Generation of enterochromaffin and pancreatic cells

Abstract

There is described herein populations of enterochromaffin cells, islet-like cells, proglucagon+ cells or ARX expressing cells, methods of generating the same from a human pluripotent stem cell-derived population of PDX1+ progenitors, and uses of the same for therapy and diagnosis.

Description

[0001] GENERATION OF ENTEROCHROMAFFIN AND PANCREATIC CELLS

[0002] RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 604826 filed on November 30, 2023, incorporated herein by reference in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The invention relates to the generation of enterochromaffin (EC)-like cells and pancreatic progenitors.

[0006] BACKGROUND OF THE INVENTION

[0007] The ability to differentiate human pluripotent stem cells (hPSCs) into pancreatic endocrine cells in vitro offers an unprecedented access to islet-like cells (hPSC-islets), both for research into their function and pathology, and for the therapy of type 1 diabetes (T1 D). Following the specification of hPSCs into pancreatic progenitors (hPSC-PPs), the final stages of hPSC-islet differentiation produce a variety of endocrine cell types, including mono-hormonal insulin-producing beta-like cells, which are likely the most important components of a therapeutic product for T1 D. Although many pathways that promote the differentiation of hPSC-PPs into pancreatic endocrine cells have been identified, little is known about the molecular cues that direct hPSC- PPs towards specific islet lineages. As a result, hPSC-PPs can be differentiated into cells with nearly ubiquitous expression of endocrine markers such as synaptophysin1, NKX2-21 2and chromogranin A (CHGA)1 3, but the allocation of these endocrine cells towards each islet lineage remains uncontrolled and difficult to predict. This is a challenge for therapeutic or research applications of hPSC-islets which may require a critical frequency of specific hormone-producing cells.

[0008] One of the endocrine fates spontaneously acquired during hPSC-islet differentiation is characterized by expression of the vesicular serotonin transporter SLC18A14. This gene is not known to be expressed in pancreatic islets, but rather is restricted (in derivatives of the definitive endoderm) to enterochromaffin (EC) cells of the stomach and intestine5. hPSC-derived EC-like cells have been found to resemble fetal beta cells6and are slowly depleted over time during prolonged culture in vitro67, suggesting that they may be long-lived precursors of beta-like cells. Seemingly at odds with this hypothesis is the fact that EC-like cells were found to persist in vivo as long as 6 months after transplantation, during which time transcriptional differences between these cells and beta-like cells continued to increase8. Little is known about the functional effects of EC-like cells, although their depletion by magnetic enrichment of CD49a+ beta-like cells enhanced hPSC-islet insulin secretion, hinting at a potential deleterious effect of EC-like cells on beta-like cell function4. The developmental and functional relationships between EC-like and beta-like cells therefore represent important gaps in our understanding of hPSC-islet biology, potentially limiting the use of this technology for downstream applications.

[0009] Our group has developed a method for the generation hPSC-PPs that leverages use of a tankyrase inhibitor to efficiently induce hPSC-PPs with high expression of the NKX6.19-11. Despite these improvements, the generation of hPSC-islets enriched in beta-like cells has remained challenging.

[0010] SUMMARY OF THE INVENTION

[0011] Protocols for the directed differentiation of human pluripotent stem cells (hPSCs) into islet-like cells (hPSC-islets) generate a variety of endocrine cell types, including SLC18A1+ enterochromaffin (EC)-like cells not normally found in primary pancreatic islets. Although the presence of these cells is believed to negatively impact the functionality of hPSC-islets, investigating this issue has been challenging due to the lack of methods to generate hPSC-islets composed of specific endocrine lineages of interest, such as beta-like or EC-like cells. Here, we compared the effects of FGF and BMP signaling in endocrine lineage allocation of hPSC-derived pancreatic progenitors (hPSC-PP). Our results demonstrate that the BMP4 and FGF2 / MEK pathways can be applied during endocrine commitment to shift hPSC-PPs between islet and non-islet endocrine lineages. Using this knowledge, we develop patterning protocols to enrich for alpha-like, beta-like, and EC-like cells. When applying these culture conditions to PDX1+S0X9+ hPSC-PPs with varying levels of NKX6-1 expression, we observe a striking degree of progenitor bias towards each endocrine lineage. In an aspect there is provided a method for generating enterochromaffin cells from a human pluripotent stem cell-derived population of PDX1 + progenitors, the method comprising: providing a population of human pluripotent stem cell-derived cells identified as PDX1+ progenitors for differentiation into enterochromaffin cells; and endocrine differentiating the population of pancreatic progenitors in the presence of an inhibitor of at least one of Fibroblast Growth Factor Receptor (FGFR), MAPK and ERK Kinase (MEK), and Extracellular signal Regulated protein Kinase (ERK) pathways.

