A method of differentiating human embryonic stem cells into beta cells via camp signalling pathway promotion

Abstract

The invention relates to a method of differentiating pancreatic bi-potent progenitor cells into beta cells comprising cultivating pancreatic endocrine precursor cells in a medium that promotes the cAMP signalling pathway that lies downstream of apical-basal polarity in endocrine precursors, which can be used to increase the differentiation of progenitor cells into beta cells. Furthermore, the present invention relates to a method of enriching and / or isolating one or more cells expressing a marker of apical-basal polarity, thereby isolating beta cells, and to a method of producing a population of beta cells from a population of human embryonic stem cells. Furthermore, the present invention relates to a population of beta cells obtainable by the method, a pharmaceutical composition comprising the beta cells, a cell culture of progenitor cells in a medium comprising a cAMP agonist, a complex comprising a beta cell bound by the binding agents specific for CD133 and CD49a, and a use of specific binding partners for isolating beta cells.

Description

New International patent applicationApplicants: Helmholtz Zentrum Munchen - Deutsches Forschungszentrum fur Gesundheit und Umwelt (GmbH)University of Copenhagen Our ref.: HEL18405PCTTITLEA method of differentiating human embryonic stem cells into beta cells via cAMP signalling pathway promotionFIELD OF THE INVENTION

[0001] The present invention provides methods for differentiating pancreatic progenitor cells into beta cells and for producing beta cells from human embryonic stem cells. Beta cells obtainable by the methods can be used in the treatment of diabetes.BACKGROUND

[0002] Fate commitment is the sum of unique intrinsic properties and extrinsic cues a progenitor / stem cell receives. While our knowledge of intrinsic properties, such as transcriptional signatures, is extensive, much less is known about how fate commitment is regulated by extrinsic cues, such as cell-cell and cell-extracellular matrix (ECM) interactions. Discerning the cues that induce cell fate within a dynamically shifting microenvironment constitutes an essential step in understanding organogenesis.

[0003] The pancreas is an endoderm-derived glandular organ composed of an exocrine and an endocrine compartment. As pancreas development proceeds, an elaborate tubular network of peripheral tip and central trunk domains forms. The tips give rise to acinar cells, whereas the trunk contributes to the duct and endocrine lineages. It has previously been demonstrated that differential cell-cell adhesion controls the fate of multipotent pancreatic progenitors by dictating their migration to either the tip or trunk niche, where they are exposed to specific fate-inducing cues. For example, changes in mechanosignalling via actin dynamics instruct the fate of bi-potent progenitors into the endocrine and duct lineages within the trunk.

[0004] Neurogenin 3 expressing (NEUROG3+) pancreatic endocrine progenitors (EPs) are specified in the trunk. Lineage tracing and loss-of-fimction experiments prove that all endocrine subtypes, including glucagon-producing alpha cells, insulin-producing beta cells, somatostatinproducing delta cells, ghrelin-producing epsilon cells, and pancreatic polypeptide-producing PP cells, go through an NEUROG3 -expressing stage. Despite this role of NEUROG3 as the master regulator of the EP program, the mechanism by which NEUROG3+cells give rise to each of the five different endocrine cell types remained uncertain, particularly with regard to fate of the two primary endocrine cell populations, alpha and beta cells.

[0005] The luminal network emerges as a result of dynamic changes of the polarization status of the progenitor epithelial cells that undergo extensive cell rearrangement. Interestingly, forced exiting of NEUR0G3+cells from the polarized luminal epithelium through conditional ablation of pl20ctn or Ecad in NEUR0G3+cells shifts their differentiation towards alpha cells at the expense of beta cells. However, far less is known about how EPs differentiate into specific endocrine cell types. There is a need in the art to better understand the differentiation of endocrine progenitor cells. There is also a need in the art for improved means and methods for differentiating endocrine progenitor cells to beta cells.SUMMARY OF THE INVENTION

[0006] The invention relates to a method of differentiating one or more pancreatic bi-potent progenitor cells into one or more beta cells comprising cultivating pancreatic bi-potent progenitor cells in a medium that comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist.

[0007] The present invention also relates to a method of enriching and / or isolating one or more beta cells, wherein the method comprises isolating and / or enriching cells expressing a marker of apical-basal polarity, wherein preferably the marker of apical-basal polarity is CD133.

[0008] The present invention also relates to a method of producing a population of beta cells from a population of human embryonic stem cells comprising Stage 1 : differentiating pluripotent stem cells to definitive endoderm cells; Stage 2: differentiating definitive endoderm cells to primitive gut tube cells; Stage 3: differentiating primitive gut tube cells to posterior foregut cells; Stage 4: differentiating posterior foregut cells to pancreatic bi-potent progenitor cells; Stage 5: differentiating pancreatic bi-potent progenitor cells to pancreatic endocrine precursor cells comprising cultivating pancreatic bi-potent progenitor cells in a medium that optionally comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist; Stage 6: differentiating pancreatic endocrine precursor cells to immature beta cells, and optionally enriching and / or isolating cells expressing CD133 and optionally CD49a; Stage 7: differentiating immature beta cells to more mature beta cells; wherein stage 5 comprises cultivating pancreatic bi-potent progenitor cells in a medium that comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist, and / or wherein stage 6 comprises enriching and / or isolating cells expressing CD133 and optionally CD49a.

[0009] The present invention also relates to a population of beta cells that is obtainable by a method of the present invention.

[0010] The present invention also relates to a pharmaceutical composition comprising a population of beta cells of the present invention.

[0011] The present invention also relates to a population of beta cells obtainable by a method of the present invention for use in therapy, wherein the therapy is preferably the treatment of diabetes.

[0012] The present invention also relates to a cell culture comprising pancreatic bi-potent progenitor cells and / or pancreatic endocrine precursor cells, in a medium that comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist.

[0013] The present invention also relates to a complex comprising a beta cell bound by a binding agent specific for CD133, wherein the binding agent is conjugated to a detectable label or a solid support.

[0014] The present invention also relates to use of a binding agent specific for CD133 for isolating and / or enriching beta cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1: Disruption of apical-basal polarity by dominant-negative CDH1 mutants in Matrigel overlay culture system or by Cdc42 knockout in the developing mouse pancreas alters beta cell specification.

[0016] (a) Design of dominant-negative CDH1 mutants with deletions in the extracellular domain (CDH1 AE) or the pl20ctn binding site (CDH1AP). mCh, IRES-coupled mCherry (red fluorescent protein) reporter.

[0017] (b) Confocal images of S5 (Day 13) differentiated cells (+ / - 72h Dox treatment). EZR (Ezrin), green. BCAT (Beta-catenin), gray. mCh (mCherry), red. Scale bar, 50 pm.

[0018] (c) Flow cytometry quantification of insulin (INS) single-positive beta cells and glucagon (GCG) positive alpha cells in mCherry+and mCherry' subpopulations after Dox treatment (S4- S6). Data represent the mean ± SD (n = 5). *, p<0.05; **, p<0.005 (two-tailed paired Student’s t- test).

[0019] (d) Maximum intensity projections (z-stack) of confocal tile scans for E16.5 dorsal pancreata stained for insulin (Ins) and glucagon (Gcg) expression. Ins, green. Gcg, red. Cdhl (E- cadherin), gray.

[0020] (e) Image quantification based on Ins+and Gcg+cell segmentation, normalized to segmented Cdhl+epithelial tissue. Data represent the mean ± SD (n = 6 embryos from 3 litters). *, p<0.05 (two-tailed unpaired Student’s t-test). Different data point symbols indicate different litters.

[0021] (f) Image quantification of beta-to-alpha ratio based on segmentation of Ins+and Gcg+cells. Data represent the mean ± SD (n = 6 embryos from 3 litters). *, p<0.05 (two-tailed unpaired Student’s t-test). Different data point symbols indicate different litters.

[0022] Figure 2. Apical-basal polarity in the human endocrine progenitor niche affects alpha versus beta cell fate.

[0023] (a) Luminal network development and endocrine progenitor (EP) generation in S5 differentiating Matrigel overlay cultures. Images show maximum intensity projections of confocal z-stacks. EZR, NEUROG3-eGFP, NEUROG3 (antibody staining), DNA (DAPI). Scale bar, 50 pm.

[0024] (b) Image quantification of apical membrane content based on segmentation of luminal EZR signal, normalized to DNA content. Data represent the mean ± SEM (n = 4). *, p<0.05 (two- tailed paired Student’s t-test).

[0025] (c) Image quantification of apical-basally polarized EPs. The percentage of NEUROG3+cells (identified by NEUROG3-eGFP+and nuclear NEUROG3+) containing EZR+membranes is shown. Data represent the mean ± SEM (n = 4). *, p<0.05 (two-tailed paired Student’s t-test).

[0026] (d) Experimental setup for assessing the differentiation potential of early- and late-induced EPs. EP factors were treated on Day 8 (early induced) or Day 11 (late induced) for 2 days. LatB, latrunculin B.

[0027] (e) Flow cytometry quantification of synchronized endocrinogenesis. The periods of treatment with pro-endocrine factors and the percentage of NEUROG3+cells are indicated.

[0028] (f) Flow cytometry quantification of the beta-to-alpha ratio after early or late EP induction. Data represent the mean ± SEM (n = 6). ***, p<0.001 (two-tailed unpaired Student’s t-test).

[0029] Figure 3. Apical-basally polarized human endocrine progenitors are primed to become beta cells.

[0030] (a) Apical membrane-specific localization of PROMI in S5 (Day 13) differentiating Matrigel overlay cultures. The image shows orthogonal views of the z-stack. PROMI (Prominin- 1, CD133), EZR, PDX1, DNA (DAPI). Scale bar, 25 pm.

[0031] (b) A subset of cells in S5 Matrigel overlay differentiation cultures retain a PROMI+membrane domain after dissociation, confirming that dissociated S5 cells can be sorted for membrane PROMI expression. Phase-contrast image and magnifications (square insert) show dissociated S5 cells. EZR is expressed in all cells but is enriched in the apical domain. Scale bar, 25 pm and 5 pm (magnifications, square insert).

[0032] (c) Flow cytometry quantification of the percentage of PROMF and PROM1+cells in NEUROG3+cell population at S5 (Day 13). Data represent the mean ± SD (n = 12).

[0033] (d) Experimental setup for assessing the differentiation potential of polarized and nonpolarized EPs by flow cytometry sorting. S5 (Day 13) cells are dissociated and sorted by NEUROG3-TagRFPt and PROMI. The sorted TagRFPt+PROMF and TagRFPt+PROMl+cells were reseeded in 2D and cultured for 4 days with basal medium.

[0034] (e) Immunocytochemistry for INS and GCG expression in sorted PROM1+and PROMF cells. Cells are sorted at Day 13 by TagRFPt and PROMI, reseeded, and cultured for 4 days with basal medium for imaging-based quantification. Confocal tile scans and magnifications (square insert). INS, GCG, DNA (DAPI). Scale bars, 500 pm (left) / 100 pm (insert, right).

[0035] (f) Imaging-based quantification of the beta-to-alpha ratio after sorting EPs for PR0M1. Data represent the mean ± SEM (n = 7). *, p<0.05 (two-tailed paired Student’s t-test).

[0036] Figure 4. Single-cell RNA sequencing reveals differentiation propensities in polarized endocrine progenitors.

[0037] (a) Setup of the CITE-seq experiment to investigate different types of EPs by hashtag conjugation. All cells were sorted for single, living (DAPI") cells before treatment with hashtag antibodies. Late-induced cell cultures were additionally sorted for apical-basal polarity (PROMF , non-polarized; PROM1+, polarized). Each sample consists of 3 biological replicates.

[0038] (b) Clustering UMAP projections showing 12 endocrine subclusters in all 4 samples.

[0039] (c) Allocation of hashtags to individual cells and subclusters. Early induced EPs, Late induced EPs, Late-induced non-polarized EPs, Late-induced polarized EPs.

[0040] (d) Comparison of fraction of cells labeled with hashtags for early / late EP induction and non-polarized / polarized status in selected subclusters.

[0041] (e) Enriched transcripts upon the comparison between early- and late-induced and between non-polarized and polarized endocrine cells (log2 fold change > 1, adjusted p-value <0.0001).

[0042] (f) Hashtag labels and trajectories based on RNA velocity analysis and predicted terminal states.

[0043] (g) Absorption probabilities for predicted terminal states for individual cells based on hashtag labels.

[0044] Figure 5. Apical-basal polarity promotes beta cell fate by suppressing ARX expression.

[0045] (A) Venn plots showing overlaps of enriched genes between polarized EPs (versus nonpolarized EPs) and early beta cells (versus early alpha cells), as well as of enriched genes between non-polarized EPs (versus polarized EPs) and early alpha cells (versus early beta cells). The scRNA-seq data from pre-beta cells versus pre-alpha cells were obtained from a published dataset.

[0046] (B and C) Validation of differential ARX expression between TagRFPt+PROMl' and TagRFPt+PROMl+cells at Day 13 by RT-qPCR (B) and western blot (C). GAPDH served as a loading control. Data represent the mean ± SD (n = 6 for RT-qPCR; n = 4 for western blot, independent repeats).

[0047] (D) Flow cytometry quantification of the percentage of ARX+PROM1‘ and ARX+PROM1+cells in S5 (Day 13) differentiating Matrigel overlay cultures. Data represent the mean ± SD (n = 6). **, p<0.001 (two-tailed paired Student’s t-test).

[0048] (E) Flow cytometry quantification of the percentage of NEUROG3+cells between mCherry+and mCherry' subpopulations during Day 10-13 after Dox treatment. Data represent the mean ± SD (n = 6). *, p<0.05 (two-tailed paired Student’s t-test).

[0049] (F) Flow cytometry quantification of the percentage of ARX+cells between mCherry+and mCherry' subpopulations during Day 10-13 after Dox treatment. Data represent the mean ± SD (n = 6). *, p<0.05; **, p<0.005; ***, p<0.001 (two-tailed paired Student’s t-test).

[0050] Figure 6. Apical-basal polarity regulates ARX expression via cAMP signalling.

[0051] (A and B) Quantification of EGR1 expression in PR0M1" and PROM1+EPs at Day 13 by RT-qPCR (A) and western blot (B). GAPDH served as a loading control, (n = 10 for RT-qPCR; n = 3 for western blot, independent repeats). *, p<0.05; *, p<0.05; **, p<0.01 (two-tailed paired Student’s t-test).

[0052] (C) Validation of EGR1 and ARX expression by RT-qPCR in NEG-siRNA and EGR1- siRNA cultures. Data represent the mean ± SD (n = 6 independent repeats). ** p<0.01 (two-tailed paired Student’s t-test).

[0053] (D) Flow cytometry quantification of the beta-to-alpha ratio in NEG-siRNA and EGR1- siRNA cultures on Day 17. Data represent the individual biological repeats (n = 6 independent repeats). *, p<0.05 (two-tailed paired Student’s t-test).

[0054] (E) ELISA quantification of cAMP level in PR0M1' and PROM1+EPs at Day 13. Data represent the mean ± SD (n = 5 independent repeats). *, p<0.05 (two-tailed paired Student’s t- test).

[0055] (F and G) Western blot quantification of phosphorylated CREB (pCREB) (F) and EGR1 (G) protein levels after Oh, 0.5h, Ih, 2h, 6h and 24h 10 pM Forskolin (FSK, Sigma-Aldrich) treatment. Data represent the mean ± SD (n = 4 for pCREB; n = 3 for EGR1, independent repeats). *, p<0.055 (two-tailed paired Student’s t-test).

[0056] (H) Western blot quantification of ARX protein levels in control and FSK treatment cells at S5 (Day 13). FSK was treated from Day 10 to Day 13. GAPDH served as a loading control. Data represent the mean ± SD (n = 5 independent repeats). ***, p<0.001 (two-tailed paired Student’s t-test).

[0057] (I) Flow cytometry quantification of the NEUROG3-eGFP+(represents the whole endocrine cells) and ARX+proportion in control and FSK-treated cells at Day 13. (NEUROG3: n = 7; ARX: n = 9, independent repeats).) ***, p<0.001 (two-tailed unpaired Student’s t-test).

[0058] (J) Flow cytometry quantification of the beta-to-alpha ratio in control and FSK-treated cultures on Day 17. Data represent the individual biological repeats (n = 9, independent repeats).***, p<0.001 (two-tailed paired Student’s t-test).

