoprotein that has been identified as a unique marker of human fetal pancreatic progenitors (Cogger et al., 2017; Ramond et al., 2017). Importantly, GP2+ enriched hPS cell-derived PP cells are more efficiently differentiating into pancreatic endocrine cells (Aghazadeh et al., 2022; Ameri et al., 2017). Direct comparison of the GP2 expression among ePP cells showed that our method resulted in passage-dependent strong GP2 upregulation and expression. This expression was nearly 50-fold higher than expression in the FN ePP cells at p 10 whereas feeder ePP cells did not express this marker to any appreciable extent (Figure 6E, corresponding Table herein, Figure 11). Robust GP2 expression in our ePP cells was confirmed by immunofluorescence (Figure 6F).

[0174] To further understand the differences among ePP cells we compared the expression of several additional pancreatic markers at the gene expression level. PDX1 expression was generally reduced after expansion but remained at levels comparable among the three procedures (Figure 6G, corresponding Table herein, FIGURE 12). SOX9 expression followed the same pattern in our VTN-N ePP cells whereas it remained stable or even increased in feeder and FN ePP cells, respectively (Figure 6G, corresponding Table herein, FIGURE 12). SOX9 is also a major driver of the ductal pancreatic program and, to assess whether higher SOX9 levels might be associated with higher levels of this program, we examined transcription of duct program transcription factors (TFs), such as PROX1, HES1, GLIS3 and ONECUT1. Expression of these genes segregates from bipotent progenitors to duct progenitors and differentiated duct cells and is excluded from the endocrine compartment (Bastidas-Ponce et al., 2017). Overall levels of these transcription factors were higher in FN ePP cells suggesting that the ductal program may remain active in these cells66RECTIFIED SHEET (RULE 91) ISA / EP(Figure 13G, corresponding Table herein, FIGURE 12). During pancreas development, both NKX6-1 and NKX6-2 are expressed in progenitor cells, acting in concert to define bipotent progenitors and to subsequently specify endocrine cells (Binot et al., 2010; Henseleit et al., 2005; Nelson et al., 2007; Pedersen et al., 2005; Schaffer et al., 2010). The antibody used here most likely recognizes both proteins, given the very high similarity of NKX6-1 and NKX6-2 at the region of the antigen (Figure 13H). Consistent with the flow cytometry experiments, our expanded cells showed upregulated and high combined NKX6-1 / 2 expression, similar to that of feeder ePP cells (Figure 6G, corresponding Table herein, FIGURE 12). Interestingly, that was due to a strong, expansion-dependent upregulation of NKX6-2 (Figure 131, corresponding Table herein, FIGURE 12). Here it is important to note that whereas NKX6-1 / 2 expression in feeder and VTN-N ePP cells is strong in all samples analysed, it is very low in all FN ePP sample with the exception of a single sample (Figure 6G, corresponding Table herein, FIGURE 12). This shows lack of reproducibility in the expansion of PDX1 + / NKX6-1 + FN ePP cells. The expression of other progenitor TF genes such as FOXA2 and RBJ remained comparable in all expanded cells although FOXA2 retained higher levels in the FN ePP cells and RBPJ retained higher levels in our ePP cells (Figure 131, corresponding Table herein, FIGURE 12). Similarly, expression of acinar TFs such as BHLHA15 and RBPJL was virtually undetectable in all expanded cells. Of note, expression of PTF1A was also undetectable in all ePP cells (corresponding Table herein, FIGURE 12).

[0175] Specification of NEUROG3+ pancreatic endocrine progenitors depends on the lengthening of G1 phase and these progenitors, once specified, divide very rarely, if at all, employing a feed-forward mechanism for their differentiation (Azzarelli et al., 2017; Krentz et al., 2017; Wang et al., 2008). Therefore, it is expected that an efficient PP expansion procedure would efficiently repress the endocrine program. Indeed, a common feature of all expansion procedures was the repression of the endocrine differentiation program. Key transcription factors such as NEUROG3 and its downstream effectors NEUROD1, NKX2-2, and INSM 1 were essentially switched off with very similar efficiency during expansion, although it is worth noting that expression of these genes was already very low in the Ma et al protocol (Ma et al., 2022) in pO cells (Figure 6G, 131, corresponding Table herein, FIGURE 12). Notable exceptions were RFX3 and RFX6, particularly the latter, substantial expression of which was retained in the FN ePP cells (Figure 6G, 131, corresponding Table herein, FIGURE 12). Expression of67RECTIFIED SHEET (RULE 91) ISA / EPterminal endocrine differentiation markers in all ePP cells was also negligible, particularly at late passages (corresponding Table herein, FIGURE 12).

[0176] We finally compared the expression of liver and gut markers. Feeder ePP cells retained only negligible expression of the liver markers AFP and HHEX. However, expression of these markers was nearly 7-fold and 10-fold higher, respectively, in the FN ePP cells compared to those expanded with our procedure, suggesting an overall decreased propensity to endocrine differentiation (Figure 6G, 131, corresponding Table herein, FIGURE 12). Finally, expression of the gut marker CDX2 was slightly higher in the feeder ePP cells but generally comparable in all three methods (Figure 131, corresponding Table herein, FIGURE 12).

[0177] In summary, the comparative transcriptome analyses suggested that our expansion procedure strengthens the PP identity while efficiently repressing the initiation of endocrine differentiation and alternative liver fate.