[0012] In an aspect there is provided a method for generating islet-like beta and / or delta cells from a human pluripotent stem cell-derived population of PDX1+ progenitors, the method comprising providing a population of human pluripotent stem cell-derived cells identified as PDX1+ progenitors for differentiation into islet-like beta and / or delta cells; and endocrine differentiating the population of progenitors in the presence of a Fibroblast Growth Factor Receptor (FGFR) agonist.

[0013] In an aspect there is provided a method for generating proglucagon+ cells from a human pluripotent stem cell-derived population of PDX1 + progenitors, the method comprising providing a population of human pluripotent stem cell-derived cells identified as PDX1+ progenitors for differentiation into proglucagon+ cells; and endocrine differentiating the population of progenitors in the presence of a Bone Morphogenetic Protein Receptor (BMPR) agonist.

[0014] In an aspect there is provided a method for generating ARX expressing cells from a human pluripotent stem cell-derived population of PDX1 +progenitors, the method comprising providing a population of human pluripotent stem cell-derived cells identified as PDX1+ progenitors for differentiation into ARX+ cells; and endocrine differentiating the population of progenitors in the presence of a Bone Morphogenetic Protein Receptor (BMPR) agonist.

[0015] In an aspect there is provided the population of enterochromaffin cells produced by the method described herein.

[0016] In an aspect there is provided the population of islet-like beta and / or delta cells produced by the method described herein.

[0017] In an aspect there is provided the population of proglucagon+ cells or ARX expressing cells produced by the method described herein. In an aspect there is provided a use of the population described herein for cell therapy in a subject in need thereof.

[0018] In an aspect there is provided a use of the population described herein for the screening of therapeutic agents.

[0019] In an aspect there is provided a use of the population described herein to investigate the effect of one or more therapeutic agents, diagnostic agents and / or conditions on said population or said population’s development or differentiation.

[0020] BRIEF DESCRIPTION OF FIGURES

[0021] These and other features of the preferred embodiments of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings wherein:

[0022] Figure 1 : The effect of endocrine induction media composition on endocrine commitment and hormone lineage specification, a) Schematic describing the sequence of stages during hPSC-islet differentiation, b) Schematic describing the sequence of factors applied during endocrine differentiation, including candidate endocrine- modulating factors, c) Changes in endocrine commitment and hormone lineage composition between days 23 and 29 of differentiation, d-e) Representative flow cytometry plots for Islet LIM homeobox 1 (ISL1) vs solute carrier family 18 member A1 (SLC18A1) (d) and NK homeobox 1 (NKX6-1) vs C-PEPTIDE (CPEP) (e) expression at day 29 of differentiation across all conditions tested, f-i) Quantification of frequency of Chromogranin A (CHGA)+ (f), NKX6-1+CPEP+ (g), Aristaless related homeobox (ARX)+ (h) and SLC18A1+ (i) cells at days 23 and 29. Statistics were done only on day 29 data using paired one-way ANOVA followed by Holm-Sidak’s post-hoc test for comparisons of each condition to control only; *, p < 0.05; **, p < 0.01 ; ****, p < 0.0001. All error bars represent the standard error of the mean. Legend: ns, non-significant; BSA, Bovine serum albumin; FGF10, Fibroblast growth factor 10; T3, triiodothyronine; ATRA, all trans retinoic acid; DBZ, Dibenzazepine; LatA, Latrunculin A; PD03, PD0325.

[0023] Figure 2: PD03 treatment during endocrine commitment promotes EC differentiation, a) Experimental design for testing the effects of PD03 treatment at different time points, b) Representative flow cytometry pseudoplots for SLC18A1 expression vs FSC in hPSC-islets exposed to PD03 between days 13 and 16, days 16 and 22 or continuously from days 13 to 22 during endocrine differentiation, c) Quantification of hPSC-islet frequencies of SLC18A1+ cells, as determined by flow cytometry, across all conditions tested. One-way ANOVA followed by Holm-Sidak’s post-hoc test, comparing each condition against the control. Error bars represent the standard error of the mean, d) Representative flow cytometry pseudoplots for SLC18A1 vs PDX1 expression in day 23 cells treated with different inhibitors between days 13 and 16. Legend: CTRL, control; ns, non-significant; PD03, PD0325; PD17, PD173074; SCH, SCH772984.