[0059] (K) Western blot quantification of ARX protein levels in control and IBMX treatment cells at S5 (Day 13). IBMX was treated from Day 10 to Day 13. GAPDH served as a loading control.Data represent the mean ± SD (n = 5, independent repeats). *, p<0.05 (two-tailed paired Student’s t-test).

[0060] (L) Flow cytometry quantification of the NEUROG3-eGFP+(represents the whole endocrine cells) and ARX+proportion in control and IBMX-treated cells on Day 13. Data represent the mean ± SD (n = 5, independent repeats). **, p<0.01 (two-tailed paired Student’s t-test).

[0061] (M) Flow cytometry quantification of the beta-to-alpha ratio in control and IBMX-treated cultures on Day 17. Data represent the individual biological repeats (n = 5, independent repeats). **, p<0.01 (two-tailed paired Student’s t-test).

[0062] (N) Model illustrating the proposed mechanism for regulation of pancreatic beta and alpha cell allocation by apical-basal polarity. Tight junctions (TJ) provide essential physical and signalling cues for establishing apical-basal polarity. Within the polarized EPs, PR0M1 facilitates the recruitment of ERM family proteins to bring PKA to membrane lipid rafts where GPCR, adenylyl cyclase (AC), and PKA substrates are in close proximity to increase the response of cAMP signals. The resulting increased activity of cAMP / PKA-CREB signalling promotes the expression of EGR1 and further inhibits the expression of ARX, thereby allowing EPs to differentiate into beta cells. In the non-polarized environment, EPs exhibit relatively low activity of cAMP / PKA-CREB signalling, subsequently diminishing EGR1 expression and permitting the expression of ARX. This shift allows EPs to differentiate into alpha cells. Forskolin and IBMX, as cAMP agonists, can increase intracellular cAMP levels and promote the generation of beta cells.

[0063] Figure 7: Dominant-negative CDH1 mutant cell line design and characterization and the Matrigel overlay differentiation protocol setup.

[0064] (a) Transgenic cassette inserted into hESCs for inducible and detectable overexpression of dominative-negative CDH1 mutants. ITR, inverted terminal repeats. TetOP, Dox-responsive promoter. IRES, internal ribosomal entry site. pA, polyadenylation signal. EFl alpha, constitutive promoter. rtTA, reverse tetracycline trans-activator. NeoR, neomycin resistance.

[0065] (b) Morphology changes upon mutant CDH1 overexpression. Phase contrast images of clonal hESC lines cultured under pluripotency-maintaining conditions. Dox, doxycycline (48h treatment). Scale bar, 100 pm.

[0066] (c) Protocol for differentiation of hESCs into alpha and beta cells inside a 3D epithelial environment. DE, definitive endoderm. PGT, primitive gut tube. PFG, posterior foregut. PE,pancreatic endoderm. EP, endocrine progenitors. MG, matrigel. i, inhibitor, a, activator. AA, activin A. VitC, vitamin C. RA, retinoic acid. SHH, Sonic Hedgehog. T3, triiodothyronine. ySec, gamma secretase.

[0067] (d) Confocal image with orthogonal views of z-stack in S5 (Day 13) differentiating Matrigel overlay cultures. EZR, ZO-1, PDX1, DNA (DAPI). Scale bar, 25 pm.

[0068] (e) Mosaic expression of the mCherry reporter in CDH1 mutant cultures at S6 (treated with Dox for 13 days). Scale bar, 75 pm.

[0069] (f) Flow cytometry quantification of transgenic (mCherry+) S6 cells (treated with Dox for 13 days). Data represent the mean ± SEM (n = 5).

[0070] Figure 8. Cdc42 knockout disrupts apical-basal polarity in the developing mouse pancreas. (Related to Figure 1)

[0071] (a) Breeding setup for the generation of pancreatic (Pdxl-C e. driven) Cdc42 knockout mice, fl, LoxP-flanked allele; R26, ROSA26 locus; YFP, yellow fluorescent protein; wt, wild-type control; KO, knockout.

[0072] (b) E16.5 wild-type and Cdc42 knockout dorsal pancreas tissue. Confocal images and maximum intensity projection (max. int. proj.) of a z-stack. Cdhl, gray. Mucl (Mucinl), red. R26- YFP, green. Scale bar, 250 pm.

[0073] Figure 9. Endocrine progenitor niche in the developing human pancreas and NEUROG3-eGFP / EZR-mKate double reporter cell line design. (Related to Figure 2)

[0074] (a) EP niche in the developing human pancreas. Confocal images of fetal pancreas sections (9.9 wpc) with orthogonal views of z-stack. Filled arrowheads, apical-basally polarized NEUROG3+cells containing an EZR+membrane. Empty arrowheads, non-polarized NEUROG3+cells. EZR, NEUROG3, BCAT, DNA (DAPI). Scale bar, 25 pm.

[0075] (b) Transgenic locus of the NEUROG3-eGFP reporter. BAC, bacterial artificial chromosome, eGFP, enhanced green fluorescent protein. pA, polyadenylation signal. CDS, coding sequence.

[0076] (c) Transgenic locus of the EZR-mKATE2 fusion reporter. GS, glycine-serine linker. mKATE2, red / far-red fluorescent protein. P2A, self-cleaving peptide. HygroR, hygromycin resistance.

[0077] Figure 10. PROMI is a surface marker of apical-basal polarity and NEUROG3- TagRFPteGFP reporter cell line design. (Related to Figure 3)

[0078] (a, b) Apical membrane-specific localization of PROMI in the developing human pancreas. Confocal images of fetal pancreas sections (10.6 wpc). (a) MUC1, LAM (laminin), DNA (DAPI). (b) EZR, aPKC (atypical protein kinase C), DNA (DAPI). Scale bars, 25 pm.

[0079] (c) Transgenic locus of the NEUROG3-TagRFPteGFP reporter. TagRFPt, red fluorescent protein. NLS, nuclear localization signal.

[0080] Figure 11. Identification and characterization of cell types by single-cell RNA sequencing. (Related to Figure 4)

[0081] (a) Clustering UMAP projections showing the entire dataset in all 4 samples. The samples are comprised of endocrine, bipotent, ductal, and PSC-like populations.

[0082] (b) Expression of selected marker genes in each identified population. Log2 fold change in count numbers relative to the average throughout the dataset is displayed.

[0083] (c) Allocation of hashtags to individual cells. Cells without hashtags or with two hashtags were excluded.

[0084] (d) Expression of selected marker genes in the endocrine subclusters. Log2 fold change in count numbers relative to the average throughout the endocrine population is displayed.

[0085] (e) Enriched transcripts in 12 endocrine subclusters compared to the average throughout the endocrine population (log2 fold change > 1, adjusted p-value < 0.0001). The 20 genes with the highest fold changes are shown.

[0086] (f) Pseudotime analysis of cells in the endocrine population.

[0087] Figure 12. Single-cell RNA sequencing data validation. (Related to Figures 4)

[0088] Validation of enriched top 3 transcripts identified in early-induced, late-induced, PROMI' (non-polarized), and PROM1+(polarized) EPs by RT-qPCR. Data represent the mean ± SD (n = 7 for early and late EPs; n = 5 for PR0M1+and PR0M1 ' EPs). *, p<0.05; **, p<0.005; ***, p<0.001 (two-tailed paired Student’s t-test).

[0089] Figure 13. ARX is a downstream target of NEUROG3 and partakes in the regulation of alpha cell generation. (Related to Figure 5)

[0090] (a and b) Exemplary staining of ARX with NEUROG3-tagRFPt and NEUROG3-eGFP (a) and quantification of co-expressing cells (b). ARX, NEUROG3-tagRFPt, NEUROG3-GFP, DAPI. Scale bars, 25 pm.

[0091] (c) Time course analysis of ARX and GCG expression levels during Day 9-16 of differentiation. Data represent the mean ± SD (n = 4).

[0092] (d) Exemplary staining shows a nearly complete overlap between GCG+cells and ARX+cells at Day 20 (the end of S6). ARX, GCG, DAPI. Scale bars, 20 pm.

[0093] (e) Flow cytometry quantification of ARX+cell proportion in GCG+cells at Day 20 (the end of S6). Data represent the mean ± SD (n = 4).

[0094] (f) Model illustrating that ARX is absent in early NEUROG3+EPs (TagRFPt+and eGFP ) but starts expression in late NEUROG3+EPs (TagRFPt+and eGFP+). Later, ARX continues to be expressed in GCG+alpha cells, which turn off TagRFPt but still express GFP.

[0095] Figure 14: Activation of the cAMP signalling pathway during the endocrine progenitor stage decreases ARX expression and increases beta cell generation.

[0096] Figure 15: Activation of the cAMP signalling pathway by Forskolin treatment during the endocrine progenitor stage decreases ARX expression and increases beta cell generation.

[0097] (a) Experimental setup for Forskolin treatment during EP stage.

[0098] (b) Western blot quantification of ARX protein levels in control and FSK treatment cells at S5 (Day 13). FSK was treated from Day 10 to Day 13. GAPDH served as a loading control. Data represent the mean ± SD (n = 9). ***, p<0.001 (two-tailed paired Student’s t-test).

[0099] (c) Flow cytometry quantification of the NEUROG3-eGFP+(right, represents the whole endocrine cells) and ARX+(left) proportion in control and FSK-treated cells at Day 13. Data represent the mean ± SD (NEUROG3: n = 5; ARX: n = 7). ***, p<0.001 (two-tailed paired Student’s t-test).

[0100] (d) Flow cytometry quantification of the beta-to-alpha ratio in control and FSK-treated cultures at Day 17-18. Data represent the individual biological repeats (n = 9). *, p<0.05 (two- tailed unpaired Student’s t-test).

[0101] Figure 16: Activation of the cAMP signalling pathway by IBMX treatment during the endocrine progenitor stage decreases ARX expression and increases beta cell generation.

[0102] (a) Experimental setup for IBMX treatment during EP stage.

[0103] (b) Western blot quantification of ARX protein levels in control and IBMX treatment cells at S5 (Day 13). IBMX was treated from Day 10 to Day 13. GAPDH served as a loading control. Data represent the mean ± SD (n = 5). ***, p<0.001 (two-tailed paired Student’s t-test).

[0104] (c) Flow cytometry quantification of the NEUROG3-eGFP+(right, represents the whole endocrine cells) and ARX+(left) proportion in control and IBMX-treated cells at Day 13. Data represent the mean ± SD (n = 7). ***, p<0.001 (two-tailed paired Student’s t-test).

[0105] (d) Flow cytometry quantification of the beta-to-alpha ratio in control and IBMX-treated cultures at Day 17-18. Data represent the individual biological repeats (n = 5). *, p<0.05 (two- tailed unpaired Student’s t-test).

[0106] Figure 17: Quantification of beta cell and GCG+cell proportion among unsorted, CD49+ / CD133hl8hand CD49+ / CD133 cells, as well as unsorted, Gl)133hi"h. NGN3+ / CD133hl8hand CD49+ / CD133

[0107] Figure 18: CD133 is a Novel Marker for Enriching SC-beta Cells in Combination with the Endogenous Neurog3 Reporter.

[0108] A, Schematic diagram of stepwise differentiation protocol to generate SC-islets from hESC. B, Quantitative real-time PCR (RT-qPCR) assessment of marker genes in sorted and unsorted group, (n = 4 independent samples). Data are expressed as mean ± SEM, *p < 0.05, **p < 0.01, P values determined by two-tailed Welch’s t-test. ns, not significant. C, insulin secretion simulation index of SC-islets by in vitro static GSIS. D, quantification of secreted insulin from SC-islets challenged sequentially with 1.67 or 16.67mM glucose with a 30-min incubation for each concentration. Data presented as mean insulin secreted normalized on insulin content ± SEM (n = 2 biologically independent samples). E, immunostaining on frozen sections for INSULIN and NKX6.1 of re-aggregated cells after NGN3 / CD 133hlghand NGN3 / CD133' sorting.

[0109] Figure 19: Utilizing CD133 as A Marker for Excluding Glucagon-Producing Cells and Improve the Purity of Stem Cell-Derived beta Cells.

[0110] SC-alpha vs SC-beta population relative position based on CD 133 expression level along S6 differentiation. Relative position is defined as (0.95 percentile alpha population - 0.25 percentile beta population) / (0.75 percentile beta population - 0.25 percentile beta population). Right, Flow cytometry analysis based on gating selection during S6 differentiation.

[0111] Figure 20: Refining SC-beta Cell Purity Using CD133 / CD49a-Based Sorting.

[0112] (A) Quantification of %SC beta cells (NKX6.1+INSULIN+) and %SC-alpha cells (GCG+) cells of sorted cells by flow cytometry (n=3 / 4 independent differentiation experiments).

[0113] (B) Flow cytometry quantification of %SC beta cells (NKX6.1+INSULIN+) and %SC- alpha cells (GCG+) cells of recovered cells after sorting and suspension culture for one more week (n= independent differentiation experiments), *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001. Data is presented as the mean ± SEM, p values calculated using 2way ANOVA.

[0114] (B and C) Insulin secretion stimulation index of by in vitro static GSIS.

[0115] (D and EE) Quantification of secreted insulin from SC-islets challenged sequentially with 1.67 or 16.67mM glucose with a 30-min incubation for each concentration. Data presented as mean insulin secreted normalized on insulin content ± SEM.DETAILED DESCRIPTION

[0116] The present invention is based on the finding that there is a connection between cell adhesion and apical-basal cell polarity in influencing the fate determination of NEUROG3+cells into the alpha and beta cell lineages. As shown in the examples, the inventors of the present invention have found that dynamic modulation of apical-basal polarity, either directly or indirectly via cell-cell adhesion, within human EPs changes the cell fate preference. It has been surprisingly demonstrated that while polarized EPs, via cAMP-mediated inhibition of ARX expression, preferentially turn into beta cells, reduced cAMP levels lead to maintenance of ARX and alpha cell differentiation in non-polarized EPs.

[0117] In early mammalian embryo development, apical-basal polarity plays a critical role in the initial lineage allocation between inner cell mass and trophectoderm. By the end of the 8-cell stage, the outer cells of embryos acquire apical-basal polarity, which promotes Yap nuclear localization, leading to the expression of Cdx2 and the commitment to the trophectoderm lineage. During organogenesis, multipotent epithelial progenitors coordinate tissue remodelling and fate decisions, and compelling evidence exists for a reciprocal governance relationship between these processes. While it is known that apical-basal polarity governs the balance between proliferation and differentiation of neural progenitors through Notch signalling, it remained unclear whether apical- basal polarity controls subsequent fate decisions of multipotent neural progenitors. The dynamic nature of tissue remodelling implies that epithelial features, such as apical-basal polarity,continuously change (for polarity from polarized to non-polarized states). A central question in cell and developmental biology is therefore how dynamic changes in multipotent epithelial progenitor features directly affect their cell lineage decisions.

[0118] The inventors of the present application have developed an hPSC-based in vitro model that mimics the human EP niche, which revealed dynamic shifts in apical-basal polarization of EPs. The inventors of the present application have surprisingly uncovered a direct role of apical-basal polarity in the fate allocation of EPs into the beta and alpha cell lineages. Specifically, the inventors of the present application demonstrate that apical-basal polarity in EPs promotes beta cell specification via cAMP / PKA-CREB-EGRl -mediated inhibition of ARX expression, while reduced cAMP levels in non-polarized EPs maintain expression of ARX leading to alpha cell differentiation.

[0119] Surprisingly, the inventors of the present application have identified the unknown mechanism for how ARX expression within EPs is dictated by environmental cues. Specifically, while non-polarized EPs maintain ARX expression leading to alpha cell specification, polarized EPs preferentially differentiate into beta cells because of reduced ARX expression.