[0178] Expansion conditions are broadly applicable and ePP cells differentiate into islet-like clusters containing functional |3-cells

[0179] Different hPS cell lines may vary in their differentiation efficiency and this complicates the clinical implementation of this technology. We asked whether the expansion conditions that we established for the male H1 human embryonic stem (hES) cells were applicable to other hPS cell lines of both sexes and whether expansion of the corresponding PP cells could also significantly increase the ratio of PDX1 + / SOX9+ / NKX6.1 + cells. This would be particularly important for hPS cell lines that differentiate less efficiently into PP cells. We used the female H9 hES cell line, a line that has a preference for neural rather than endoderm differentiation, and a male iPS cell line derived in the CRTD (CRTD1, hPSCreg: CRTDi004-A) with unknown lineage preference. Cells were differentiated into PP cells in monolayer culture (corresponding Table herein, Figure 1 1) and PP cells were expanded in C6 for at least ten passages. Expansion efficiency was comparable as assessed by the growth curves with a Td of 2.3 and 2.2 days for H9- (H9-PP) and CRTD1- (CRTD1-PP) derived PP cells, respectively. Expansion in VTN-N coated cell culture surface was at least equally efficient as expansion on Matrigel (Figure 7A, B).

[0180] We then compared the maintenance of the PP identity during expansion ofH9-PP and CRTD1-PP cells to that of H1 -PP cells first by qPCR for gene expression levels at68RECTIFIED SHEET (RULE 91) ISA / EPpO, p5 and plO. Expression of PDX1, NKX6.1 and SOX9 in H9 derived PP cells was strikingly similar to that of H1 derived PP cells at pO but also following expansion. Expression of these markers in CRTD1-PP cells diverged with decreased PDX1 expression, at pO and plO, but increased NKX6.1 expression at p5 and pl 0 (FIGURE 14, Figure 14A-C). Expression of FOXA2 and PTF1A in H9-PP and CRTD1 -PP pO and expanded cells was also very similar to that in H1 - PP cells with the exception of a transient increase in FOXA2 expression at p5 (FIGURE 14, Figure 14D, E). Expression of AFP and CDX2 were also very similar in H9-PP and CRTD1-PP cells as compared to H1 -PP cells except a transient CDX2 upregulation in CRTD1 -PP cells at p5 (FIGURE 14, Figure 14F, G).

[0181] Finally, we evaluated the presence of PDX1 + / SOX9+ andPDX1 + / SOX9+ / NKX6.1 + cells in H9-PP and CRTD1 -PP cells at pO, p5 and plO using flow cytometry. H9 cells were less efficiently differentiated in comparison to either H1 or CRTD1 cells giving rise to 77%±8% PDX1 + / SOX9+ PP cells as opposed to 98%±2% and 89%±5% PDX1 + / SOX9+ PP cells for H1 - and CRTD1 -PP cells, respectively. However, this percentage was 91% by plO, similar to that for H1- (98%±1%) and CRTD1 - (95%±6%) PP cells at the same passage (Figure 5H, 7C, D). Importantly, the % of PDX1 + / SOX9+ / NKX6.1 + PP cells increased from 45%±29% and 39%±6% at pO to 90% and 85%±6% at p10 for H9- and CRTD1 -derived PP cells respectively. This was a very similar increase to that for H1 -derived PP cells, which was from 48%± 11% at pO to 89%± 13% at plO (Figure 5H, 7C, D). Chromosomal stability, following expansion, was confirmed since no alterations were found after analysis of the G-banding of at least 20 metaphases for each line at pl 2 (H9-PP cells) or p13 (CRTD1-PP cells) (FIGURE 14, Figure 14H, I).

[0182] Having established that our PP expansion procedure can be applied with very similar efficiency in PP cells derived from different hPS cell lines, we asked whether expanded cells can be differentiated equally efficiently into endocrine cells. H 1 -PP cells from direct differentiations (dPP) or H1-PP, H9-PP and CRTD1 -PP cells expanded (ePP) for at least ten passages were clustered in micropatterned wells and differentiated using an adaptation of published media (Mahaddalkar et al., 2020; Rezania et al., 2014; Shi et al., 2017) (corresponding Table herein, Figure 1 1) to generate hPS cell derived SC-islets. Both H1 dPP and ePP cells gave rise to similar clusters containing INS+ / NKX6.1 +, INS+ / MAFA+, GCG+ and SST+ endocrine cells (Figure 7E, F and FIGURE 14J, K) as well as similar percentages of INS+, INS+ / GCG+ and GCG+ cells (Figure 7G). The total number of INS+ and GCG+ cells was 69RECTIFIED SHEET (RULE 91) ISA / EPbetween 50% and 55% in both H1 dPP and ePP derived SC-islets but ePP cells might have a tendency to give rise to more INS+ / GCG+ (Figure 7G).

[0183] Gene expression levels for differentiated [3-cell markers such as NKX6.1, PDX1 and SLC30A8, but also SST, were very similar between SC-islets derived from H1 dPP cells and ePP cells and comparable to those derived H9 ePP and CRTD1 ePP cells. Altogether, they were also similar to expression levels detected in human islets (Figure 7H, FIGURE 14N). A similar pattern was observed when comparing the expression of INS, GCG and MAFA among SC-islets of different origin but for these markers, expression in human islets was much higher, presumably reflecting the very different maturation status of SC-islets (FIGURE 14, Figure 14N).

[0184] Finally, we compared the functionality of the p-cells in dPP- (from H1 hPS cells) and ePP- (from H1, H9 and CRTD1 hPS cells) derived SC-islets in static GSIS assays where SC- islets were sequentially incubated in Kreb's buffer containing low glucose levels (2.8mM), Kreb's buffer containing high glucose levels (16.7 mM) and finally a depolarizing Kreb's buffer containing high glucose levels (16.7 mM) and KCI (30 mM). As a comparison, human islets were processed in a similar manner. Human C-pep levels were measured from the supernatant of successive incubations and used to calculate the fold-stimulation. These experiments showed that dPP and ePP derived SC-islets contain p-cells of very similar functionality (Figure 7I) and, as expected, close, but not similar, functionality to human islets.

[0185] In summary, non-expanded and expanded PP cells, derived form different hPS cell lines, differentiate into SC-islets with essentially the same efficiency that contain p-cel Is of similar functionality.