[0024] Figure 3: Phenotypic characterization of EC-like cells: Comparison of day 23 with day 29 hPSC-islets for frequencies of NKX6-1+CPEP+ (a), total CPEP+ (b), NKX6-1+ (c), and ISL1+ (d) cells within the SLC18A1+ population, as determined by flow cytometry, across all LatA / WIKI4 / PD03 conditions (Fig. 1 b). Parent gate shown on far left. All comparisons were done using paired one-way ANOVA followed by Holm-Sidak’s post- hoc test, comparing each condition against the control; ****, p < 0.0001. Error bars represent the standard error of the mean.

[0025] Figure 4: FGF2 and Betacellulin (BTC) suppress the differentiation of EC-like cells: a) Experimental design for testing the effects of FGF2 and BTC. b-d) Quantification of hPSC-islet frequencies of NK2 homeobox 2 (NKX2-2)+ (b), SLC18A1+ (c), and ISL1 + cells (d), as determined by flow cytometry, across all conditions tested, e) Representative flow cytometry pseudoplots for ISL1 vs SLC18A1 expression in hPSC- islets exposed to FGF2 and BTC for short or long durations during endocrine differentiation, f-i) Quantification of hPSC-islet frequencies of SLC18A1-NKX6- 1+CPEP+ (f), ARX+ (g), CPEP- Glucagon (GCG)- Somatostatin (SST)+ (h), and Hematopoietically expressed homeobox (HHEX)+SST+ cells, as determined by flow cytometry, across all conditions tested, j) Representative flow cytometry pseudoplots for HHEX vs SST expression in hPSC-islets exposed to FGF2 and BTC for short or long durations during endocrine differentiation. All comparisons were done using paired one-way ANOVA followed by Holm-Sidak’s post-hoc test, comparing each condition against the control; *, p < 0.05; **, p < 0.01 ; ***, p < 0.001. Error bars represent the standard error of the mean. Legend: BTC, betacellulin; CTRL, control; F2, FGF2; ns, non-significant. Figure 5: BMP4 signaling suppresses the differentiation of EC-like cells, a) Experimental design for testing the effects of BMP4 and BMP signaling antagonists (LDN and NOGGIN), b-d) Quantification of hPSC-islet frequencies of NKX2-2+ (b), SLC18A1+ (c), and ISL1+ cells (d), as determined by flow cytometry, across all conditions tested, e) Representative flow cytometry pseudoplots for ISL1 vs SLC18A1 expression in hPSC-islets exposed to BMP4 at various timepoints throughout endocrine differentiation, f-g) Quantification of hPSC-islet frequencies of ARX+ (f), CPEP-GCG-SST+ (g) cells, as determined by flow cytometry, across all conditions tested, h) Representative flow cytometry pseudoplots for ARX expression vs FSC in hPSC-islets exposed to BMP4 at various timepoints throughout endocrine differentiation, i) Quantification of hPSC-islet frequencies of SLC18A1-NKX6- 1+CPEP+ cells, as determined by flow cytometry, across all conditions tested, j) Representative flow cytometry pseudoplots for NKX6-1 vs CPEP expression in hPSC- islets exposed to BMP4 at various timepoints throughout endocrine differentiation. All comparisons were done using paired one-way ANOVA followed by Holm-Sidak’s post- hoc test, comparing each condition against the control; *, p < 0.05; **, p < 0.01 ; ***, p < 0.001 ; ****, p < 0.0001. Only significant comparisons are shown. Error bars represent the standard error of the mean.