[0120] To resolve the underlying molecular mechanism for how cell polarity controls ARX expression, the inventors of the present application first identified EGR-1 as a negative regulator of ARX expression. EGR-1 gene transcription is regulated by G-protein coupled receptors (GPCRs) via the cAMP / PKA-CREB signalling pathway in pancreatic beta cells. However, none of the GPCRs expressed in EPs are differentially expressed in polarized versus non-polarized EPs. Another potential regulator of this pathway is CD133 (PR0M1). First, PR0M1 is specifically expressed in polarized EPs (Figure 3 A, 10A and 10B). Secondly, PR0M1 regulates hepatic gluconeogenesis and adipogenesis via membrane recruitment of ERM family proteins (Ezrin (EZR), Radixin, and Moesin), thereby bringing PKA in close proximity to GPCRs, adenylyl cyclase (AC), and PKA substrates within membrane lipid rafts. The fact that PR0M1 and EZR are specifically localized at the apical membrane of polarized EPs (Figure 3A and 10B) suggests a similar role of PR0M1 in the regulation of the cAMP / PKA-CREB signalling pathway in polarized EPs. Consistently, cAMP levels are higher in PROM1+polarized EPs compared to PROMT nonpolarized EPs.

[0121] Altogether, the inventors of the present application have found that apical-basal polarity as a key regulator of the transcriptional machinery involved in the fate allocation of EPs into the betaand alpha cell lineages. The present application shows that the cAMP / PKA-CREB signalling pathway is more active in polarized EPs, which will give rise to beta cells, compared to nonpolarized EPs, which are destined to become alpha cells. The unique expression of PR0M1 and EZR on the apical membrane of polarized EPs together with the connection between PR0M1 and ERM proteins in activation of the cAMP / PKA-CREB signalling pathway suggest a mechanism for the regulation of this pathway in EPs by apical-basal polarity. The inventors of the present invention have found that the key link between the cAMP / PKA-CREB signalling pathway and the transcriptional machinery involved in beta and alpha cell specification is mediated by EGR1 and ARX. In polarized EPs, activation of cAMP / PKA-CREB signalling increases expression of EGR1, leading to reduced expression of ARX and beta cell specification. In non-polarized EPs, cAMP / PKA-CREB signalling is below the threshold to maintain sufficient levels of EGR1 to inhibit expression of ARX, resulting in sustained ARX expression and the birth of alpha cells (Figure 6K). Considering the dynamic shift of EPs between polarized and a non-polarized states and the short half-life of cAMP, the inventors of the present application postulate that only when apical-basal polarity stabilizes will PR0M1ZEZR proteins be able to bring all the components of the cAMP / PKA-CREB signalling pathway in proximity on the apical membrane. This enables activation of the pathway to ultimately induce beta cell specification via reduced expression of ARX. These findings elucidate how cell polarization guides EPs to enter the beta or alpha cell lineages, offering insights into the environmental cues and mechanisms governing multipotent progenitors' acquisition of distinct fates within their dynamic niche.

[0122] Based on these findings, the inventors of the present application have further found that beta cell differentiation can be enhanced, by enhancing the downstream signalling pathway and / or transcription machinery that is regulated by apical-basal polarity. This downstream signalling pathway and / or transcription machinery can be activated by contacting pancreatic bi-potent progenitor cells with one or more compounds that promote cAMP signalling pathway, such as a cAMP agonist.

[0123] Further, the inventors of the present application have also found that the PR0M1 (CD133) can be used as a marker for beta cells. By isolating cells that are PROM1+, it is possible to enrich beta cells and deplete alpha cells in a mixture comprising both types of cells. This selective enrichment of beta cells is useful for the preparation of a cell population having high beta cell content. Such cell population with high beta cell content can be useful as a therapy for diabetes.

[0124] Accordingly, the present invention relates to a method of differentiating one or more pancreatic bi-potent progenitor cells into one or more beta cells comprising cultivating pancreatic bi-potent progenitor cells in a medium that comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist. The suitable amount of time for cultivating pancreatic bipotent progenitor cells in a medium that comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist, is at least about 30 mins to about 6 d, preferably 3 d. Thus, the suitable amount of time may be about 30 mins, about 60 mins, about 2 hours, about 4 hours, about 8 hours, about 12 hours, about 16 hours, about 20 hours, about 24 hours, about 28 hours, about 32 hours, about 36 hours, about 40 hours, about 44 hours, about 2 d, about 2.5 d, about 3 d, about 3.5 d, about 4 d, about 4.5 d, about 5 d, about 5.5 d, about 6 d or even longer than 6 days.

[0125] As used herein, “pluripotent stem cells” refer to cells that can differentiate into any cell type in the body, including cells of all three germ layers ectoderm, mesoderm, and endoderm, except for extra-embryonic tissues like the placenta. They include embryonic stem cells (derived from early embryos) and induced pluripotent stem cells (adult cells reprogrammed to a pluripotent state). Examples of human pluripotent stem cell lines that can be used by the present invention include SA121, HUES4, and H9, just to name a few.

[0126] As used herein, “definitive endoderm cells” refer to are a specific layer of cells that arise during embryonic development and give rise to the internal organs and tissues of the body. These cells originate from the inner cell mass of the blastocyst and are crucial for forming the endoderm layer, which eventually differentiates into structures such as the liver, pancreas, lungs, and gastrointestinal tract. Definitive endoderm cells are a more specialized stage compared to the earlier primitive endoderm, with the potential to develop into various essential organ systems.

[0127] As used herein, “primitive gut tube cells” refer to early developmental cells that form the initial structure of the gastrointestinal tract in the embryo. They arise from the definitive endoderm and fold to create the primitive gut tube, which eventually differentiates into the entire digestive system, including the esophagus, stomach, intestines, and associated organs. During embryogenesis, the primitive gut tube undergoes further specialization and segmentation to develop into the various parts of the digestive tract. Primitive gut tube cells are essential for establishing the basic framework of the gut and for the subsequent development of more specialized cells and tissues within the gastrointestinal system.

[0128] As used herein, “posterior foregut cells” refer to a subset of cells within the developing foregut that contribute to the formation of the pancreas. During embryonic development, the foregut is divided into anterior, middle, and posterior regions, with the posterior foregut playing an important role in pancreatic development.

[0129] As used herein, “pancreatic bi-potent progenitor cells” refer to specialized cells within the developing pancreas that have the potential to differentiate into two key pancreatic cell types: endocrine cells, which include insulin-producing beta cells, and exocrine cells, which are responsible for producing digestive enzymes. These progenitor cells lead to the formation of the islets of Langerhans and the acinar tissue.

[0130] As used herein, “pancreatic endocrine precursor cells” refers to a group of pancreatic progenitor cells originating from the developing fore-gut endoderm which have the ability to differentiate into the endocrine precursor cells. Pancreatic endocrine precursor cells are a committed group of progenitors that develop into all of the endocrine cells in the pancreas including delta cells, PP-cells, epsilon cells, beta cells and alpha cells. PP-cells produce pancreatic polypeptide which is a regulator of endocrine and exocrine secretions in the pancreas and gut. Delta cells produce somatostatin which is a growth hormone inhibiting hormone. Epsilon cells produce Ghrelin (hunger hormone) which is a neuropeptide that acts on the hypothalamic center of the brain and mediates hunger.

[0131] As used herein, “beta cells” refer to specialized cells located in the pancreas, specifically within the islets of Langerhans. They play a crucial role in regulating blood sugar levels by producing and secreting insulin, a hormone that serves to lower blood glucose. When blood sugar levels rise, such as after eating, beta cells release insulin into the bloodstream, which facilitates the uptake of glucose by cells for energy or storage. Dysfunction or destruction of beta cells, as seen in conditions like type 1 and type 2 diabetes, leads to impaired insulin production and regulation of blood glucose levels. As used herein, the term “beta cells” encompasses both mature beta cells as well as immature beta cells.

[0132] As used herein “mature beta cells” refer to beta cells containing more insulin granules and secrete more insulin when stimulated with high levels of glucose compared to immature beta cells. As used herein "immature beta cells” refer to beta cells arising during embryonic development with an immature phenotype, which is characterized by lower insulin content and lower insulin secretion during high glucose challenge than mature beta cells. Immature beta cells also lackinsulin in low glucose conditions. In addition, the expression levels of some beta cell marker genes (e.g., MAFA, SIX2, SIX3, IAPP, UCN3) are also lower in immature beta cells than in mature beta cells.

[0133] Preferably, the one or more pancreatic bi-potent progenitor cells are cultivated in an airliquid interface, preferably in a 2D or 3D culture, optionally in a Matrigel overlay culture. As used herein, “Matrigel” refers to a commercial, well-known, and widely used cell culture matrix composed of natural polymers (laminin, collagen IV, and entactin) and growth factors secreted by Engelbreth-Holm- Swarm mouse sarcoma cells. Matrigel is typically used in research for culturing cells that require a 3D environment, such as stem cells, organoids, and cancer cells, since it mimics the natural extracellular environment found in tissues. It supports cell attachment, growth, differentiation, and migration, making it useful in creating 3D cultures that more closely resemble in vivo conditions. Other types of 2D and 3D cultures are known to the skilled person and include cells on multi-well plates or in flasks (2D) and suspension culture of cells either on low-attachment 6-well plates using orbital shakers or in spinner flasks (3D).

[0134] Preferably, the cell culture comprising air-liquid interface promotes the establishment of apical-basal polarity in said cells. Thus, culturing pancreatic bi-potent progenitor cells and / or pancreatic endocrine precursor cells in said cell culture directly promotes the differentiation of said cells into beta cells. The establishment of apical-basal polarity leads to activation of cAMP signalling pathway, thereby promoting beta cell fate by suppressing ARX expression as described elsewhere herein. The use of specific culture conditions to promote apical-basal polarity of endocrine precursor cells is therefore another way of generating beta cells, alternative to the stimulation of cAMP signalling pathway, which lies downstream of the signalling pathway of apical-basal polarity in endocrine precursor cells, using e.g. cAMP agonists as described elsewhere herein.

[0135] It is envisaged by the present invention that the promotion of apical-basal polarity of endocrine precursor cells with specific cell culture conditions may be used to generate beta cells in a medium that does not comprise a compound that promotes cAMP signalling pathway, e.g. in the absence of cAMP agonists. It is also envisaged by the present invention that the precursor cells, i.e. pancreatic bi-potent progenitor cells and / or pancreatic endocrine precursor cells, are grown in medium which comprises beads, wherein the cells are seeded onto the surface of the beads, wherein growth of the cells on the beads promotes the establishment of apical-basal polarity insaid cells leading to activation of cAMP signalling pathway, thereby promoting beta cell fate by suppressing ARX expression as described elsewhere herein.

[0136] Preferably, the one or more pancreatic bi-potent progenitor cells are obtainable or obtained according to Stage 4, Stage 3-4, Stage 2-4, and / or Stage 1-4 as defined elsewhere herein. Preferably, the method comprises conducting Stage 1, Stage 2, Stage 3, and / or Stage 4 as defined elsewhere herein, prior to cultivating the pancreatic endocrine precursor cells for about 3 d in a medium that comprises a compound that promotes cAMP signalling pathway. Preferably, the method comprises conducting Stage 6 and / or Stage 7 as defined elsewhere herein, after cultivating the pancreatic endocrine precursor cells for about 3 d in a medium that comprises a compound that promotes cAMP signalling pathway.

[0137] As used herein, “cAMP” refers to cyclic adenosine monophosphate (cAMP), which is a secondary messenger molecule that plays a crucial role in various biological processes by mediating the effects of primary messengers, such as hormones. It is synthesized from adenosine triphosphate (ATP) by the enzyme adenylate cyclase in response to extracellular and intracellular signals. cAMP activates protein kinase A (PKA) and other signalling pathways, leading to the regulation of cellular functions such as gene expression, metabolism, and cell proliferation. Thus, cAMP is a regulatory molecule involved in intracellular signal transduction mechanisms.

[0138] As used herein, “cAMP signalling pathway” refers to key cellular communication system that regulates various physiological processes. It begins when a signalling molecule binds to a G protein-coupled receptor (GPCR) on the cell surface, activating a G protein. The G protein then stimulates adenylate cyclase to convert ATP into cAMP. Elevated cAMP levels activate protein kinase A (PKA), which phosphorylates target proteins, influencing processes such as gene expression, metabolism, and cell growth. Phosphodiesterases (PDEs) terminate the signal by breaking down cAMP.

[0139] Preferably, the cAMP agonist is selected from the group consisting of forskolin, IBMX, Dibutyryl-cAMP, 8-MA-cAMP, 6-MB-cAMP, exendin-4. More preferably, the cAMP agonist is forskolin, wherein the forskolin is preferably present at a concentration of about 5 pM to about 40 pM, more preferably about 10 pM. More preferably, the cAMP agonist is IBMX, wherein the IBMX is preferably present at a concentration of about 50 pM to about 200 pM, more preferably about 100 pM.

[0140] Preferably, the medium comprises about 20 mM glucose, SANT-1, about 0.05 pM retinoic acid, an ALK5 inhibitor, T3 and LDN193189. Preferably, the medium comprises sodium bicarbonate, Glutamax, glucose, BSA, SANT-1, retinoic acid, LDN193189, ITS-X, T3 (3,3’ ,5- Triiodo-l-thyronine sodium salt), ALK5 inhibitor II, zinc sulfate, and heparin. More preferably, the medium comprises about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 20 m glucose, about 2% BSA, about 0.25 pM SANT-1, about 0.05 pM retinoic acid, about 100 nM LDN193189, about 0.5% ITS-X, about 1 pM T3 (3,3’,5-Triiodo-l-thyronine sodium salt), about 10 pM ALK5 inhibitor II, about 10 pM zinc sulfate, and about 10 pg / ml heparin. Preferably, the medium is MCDB131 medium.

[0141] Preferably, the method increases differentiation of pancreatic bi-potent progenitor cells into beta cells as compared to a reference method, wherein the reference method preferably does not comprise the use of said cAMP agonists, but is otherwise identical. Preferably, the method decreases differentiation of pancreatic bi-potent progenitor cells into alpha cells as compared to a reference method, wherein the reference method preferably does not comprise the use of said cAMP agonists, but is otherwise identical. For example, the reference method is a method comprising at least Stage 5 as disclosed herein, with the exception that the reference method does not comprise the use of a cAMP agonist in Stage 5.

[0142] Preferably, the method comprises a step of enriching and / or isolating one or more beta cells as defined elsewhere herein at the end of Stage 6.

[0143] The present invention also relates to a method of enriching and / or isolating one or more beta cells, wherein the method comprises isolating and / or enriching cells expressing a marker of apical-basal polarity, wherein preferably the marker of apical-basal polarity is CD 133. CD 133, also known as Prominin- 1 or PR0M1, is a transmembrane glycoprotein that serves as a marker of stem cells, particularly hematopoietic stem cells and cancer stem cells. It is expressed on the surface of a variety of stem and progenitor cells, as well as on certain cancer cells. CD133 has been implicated in various cellular processes, including cell proliferation and apoptosis. CD133 is located specifically in the apical domain of polarized epithelial cells. Thus, in the context of the present invention, CD 133 is used as a cell surface marker of apical-basal polarity. The skilled person understands from the disclosure of the present application that other apical-basal polarity surface markers expressed in endocrine precursors may be used in the same way as CD 133 to enrich beta cells in the method of the present invention.

[0144] Preferably, the method comprises isolating and / or enriching one or more cells expressing CD133 from a mixture comprising beta cells and alpha cells.

[0145] Preferably, the method of enriching and / or isolating comprises contacting the one or more beta cells with a binding agent that specifically binds CD 133 and isolating the beta cell bound to the binding agent.

[0146] The term "binding agent" characterizes in connection with the present invention a compound which is capable of specifically binding to / interacting with / recognizing a given target epitope or a given target site on the target molecules (antigens), such as CD 133. The structure and / or function of the binding agent is preferably based on the structure and / or function of an antibody. The term "antibody" generally refers to a molecule in which the structure and / or function is / are based on the structure and / or function of an antibody. Typical examples of an antibody are immunoglobulins, as well as derivatives or functional fragments thereof which still retain the binding specificity. Techniques for the production of antibodies are well known in the art. The term "antibody" includes immunoglobulins (Ig's) of different classes (i.e. IgA, IgG, IgM, IgD, IgE, IgY etc.) and subclasses (such as IgGl, lgG2 etc.), as well as heavy chain only antibodies (such as hcIgGm IgNAR, etc.), even if recombinantly produced in foreign hosts using techniques known to those skilled in the arts. Illustrative examples of an antibody are full length immunoglobulins, Fab fragments, F(ab')2, Fv fragments, single-chain Fv fragments (scFv), diabodies, or domain antibodies (Holt LJ et al., Trends Biotechnol. 21(11), 2003, 484-490). Domain antibodies may be single domain antibodies, single variable domain antibodies or immunoglobulin single variable domain having only one variable domain, which may be VH or VL, that specifically bind an antigen or epitope independently of other V regions or domains. A particularly preferred single domain antibody is a VHH domain heavy chain only antibody. The definition of the term "antibody" thus also includes embodiments such as chimeric, single chain and humanized antibodies. The term “antibody” may also include fragments of antibodies.