[0186] Example 2: Reduction of liver and gut specification in expanding PP cells

[0187] MATERIALS AND METHODS

[0188] Expansion and cryopreservation of PP cells

[0189] The monolayer of PP cells was dissociated using TrypLE Express (Gibco, 12604- 013) and cells were used for expansion. Expansion cultures were maintained on polystyrene cell culture plates (Corning, CLS3516) coated with Matrigel (Corning, 354277) or Cultrex (R&D systems, 3434-005-02), diluted 1 :50 in DMEM / F-12 or with 20 ug / mL recombinant truncated vitronectin (VTN-N) (Thermo Fischer Scientific, A31804) diluted in DMEM / F-12 (Gibco, 21331 -70RECTIFIED SHEET (RULE 91) ISA / EP020) for 1 hr at room temperature. PP cells were re-suspended in PP expansion media C6 (Figure 8) and seeded at a density of 2.1 x 105 / cm2. Expansion media were supplemented, during the first day, with 10 pM ROCKi. The expansion medium was then changed daily, and cells were typically passaged every 4thday using TrypLE Express dissociation into single cells. Karyotyping for expanded PP cells and mycoplasma testing was as described above for the hPS cell lines.

[0190] Expanding PP cells were routinely frozen at later passages using mFreSR (Stem Cell technologies, 05854), supplemented with 20 pM ROCKi, at a density of 10 million cells / ml. To ensure proper controlled freezing (- 1 °C / min), cryotubes were placed in Mr. Frosty™ Freezing Container (Thermo Fischer Scientific, 5100-0001) at - 80 °C. After 24 hours, cryotubes were transferred to a liquid nitrogen chamber. For thawing, frozen cells were placed at 37°C and then transferred to 6ml DMEM / F-12 at room temperature for centrifugation. After spinning down the cells at 600xg, the pellet was resuspended using PP expansion media and cells were counted using the Countess li Automated Cell Counter (Thermo Fischer Scientific) and Trypan blue. Typical recovery rates were above 85%. Cells were then seeded as described above for expansion.

[0191] Differentiation of PP cells into SC-islets using micropatterned wells

[0192] PP cells were dissociated using TrypLE Express at 37°C for 2-3 mins and seeded in microwells of AggreWell 800 plates (STEMCELL Technologies, 34825), using the recommended procedure by the supplier. Directly differentiated and expanded PP cells were seeded at a density of 5000 or 2000 cells per microwell, respectively. Media were an adaptation of published procedures (Balboa et al., 2022; Mahaddalkar et al., 2020; Rezania et al., 2014; Shi et al., 2017) (Figure 11). Expanded PP cells were kept in expansion media for 24 hours and, during the first day of FIGURE 12, the medium was supplemented with one fourth of the concentration of C6 additional factors. Media of FIGURE 12-FIGURE 14 were as described in Figure 11 and the medium was changed daily.

[0193] Fluorescence-activated cell sorting (FACS) of hPS cell derived PP and endocrine cells

[0194] Cells were first washed with 2% BSA in DMEM-F12 and then dissociated into single cells using TrypLE Express (Gibco, 12604-013). Following dissociation, the cells were counted using the Countess II Automated Cell Counter (Thermo Fischer Scientific) and Trypan71RECTIFIED SHEET (RULE 91) ISA / EPblue. Then, cells were washed with PBS and fixed at a concentration of 4 million / ml in in 4% PFA for 10 min at 4°C. For staining with transcription factor antibodies, cells were washed with PBS and permeabilized using the Foxp3 Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00) for 1 h in dark at 4°C. Cells were then washed again using the 1x permeabilization buffer. Thereafter, 2x106cells / sample were blocked using 100 pl of 5% serum in 1x permeabilization buffer and then incubated with primary antibodies at the appropriate concentration (FIGURE 12) in the same buffer overnight at 4°C. Then, they were washed twice with 1x permeabilization buffer and incubated with secondary antibodies at the appropriate concentration (FIGURE 14) in the same buffer at room temperature for 1 hour in dark. For cytoplasmic factors, 2x106cells / sample were blocked for 30 mins at RT in 100 pl PBS containing 2% serum and 0.3% Triton X-100. Then cells were washed with 0.1% Triton X- 100 in PBS solution and incubated with primary antibodies at the appropriate concentrations (FIGURE 12) in the same buffer overnight. Thereafter, cells were washed twice with 0.1% Triton X-100 in PBS solution and incubated with secondary antibodies at the appropriate concentration (FIGURE 14) at room temperature in the dark for 1 hour. For conjugated antibodies, the appropriate number of cells was used, as directed by the manufacturer, for both the isotype control and staining sample. Samples were stained overnight with conjugated antibodies and then washed with 0.1% Triton X-100 in PBS solution. FACS data were acquired using BD FACSCanto™ II and analysed using the FlowJo software.

[0195] RNA isolation and qRT-PCR (q PCR)

[0196] Total RNA was prepared using the RNeasy kit with on-column genomic DNA digestion (Qiagen, 74004) following the manufacturer's instructions. First strand cDNA was prepared using the TAKARA PrimeScript RT Master Mix (TAKARA RR036A). Real-time PCR primers (corresponding Table herein) were designed using the Primer 3 software (SimGene), their specificity was ensured by in silica PCR and they were further evaluated by inspection of the dissociation curve. Reactions were performed with the FastStart Essential DNA Green Master mix (Roche 06924204001) using the Roche LightCycler 480 and primary results were analyzed using the on-board software. Reactions were carried out in technical triplicates from at least three independent biological samples. Relative expression values were calculated using the AACt method by normalizing to H1 undifferentiated expression levels and the TBP housekeeping gene.72RECTIFIED SHEET (RULE 91) ISA / EP

[0197] Titration of the effects of the BMP receptor inhibitor K02288 in expanding PP cells was carried out with the following result:

[0198] PP cells derived from the differentiation of H1 hPS cells were expanded for five passages as described herein in the absence or presence of different amounts of K02288. At the end of that period expression of the PP markers PDX1, NKX6.1 and SOX9 as well as of the liver marker AFP and the gut marker CDX2 was assessed by qRT-PCR. It was determined that K02288 at 0.25 uM is effective in repressing expression of the liver and gut markers without affecting the expression of the PP markers, p < 0.05 (*), p < 0.005 (**), p < 0.0005 (***), p < 0.0001 (****).