[0026] Figure 6: ISX9, PD03 and LDN pattern hPSC-PPs towards the EC lineage, a) Experimental schematic for determining the additive effects of PD03 and LDN on EC- like cell differentiation, b) Representative flow cytometry pseudocolour plots for SLC18A1 expression vs. FSC of d29 cultures differentiated with PD03 and / or LDN. c-f) Quantification of SLC18A1+ (c), ISL1+ (d), SLC18A1-NKX6-1+CPEP+ (e) and ARX+ (f) cells, as assessed by flow cytometry at d29 cultures differentiated with PD03 and / or LDN. Populations compared by paired one-way ANOVA followed by Holm-Sidak’s post-hoc test; *, p < 0.05; **, p < 0.01 ; ***, p < 0.001 ; ****, p < 0.0001. Error bars represent the standard error of the mean. Legend: CTRL, control; EC, enterochromaffin; DBZ, Dibenzazepine; ISX9, isoxazole-9; LatA, Latrunculin A. PD03, PD0325; LDN, LDN193189

[0027] Figure 7: The phenotype of hPSC-PPs derived from different methods of endodermal patterning, a) Experimental design for endodermal patterning, b-c) Representative flow cytometry pseudocolour plots for PDX1 vs. NKX6-1 (b), and SOX9 vs. NKX2-2 expression at day 13 of differentiation, d-f) Quantification of frequencies of PDX1+NKX6-1+ (d), PDX1+SOX9+ (e), and NKX2-2+ (f) cells at day 13 of differentiation. Populations were compared by paired one-way ANOVA followed by Holm-Sidak’s post-hoc test; *, p < 0.05; **, p < 0.01 ; ***, p < 0.001 ; ****, p < 0.0001. Error bars represent the standard error of the mean. Legend: EC, enterochromaffin.

[0028] Figure 8: hPSC-islet endocrine composition is influenced by initial hPSC-PP phenotype and patterning factors applied during endocrine differentiation, a) Experimental design for studying the effect of progenitor patterning versus endocrine patterning in determining final hPSC-islet endocrine composition, b) Representative flow cytometry pseudocolour plots for hPSC-cultures derived from NKX6-1 -suppressed (top row), control (middle row) and NKX6-1 -enhanced (bottom row) hPSC-PPs subjected to either alpha (left column), beta (middle column), or EC (right column) patterning. For reference, the phenotype of associated hPSC-PPs is shown in the leftmost column, c-j) Quantification of day 26 hPSC-islet frequencies of ARX+ (c), ARX+GCG+ (d), ARX+CPEP+ (e), ARX+ Pancreatic polypeptide (PPY)+ (f), SLC18A1- NKX6-1+CPEP+ (g), HHEX+SST+ (h), SLC18A1+ (i), and ISL1+ (j) cells derived from each of the progenitor-patterning combinations. Populations were compared by oneway ANOVA followed by Holm-Sidak’s post-hoc test; *, p < 0.05; **, p < 0.01 ; ***, p < 0.001 ; ****, p < 0.0001. Error bars represent the standard error of the mean, k) Quantification of total beta-like cells (SLC18A1-NKX6-1+CPEP+) generated per well at day 23 of differentiation in each experimental combination of NKX6-1-low (NKX6-1 <10%, “Low”), control (NKX6-1 70-80%, “CTL”), or NKX6-1-high (NKX6-1 >90%, “High”) progenitors exposed to either alpha, beta or EC endocrine patterning. Cell numbers were compared by one-way ANOVA followed by Sidak’s post-hoc test, comparing the mean of each group against the mean of the High-Beta condition; *, p < 0.05; **, p < 0.01 ; ***, p < 0.001 ; ****, p < 0.0001. Error bars represent the standard error of the mean. I) Comparison of total numbers of day 13 NKX6-1+ hPSC-PPs and day 26 SLC18A1-NKX6-1+CPEP+ beta-like cells derived from beta patterning of NKX6-1-suppressed, control and NKX6-1 -enhanced hPSC-PPs. Populations compared by paired mixed-effects analysis with Geisser-Greenhouse correction for repeated measures followed by Sidak’s post-hoc test; *, p < 0.05; **, p < 0.01 ; ***, p < 0.001 ; ****, p < 0.0001. Error bars represent the standard error of the mean. Legend: EC, enterochromaffin; BLC. Beta-like cells; ns, non-significant.

[0029] Figure 9: Comparing the functionality of hPSC-islets enriched in beta-like and EC-like cells. Comparison of hPSC-islets generated from an EC-enriched protocol or a beta-enriched protocol with regards to insulin secretion in response to either 30 mM KCI (a) or 20 mM glucose (b). Unpaired t test was used to compare populations; **, p < 0.01. Error bars represent the standard error of the mean. Legend: EC, enterochromaffin; KCI, potassium chloride.