[0147] The term "specifically binding", as used herein means that the binding domain preferentially binds or recognizes the target even when the binding partner is present in a mixture of other molecules or other structures. The binding may be mediated by covalent or non-covalent interactions or a combination of both.

[0148] Antibodies that are specific for CD 133 are widely commercially available, such as antihuman CD133 Antibody (Miltenyi Biotec, #130-113-668), and BD Pharmingen™ APC MouseAnti-Human CD133 (BD Bioscience, #566596), further antibodies specific for CD133 are, e.g., described in WO2022022718, W02021080542, WO2011149493, or W02010057110.

[0149] Preferably, the method of enriching and / or isolating comprises isolating and / or enriching one or more cells expressing a marker of endocrine cells, wherein preferably the marker of endocrine cells is CD49a. CD49a, also known as integrin alpha-1 (ITGA1), is a transmembrane protein that functions as a cell surface receptor involved in cell adhesion, migration, and signal transduction. It is a part of the integrin family of proteins and pairs with the beta-1 integrin subunit (CD29) to form the alphalbetal integrin complex, also known as the VLA-1 (Very Late Antigen- 1) receptor. In the context of the present invention, CD49a is used as a general pancreatic endocrine cell marker. The use of CD49a as a general pancreatic endocrine cell marker reduces the population of non-endocrine cells, such as duct cells. Thus, it is understood that the method of cell enriching preferred by the present invention comprises a combination of CD133 and CD49a markers. The method of enriching and / or isolating may also comprise isolating and / or enriching one or more cells expressing alternative endocrine cell markers, such as NGN3 (neurogenin-3).

[0150] Antibodies that are specific for CD49a are widely commercially available, such as CD49a antibody, anti-human, PE, REAfinity™ (Miltenyi Biotec, #130-119-306) or BD Pharmingen™ PE Mouse Anti-Human CD49a (BD Bioscience, #55959), further antibodies specific for CD49a are, e.g., described in W02017094001, WO2013123114, WO2019018688, and US20160017043.

[0151] Preferably, the method reduces the population of glucagon-producing cells. Preferably, the method reduces the population of non-endocrine cells.

[0152] The term “glucagon-producing cells” as used herein refers to alpha cells, which are located in the islets of Langerhans within the pancreas. Alpha cells are responsible for synthesizing and secreting glucagon, a hormone that plays a crucial role in maintaining blood glucose levels, particularly during fasting or periods of low blood sugar. Preferably, the isolation and / or enriching is conducted by cell sorting. More preferably, the isolation and / or enrichment is conducted by means of fluorescence activated cell sorting (FACS) or magnetic-activated cell sorting (MACS).

[0153] The method preferably comprises, prior to isolation and / or enrichment, cultivating beta cells in an air-liquid interface in a medium comprising about 20 mM glucose, ALK5 inhibitor II, T3, LDN193189, and gamma-secretase inhibitor XX for 7-15 d, preferably 7 d. The method preferably comprises, prior to isolation and / or enrichment, cultivating beta cells in an air-liquid interface in in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose,BSA, LDN193189, ITS-X, T3, ALK5 inhibitor II, zinc sulfate, gamma-secretase inhibitor XX and of heparin, for 7-15 d, preferably 7d, preferably as defined by the present invention for Stage 6.

[0154] Preferably, the method further comprises differentiating cells to more mature beta cells after the isolation and / or enrichment, for example as disclosed herein for stage 7. Preferably, the final differentiation is conducted in a 3D culture in a medium comprising ALK inhll, T3, N-Cys, and AXL inh for about 7-15 d, preferably about 7 d.

[0155] Preferably, the one or more pancreatic bi-potent progenitor cells are obtainable or obtained according to Stage 5, Stage 4-5, Stage 3-5, Stage 2-5, and / or Stage 1-5 as defined by the present invention.

[0156] Preferably, the method comprises conducting Stage 1, Stage 2, Stage 3, Stage 4, Stage 5 and / or Stage 6 as defined by the present invention, prior to isolating and / or enriching one or more cells expressing CD133 and optionally CD49a from a mixture comprising beta cells and alpha cells.

[0157] Preferably, the method comprises conducting Stage 7 as defined by the present invention, after isolating and / or enriching one or more cells expressing CD133 and optionally CD49a.

[0158] It is understood that the method of differentiating one or more pancreatic bi-potent progenitor cells of the disclosure can be combined with the method of enriching and / or isolating one or more beta cells of the disclosure. By combining both methods, the yield of beta cells can be further enhanced.

[0159] Present invention also relates to a method of producing a population of beta cells from a population of human embryonic stem cells comprising Stage 1 : differentiating pluripotent stem cells to definitive endoderm cells; Stage 2: differentiating definitive endoderm cells to primitive gut tube cells; Stage 3: differentiating primitive gut tube cells to posterior foregut cells; Stage 4: differentiating posterior foregut cells to pancreatic bi-potent progenitor cells; Stage 5: differentiating pancreatic bi-potent progenitor cells to pancreatic endocrine precursor cells comprising cultivating pancreatic bi-potent progenitor cells in a medium that optionally comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist, preferably as defined in the present invention; Stage 6: differentiating pancreatic endocrine precursor cells to immature beta cells, and optionally enriching and / or isolating cells expressing CD133 and optionally CD49a, preferably as defined in the present invention; and Stage 7: differentiating immature beta cells to more mature beta cells; wherein stage 5 comprises cultivating pancreaticbi-potent progenitor cells in a medium that comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist, and / or wherein stage 6 comprises enriching and / or isolating cells expressing CD 133 and optionally CD49a.

[0160] Preferably, stage 1 is conducted as follows: culturing pluripotent stem cells in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose, bovie serum albumin (BSA), Activin A, and CHIR-99021 on day 1; culturing the cells in MCDB131 medium, which comprises BSA, sodium bicarbonate, Glutamax, glucose, Activin A, and CHIR-99021 on day 2; and culturing the cells in MCDB131 medium, which comprises BSA, sodium bicarbonate, Glutamax, glucose and Activin A on day 3. Preferably, stage 1 is conducted as follows: culturing pluripotent stem cells in MCDB131 medium, which comprises about 1.5 mg / ml sodium bicarbonate, about IX Glutamax, about 10 mM glucose, about 0.5% bovine serum albumin (BSA), about 100 ng / ml Activin A, and about 3 pM of CHIR-99021 on day 1; culturing the cells in MCDB131 medium, which comprises about 0.5% BSA, about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 10 mM glucose, about 100 ng / ml Activin A, and about 0.3 pM CHIR-99021 on day 2; and culturing the cells in MCDB131 medium, which comprises about 0.5% BSA, about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 10 mM glucose and about 100 ng / ml Activin A. Preferably, stage 1 is conducted for about 3 d.

[0161] Preferably, stage 2 is conducted as follows: rinsing definitive endoderm cells and / or cells obtainable or obtained in stage 1 with DPBS; and subsequently culturing the cells in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose, BSA, ascorbic acid and FGF7 for about 2 d. Preferably, stage 2 is conducted as follows: rinsing definitive endoderm cells and / or cells obtainable or obtained in stage 1 with about IX DPBS (without Mg2+and Ca2+); and subsequently culturing the cells in MCDB131 medium, which comprises about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 10 mM glucose, about 0.5% BSA, about 0.25 mM ascorbic acid and about 50 ng / ml of FGF7 for about 2 d. Preferably, stage 2 is conducted for about 2d.

[0162] Preferably, stage 3 is conducted as follows: culturing primitive gut tube cells and / or cell obtained or obtainable in stage 2 in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose, 2BSA, ascorbic acid, FGF7, SANT-1, retinoic acid, LDN193189, ITS-X, and TPB, for about 2 d. Preferably, stage 3 is conducted as follows: culturing primitive gut tube cells and / or cell obtained or obtainable in stage 2 in MCDB131 medium, which comprises about 2.5 g / 1 sodium bicarbonate, about IX Glutamax, about 10 mM glucose, about 2% BSA, about 0.25 mMascorbic acid, about 50 ng / ml FGF7, about 0.25 pM SANT-1, about 1 pM retinoic acid, about 100 nM LDN193189, about 0.5% ITS-X (v / v), and about 200 nM TPB, for about 2 d. Preferably, stage3 is conducted for about 2 d.

[0163] Preferably, stage 4 is conducted as follows: culturing posterior foregut cells and / or cell obtained or obtainable in stage 3 in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose, BSA, ascorbic acid, FGF7, SANT-1, retinoic acid, LDN193189, ITS-X, and TPB, for about 3 d. Preferably, stage 4 is conducted as follows: culturing posterior foregut cells cells and / or cell obtained or obtainable in stage 3 in MCDB131 medium, which comprises about 2.5 g / 1 sodium bicarbonate, about IX Glutamax, about 10 mM glucose, about 2% BSA, about 0.25 mM ascorbic acid, about 2 ng / ml of FGF7, about 0.25 pM SANT-1, about 0.1 pM retinoic acid, about 200 nM LDN193189, about 0.5% ITS-X, and about 100 nM TPB, for about 3 d. Preferably, stage 4 is conducted for about 3 d.

[0164] Preferably, stage 5 is conducted as follows: culturing pancreatic bi-potent progenitor cells and / or cell obtained or obtainable in stage 4 in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose, BSA, SANT-1, retinoic acid, LDN193189, ITS-X, T3 (3,3’ ,5- Triiodo-l-thyronine sodium salt), ALK5 inhibitor II, zinc sulfate and heparin, for about 3 d. Preferably, stage 5 is conducted as follows: culturing pancreatic bi-potent progenitor cells and / or cell obtained or obtainable in stage 4 in MCDB131 medium, which comprises about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 20 mM glucose, about 2% BSA, about 0.25 pM SANT-1, about 0.05 pM retinoic acid, about 100 nM LDN193189, about 0.5% ITS-X, about 1 pM T3 (3,3’,5-Triiodo-l-thyronine sodium salt), about 10 pM ALK5 inhibitor II, about 10 pM zinc sulfate and about 10 pg / ml heparin, and a compound that promotes cAMP signalling pathway, for about 3 d. Preferably, stage 5 is conducted as defined in the present invention. Preferably, stage 5 is conducted for about 3 d.

[0165] Preferably, the compound that promotes cAMP signalling pathway is a cAMP agonist. Preferably, the cAMP agonist is selected from the group consisting of forskolin, IBMX, Dibutyryl- cAMP, 8-MA-cAMP, 6-MB-cAMP, exendin-4. More preferably, the cAMP agonist is forskolin, wherein the forskolin is preferably present at a concentration of about 5 pM to about 40 pM, more preferably about 10 pM. Preferably, the cAMP agonist is IBMX, wherein the IBMX is preferably present at a concentration of about 50 pM to about 200 pM, more preferably about 100 pM.

[0166] Preferably, stage 6 is conducted as follows: culturing pancreatic endocrine precursor cells and / or cell obtained or obtainable in stage 5 of the differentiation in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose, BSA, LDN193189, ITS-X, T3, ALK5 inhibitor II, zinc sulfate, gamma- secretase inhibitor XX and of heparin, for about 7-15 d, preferably about 7d.

[0167] Preferably, stage 6 is conducted as follows: culturing pancreatic endocrine precursor cells and / or cell obtained or obtainable in stage 5 of the differentiation in MCDB131 medium, which comprises about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 20 mM glucose, about 2% BSA, about 100 nM LDN193189, about 0.5% ITS-X, about 1 pM T3, about 10 pM ALK5 inhibitor II, about 10 pM zinc sulfate, about 100 nM gamma-secretase inhibitor XX and about 10 pg / ml of heparin, for about 7-15 d, preferably about 7d. Preferably, stage 6 comprises at the end of cultivation a step of enriching and / or isolating one or more beta cells according to the method of the present invention. Preferably, stage 6 is conducted for about 7-15 d, more preferably about 7 d.

[0168] Preferably, stage 7 is conducted as follows: culturing immature beta cells and / or cell obtained or obtainable in stage 6 of the differentiation in MCBD131 medium, which comprises sodium bicarbonate, Glutamax, glucose, BSA, ITS-X, T3, ALK5 inhibitor II, zinc sulfate, N-acetyl cysteine, Trolox, R428, and heparin, for about 7-15 d. Preferably, stage 7 is conducted as follows: culturing immature beta cells and / or cell obtained or obtainable in stage 6 of the differentiation in MCBD13 1 medium, which comprises about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 20 m glucose, about 2% BSA, about 0.5% ITS-X, about 1 pM T3, about 10 pM ALK5 inhibitor II, about 10 pM zinc sulfate, about 1 mM N-acetyl cysteine, about 10 pM Trolox, about 2 pM R428 and about 10 pg / ml heparin, for about 7-15 d. Preferably, stage 7 is conducted for about 7- 15 d.

[0169] Preferably, the cultivation in any one of stages 1-7 is in an air-liquid interface, preferably in a 2D or 3D culture, optionally in a Matrigel overlay culture. Preferably, the cultivation in stage 7 is in a 3D culture.

[0170] Preferably, cells disclosed herein are of human origin. A cell of the disclosure is thus preferably a human cell.

[0171] Preferably, a cell / cells of the disclosure is / are derived from a pluripotent stem cell, such as an embryonic stem cell, or an induced pluripotent stem cell, preferably a human pluripotent stem cell, such as a human embryonic stem cell or a human induced pluripotent stem cell.

[0172] The present invention also relates to a population of beta cells that is obtainable by the method of the invention. The population of beta cells is preferably a population of stem cell- derived beta cells. The stem cell-derived beta cells disclosed herein share many distinguishing features of native beta cells, but are different in certain aspects (e.g., gene expression profiles). The stem cell-derived beta cell may be non-native or non-naturally occurring. As used herein, "non-native" or 'non-naturally occurring" means that the beta cell (e.g., stem cell-derived beta cell) is markedly different in certain aspects from beta cells which exist in nature, i.e., native beta cells. In should be appreciated, however, that these marked differences typically pertain to structural features which may result in the stem cell-derived beta cells exhibiting certain functional differences, e.g., although the gene expression patterns of stem cell-derived beta cells differ from native beta cells, the stem cell-derived beta cells behave in a similar manner to native beta cells but certain functions may be altered (e.g., improved) compared to native beta cells.

[0173] The population of, preferably stem cell-derived, beta cells of the disclosure share many characteristic features of native beta cells which are important for normal beta cell function. The stem cell-derived beta cell may exhibit a glucose-stimulated insulin secretion (GSIS) response in vitro. The stem cell-derived beta cell may exhibit a GSIS response in vivo. The stem cell-derived beta cell may exhibit in vitro and in vivo GSIS responses. The GSIS responses resemble the GSIS responses of endogenous pancreatic beta cells. The GSIS response may be observed upon transplanting the cell into a human or animal.

[0174] The present invention also relates to a pharmaceutical composition comprising the population of beta cells of the present invention.

[0175] A pharmaceutical composition may generally include one or more pharmaceutically acceptable and / or approved additives like carriers, antibiotics, preservatives, adjuvants, diluents and / or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like. As used herein, the term "pharmaceutically acceptable" means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the population of beta cells according to the present invention.