[0199] Example 3: Expansion of hPS cell derived pancreatic progenitors

[0200] In this example a further expansion of hPS cell derived pancreatic progenitors has been carried out using a modified protocol (Figure 16) compared to, for example, results / protocol as outlined in Figure 15. Accordingly, Figure 16 shows that PP cells derived from the differentiation of H1 hPS cells are expanded for five passages as described in the modified SOP described below in the absence or presence of different amounts of K02288. At the end of that period expression of the PP markers PDX1, NKX6. 1 and SOX9 as well as of the liver marker AFP and the gut marker CDX2 are assessed by qRT-PCR as previously described. It was determined that K02288 at 0.25 pM effectively represses the expression of the liver and gut markers without affecting the expression of the PP markers. Said modified protocol (SOP) of expansion of hPS cell derived pancreatic progenitors has been carried out as outlined below. Otherwise Example 3 is carried out as previously described herein above.

[0201] A. Initiating de novo expansion from pancreatic progenitor (PP) cells

[0202] 1 . Estimating cell yield from differentiation (~6 million per well of a 6 well plate (6wpw) and coating appropriate number of wells with Matrigel diluted in DMEM / F12 (1 :50) or Vitronectin-N (20 pg / ml) diluted in DMEM / F12 at least one hour before proceeding, considering that each 6wpw should be seeded with 3 million cells for a successful expansion.

[0203] 2. Washing the wells of PP cells with 2 ml of DPBS and incubating with 1 ml TryPLE, warmed at 25° C, at 37 °C for 2-3 minutes until the rounding up cells can be seen.73RECTIFIED SHEET (RULE 91) ISA / EP

[0204] 3. Further breaking up cells by gently triturating 3x with the P1000 tip.Stopping enzyme activity by immediately adding 1 ml of 2% BSA in DMEM / F-12 containing 20 pM of ROCKi. Processing in parallel of only up to three wells.

[0205] 4. Once all the wells are processed, gently triturating the cells a further 3X using a P1000 in all the wells before passing through a 40 urn strainer (Corning, FALC352340) into an empty 6wpw.

[0206] 5. Transferring the cell suspension to a 15 ml falcon, washing each well with 1 ml 2% BSA in high glucose DMEM 10 pM ROCKi and adding it to the corresponding falcon before spinning down for 4 min at 300xg.

[0207] 6. Aspirating the supernatant and re-suspending the pellet in 1 ml of expansion media containing 10 pM of ROCKi. Taking 25 pl of the cell solution and mixing with 25 pl of Trypan blue solution. Adding 10 pl of the well-mixed cell / trypan blue solution to a chamber of the Cell Countess™ counting chambers and proceeding to count using the Cell Countess™ II (see note 1).

[0208] 7. Seeding 3 million (see note 2) cells per 6wpw, coated as above, in a total volume of 2 ml (cell suspension + expansion media) media containing 10 pM ROCKi.

[0209] 8. Next day carrying out a 2 ml DPBS wash and adding 2 ml of fresh expansion media. Carrying out daily washes and media changes.

[0210] 9. For subsequent passages (see note 3) seeding 2 million (see note 2) cells per 6wpw, coated as above, in a total of 2 ml of expansion media containing 10 pM ROCKi.

[0211] Note l: To count cells, placing lOpI of trypan blue I cell solution into each of the counting chambers of the slide for Cell Countess II. Taking both counts and an average of the counts for a better accuracy. If values differ by 10% or more, mixing cell suspension again as above, retaking both measurements and discarding the outlier before calculating the average number.

[0212] Note 2: When seeding cells, advantageously making sure that the seeding density is adjusted to the cell death rate. For instance, if the total cell count is 3 million cells, with 2.7 million live cells and 0.3 million dead cells (10% death rate), the seeding is carried out with 2.2 million live cells (2 million + 2 million x % of dead cells).74RECTIFIED SHEET (RULE 91) ISA / EP

[0213] Note 3: Passaging is typically done every four days, however also possible that cells can be split earlier. Cells are not typically left for more than four days before passaging because if cells become over-confluent they might start losing PP identity. TryPLE digestion of a good culture does not typically exceed 3-4 minutes.

[0214] B. Cryopreservation of expanding PP cells:

[0215] 1. Warming TryPLE to 25°C.

[0216] 2. Aspirating medium from the well and wash once with 2 mL of DPBS.

[0217] 3. Adding 1 mL of TryPLE per 6wpw and incubating at 37°C for 2 minutes and checking to see if the cells are rounded up. Increasing incubation time if necessary until cells are rounded up.

[0218] 4. Gently mixing by pipetting 3x with P1000 to break up cells and removing from the well and immediately adding 2% BSA / DMEM / F-12 to each well to stop the reaction. Pipetting further to obtain single-cell suspension. Transferring it to a 15 mL conical tube. Rinsing the well with an additional 1 mL of DMEM to collect any remaining cells.

[0219] 5. Adding 25 pl of cell solution to 25 pl of 1x trypan blue. Mixing well and placing lOpI of solution into counting chamber for Cell Countess II, counting cells (see note 1) while the falcon is spinning down.

[0220] 6. Centrifuging the falcon at 300xg for 4 minutes before the counting step.