[0030] DETAILED DESCRIPTION

[0031] In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it is understood that the invention may be practiced without these specific details.

[0032] Herein, we describe our efforts to understand mechanisms regulating endocrine commitment and hormone lineage allocation using a marker-directed strategy. We identify small molecule inhibitors of the MAPK / ERK and BMP pathways as major drivers of EC-like differentiation in our protocol, while agonists of these pathways inhibit SLC18A1 expression in favour of other islet lineages. Using this knowledge, we design refined protocols that bias hPSC-PPs towards the beta, alpha, and EC lineages, and find that NKX6-1 expression at the hPSC-PP stage also associates with the final hPSC-islet composition. Using this knowledge, we compare hPSC-islets enriched in either beta-like or EC-like cells and confirm that enhancing differentiation into islet lineages at the expense of EC-like cells enhances insulin-secreting functionality of hPSC-islets in vitro. These findings provide a valuable framework for directing hPSCs towards specific endocrine subtypes, ultimately contributing to the generation of hPSC-islets that better recapitulate the functionality and composition of their primary counterparts.

[0033] In the methods described herein, whether to generate enterochromaffin cells, islet-like cells, proglucagon+ cells or ARX expressing cells, these cells are expected to be capable of being derived from any human pluripotent stem cell-derived population of PDX1+ progenitors,

[0034] In an aspect there is provided a method for generating enterochromaffin cells from a human pluripotent stem cell-derived population of PDX1 + progenitors, the method comprising: providing a population of human pluripotent stem cell-derived cells identified as PDX1+ progenitors for differentiation into enterochromaffin cells; and endocrine differentiating the population of pancreatic progenitors in the presence of an inhibitor of at least one of Fibroblast Growth Factor Receptor (FGFR), MAPK and ERK Kinase (MEK), and Extracellular signal Regulated protein Kinase (ERK) pathways. The term "pluripotent stem cell" as used herein refers to a cell that has the capacity to self-renew by dividing, and to develop or differentiate, under different conditions, to more than one differentiated cell type, for example into one or more cell types characteristic of the three germ cell layers, and includes embryonic stem cells and induced pluripotent stem cells. Embryonic stem cells and induced pluripotent stem cells are examples of pluripotent stem cells. Pluripotent cells are characterized by their ability to differentiate to more than one cell type using, for example, a nude mouse teratoma formation assay. Pluripotency is also evidenced by the expression of embryonic stem (ES) cell marker.

[0035] In the context of a cell, the term "differentiated", or "differentiating" is a relative term and a "differentiated cell" is a cell that has progressed further down the developmental pathway than the cell it is being compared with, and in some cases a “differentiated cell” is more specialized. In some cases, a “differentiated cell” is a cell that changed from one cell type to another. Thus, stem cells can differentiate to lineage-restricted precursor cells (such as an endodermal progenitor cell), which in turn can differentiate into other types of precursor cells further down the pathway and then to an end-stage differentiated cell, which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.

[0036] The term "cell culture medium" (also referred to herein as a "culture medium" or "medium") as referred to herein is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation and optionally differentiation. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, vitamins etc. Cell culture media ordinarily used for particular cell types are known to those skilled in the art.

[0037] The term “contacting” refers to any suitable means by which the cell is cultured or incubated with the component(s) together in vitro. For example, the compound is added to the cells in culture, or is transferred to or mixed with culture medium containing the compound. For example the cells may be treated in adherent culture, or in suspension culture, the components can be added temporally substantially simultaneously (e.g. together in a cocktail) or sequentially (e.g. within 1 hour from an addition of a first component). The cells can also be contacted with another agent such as a growth factor or other differentiation agent or environments to stabilize the cells, or to differentiate the cells further and include culturing the cells under conditions known in the art for example for culturing the pluripotent (and / or differentiated) population.

[0038] The term "progenitor cell” refers to cells that have a cellular phenotype that is at an earlier step along a developmental pathway or progression than is a fully differentiated cell relative to a cell which it can give rise to by differentiation. Progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.