[0176] The present invention also relates to a population of beta cells of the invention for use in therapy, wherein the therapy is preferably the treatment of diabetes, such as type I diabetes and / or type II diabetes. Type I diabetes is an autoimmune disorder characterized by the destruction of insulin-producing beta cells in the pancreas, leading to a severe deficiency in insulin production. As a result, individuals with Type I diabetes require lifelong insulin therapy to regulate blood glucose levels. The onset is typically in childhood or early adulthood, and the exact cause is thought to involve genetic predisposition and environmental factors. Type II Diabetes is a metabolic disorder primarily characterized by insulin resistance, where the body’s cells do not respond effectively to insulin, combined with a relative decline in insulin production over time. This leads to elevated blood glucose levels. Type II diabetes is more common in adults and is often associated with obesity, sedentary lifestyle, and genetic factors. Management typically involves lifestyle changes, oral medications, and, in some cases, insulin therapy. The present invention also relates to a cell culture comprising pancreatic bi-potent progenitor cells and / or pancreatic endocrine precursor cells, in a medium that comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist. Preferably, the medium comprises about 20 mM glucose, SANT-1, about 0.05 pM retinoic acid, an ALK5 inhibitor, T3 and LDN193189. Preferably, cell culture comprises cells in an air-liquid interface, preferably in a 2D or 3D culture, optionally in a Matrigel overlay culture. Preferably, the cell culture comprising air liquid interface promotes the establishment of apical basal polarity in said cells. As disclosed elsewhere herein, use of specific culture conditions to promote apical-basal polarity of endocrine precursor cells may be used in the absence of compounds promoting cAMP signalling pathway, which lies downstream of the signalling pathway of apical-basal polarity in endocrine precursor cells. Preferably, the cAMP agonist is selected from the group consisting of forskolin, IBMX, Dibutyryl-cAMP, 8-MA-cAMP, 6-MB-cAMP, exendin-4. Preferably, the cAMP agonists is forskolin, wherein the forskolin is preferably present at a concentration of about 5 pM to about 40 pM, more preferably about 10 pM. Preferably, the cAMP agonist is IBMX, wherein the IBMX is preferably present at a concentration of about 50 pM to about 200 pM, more preferably about 100 pM. Preferably, the medium is MCDB131 medium. Preferably, the medium comprises sodium bicarbonate, Glutamax, glucose, BSA, SANT-1, retinoic acid, LDN193189, ITS-X, T3 (3,3’,5-Triiodo-l-thyronine sodium salt), ALK5 inhibitor II, zinc sulfate and heparin. Preferably, the medium comprises about 1.5 g / 1sodium bicarbonate, about IX Glutamax, about 20 mM glucose, about 2% BSA, about 0.25 pM SANT-1, about 0.05 pM retinoic acid, about 100 nM LDN193189, about 0.5% ITS-X, about 1 pM T3 (3,3’,5-Triiodo-l-thyronine sodium salt), about 10 pM ALK5 inhibitor II, about 10 pM zinc sulfate and about 10 pg / ml heparin.

[0177] The present invention also relates to a complex comprising a beta cell bound by a binding agent specific for CD133, wherein the binding agent is conjugated to a detectable label or a solid support. Preferably, the complex further comprises a binding agent specific for CD49a. Preferably, the binding agent specific for CD 133 is an antibody specific for CD 133 and / or the binding agent specific for CD49a is an antibody specific for CD49a.

[0178] The present invention also relates to use of a binding agent specific for CD 133 for isolating and / or enriching beta cells. Preferably, the binding agent is an antibody specific for CD133. Preferably, the use further comprises a binding agent specific for CD49a. Preferably, the binding agent is an antibody specific for CD49a.

[0179] The binding agent disclosed herein may be conjugated to a detectable label. Such a “detectable label” may be any appropriate chemical substance or enzyme, which directly or indirectly generates a detectable compound or signal in a chemical, physical, optical, or enzymatic reaction. A preferred detectable label is an optically detectable label, such as fluorescent label. Non-exhaustive examples for suitable fluorescent labels are “green” emitters (Atto488, Alexa488, Cy2, etc.), “orange” emitters (Atto542, alexa555, Cy3, etc.), “Red-far-Red” emitters (Alexa633, Atto 647N, Cy5, etc.), infrared emitters (Atto700, LiCor IRDye700, LiCor IRDye800, etc.), ultraviolet absorbing fluorescent dyes (Atto390 or Alexa405). A fluorescent label may also be a fluorescent protein, such as GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, or near-infrared fluorescent proteins. A fluorescent label is preferably useful for FACS application.

[0180] The binding agent disclosed herein may also be conjugated to a solid support. The term "solid support" or in the context of the present invention refers to any type of carrier material that can be used for immobilization of affinity ligands such as a cell or parts thereof and it can refer to material in particulate (e. g. beads or granules, generally used in extraction columns) or in sheet form (e. g. membranes or filters, glass or plastic slides, microtiter assay plates, dipstick, capillary fill devices or such like) which can be flat, pleated, or hollow fibers or tubes. Suitable matrices are well known to the skilled person. A solid support can be a magnetic bead or polymeric bead or a chromatographic stationary phase. A magnetic bead is preferably useful for MACS applications.

[0181] It must be noted that as used herein, the singular forms "a", "an" and "the" include plural references and vice versa unless the context clearly indicates otherwise.

[0182] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series.

[0183] 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.

[0184] 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".

[0185] 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.

[0186] Throughout this specification and the claims, 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”.

[0187] 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.

[0188] 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. For example, when disclosure uses the term “comprising”, the disclosure also encompasses the replacement of the term “comprising” with the terms “consisting essentially of’ as well as “consisting of’ and vice versa. E.g., the term "comprising" is meant to provide explicit support also for its replacement with "consisting essentially of' and / or "consisting of', the term "consisting essentially of' is meant to provide explicit support also for its replacement with "comprising" and / or "consisting of', and the term "consisting of' is meant to provide explicit support also for its replacement with "consisting essentially of' and "comprising". The possibility to replace terms with each other is not to beunderstood that these terms are synonymous. For the avoidance of doubt, the term "comprising", "consisting essentially of' or "consisting of that is explicitly recited in the respective context is the preferred term, while its replacement with any one of the other two terms is less preferred.

[0189] The invention is further characterized by the following items.1. A method of differentiating one or more pancreatic bi-potent progenitor cells into one or more beta cells comprising cultivating pancreatic bi-potent progenitor cells in a medium that comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist.2. The method of item 1, wherein the medium comprises about 20 mM glucose, SANT-1, about 0.05 pM retinoic acid, an ALK5 inhibitor, T3 and LDN193189, and wherein the suitable amount of time for cultivating pancreatic bi-potent progenitor cells in a medium that comprises a compound that promotes cAMP signalling pathway is at least about 30 mins to about 6 d, preferably about 3 d.3. The method of item 1 or 2, wherein the one or more pancreatic bi-potent progenitor cells are cultivated in an air-liquid interface, preferably in a 2D or 3D culture, optionally in a Matrigel overlay culture.4. The method of any one of the preceding items, wherein the cAMP agonist is selected from the group consisting of forskolin, IBMX, Dibutyryl-cAMP, 8-MA-cAMP, 6-MB-cAMP, exendin-4.5. The method of item 4, wherein the cAMP agonist is forskolin, wherein the forskolin is preferably present at a concentration of about 5 pM to about 40 pM.6. The method of item 4, wherein the cAMP agonist is IBMX, wherein the IBMX is preferably present at a concentration of about 50 pM to about 200 pM.7. The method of any one of the preceding items, wherein the method increases differentiation of pancreatic bi-potent progenitor cells into beta cells as compared to a reference method, wherein the reference method preferably does not comprise the use of said cAMP agonists, but is otherwise identical.8. The method of any one of the preceding items, wherein the method decreases differentiation of pancreatic bi-potent progenitor cells into alpha cells as compared to a reference method, wherein the reference method preferably does not comprise the use of said cAMP agonists, but is otherwise identical.9. The method of any one of the preceding items, wherein the medium is MCDB 131 medium.10. The method of any one of the preceding items, wherein the medium comprises sodium bicarbonate, Glutamax, glucose, BSA, SANT-1, retinoic acid, LDN193189, ITS-X, T3 (3,3’ ,5- Triiodo-l-thyronine sodium salt), ALK5 inhibitor II, zinc sulfate, and heparin.11. The method of any one of the preceding items, wherein the medium comprises about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 20 mM glucose, about 2% BSA, about 0.25 pM SANT-1, about 0.05 pM retinoic acid, about 100 nM LDN193189, about 0.5% ITS-X, about 1 pM T3 (3,3’,5-Triiodo-l-thyronine sodium salt), about 10 pM ALK5 inhibitor II, about 10 pM zinc sulfate, and about 10 pg / ml heparin.12. The method of any one of the preceding items, wherein the one or more pancreatic bipotent progenitor cells are obtainable or obtained according to Stage 4, Stage 3-4, Stage 2-4, and / or Stage 1-4 as defined in any one of items 32-61.13. The method of any one of the preceding items, comprising conducting Stage 1, Stage 2, Stage 3, and / or Stage 4 as defined in any one of items 32-61, prior to cultivating the pancreatic endocrine precursor cells for about 3 d in a medium that comprises a compound that promotes cAMP signalling pathway.14. The method of any one of the preceding items, comprising conducting Stage 6 and / or Stage 7 as defined in any one of items 32-61, after cultivating the pancreatic endocrine precursor cells for about 3 d in a medium that comprises a compound that promotes cAMP signalling pathway.15. The method of item 14, comprising a step of enriching and / or isolating one or more beta cells as defined in any one of items 32-61 at the end of Stage 6.16. A method of enriching and / or isolating one or more beta cells, wherein the method comprises isolating and / or enriching cells expressing a marker of apical-basal polarity, wherein preferably the marker of apical-basal polarity is CD133.17. The method of item 16, wherein the method comprising isolating and / or enriching one or more cells expressing CD133 from a mixture comprising beta cells and alpha cells.18. The method of item 16 or 17, wherein the method comprises contacting the one or more beta cell with a binding agent that specifically binds CD 133 and isolating the beta cell bound to the binding agent.19. The method of any one of items 16-18, wherein the method further comprises isolating and / or enriching one or more cells expressing a marker of endocrine cells, wherein preferably the marker of endocrine cells is CD49a.20. The method of any one of items 16-18, wherein the method reduces the population of glucagon-producing cells.21. The method of any one of items 16-20, wherein isolation and / or enriching is conducted by cell sorting.22. The method of item 21, wherein isolation and / or enriching is conducted by means of fluorescence activated cell sorting (FACS) or magnetic-activated cell sorting (MACS).23. The method of any one of items 16-22, wherein the method comprises, prior to isolation and / or enrichment, cultivating beta cells in an air-liquid interface in a medium comprising about 20 mM glucose, ALK5 inhibitor II, T3, LDN193189, and gamma- secretase inhibitor XX for 7-15 d, preferably 7 d.24. The method of any one of items 16-22, wherein the method comprises, prior to isolation and / or enrichment, cultivating beta cells in an air-liquid interface in in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose, BSA, LDN193189, ITS-X, T3, ALK5 inhibitor II, zinc sulfate, gamma- secretase inhibitor XX and of heparin, for 7-15 d, preferably 7d, preferably as defined for Stage 6 according to any one of items 32-61.25. The method of any one of items 16-24, further comprising differentiating cells to more mature beta cells after the isolation and / or enrichment.26. The method of item 25, wherein final differentiation is conducted in a 3D culture in a medium comprising ALK5 inhibitor II, T3, N-Cys, and AXL inhibitor for about 7-15 d, preferably about 7 d.27. The method of any one of items 16-24, wherein the one or more pancreatic bi-potent progenitor cells are obtainable or obtained according to Stage 5, Stage 4-5, Stage 3-5, Stage 2-5, and / or Stage 1-5 as defined in any one of items 32-61.28. The method of any one of items 16-25, comprising conducting Stage 1, Stage 2, Stage 3, Stage 4, Stage 5 and / or Stage 6 as defined in any one of items 32-61, prior to isolating and / or enriching one or more cells expressing CD133 and optionally CD49a from a mixture comprising beta cells and alpha cells.29. The method of any one of items 16-26, comprising conducting Stage 7 as defined in any one of items 32-61, after isolating and / or enriching one or more cells expressing CD133 and optionally CD49a.30. The method of any one of the preceding items, wherein the method comprises conducting the method of any one of items 1-15 and conducting the method of any one of items 16-29.31. The method of item 30, wherein the method is a method of differentiating and enriching of beta cells.32. A method of producing a population of beta cells from a population of human embryonic stem cells comprising a. Stage 1 : differentiating pluripotent stem cells to definitive endoderm cells; b. Stage 2: differentiating definitive endoderm cells to primitive gut tube cells; c. Stage 3 : differentiating primitive gut tube cells to posterior foregut cells; d. Stage 4: differentiating posterior foregut cells to pancreatic bi-potent progenitor cells; e. Stage 5: differentiating pancreatic bi-potent progenitor cells to pancreatic endocrine precursor cells comprising cultivating pancreatic bi-potent progenitor cells in a medium that optionally comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist, preferably as defined in any one of items 1-15. f. Stage 6: differentiating pancreatic endocrine precursor cells to immature beta cells, and optionally enriching and / or isolating cells expressing CD133 and optionally CD49a, preferably as defined in any one of items 16-29; and g. Stage 7: differentiating immature beta cells to more mature beta cells; wherein stage 5 comprises cultivating pancreatic bi-potent progenitor cells in a medium that comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist, and / or wherein stage 6 comprises enriching and / or isolating cells expressing CD133 and optionally CD49a.33. The method of item 32, wherein stage 1 is conducted for about 3 d.34. The method of item 32 or 33, wherein stage 1 is conducted as follows:a. culturing pluripotent stem cells in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose, bovine serum albumin (BSA), Activin A, and CHIR-99021 on day 1; b. culturing the cells in MCDB131 medium, which comprises BSA, sodium bicarbonate, Glutamax, glucose, Activin A, and CHIR-99021 on day 2; c. culturing the cells in MCDB131 medium, which comprises BSA, sodium bicarbonate, Glutamax, glucose and Activin A on day 3.35. The method of any one of items 32-34, wherein stage 1 is conducted as follows: a. culturing pluripotent stem cells in MCDB131 medium, which comprises about 1.5 mg / ml sodium bicarbonate, about IX Glutamax, about 10 mM glucose, about 0.5% (w / v) bovine serum albumin (BSA), about 100 ng / ml Activin A, and about 3 pM of CHIR-99021 on day 1; b. culturing the cells in MCDB131 medium, which comprises about 0.5% BSA, about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 10 mM glucose, about 100 ng / ml Activin A, and about 0.3 pM CHIR-99021 on day 2; c. culturing the cells in MCDB131 medium, which comprises about 0.5% BSA, about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 10 mM glucose and about 100 ng / ml Activin A.36. The method of any one of items 32-35, wherein stage 2 is conducted for about 2d.37. The method of any one of items 32-36, wherein stage 2 is conducted as follows: a. rinsing definitive endoderm cells and / or cells obtainable or obtained in stage 1 with DPBS; b. subsequently culturing the cells in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose, BSA, ascorbic acid and FGF7 for about 2 d.38. The method of any one of items 32-37, wherein stage 2 is conducted as follows: a. rinsing definitive endoderm cells and / or cells obtainable or obtained in stage 1 with about IX DPBS (without Mg2+ and Ca2+); b. subsequently culturing the cells in MCDB131 medium, which comprises about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 10 mM glucose, about 0.5% BSA, about 0.25 mM ascorbic acid and about 50 ng / ml of FGF7 for about 2 d.39. The method of any one of items 32-38, wherein stage 3 is conducted for about 2 d.40. The method of any one of items 32-39, wherein stage 3 is conducted as follows: culturing primitive gut tube cells and / or cell obtained or obtainable in stage 2 in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose, BSA, ascorbic acid, FGF7, SANT-1, retinoic acid, LDN193189, ITS-X, and TPB, for about 2 d.41. The method of any one of items 32-40, wherein stage 3 is conducted as follows: culturing primitive gut tube cells and / or cell obtained or obtainable in stage 2 in MCDB131 medium, which comprises about 2.5 g / 1 sodium bicarbonate, about IX Glutamax, about 10 mM glucose, about 2% BSA, about 0.25 mM ascorbic acid, about 50 ng / ml FGF7, about 0.25 pM SANT-1, about 1 pM retinoic acid, about 100 nM LDN193189, about 0.5% (v / v) ITS-X, and about 200 nM TPB, for about 2 d.42. The method of any one of items 32-41, wherein stage 4 is conducted for about 3 d.43. The method of any one of items 32-42, wherein stage 4 is conducted as follows: culturing posterior foregut cells and / or cell obtained or obtainable in stage 3 in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose, BSA, ascorbic acid, FGF7, SANT-1, retinoic acid, LDN193189, ITS-X, and TPB, for about 3 d.44. The method of any one of items 32-43, wherein stage 4 is conducted as follows: culturing posterior foregut cells cells and / or cell obtained or obtainable in stage 3 in MCDB131 medium, which comprises about 2.5 g / 1 sodium bicarbonate, about IX Glutamax, about 10 mM glucose, about 2% BSA, about 0.25 mM ascorbic acid, about 2 ng / ml of FGF7, about 0.25 pM SANT-1, about 0.1 pM retinoic acid, about 200 nM LDN193189, about 0.5% ITS-X, and about 100 nM TPB, for about 3 d.45. The method of any one of items 32-44, wherein stage 5 is conducted as defined in any one of items 1-15.46. The method of any one of items 32-45, wherein stage 5 is conducted for about 3 d.47. The method of any one of items 32-46, wherein stage 5 is conducted as follows: culturing pancreatic bi-potent progenitor cells and / or cell obtained or obtainable in stage 4 in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose, BSA, SANT-1, retinoic acid, LDN193189, ITS-X, T3 (3,3’,5-Triiodo-l-thyronine sodium salt), ALK5 inhibitor II, zinc sulfate and heparin, for about 3 d.48. The method of any one of items 32-47, wherein stage 5 is conducted as follows: culturing pancreatic bi-potent progenitor cells and / or cell obtained or obtainable in stage 4 in MCDB131 medium, which comprises about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 20 mM glucose, about 2% BSA, about 0.25 pM SANT-1, about 0.05 pM retinoic acid, about 100 nM LDN193189, about 0.5% ITS-X, about 1 pM T3 (3,3’,5-Triiodo-l-thyronine sodium salt), about 10 pM ALK5 inhibitor II, about 10 pM zinc sulfate and about 10 pg / ml heparin, and a compound that promotes cAMP signalling pathway, for about 3 d.49. The method of any one of items 32-48, wherein the compound that promotes cAMP signalling pathway is a cAMP agonist.50. The method of item 49, wherein the cAMP agonist is selected from the group consisting of forskolin, IBMX, Dibutyryl-cAMP, 8-MA-cAMP, 6-MB-cAMP, exendin-4.51. The method of item 49 or 50, wherein the cAMP agonists is forskolin, wherein the forskolin is preferably present at a concentration of about about 5 pM to about 40 pM.52. The method of item 49 or 50, wherein the cAMP agonist is IBMX, wherein the IBMX is preferably present at a concentration of about 50 pM to about 200 pM.53. The method of any one of items 32-52, wherein stage 6 is conducted for about 7-15 d, preferably about 7 d.54. The method of any one of items 32-54, wherein stage 6 is conducted as follows: culturing pancreatic endocrine precursor cells and / or cell obtained or obtainable in stage 5 of the differentiation in MCDB131 medium, which comprises sodium bicarbonate, Glutamax, glucose, BSA, LDN193189, ITS-X, T3, ALK5 inhibitor II, zinc sulfate, gamma-secretase inhibitor XX and of heparin, for about 7-15 d, preferably about 7d.55. The method of any one of items 32-53, wherein stage 6 is conducted as follows: culturing pancreatic endocrine precursor cells and / or cell obtained or obtainable in stage 5 of the differentiation in MCDB131 medium, which comprises about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 20 mM glucose, about 2% BSA, about 100 nM LDN193189, about 0.5% ITS- X, about 1 pM T3, about 10 pM ALK5 inhibitor II, about 10 pM zinc sulfate, about 100 nM gamma-secretase inhibitor XX and about 10 pg / ml of heparin, for about 7-15 d, preferably about 7d.56. The method of any one of items 32-55, wherein stage 6 comprises at the end of cultivation a step of enriching and / or isolating one or more beta cells according to any one of items 16-29.57. The method of any one of items 32-56, wherein stage 7 is conducted for about 7-15 d.58. The method of any one of items 32-57, wherein stage 7 is conducted as follows: culturing immature beta cells and / or cell obtained or obtainable in stage 6 of the differentiation in MCBD131 medium, which comprises sodium bicarbonate, Glutamax, glucose, BSA, ITS-X, T3, ALK5 inhibitor II, zinc sulfate, N-acetyl cysteine, Trolox, R428, and heparin, for about 7-15 d.59. The method of any one of items 32-58, wherein stage 7 is conducted as follows: culturing immature beta cells and / or cell obtained or obtainable in stage 6 of the differentiation in MCBD131 medium, which comprises about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 20 mM glucose, about 2% BSA, about 0.5% ITS-X, about 1 pM T3, about 10 pM ALK5 inhibitor II, about 10 pM zinc sulfate, about 1 mM N-acetyl cysteine, about 10 pM Trolox, about 2 pM R428 and about 10 pg / ml heparin, for about 7-15 d.60. The method of any one of items 32-59, wherein the cultivation in any one of stages 1-7 is in an air-liquid interface, preferably in a 2D or 3D culture, optionally in a Matrigel overlay culture.61. The method of any one of items 32-60, wherein the cultivation in stage 7 is in a 3D culture.62. The method of any one of the preceding items, wherein the cell is a human cell.63. The method of any one of the preceding items, wherein the cell is / are derived from human embryonic stem cells.64. A population of beta cells that is obtainable by the method of the preceding items.65. A pharmaceutical composition comprising the population of beta cells of item 64.66. A population of beta cells of item 64 for use in therapy, wherein the therapy is preferably the treatment of diabetes.67. A cell culture comprising pancreatic bi-potent progenitor cells and / or pancreatic endocrine precursor cells, in a medium that comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist.68. The cell culture of item 67, wherein the medium comprises about 20 mM glucose, SANT- 1, about 0.05 pM retinoic acid, an ALK5 inhibitor, T3 and LDN193189.69. The cell culture of item 67 or 68, wherein the cell culture comprises cells in an air-liquid interface, preferably in a 2D or 3D culture, optionally in a Matrigel overlay culture.70. The cell culture of item 69, wherein the cell culture comprising air liquid interface promotes the establishment of apical basal polarity in said cells.71. The cell culture of any one of items 67-70, wherein the cAMP agonist is selected from the group consisting of forskolin, IBMX, Dibutyryl-cAMP, 8-MA-cAMP, 6-MB-cAMP, exendin-4.72. The cell culture of any one of items 67-71, wherein the cAMP agonists is forskolin, wherein the forskolin is preferably present at a concentration of about 5 pM to about 40 pM.73. The cell culture of any one of items 67-72, wherein the cAMP agonist is IBMX, wherein the IBMX is preferably present at a concentration of about 50 pM to about 200 pM.74. The cell culture of any one of items 67-73, wherein the medium is MCDB131 medium.75. The cell culture of any one of items 67-74, wherein the medium comprises sodium bicarbonate, Glutamax, glucose, BSA, SANT-1, retinoic acid, LDN193189, ITS-X, T3 (3,3’ ,5- Triiodo-l-thyronine sodium salt), ALK5 inhibitor II, zinc sulfate and heparin.76. The cell culture of any one of items 67-75, wherein the medium comprises about 1.5 g / 1 sodium bicarbonate, about IX Glutamax, about 20 mM glucose, about 2% BSA, about 0.25 pM SANT-1, about 0.05 pM retinoic acid, about 100 nM LDN193189, about 0.5% ITS-X, about 1 pM T3 (3,3’,5-Triiodo-l-thyronine sodium salt), about 10 pM ALK5 inhibitor II, about 10 pM zinc sulfate and about 10 pg / ml heparin.77. A complex comprising a beta cell bound by a binding agent specific for CD133, wherein the binding agent is conjugated to a detectable label or a solid support.78. The complex of item 77, further comprising a binding agent specific for CD49a.79. The complex of item 77 or 78, wherein the binding agent specific for CD133 is an antibody specific for CD133 and / or the binding agent specific for CD49a is an antibody specific for CD49a.80. Use of a binding agent specific for CD133 for isolating and / or enriching beta cells.81. The use of item 80, wherein the binding agent is an antibody specific for CD133.82. The use of item 80, further comprising a binding agent specific for CD49a.83. The use of item 82, wherein the binding agent is an antibody specific for CD49a.84. The use of any one of items 80 to 83, wherein the binding agent is conjugated to a detectable label.85. The use of any one of items 80-84, wherein the binding agent is conjugated to a solid support.EXAMPLESExample 1: Cell-cell adhesion is linked to apical-basal polarity and cell fate in the human endocrine progenitor niche.