[0221] 7. Centrifuging the cells, gently re-suspending them in mFreSR at a concentration of 10x106cells / ml and transferring the cell suspension in 1.2 ml labelled cryovials. If the cell number is less then freezing proportionally. Cell concentration is observed. If not enough cells for 1 ml, adjusting the volume accordingly. Adding ROCKi at final concentration of 20 pM.

[0222] 8. Freezing vials using a standard slow rate-controlled cooling protocol that reduces temperatures at approximately 1 °C / min. Placing the vials in the Thermo Mr Frosty filled with fresh absolute isopropanol up to the filling limit at room temperature and then store at -80 C, followed by long-term storage at liquid nitrogen the next day.

[0223] C. Thawing Cryopreserved PP Cells75RECTIFIED SHEET (RULE 91) ISA / EP

[0224] Note: The thawing procedure is advantageously carried out as quickly as possible.

[0225] 1. Adding 5mL of warm DMEM high glucose to a 15 mL tube.

[0226] 2. Quickly thawing the cells in a 37°C water bath by gently and continuously shaking the cryovial until a very small frozen cell pellet remains.

[0227] 3. Transferring the contents of the cryovial to the 15 mL conical tube and centrifuging cells at 300xg for 4 minutes at room temperature.

[0228] 4. Aspirating the supernatant with the vacuum pump until <200ul are left and the remaining with the P200 as not to disturb the pellet. Re-suspending the pellet in 1 ml of PP Expansion medium. Mixing by pipetting and counting the cells by mixing 25 pl of the cells with 25 pl Trypan blue (0.4 %) placing lOpI of solution into both counting chambers of the slide for Cell Countess II to count using the Cell Countess™ II (see note 1).

[0229] 5. Seeding 3x105cells / well in hES qualified Corning Matrigel (1 :50) precoated 6-well plate as above containing 2 ml of PP Expansion medium + Rock Inhibitor 10 pM.

[0230] 6. Placing the plate in a 37°C incubator. Changing media as soon as possible the next morning.

[0231] 7. Performing daily medium changes using 2 ml / well of PP Expansion medium.

[0232] D. Differentiation of expanded PP cells

[0233] 1. Re-suspending cells in 1 ml of Expansion medium + ROCKi 20 pM and counting them with Cell Countess II (please see note 1). Taking two counts and an average of the counts for a better accuracy. Adjusting the cell concentration at 0.6 million cells / ml (for 2000 cells / microwell of Aggrewell 800) with expansion medium.

[0234] 2. Adding 1 mL of the cell suspension in each well previously prepared with 1 ml of full expansion medium.

[0235] 3. After the first day incubation with expanding medium, completely replacing 2ml expansion medium with PEP medium + 1X PEP factors + 0.25X each of EGF, bFGF and FGF18 used in expansion.RECTIFIED SHEET (RULE 91) ISA / EP

[0236] 4. After the end of the second day continuing differentiation with PEP medium as for direct PP cells.

[0237] E. Preparation of PP Expansion medium

[0238] Table 6. Media.

[0239] Table 7. Factor solutions.77RECTIFIED SHEET (RULE 91) ISA / EP

[0240] Table 7 Note: * All stock solutions are kept in -80°C. Working stocks kept at 4°C, except ALK5i II which is kept at -20°C. ** Use tight-capped tubes.

[0241] F. Reagents

[0242] Table 8. Reagents.78RECTIFIED SHEET (RULE 91) ISA / EP

[0243] G. Storage and preparation of various reagents

[0244] TrypLE Express (Life Technologies Cat. No. 12604013)

[0245] Storing at 4°C, warming the required amount to 25°C before use.

[0246] Trypan Blue Solution, 0.4% (Life Technologies T10282)

[0247] Passing through a 22|jm filter and storing at room temperature.

[0248] ROCKi (Miltenyi Cat. No. 130-104-169)

[0249] 20 mM in H2O, long term storage at -20 °C, working aliquot at 4°C.

[0250] Matrigel stock preparation and use

[0251] Diluting 1:5 with DMEM / F12 and storing as 600 pl aliquots in -20 °C.

[0252] Advantageously keeping on ice when thawing and handling to prevent gelling

[0253] After thawing, diluting stock a further 1 :10 with DMEM / F12 (1:50 final) before using.

[0254] Vitronectin stock preparation and use

[0255] Diluting 1:5 with DMEM / F12 and storing as 500 pl aliquots in -20 °C.

[0256] Advantageously keeping on ice when thawing and handling to prevent gelling.

[0257] After thawing, diluting stock a further 1 :5 with DMEM / F12 (1:25 final) before using.

[0258] Final concentration: 20pg / ml.79RECTIFIED SHEET (RULE 91) ISA / EP

[0259] Working stocks at 4°C or -20°C can be typically used for 2-3 weeks.

[0260] Stocks at -80°C can be typically used for a year.

[0261] PP expansion basal media

[0262] Table 9. Expansion basal media.RECTIFIED SHEET (RULE 91) ISA / EP

[0263] PP expansion factors to add to basal media

[0264] Table 10. Expansion factors.RECTIFIED SHEET (RULE 91) ISA / EP

[0265] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds described herein are presently representative of certain embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the 82RECTIFIED SHEET (RULE 91) ISA / EPart which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0266] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0267] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0268] Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0269] REFERENCES

[0270] Balboa, D., Barsby, T., Lithovius, V., Saarimaki-Vire, J., Omar-Hmeadi, M., Dyachok, O., Montaser, H., Lund, P.E., Yang, M., Ibrahim, H.zet al. (2022). Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells. Nature biotechnology.83RECTIFIED SHEET (RULE 91) ISA / EP

[0271] Cheung, A.Y., Horvath, L.M., Grafodatskaya, D., Pasceri, P., Weksberg, R., Hotta, A., Carrel, L., and Ellis, J. (2011). Isolation of MECP2-null Rett Syndrome patient hiPS cells and isogenic controls through X-chromosome inactivation. Hum Mol Genet 20, 2103-2115.