[0190] To investigate the role of cell-cell adhesion for alpha and beta cell specification inside the human EP niche, human embryonic stem cell (hESC) lines have been generated that overexpress dominant-negative E-cadherin (CDH1) mutants, CDH1AE and CDH1 AP, in a doxycycline (Dox)- inducible manner (Figure 7a). Overexpression of these mutants therefore lowers the adhesive strength between epithelial cells to different degrees. As expected, colonies of CDH1AE and CDH1AP undifferentiated hESCs lost their epithelial morphology upon Dox treatment and grew as disconnected, spindle-like cells (Figure 7b).

[0191] To mimic the in vivo epithelial niche of human EPs in vitro, the inventors of the present application developed a Matrigel overlay culture system based on a previously developed differentiation protocol. In brief, hESCs are seeded in medium supplemented with Matrigel, which then forms a gelatinous ECM layer covering the cells throughout the subsequent six differentiation stages (S1-S6; Figure 7c). This allows the cells to efficiently differentiate into a pancreatic endoderm-like epithelium that grows in multiple layers and forms expanding and coalescing luminal cavities with well-defined localized expression of apical-basal polarity markers (Figure 7d).

[0192] The expression of the inducible CDH1 mutant transgenes can be monitored by an IRES- coupled mCherry fluorescent reporter (Figure 7a). Dox treatment during progressed stages of the differentiation (S4 and later) resulted in a mosaic expression pattern with an average of 40% and 20% expression of mCherry (mCh) in CDH1AE and CDH1AP cells, respectively (Figure le and f). Dominant-negative E-cadherin overexpression was induced shortly before the first NEUROG3+progenitors arise until after the hormone subtypes are specified (S4-S6) and quantified INS and GCG expressing cells by flow cytometry. GCG+INS+polyhormonal cells have previously been shown to mature into alpha cells and will, together with GCG+INS' cells, be referred to as alpha cells, while GCGTNS+cells are considered to represent beta cells. The differentiating mutant cellsonly formed very few microlumens that failed to expand into a network of larger connected lumens as observed in the untreated control cultures (Figure lb). Strikingly, the ratio of beta to alpha cells (beta-to-alpha) was reduced by almost 4-fold in CDH1 AE and CDH1 AP expressing cells compared to their transgene non-responsive counterparts (Figure 1c). These results strongly suggest that lowered cell-cell adhesion interferes with apical-basal polarity in the human EP niche favouring commitment to an alpha fate over a beta cell fate.Example 2: Disruption of apical-basal polarity in the developing mouse pancreas alters endocrine cell specification.

[0193] To test if apical-basal polarity in EPs controls alpha versus beta cell fate, conditional Cdc42 knockout mice were analysed. Ablation of the small Rho-family GTPase Cdc42 in the pancreatic epithelium causes a severe perturbation of apical-basal polarity, lumen expansion, and overall endocrinogenesis. E16.5 fetal pancreata of 7A&7-Cre; Ct / c42fl / fl; R26-YFP mice were dissected (homozygous pancreatic Cdc42 knockout) and compared to their Pdxl-C Q litter mates as a wildtype control (Figure 8a). More than 95% of the epithelial cells in the knockout expressed YFP, indicating a high recombination efficiency (Figure 8b). Consistent with the reported knockout phenotype, only very small microlumens but no tubular network were visible, and the apical membrane-specific marker Mucl was abundantly mislocated in intracellular aggregates (Figure 8b). As previously described, the overall number of hormone cells (Ins+or Gcg+) was dramatically reduced in the knockout tissue (Figure Id and e). However, within the remaining endocrine cell population it is now shown that the relative impact on beta cell formation was more significant than on alpha cell formation, leading to a more than 5-fold reduced beta-to-alpha ratio in the Cdc42 knockout (Figure If). In conclusion, disruption of apical-basal polarity in the mouse pancreatic epithelium inhibits endocrine differentiation overall but stimulates the remaining EPs to adopt an alpha rather than a beta cell fate.Example 3: A dynamically changing apical-basal polarity status of human endocrine progenitors affects alpha versus beta cell fate.

[0194] The observation that more alpha cells are generated from non-polarized EPs while efficient beta cell generation seems to require a niche with proper apical-basal polarity is intriguing. Activation of the endocrine program early (E8.5) in development, when most of the pancreatic epithelium is non-polarized, almost exclusively yielded alpha cells. In contrast, late (E14.5) endocrine induction, when the progenitor niche consists of a more complex luminal plexus wheremore EPs have acquired apical-basal polarity, resulted in increased commitment of beta cells. Far less is known about morphological changes inside the EP niche and the temporal occurrence of hormone cell types during human fetal development. Human fetal pancreas material was used to examine the apical-basal polarization of EPs. At 9.9 weeks post conception (wpc), roughly equal numbers of polarized and non-polarized EPs could be distinguished in sections stained for NEUR0G3 and EZR (Figure 9a).

[0195] In the Matrigel overlay hESC model, a plexus-like luminal network, similar to the corresponding luminal network in vivo, develops over time from expanding and fusing lumens. Within the three days when most EPs are specified (Day 10-13; S5), the apical membrane content increases on average by almost 4-fold (Figure 2a and b). To quantify the EP polarization status, a NEUROG3 reporter was utilized that allows EP segmentation based on a cytosolic fluorescence signal (eGFP under the control of the endogenous NEUROG3 locus; Figure 9b). Due to different protein synthesis and turnover rates, the eGFP and NEUROG3 expression do not entirely overlap in this reporter (Figure 2a). Only eGFP+cells that also stained positive for a NEUROG3 antibody were therefore segmented and analysed. Significantly more NEURO G3+ / eGFP+cells featured an apical (EZR+) membrane domain on day 13 than on day 10 (Figure 2c). The concomitant lumen expansion and EP appearance were further analysed by combining the NEUROG3-eGFP reporter with an EZR-mKATE2 fusion reporter (Figure 9c). Long-term (72h) live-imaging experiments during S5 revealed a highly dynamic epithelial environment in which the EPs dynamically shifted from being integrated within remodeling lumens and moving around outside the lumens, presumably as a result of a constant change in their apical-basal polarity status.

[0196] These results indicate that EPs spend less time in a polarized state before committing to a specific hormone cell type if they arise early during the differentiation. If apical-basal polarization indeed enhances the likelihood for EPs to differentiate into beta cells, one would expect that early- induced EPs give rise to more alpha cells than late-induced EPs. To test this hypothesis directly, the 3D differentiation protocol was modified to achieve a more synchronized induction of endocrinogenesis at the earliest time when few EPs normally occur (after the first day of S4). To this end, a set of EP-promoting factors was added for two days (Day 8-9) to boost early differentiation into EPs, followed by seven days of culture in basal medium without differentiation factors. A combination of a Notch inhibitor, an ALK5 inhibitor, and the actin polymerization inhibitor latrunculin B gave the best results, leading to a wave of NEUROG3 cells that peaked at Day 11 and then rapidly subsided (Figure 2d and e). For comparison, EP formation was inducedthe same way three days later. Here, the S4 stage was extended to four days before adding pro- endocrine factors to limit early EP induction and to allow maturation of the epithelial niche (luminal network) without altering the exposure to other factors. Indeed, a delayed defined EP wave peaking at Day 13 was accomplished using this setup (Figure 2d and e). The resulting alpha and beta cells from the early and late EP protocols were then quantified by flow cytometry. Strikingly, the beta-to-alpha ratio was, on average, more than 10-fold higher in hormone cell populations derived from EPs that originated in a more mature environment characterized by a more apical-basally polarized epithelium (Figure 2f).Example 4: Apical-basally polarized human endocrine progenitors are primed to become beta cells.

[0197] Next, the inventors set out to test more directly whether alpha and beta cell fate depends on the apical-basal polarity status of EPs by a cell sorting approach. Prominin-1 (PROMI, CD133) was identified as a specific marker of apical membranes / lumens in the Matrigel overlay culture system (Figure 3a). Moreover, PROMI is exclusively expressed in apical membrane domains and co-localizes with other apical markers (MUC1, EZR and aPKC) in 10.6 wpc human fetal pancreas tissue (Figure 10a and b). PROMI is a highly evolutionary conserved transmembrane glycoprotein located within the apical microvilli of embryonic and adult mammalian epithelial cells. Importantly, it contains two large extracellular loops, rendering it a potential marker for antibodybased cell sorting for apical-basally polarized cells.