[0272] Ignatiadis, N., Klaus, B., Zaugg, J.B., and Huber, W. (2016). Data-driven hypothesis weighting increases detection power in genome-scale multiple testing. Nature methods 13, 577-580.

[0273] Kuleshov, M.V., Jones, M.R., Rouillard, A.D., Fernandez, N.F., Duan, Q., Wang, Z., Koplev, S., Jenkins, S.L, Jagodnik, K.M., Lachmann, A., et al. (2016). Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic acids research 44, W90-97.

[0274] Liao, Y., Smyth, G.K., and Shi, W. (2014). featurecounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923- 930.

[0275] Love, M.L, Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome biology 15, 550.

[0276] Ma, X., Lu, Y„ Zhou, Z„ Li, Q„ Chen, X., Wang, W„ Jin, Y„ Hu, Z„ Chen, G„ Deng, Q.zet al. (2022). Human expandable pancreatic progenitor-derived beta cells ameliorate diabetes. Sci Adv 8, eabk1826.

[0277] Mahaddalkar, P.U., Scheibner, K., Pfluger, S., Ansarullah, Sterr, M., Beckenbauer, J., Irmler, M., Beckers, J., Knobel, S., and Lickert, H. (2020). Generation of pancreatic beta cells from CD177(+) anterior definitive endoderm. Nature biotechnology 38, 1061-1072.

[0278] Nakamura, A., Wong, Y.F., Venturato, A., Michaut, M., Venkateswaran, S., Santra, M., Goncalves, C., Larsen, M., Leuschner, M., Kim, Y.H., et al. (2022). Long-term feeder- free culture of human pancreatic progenitors on fibronectin or matrix-free polymer potentiates beta cell differentiation. Stem Cell Reports.

[0279] Ramond, C., Beydag-Tasoz, B.S., Azad, A., van de Bunt, M., Petersen, M.B.K., Beer, N.L., Glaser, N., Berthault, C., Gloyn, A.L., Hansson, M., et al. (2018). Understanding human fetal pancreas development using subpopulation sorting, RNA sequencing and single-cell profiling. Development 745.

[0280] Rezania, A., Bruin, J.E., Arora, P., Rubin, A., Batushansky, I., Asadi, A., O'Dwyer, S., Quiskamp, N., Mojibian, M., Albrecht, T.zet al. (2014). Reversal of diabetes with insulin-84RECTIFIED SHEET (RULE 91) ISA / EPproducing cells derived in vitro from human pluripotent stem cells. Nature biotechnology 32, 1121-1133.

[0281] Shi, Z.D., Lee, K„ Yang, D„ Amin, S„ Verma, N„ Li, Q.V., Zhu, Z„ Soh, C.L., Kumar, R., Evans, T.zet al. (2017). Genome Editing in hPSCs Reveals GATA6 Haploinsufficiency and a Genetic Interaction with GATA4 in Human Pancreatic Development. Cell stem cell 20, 675-688 e676.

[0282] Wolf, C., Rapp, A., Berndt, N., Staroske, W., Schuster, M., Dobrick-Mattheuer, M., Kretschmer, S., Konig, N., Kurth, T., Wieczorek, D., et al. (2016). RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA. Nature communications 7, 11752.

[0283] Wu, T.D., and Nacu, S. (2010). Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26, 873-881.

[0284] Young, L., Sung, J., Stacey, G., and Masters, J.R. (2010). Detection of Mycoplasma in cell cultures. Nature protocols 5, 929-934.85RECTIFIED SHEET (RULE 91) ISA / EP

Claims

CLAIMS1. An in vitro or ex vivo method for expanding pluripotent stem cells-derived (e.g., induced pluripotent stem cells-derived) pancreatic progenitors (PP), preferably human PPs (e.g., expressing PDX1+, SOX9+and NKX6.1+genes / proteins): said method comprising:(i) providing pluripotent stem cells-derived pancreatic progenitors (PP), e.g., providing H1-PP (e.g., H1 cells-derived PPs, e.g., H 1 cells having accession number: WA01 (hPSCReg-ID: WAe001-A)), H9-PP (e.g., H9 cells derived PPs, e.g., H9 cells having accession number: WA09 (hPSCReg-ID: WAe009-A) or CRTD1 -PP cells (e.g., CRTD1 cells-derived PPs, e.g., CRTD1 cells having accession number: CRTDi004-A (hPSCreg- ID: CRTDi004-A); a) activating MAPK (Mitogen -activated protein kinase) signaling pathways in said cells by the means of adding one or more of the MAPK activators comprising: (i') a combination of one or more FGF18 molecules (e.g., having UniProt Accession Number: 076093), one or more EGF molecules (e.g., having UniProt Accession Number: P01133) and one or more FGF2 (e.g., having UniProt Accession Number: P09038) molecules; (ii') a combination of one or more FGF molecules (preferably FGF18, e.g., having UniProt Accession Number: 076093) and one or more EGF (Epidermal growth factor) (e.g., having UniProt Accession Number: P01133) molecules and / or (iii') one or more FGF (Fibroblast growth factor) molecules, preferably comprising one or more FGF18 molecules, e.g., having UniProt Accession Number: 076093); b) inhibiting and / or disabling and / or modulating retinoic acid signaling in said cells, preferably by the means of using a serum-free and / or animal product free (e.g., GMP-compliant) supplement without Vitamin A for cell cultivation;c) inhibiting and / or disabling and / or modulating TGF-[3 (Transforming growth factor beta) signaling pathway in said cells, e.g., by the means of adding one or more inhibitor / s that block TGF- / 31, -[32, and / or -[33 ligands (e.g., StemMACS RepSox inhibitor) and adding one or more inhibitor / s that block BMP-2 (Bone Morphogenetic Protein-2, e.g., having UniProt Accession Number: P12643), BMP-4 (Bone Morphogenetic Protein-4, e.g., having UniProt Accession Number: P12644) and / or BMP-7 (Bone Morphogenetic Protein-7, e.g., having UniProt Accession Number: P18075) signaling (e.g., K02288 inhibitor) and d) inhibiting Wnt signaling pathway (e.g., canonical Wnt signaling) in said cells, preferably by the means of adding one or more Wnt / |3-Catenin inhibitors (e.g., Endo-IWR-1); ii) expanding said cells, preferably said expanding is carried out using a recombinant truncated vitronectin (VTN-N) coating, further preferably said expanding is carried out using fibronectin coating.

2. The method of any one of the preceding claims, wherein said PPs are human PPs expressing PDX1+ / SOX9+ / NKX6.1+(e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426), preferably said PPs are derived from H1, H9 or CRTD1 cell lines: said method comprising:(i) providing hPS cells derived pancreatic progenitors (PP), preferably HI PP, H9-PP or CRTD1-PP cells; a) activating MAPK signaling pathways in said cells by the means of adding one or more of the MAPK activators, preferably EGF and / or FGF18 and / or FGF2 molecules (e.g., EGF and FGF18 and FGF2); b) inhibiting retinoic acid signaling in said cells, preferably by the means of using a serum-free supplement without Vitamin A for cell cultivation; c) inhibiting TGFp signaling pathway in said cells, preferably by the means of adding one or more inhibitor / s that block TGF-filligands (e.g., StemMACS RepSox inhibitor) and / or one or more BMP-2 inhibitors (e.g., K02288 inhibitor) and d) inhibiting Wnt signaling pathway in said cells, preferably by the means of adding one or more Wnt / |3-Catenin inhibitors (e.g., Endo-IWR-1); ii) expanding said cells, preferably said expanding is carried out using a recombinant truncated vitronectin (VTN-N) coating.

3. The method of any one of the preceding claims, wherein said method is capable of: a) upregulating the expression of a PP marker GP2 (Pancreatic secretory granule membrane major glycoprotein GP2, e.g., having UniProt Accession Number: P55259) in said cells, preferably said upregulating is detectable on the RNA level (e.g., up to 140-fold induction) and / or on the protein level (e.g., from about 5% to about 70% upregulation); and / or b) producing (e.g., produces) PDX1+ / SOX9+ / NKX6.1+ / GP2+(e.g., having corresponding UniProt Accession Numbers:P52945 / P48436 / P78426 / P55259) PP cells.

4. The method of any one of the preceding claims, wherein said method is characterized by one or more of the following: a) said method produces and / or expands (e.g., selectively expands) and / or enriches (e.g., selectively enriches) PDX1+ / SOX9+ / NKX6.1+PP cells; b) said method is capable of enriching (e.g., increasing the amount / concentration) of the PDX1+ / SOX9+ / NKX6.1+PP cells in pancreatic progenitors (PP) cell culture; c) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are pancreatic progenitors (PPs) capable of proliferation (e.g., their proliferation ability is active / un-inhibited) while inhibiting differentiation and / or inhibiting endocrine differentiation of said PPs; d) said progenitor cells are human pluripotent stem (hPS), preferably iPSs, cells derived pancreatic progenitors (PP), wherein said method is capableof expanding said hPS cell derived PP cells while suppressing their differentiation; e) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into islet-like clusters (SC-islets), preferably insulin producing SC-islets (e.g., said SC- islets are capable of producing the essentially same amount of insulin as in vitro derived non-expanded PPs as defined in claim 1 (i) and / or having the essentially same amount of differentiated endocrine cells in said SC- islets; f) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into insulinproducing cells, preferably said cells are capable of secreting insulin in the presence of glucose; g) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into endocrine cells, preferably pancreatic endocrine cells, including insulinproducing beta cells; h) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of cryopreservation (e.g., at least about -195°C); i) said method does not comprise the use of a feeder layer (e.g., GMP- compliant); j) said method comprises the use of PP grown in a monolayer; k) said method is capable of producing (e.g., produces) PP cells capable of differentiating in vitro into SC-islets, (e.g., which can be subsequently transplanted); l) said method is capable of producing (e.g., produces) PP cells that do not produce insulin and / or capable of differentiating into islet cells (e.g., including beta cells, e.g., after transplantation).

5. The method of any one of the preceding claims, wherein said progenitor cells are human pluripotent stem (hPS) cell derived pancreatic progenitors (PP).The method of any one of the preceding claims, wherein said method is one or more of the following: a) said method is not limited by a number of cell divisions of said progenitor cells; b) said method is a method for unlimited expansion of said progenitor cells; c) said method is capable of producing (e.g., produces) progenitor cells that are capable of avoiding replicative senescence and / or replicative exhaustion, preferably continuously avoiding replicative senescence and / or replicative exhaustion; d) said method is capable of producing (e.g., produces) progenitor cells that are capable of avoiding arrest of cell division, preferably continuously avoiding arrest of cell division; e) said method is capable of producing (e.g., produces) progenitor cells that are capable of proliferation, preferably continuous proliferation; f) said method is capable of producing (e.g., produces) progenitor cells that are capable of self-renewal, preferably continuous self-renewal; g) said method is capable of producing (e.g., produces) progenitor cells that are capable of indefinitely renewing itself; h) said method is capable of producing (e.g., produces) progenitor cells that are capable of expansion, preferably continuous expansion; i) said method is capable of producing (e.g., produces) progenitor cells that are capable of differentiation (e.g., into pancreatic endocrine cells), while suppressing their differentiation; j) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are capable of differentiating into islet-like clusters (SC-islets); k) said method is capable of producing (e.g., produces) progenitor cells, wherein said progenitor cells are pancreatic progenitors capable of proliferation (e.g., their proliferation ability is active / un-inhibited), wherein said method is simultaneously capable of inhibiting the endocrine, hepatic and / or gut differentiation in said cells;l) said method is capable of producing (e.g., produces) progenitor cells that are capable of maintaining their differential potential, preferably continuously maintaining their differential potential; m) said method does not comprise the use of artificial modification (e.g., siRNA) of the genome of said progenitor cells; n) said method is capable of producing (e.g., produces) PP cells that do not produce insulin and / or capable of differentiating into islet cells (e.g., including beta cells, e.g., after transplantation); o) said method is capable of producing (e.g., produces) PP cells capable of differentiating in vitro into SC-islets, (e.g., which can be subsequently transplanted).