[0198] Interestingly, a subset of dissociated S5 cells stained with a PROMI antibody featured a cap-like PROM1+structure on one side of the cell. This PROM1+membrane area was also enriched in EZR, suggesting that it represents the remnant of the apical domain in previously polarized cells (Figure 3b). In contrast, other cells showed no PROMI staining and homogeneous membrane and intracellular EZR distribution, presumably representing previously non-polarized cells (Figure 3b). Furthermore, PROM1+and PROMF cell populations could be separated from S5 Matrigel overlay differentiation cultures by flow cytometry. To specifically sort EPs based on their apical-basal polarization status (PROM1+ / PROMF), an NEUROG3-TagRFPt fusion reporter was taken advantage of (Figure 10c). At S5, the NEUROG3-TagRFPt+cell population contained around 39% PROMT and 61% PROM1+cells (Figure 3c).

[0199] After completion of the EP stage (S5), Matrigel overlay-differentiated NEUROG3hlghcells were sorted for membranous PROMI expression, reseeded at low density onto Matrigel-coatedsurfaces, and allowed to differentiate for an additional four days without any specific factors (Figure 3d). These cultures were then stained for INS and GCG expression and quantified by image analysis (Figure 3e). The beta-to-alpha ratio was more than twice as high in hormone cell populations derived from PROM1+EPs than in those derived from PROMF EPs (Figure 3f), suggesting that EPs that were polarized at the time of sorting are predisposed to adopt a beta cell fate.Example 5: Single-cell RNA sequencing reveals differentiation propensities in polarized endocrine progenitors.

[0200] To gain further insight into the link between the apical-basal polarity status of EPs and fate allocation, single-cell RNA sequencing (scRNA-seq) was used. To capture EPs from early and late induction of endocrinogenesis as well as polarized and non-polarized EPs from late induction the following four different mixed populations were created: (1) early-induced EPs, (2) late- induced EPs, (3) PROMT late-induced non-polarized EPs and (4) PROM1+late-induced polarized EPs (Figure 4a). These samples were then labelled with four different antibodies conjugated to barcoded oligonucleotides (“hashtags”) before pooling and sequencing all samples together (Figure 4a).

[0201] This CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing) allowed for allocating each single-cell transcription profile to the cell’s original characteristics (apical- basal polarization and time of endocrine induction). After quality filtering for viability parameters and normalization, transcriptional data from 3,811 cells were obtained, including counts for 25,917 genes. Based on clustering and marker gene expression (Figure I l a and b), populations of bipotent pancreatic progenitors, ductal cells, pancreatic stellate cell (PSC)-like cells were identified, as well as a big supercluster corresponding to cells of the endocrine lineage. The majority of the latter expressed the EP-state marker NEUROG3 and pan-endocrine genes like NEURODI, NKX2-2, and CHGA. While a subset of cells also expressed markers of early hormone cells like MAFB and INS, markers of more mature hormone4cells like MAFA were absent (Figure 11 b). These signatures suggest that, as intended, different EP stages were captured, including both unspecified progenitors and cells that have already committed to specific endocrine subtypes. Moreover, 3,281 cells (86%) labelled with a single hashtag, with a roughly equal contribution by the four barcodes were identified (Figure 11c). Hashtag-negative and double-labelled cells / doublets were excluded from all subsequent analyses.

[0202] Sub-clustering of the endocrine cell population yielded 12 distinguishable populations (Figure 4b). The transcriptional profiles (Figure l id and e) and pseudo time analysis (Extended Figure 1 If) suggest that clusters 9 and 11 contain unspecified early state EPs. The clusters 8 (“prealpha 1”), and 12 (“pre-alpha 2”) have increasingly specific alpha-like signatures, while 2, 3, 1 (“pre-beta 1”), and 10 (“pre-beta 2”) appear to represent a distinct differentiation trajectory leading to beta-like transcriptional profiles. Cluster 7 comprises endocrine cells committed to the delta lineage (“pre-delta”).

[0203] Next, the distribution of the four hashtags throughout the endocrine population was analysed (Figure 4c and d). The same number of endocrine cells originated from early and late induction cultures. Early-induced EPs mainly contributed to clusters 4, 5, and 9 as well as clusters pre-alpha 1 and pre-delta. While late-induced EPs contributed to all clusters, the pre-beta 1 and pre-beta 2 clusters were almost exclusively comprised of late-induced cells. The majority of all endocrine cells were non-polarized. Polarized endocrine cells were mostly confined to fate- specified clusters. The pre-beta 1 and pre-beta 2 clusters contained significantly more polarized cells (52% and 89%, respectively) than the pre-alpha 1, pre-alpha 2, and pre-delta clusters (4%, 33%, and 23%, respectively). We directly compared the transcriptional profiles between early- and late-induced and between non-polarized and polarized endocrine cells and observed an overlap with genes associated with beta cell differentiation in the late-induced and polarized EP signatures (e.g. DLK1, CHGA, INS) (Figure 4e). The top differentially expressed genes in early-induced versus late-induced and non-polarized versus polarized endocrine cells were validated by RT- qPCR analysis (Figure 12).

[0204] For a more detailed understanding of the differentiation trajectories, unspliced and spliced mRNAs were analysed to derive information about RNA velocity, which predicts the future state of individual cells on a timescale of hours (Figure 4f). This analysis predicted four principal terminal states corresponding to the alpha lineage (approx, pre-alpha 2), the beta lineage (subset within pre-beta 1), the delta lineage (approx, pre-delta), and a fate-unspecified early EP state (approx, clusters 9 and 11). Next, the probabilities for each cell to end up in one of these terminal states were calculated and it was analysed how they were correlated with the hashtag information about induction time and polarization (Figure 4g). The alpha state was the most likely prediction for cells in clusters pre-alpha 2 and pre-alpha 1. While it was similarly likely for early- and late- induced cells to end up in the alpha state, it was far less likely for polarized cells than for nonpolarized cells. The terminal beta state was only probable for a subset of pre-beta 1 cells, mostlylate-induced EPs. Remarkably, polarized cells had a much higher probability of reaching the terminal beta state, while non-polarized pre-beta 1 cells had trajectories toward a less committed EP state (cluster 3). The terminal delta stage was most likely for pre-delta cells but also possible for cells from other clusters, including pre-beta 2, pre-beta 1, and pre-alpha 1. Early- and late- induced as well as non-polarized and polarized cells had similar probabilities of ending up in the delta state.

[0205] In conclusion, the CITE-seq data indicate that most alpha cells are generated from nonpolarized EPs (either early- or late-induced), while beta cells arise predominantly from late- induced, polarized EPs.Example 6: Apical-basal polarity promotes beta cell fate by suppressing ARX expression.

[0206] To identify potential candidate genes relevant for alpha versus beta cell fate allocation, the inventors integrated our CITE-seq data of non-polarized versus polarized EPs with pre-beta and pre-alpha scRNA-seq data. This analysis revealed 10 genes that were consistently upregulated in polarized EPs and early beta cells (DLK1, CRYBA2, TAGLN2, SCGN, FABP7, TSPAN1, AIF1, PAM, CHGA, and SLC8A8), and 2 genes that were consistently upregulated in non-polarized EPs and early alpha cells (ARX and C7) (Figure 5a). Notably, ARX is a downstream target of NEUROG3 and regulates the generation of alpha cells. In line with that, we confirmed the absence of ARX expression in early EPs (NEUROG3-TagRFPt+eGFP ) by immunocytochemistry, contrasting with its later expression (NEUROG3-TagRFPt+eGFP+and NEUROG3-TagRFPt' eGFP+) (Figure 13a and b). Additionally, RT-qPCR data showthat ARX expression precedes GCG expression (Figure 13c). Both immunofluorescence and flow cytometry analyses reveal a nearly complete overlap between GCG+cells and ARX+cells (Figure 13d and e). Taken together, these findings suggest that ARX, as a downstream component of NEUROG3, may influence the fate determination of alpha cells (Figure 13f). In alignment with the scRNA-seq data, RT-qPCR and Western blot analysis confirmed elevated expression of ARX in PROMT EPs compared to PROM1+EPs (Figure 5b and c). Moreover, flow cytometry analysis showed that PROMT cells exhibited a higher proportion of ARX+cells when compared to PROM1+cells (Figure 5d). Collectively, these findings suggest that a non-polarized status of EPs maintains ARX expression.

[0207] To further validate these findings, we utilized our previous observation that inhibition of cadherin function in EPs interferes with apical-basal polarity promoting alpha cell differentiation (Figure 1c). While the ratio of NEUROG3+cells was not significantly different between CDH1 AEand CDH1AP expressing (mCherry+) cells and their transgene non-responsive counterparts (mCherry') (Figure 5e), which indicates that CDH1 mutations do not affect the whole EP differentiation. In contrast with NEUR0G3, the ratio of ARX+cells was higher in CDH1 AE and CDH1 AP mCherry+cells compared to their mCherry' cells (Figure 5f). These results suggest that lowered cell-cell adhesion in the human EP niche promotes maintenance of ARX expression and an alpha cell fate, presumably by promoting a non-polarized status of EPs.

[0208] Arx-deficient mice exhibit an increased population of beta and delta cells at the expense of alpha cells, indicating the importance of Arx for alpha cell differentiation. In mice, Pax4 promotes the formation of beta cells by mutually inhibiting Arx. However, recent findings suggest that while PAX4 plays a role in regulating human beta cell function, it is not crucial for beta cell specification. In contrast to PAX4, the deletion of ARX in hESC leads to the complete removal of alpha cells and a reduction in the number of beta cells. Temporary rescue of ARX expression in ARX- knockout pancreatic progenitors restores the specification of beta cells but not alpha cells, suggesting that sustained ARX expression is vital for alpha cell formation. The duration of ARX expression dictates whether EPs differentiate into alpha or beta cells, which might be linked to the influence of EPs' residence time in the polarized state. Based on this, the inventors of the present application propose that apical-basal polarity in EPs might suppress ARX expression, thereby enhancing beta cell specification. Conversely, a non-polarized status of EPs might support alpha cell specification by maintaining ARX expression.Example 7: Apical-basal polarity promotes beta cell fate by suppressing ARX expression.

[0209] To further explore how apical-basal polarity regulates ARX expression, an in silico transcription factor (TF) motif analysis of the ARX locus was performed to predict which TFs can bind within 2 kb of the transcriptional start site of ARX (Extended Table 3). The only TF with a binding motif in this region of the ARX locus that was differentially expressed in polarized versus non-polarized EPs was EGR1 (Extended Tables 3 and 4). Consistently, studies in mice have shown that EGR1 binds to Pdxl and Arx, positively regulating Pdxl while negatively regulating Arx expression. Moreover, high-fat-fed EgrP'~ mice exhibit decreased beta cell and increased alpha cell numbers. Consistent with the scRNA data, an elevated expression of EGR1 in PROM1+EPs were detected compared to PROMT EPs by RT-qPCR and Western blot analyses (Figure 6a and b). To test if EGR1 expression is linked to alpha and beta cell fate choice, we knocked down EGR1 expression in S5 cells using siRNAs. Based on the RT-qPCR results, we confirm that EGR1negatively regulates ARX expression (Figure 6c). Furthermore, we observed a decreased beta-to- alpha cell ratio in EGR1 knockdown (EGR1 -siRNA) cells compared to the negative-siRNA (NEG- siRNA) control group (Figure 6d). Together, these results indicate that ARX expression may be downregulated by EGR1 in polarized hESC-derived EPs.Extended Table 3. In silico transcription factor binding motif analysis 2,000 base pairs upstream and downstream of the transcriptional start site of ARX. (Related to Figures 6)Extended Table 4. Differentially expressed genes list between early and late induced EPs and between polarized and non-polarized EPs. (Related to Figures 4-6)

[0210] The cAMP / PKA-CREB signalling pathway can enhance EGR-1 gene transcription. By ELISA analysis, the inventors found cAMP levels to be higher in PROM1+polarized EPs compared to PROMT non-polarized EPs (Figure 6e). To more directly test if increased cAMP activity promotes beta cell differentiation, we treated EP stage cells (S5, Day 10-13) with the cAMP agonists Forskolin (FSK, an adenylate cyclase activator) and 3 -Isobutyl- 1-methylxanthin (IBMX, a competitive non-selective phosphodiesterase inhibitor), separately. FSK treatment led to a pronounced and transient elevation in the phosphorylation of CREB (cAMP response elementbinding protein), peaking within 1-2 hours (Figure 6f). Consistently, the expression level of EGR1 also exhibited a pronounced and transient increase, reaching its maximum at 2 hours (Figure 6g). Conversely, the expression level of ARX decreased after 3 days of FSK treatment (Figure 6h). Importantly, while FSK treatment did not alter the proportion ofNEUROG3-GFP+endocrine cells, it significantly reduced the fraction of ARX+cells (Figure 6i), resulting in an elevated beta-to- alpha ratio (Figure 6j). Consistently, IBMX treatment yielded comparable results to FSK treatment in reducing ARX expression and increasing the beta-to-alpha ratio (Figures 6k-m). Thus, this data not only identifies apical-basal polarity as the epithelial feature that controls the specification of EPs into alpha and beta cells, but also how.Example 8: Identification of cAMP signalling pathway activators for promoting beta cell differentiation

[0211] Based on the previous findings, the inventors of the present application identified molecules that can be used for promoting beta cell differentiation. In addition to Forskolin, which is an adenylate cyclase activator, the inventors identified: (a) IBMX: a competitive non-selective phosphodiesterase inhibitor; (b) Exendin-4: a GLP-1 receptor agonist, which increases cAMP level via G protein coupled receptor; and (c) Dibutyryl-cAMP, 8-MA-cAMP and / or 6-MB-cAMP: which are cAMP analogs. An overview of how these molecules activate the cAMP signalling pathway during the endocrine progenitor stage is shown in Figure 14.Example 9: Increasing beta cell differentiation with Forskolin and IBMX

[0212] For cAMP agonist treatment, S5 medium was supplemented for 3 days with 10 pM Forskolin or 100 pM IBMX from Day 10 to Day 13, followed by 4-7 days medium without Forskolin or IBMX. At the end of differentiation, samples were collected for flow cytometry analysis to quantify INS and GCG expression. Forskolin directly activates adenylyl cyclase (TmAC), the enzyme that produces cAMP, which as a result raises cAMP levels in the cell. IBMX is an inhibitor of cyclic nucleotide phosphodiesterases (PDEs). By inhibiting PDEs, IBMX increases cellular cAMP levels. The experimental protocol and the results are shown in Figure 15 and Figure 16.Example 10: Using polarized surface marker (CD133, PROMI) combined with endocrine cell surface maker (CD49a, ITGA1) to enrich beta cells

[0213] To further increase the yield of beta cells, the inventors of the present application employed the apical-basal polarity surface marker CD133 (Prominin 1, PR0M1) to selectively isolate beta cells from stem cell-derived islets (SC-islets). The efficacy of this approach was validated through comprehensive flow cytometry, quantitative polymerase chain reaction (qPCR), and immunocytochemistry analyses.

[0214] At the end of S6, cells were dissociated by Accutase treatment and straining (50 pM). Sorting of living cells was performed on a BD FACS Aria III sorter after staining with an APC- conjugated PR0M1 -antibody and PE-conjugated CD49a-antibody with gating for single (scatter characteristics), living (DAPF) cells based on single-color isotype and unstained controls in 0.5% fatty acid-free BSA / PBS.

[0215] The sorted cells were re-aggregated in the AggreWell 2 days to form clusters, then the clusters were placed on a low-attached 6-well plate on a shaker and cultured for an additional 5- 10 days for flow cytometry analysis. The results are show in Figure 17.