7. The method of any one of the preceding claims, wherein said method is capable of producing (e.g., produces) at least 100-fold (e.g., 500, 700, 800, 900, 1000-fold, or 2000- fold) expansion of progenitor cells (e.g., PP cells), preferably said expansion is carried out during about at least 5 passages (e.g., 10 passages) and / or in a period from about 20 (e.g. 40) to about 25 (e.g., 45) days) and / or said progenitor cells comprising at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%) of PDX1+ / SOX97NKX6.1+cells (e.g., PDX1+ / SOX97NKX6.17GP2+).

8. An isolated PP cell (e.g., isolated expanded PP (ePP) cells) or an isolated population of PP cells (e.g., isolated population of expanded PP (ePP) cells), wherein said cell / s are PDX17SOX97NKX6.1+cell / s (e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426), wherein said cells are capable of indefinitely renewing themselves, wherein said cell / s further having one or more of the following characteristics: a) said cell / s expressing PP marker GP2 (Pancreatic secretory granule membrane major glycoprotein GP2, e.g., having UniProt Accession Number: P55259); b) said cells are capable of proliferation while having their differentiation inhibited / repressed; c) said cells are capable of unlimited expansion;d) optionally, said cells are obtainable (e.g., obtained) from pluripotent stem cells (e.g., iPSs), preferably providing human pluripotent stem (hPS) cells, further preferably providing hPS cells derived pancreatic progenitors (PP), e.g., H1 -PP, H9-PP or CRTD1 -PP cell lines.

9. A population of progenitor cells or a progenitor cell produced and / or expanded and / or modified by method according to any one of the preceding claims, preferably said cells are PDX1+ / SOX9+ / NKX6.1+cells (e.g., having corresponding UniProt Accession Numbers: P52945 / P48436 / P78426).

10. The population of progenitor cells according to any one of the preceding claims, wherein population comprising at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%) of PDX1+ / SOX9+ / NKX6.1+cells, preferably said method comprising activating MAPK signaling pathways in said cells by the means of adding MAPK activators EGF, FGF18 and FGF2.

11. A composition, preparation or kit comprising the population of progenitor cells (e.g., isolated) or the progenitor cell (e.g., isolated) according to any one of preceding claims.

12. The composition, preparation or kit according to any one of the preceding claims, wherein said composition, preparation or kit is a pharmaceutical and / or diagnostic composition, preparation or kit.

13. The population of progenitor cells (e.g., isolated), a progenitor cell (e.g., isolated), composition, preparation or kit according to any one of the preceding claims, for use as a medicament and / or in therapy.

14. The population of progenitor cells (e.g., isolated), a progenitor cell (e.g., isolated), composition, preparation or kit according to any one of the preceding claims, for use in one or more of the following methods:a) method of treatment, amelioration, prophylaxis and / or diagnostics of an endocrine disease (e.g., Diabetes, Type 1 Diabetes, Type 2 Diabetes, Gestational Diabetes, Mature Onset Diabetes of the Young (MODY)); b) method of treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; c) method for monitoring development and / or assessing the efficacy of prophylaxis and / or therapy of Diabetes, preferably Type 1 Diabetes; d) method for screening a candidate compound for use in method of treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; e) method of regenerative treatment of a cell, tissue, organ and / or body; f) method of transplantation (e.g., beta-cell transplantation); g) method of cell therapy (e.g., beta-cell therapy); h) disease modelling (e.g., diabetes modelling) i) method of insulin production and / or insulin supplementation; j) method of producing PP cells that do not produce insulin and / or capable of differentiating into islet cells (e.g., including beta cells, e.g., after transplantation); k) method of producing PP cells capable of differentiating in vitro into SC- islets, (e.g., which can be subsequently transplanted) l) method according to any one of the preceding claims; m) any combination of methods according (a)-(l); n) method of any of (a)-(m), wherein said method is an in vitro, ex vivo or in vivo method.

15. Use of the population of progenitor cells (e.g., isolated), a progenitor cell (e.g., isolated), composition, preparation or kit according to any one of the preceding claims for one or more of the following: a) for treatment, amelioration, prophylaxis and / or diagnostics of an endocrine disease (e.g., Diabetes, Type 1 Diabetes, Type 2 Diabetes, Gestational Diabetes, Mature Onset Diabetes of the Young (MODY));b) for treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; c) for monitoring development and / or assessing the efficacy of prophylaxis and / or therapy of Diabetes, preferably Type 1 Diabetes; d) for screening a candidate compound for use in method of treatment, amelioration, prophylaxis and / or diagnostics of Diabetes, preferably Type 1 Diabetes; e) for regenerative treatment of a cell, tissue, organ and / or body; f) for transplantation (e.g., beta-cell transplantation); g) for cell therapy (e.g., beta-cell therapy); h) for insulin production and / or insulin supplementation; i) for producing PP cells that do not produce insulin and / or capable of differentiating into islet cells (e.g., including beta cells, e.g., after transplantation); j) for producing PP cells capable of differentiating in vitro into SC-islets, (e.g., which can be subsequently transplanted); k) disease modelling (e.g., diabetes modelling) l) use according to any one of the preceding claims; m) any combination of methods according (a)-(l); n) for any of (a)-(m), wherein said use is an in vitro, ex vivo or in vivo use.

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