[0216] It was found that nascent or early-stage beta cells predominantly retain apical-basal polarity before maturation, in contrast to glucagon- and somatostatin-secreting cells. By integrating CD 133 with the endogenous reporter of the endocrine progenitor marker Neurog3, a substantial 2.5-fold increase in beta cell enrichment was achieved (from 29.8% to 73.7%) and a significant 6.0-fold reduction in alpha cell proportion (from 18.4% to 3.1%). Moreover, the combination of CD133 and CD49a sorting further augmented beta cell enrichment by 2.7-fold (from 28.0% to 75.5%) anddecreased alpha cells by 4.3-fold (from 13.8% to 3.2%). The enriched SC-beta clusters exhibited enhanced insulin secretion in response to glucose challenge.MethodsGeneration of transgenic Human embryonic stem cell (hESC) lines

[0217] CDH1 mutant cell lines: The mutant CDH1 sequences (Addgene #45770 and #45773) were cloned into the piggyBac destination vector PB-TAC-ERN (Addgene #80475) for doxycycline induction and mCherry co-expression using Gateway technology (Thermo Fisher). A piggyBac transposase vector (System Biosciences) was co-transfected for the generation of CDH1AE and CDH1AP.

[0218] EZR-mKATE2 / NEUROG3-eGFP double reporter cell line: The EZR sequence, including 1,000 bp homology arms, was amplified from human genomic DNA and cloned into a pBluescriptSK vector backbone using Seamless cloning (Thermo Fisher) to generate the EZR- mKATE2 fusion cassette. Two guide-RNA sequences targeting the EZR locus were cloned into a Cas9 expression vector (pX458-HFl, Addgene #48138) and co-transfected.

[0219] Plasmids (1-5 pg / 106cells) were transfected into hESCs by the P3 Primary Cell Kit (Lonza) using the CA137 program on the Lonza 4D-Nucleofector. Transfected cells were selected by 100 pg / ml G418 (Thermo Fisher) or 50 pg / ml hygromycin (Thermo Fisher) for 1-3 days, and single colonies were manually picked to generate clonal cell lines. Inducible cell lines were treated with 2 pg / ml doxycycline (Sigma-Aldrich).Human fetal pancreas

[0220] Fixed human fetal pancreatic tissue (gender unknown) was supplied by Raphael Scharfmann, French Institute of Health and Medical Research, Paris, France. The use of human tissue was approved by “ French Agence de Biomedecine” (accreditation # PFS08-011) and informed consent was obtained from all donors.The use of animals

[0221] Mouse pancreatic dissection, fixation, sectioning, and embedding were performed as previously described (Kesavan, G., et al. 2009. Cell 139, 791-801). Mice were housed at the University of Copenhagen, and all experiments were performed according to guidelines and ethics approved by the Danish Animal Experiments Inspectorate (Dyreforsogstilsynet). Data were collected from both male and female embryos.hESC culture and differentiation

[0222] hESCs were cultivated on dishes coated with hESC-qualified Matrigel (Corning) in mTeSRl medium (Stemcell Technologies) at 37 °C and 5% CO2 and passaged using TrypLE (Thermo Fisher) and 10 pM ROCK inhibitor Y-27632 (Merck Millipore).

[0223] Unless otherwise stated, all experiments were conducted with the wild-type hESC cell line SA121 (Takara Bio), which was also used to generate the inducible overexpression cell lines CDH1AE and CDH1 AP. The NEUROG3-eGFP reporter is likewise based on SA121 and was used to generate the EZR-mKATE2 / NEUROG3-eGFP double reporter cell line. NEUROG3- tagRFPt / eGFP double reporter cell line generated based on wild-type hESC cell line Hl.

[0224] For Matrigel overlay differentiation, 130,000-150,000 cells / cm2were seeded in an ice-cold dilution of 33% (v / v) growth factor reduced Matrigel (Coming) in mTESRl medium plus 10 pM ROCK inhibitor and placed at 4°C for 20-30 min to allow the cells to settle before matrix solidification was induced at 37°C. The next day, liquid mTESRl medium was added. Daily media changes were performed manually with great caution to avoid disturbing the delicate matrix, and a washing step with PBS (Thermo Fisher) was included.

[0225] The directed differentiation toward pancreatic endocrine cells can be divided into six stages (S1-S6) with distinct media based on a previous study with modifications as indicated. Two days after cell seeding, the medium was shifted to SI differentiation medium for 3 days, containing 100 ng / ml Activin A (PeproTech; replacing GDF8) and CHIR-99021 (AxonMedChem; 3 pM on day 1, 0.3 pM on day 2, withdrawal on day 3). Unless otherwise stated, the remaining stages followed the standard durations and media compositions: S2 (2 days), S3 (2 days), S4 (3 days), S5 (3 days), S6 (4-7 days). For synchronized endocrinogenesis, S4 medium was supplemented for 2 days with 0.1 pM ySec-iXX (Merck Millipore), 1 pM Latrunculin B (Sigma- Aldrich), and 10 pM ALK5iII (Santa Cruz) after either one day (early induction) or four days (late induction) in standard S4 medium, followed by 7 days of basal S4 medium without cytokines.Flow cytometry analysis and sorting

[0226] Cells were retrieved from Matrigel overlay cultures using Cell Recovery Solution (Coming) and dissociated by TrypLE treatment and straining (50 pM). Sorting of living cells was performed on a BD FACS Aria III sorter after staining with an APC-conjugated PROMI -antibody (1 : 10) with gating for single (scatter characteristics), living (DAPL) cells based on single-color isotype and unstained controls in 0.5% fatty acid-free BSA / PBS.

[0227] Flow cytometry quantification of fixed, stained cells was performed as previously described on a BD LSR Fortessa analyzer or Miltenyi MACSQuant analyzer. Antibodies are listed in the Key Resource Table.Immunofluorescence staining

[0228] Fixation and immunological staining of hESC and mouse material were performed as previously described (Mamidi, A., et al. (2018). Nature 564, 114-118, Lof-Ohlin, Z.M., et al. (2017). Nat Cell Biol 19, 1313-1325). Human fetuses (9-12.5 weeks post conception) were staged by foot length. The pancreas was dissected from the gut and stomach, fixed in 3.7% formalin for 72h, and embedded in paraffin. Longitudinal sections (4-8 pm) were cut on a microtome. For immunological staining, sections were cleared of paraffin by sequential washing in xylene, ethanol, and water. Antigen retrieval was performed in TE buffer (10 mM Tris, ImM EDTA, 0.05% Tween20, pH 9.0) at 95 °C for 20 min, followed by quenching with 3% hydrogen peroxidase for 7 min, permeabilization by 0.3% Triton X-100 and blocking for 1 h in a TSA amplification buffer (Perkin Elmer). Primary antibodies are listed in the Key Resource Table. All Alexa Fluor-conjugated secondary antibodies (Thermo Fisher) were used as 1 : 500 dilutions.

[0229] Samples were imaged with Zeiss LSM780, Zeiss LSM880, CellDiscoverer 7, or Leica SP8 confocal microscopes. Image analysis, segmentation, and quantification were performed with Fiji (Image J) and Imaris (Oxford Instruments).RT-qPCR

[0230] Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat#74106) or RNeasy Micro Kit (Qiagen, Cat#74004). According to the manufacturer's instructions, reverse transcription was performed with iScript cDNA Synthesis Kit (BIO-RAD, Cat#1708891). Realtime PCR measurements were performed in technical duplicates using the QuantStudio 7 Flex Real-Time-PCR-System (Thermo Fisher) with TaqMan FAM probes (Thermo Fisher) with TaqMan Master Mix (Thermo Fisher, Cat#4364103) or primers (Integrated DNA Technologies) with SYBR Green PCR Master Mix (Thermo Fisher, Cat#4309155). Relative gene expression was determined using the housekeeping genes GAPDH. TaqMan probes and primers are listed in Table SI.Western blot

[0231] Cells were washed twice in PBS, collected, and lysed in RIPA buffer (Thermo Fisher, Cat#8990) on ice for 30 min and then sonicated for 1 min at 5 s intervals. After centrifugation at 10,000 g at 4 °C for 10 min, the supernatant was transferred into new tubes. The concentration of the cell protein sample was measured by bicinchoninic acid, and protein samples were boiled in an SDS sample buffer at 95 °C for 5 min. The cell extract sample was resolved by 10% Acr-Bis SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF, Merck Millipore). Nonspecific binding was blocked by incubation in 3% non-fat milk in TBS at room temperature for 1 h. Blots were then probed with primary antibodies overnight by incubation at 4 °C with anti- ARX, anti-EGRl, anti-CREB or anti-pCREB. GAPDH served as a loading control. Immunoreactivity bands were then probed for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies. Protein bands were detected by Chemiluminescent HRP substrate (Thermo Fisher, Cat#34579) or l-step ultra TMB blotting solution (Thermo Fisher, Cat#37574). Protein bands were quantified by densitometry using Fiji (ImageJ) software. scRNA-seq sample preparation, library building, and analyses

[0232] The sample preparation was performed as previously described (Stoeckius, M., et al. (2017) Nat Methods 74, 865-868.). Briefly, non-polarized and polarized cells from three independent early- or late-induced differentiation cultures were purified by flow cytometry for PROMT Purified cells were then barcoded with unique Oligonucleotide Hash Tags (early, CAG TAG TCA CGG TCA; late, ATT GAC CCG CGT TAG; polarized, TAA CGA CCA GCC ATA; nonpolarized, AAA TCT CTC AGG CTC). After Hash- and CITE-tagged cells were counted and checked for viability using a hematocytometer, 25,000 cells were loaded into a Chromium Controller (10X Genomics). The library was prepared using the Chromium Single Cell 3’ v2 protocol (10X Genomics). Indexed cDNAs were then pooled and amplified by PCR according to the Chromium Single Cell 3 ’ v2 protocol (10X Genomics) and with specific primers amplifying the antibody-derived tags. The single cell 3’ v2 protocol (10X Genomics) was used for the mRNA- derived cDNA library preparation, and the KAPA HiFi HotStart Library Amplification Kit (Roche) was used for the antibody-derived tagged library. Following the final bead purification, all the libraries were pooled together and sequenced using Illumina NextSeq 500.

[0233] For analysis, raw reads were pre-processed using the 10X genomics Cell Ranger pipeline. The reads were aligned to the GRCh38 2020- A genome. cDNA reads were used to construct acount matrix, while HTO reads were used to count HTO tags using the CITE-seq-Count-1.4.3 tool. Further downstream analysis with integration was done using both Seurat and Scanpy.In Silico transcription factor binding analysis

[0234] The Gene Search function of the online tool at http: / / motifmap.ics.uci.edu was employed to search for transcription factor binding sites 2,000 base pairs upstream and downstream of the transcriptional start site of ARX. siRNA knockdown of EGR1

[0235] The end of S4 cells (at day 10) were dissociated to single cells as described above and reverse transfected with 50 nM of three siRNA against EGR1 (Thermo Fisher, ID# S4537 and S4538) or 100 nM negative control siRNA (Thermo Fisher, Cat# 4390843) using Lipofectamine RNAiMAX (Thermo Fisher, Cat# 13778030). In brief, in a 24-well format, 2 pl RNAiMAX was premixed with siRNA(s) in 100 pl OptiMEM medium (Thermo Fisher, Cat# 31985070), and 20- 30 minutes later, 500,000 cells per cm2were added in 400 pl S5 medium supplemented with 10 pM ROCK inhibitor. 24-48 hrs after transfection, cells were harvested for transcriptional analyses. On Day 13, the S5 medium was switched to basal medium for 4 days for flow cytometry analyses. cAMP ELISA

[0236] The sample preparation was performed according to the protocol instructions of the cAMP XP@ Assay Kit (Cell Signaling). Briefly, sorted cells were rinsed with 200 pl ice-cold PBS and then added 100 pl IX lysis buffer. The samples were incubated on ice for 10 min for analysis.Quantification and Statistical Analysis

[0237] Statistical analyses were performed with GraphPad Prism (version 10.0, GraphPad Software). Unless otherwise noted, a paired nonparametric test (Wilcoxon matched-pairs signed- rank test) was used to assess significance. Asterisks denote p-values as follows: * p < 0.05; ** p < 0.01; *** p < 0.001. Each n represents a biological replicate (mice or independent experiments). Data figures illustrate the mean with standard deviation and the values of individual biological replicates.Key Resources Table

[0238] Embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, 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 embodiments have been specifically disclosed by preferred embodiments and optional features, modification and variations thereof 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. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms 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. In addition, where features are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0239] Equivalents: 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 following claims.

[0240] 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 claims.

[0241] 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.

[0242] Further embodiments will become apparent from the following claims.

Claims

CLAIMS1. A method of enriching and / or isolating one or more beta cells, wherein the method comprises isolating and / or enriching cells expressing a marker of apical-basal polarity, wherein preferably the marker of apical-basal polarity is CD 133.

2. The method of claim 1, wherein the method comprises isolating and / or enriching one or more cells expressing CD133 from a mixture comprising beta cells and alpha cells, and wherein preferably the method reduces the population of glucagon-producing cells.

3. The method of any one of claims 1 or 2, wherein the method further comprises isolating and / or enriching one or more cells expressing a marker of endocrine cells, wherein preferably the marker of endocrine cells is CD49a, and wherein preferably the method reduces the population of non-endocrine cells.

4. The method of any one of claims 1-3, wherein isolation and / or enriching is conducted by cell sorting, wherein isolation and / or enriching is conducted by means of fluorescence activated cell sorting (FACS) or magnetic-activated cell sorting (MACS).

5. A method of differentiating one or more pancreatic bi-potent progenitor cells into one or more beta cells comprising cultivating pancreatic bi-potent progenitor cells in a medium that comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist.

6. The method of claim 5, wherein the suitable amount of time for cultivating pancreatic bipotent progenitor cells in the medium that comprises a compound that promotes cAMP signalling pathway is at least about 30 mins to about 6 d, preferably 3 d.

7. The method of claim 5 or 6, wherein the cAMP agonist is selected from the group consisting of forskolin, IBMX, Dibutyryl-cAMP, 8-MA-cAMP, 6-MB-cAMP, exendin-4, and wherein preferably the cAMP agonist isa. forskolin, wherein the forskolin is preferably present at a concentration of about 5 pM to about 40 pM, or b. IBMX, wherein the IBMX is preferably present at a concentration of about 50 pM to about 200 pM.

8. The method of any one of claims 5-7, wherein the method increases differentiation of pancreatic bi-potent progenitor cells into beta cells and / or decreases differentiation of pancreatic bi-potent progenitor cells into alpha cells as compared to a reference method, wherein the reference method preferably does not comprise the use said cAMP agonists, but is otherwise identical.

9. A method of producing a population of beta cells from a population of human embryonic stem cells comprisingStage 1 : differentiating pluripotent stem cells to definitive endoderm cells;Stage 2: differentiating definitive endoderm cells to primitive gut tube cells;Stage 3: differentiating primitive gut tube cells to posterior foregut cells;Stage 4: differentiating posterior foregut cells to pancreatic bi-potent progenitor cells;Stage 5: differentiating pancreatic bi-potent progenitor cells to pancreatic endocrine precursor cells comprising cultivating pancreatic bi-potent progenitor cells in a medium that optionally comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist, preferably as defined in any one of claims 1-4;Stage 6: differentiating pancreatic endocrine precursor cells to immature beta cells, and optionally enriching and / or isolating cells expressing CD133 and optionally CD49a, preferably as defined in any one of claims 5-8; andStage 7: differentiating immature beta cells to more mature beta cells; wherein stage 5 comprises cultivating pancreatic bi-potent progenitor cells in a medium that comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist, and / or wherein stage 6 comprises enriching and / or isolating cells expressing CD 133 and optionally CD49a.

10. A population of beta cells that is obtainable by the method of the preceding claims.6811. A pharmaceutical composition comprising the population of beta cells of claim 10.

12. A population of beta cells of claim 11 for use in therapy, wherein the therapy is preferably the treatment of diabetes.

13. A cell culture comprising pancreatic bi-potent progenitor cells and / or pancreatic endocrine precursor cells, in a medium that comprises a compound that promotes cAMP signalling pathway, preferably a cAMP agonist.

14. A complex comprising a beta cell bound by a binding agent specific for CD133, wherein the binding agent is conjugated to a detectable label or a solid support, and wherein optionally the complex further comprises a binding agent specific for CD49a15. Use of a binding agent specific for CD 133 for isolating and / or enriching beta cells, wherein optionally the use further comprises a binding agent specific for CD49a.

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