[0039] The term "pancreatic progenitor cell" refers to a cell which is capable of forming any of: pancreatic endocrine cells (such as glucagon producing alpha cells, insulin producing beta cells, somatostatin producing delta cells, ghrelin producing epsilon cells and pancreatic polypeptide producing cells), or pancreatic acinar cells (e. g. amylase producing- and / or trypsin producing-pancreatic cells) or pancreatic ductal cells. Formation or development of one more of pancreatic endocrine cells, pancreatic acinar cells or pancreatic ductal cells may be induced by addition of components such as a combination of a thyroid hormone, ALK5 inhibition, notch inhibition, retinoic acid, BMP and SHH inhibition and / or resulting from the environment in which the progenitor cell develops. Similarly, injection of PDX1+ / NKX6-1+ pancreatic progenitor cells in vivo can result in development of endocrine, acinar and ductal cells.

[0040] As used herein, the term “insulin producing cell” refers to a cell differentiated from a pancreatic progenitor, which secretes insulin. An insulin producing cell includes functional pancreatic beta-cells, as well as pancreatic beta-like cells that synthesize, express, or secrete insulin in a constitutive or inducible manner. A population of insulin producing cells, e.g. produced by differentiating endodermal cells to pancreatic progenitors and subsequent differentiation into insulin producing cells according to the methods described herein, can be functional pancreatic beta-cells or beta-like cells (e.g., cells that have at least two characteristics of an endogenous functional betacell). It is also contemplated that the population of insulin producing cells, e.g. produced by the methods as disclosed herein, can comprise pancreatic beta-cells or pancreatic beta-like cells, and can also contain non-insulin producing cells (e.g. cells with a beta-cell like phenotype with the exception that they do not produce or secrete insulin).

[0041] In some embodiments, the PDX1+ progenitors are NKX6-1+.

[0042] In some embodiments, the inhibitor is a FGFR inhibitor. Preferably, the FGFR inhibitor is FIIN1-hydrochloride, PD173074, Erdafitinib, pemigatinib, envatinib (E7080), ponatinib (AP24534), regorafenib (BAY 73-4506), dovitinib (TKI258), lucitanib (E3810), cediranib (AZD2171), nintedanib (BIBF 1120), brivanib (BMS-540215), LY2874455, Zoligratinib (Debio-1347), Dovitinib, (TKI258) Lactate monohydrate, FIIN-2, S49076, PRN1371 , Derazantinib(ARQ-087), ASP5878, Futibatinib (TAS-120), ODM-203, or Infigratinib (BGJ398), preferably FIIN1-hydrochloride, or PD173074, or alternatively an FGFR3 inhibitor.

[0043] In some embodiments, the inhibitor is a MEK inhibitor. Preferably, the MEK inhibitor is PD0325901 , trametinib (Mekinist), cobimetinib (Cotellic), binimetinib (Mektovi), Selumetinib (AZD6244), U0126-EtOH, PD184352 (CI-1040), PD98059, TAK-733, AZD8330, Refametinib (RDEA119), or GDC-0623, preferably PD0325901 , U0126- EtOH or PD98059.

[0044] In some embodiments, the inhibitor is a ERK inhibitor. Preferably, the ERK inhibitor is SCH772984, Ravoxertinib (GDC-0994), Temuterkib (LY3214996), Pluripotin (SC1), VX-11e, or Ulixertinib (BVD-523), preferably SCH772984.

[0045] In some embodiments, the inhibitor is added for between day 13-22 of differentiation from hPSCs or alternatively between 0-9 days of endocrine differentiation from pancreatic progenitors.

[0046] In some embodiments, the population of pancreatic progenitors is differentiated additionally in the presence of Latrunculin A.

[0047] In some embodiments, the population of pancreatic progenitors is differentiated additionally in the presence of at least one of WIKI4, DBZ and Repsox.

[0048] In some embodiments, the population of pancreatic progenitors is differentiated additionally in the presence of ISX9. In an aspect there is provided a method for generating islet-like beta and / or delta cells from a human pluripotent stem cell-derived population of PDX1+ progenitors, the method comprising providing a population of human pluripotent stem cell-derived cells identified as PDX1+ progenitors for differentiation into islet-like beta and / or delta cells; and endocrine differentiating the population of progenitors in the presence of a Fibroblast Growth Factor Receptor (FGFR) agonist.

[0049] In some embodiments, the PDX1+ progenitors are NKX6-1+.

[0050] In some embodiments, the method is for generating islet-like beta cells and the PDX1 + progenitors are NKX6-1h'9h.

[0051] In some embodiments, the method is for generating islet-like delta cells and the PDX1+ progenitors are NKX6-1|OW.

[0052] In some embodiments, the FGFR agonist is added for between day 13-16 or day 13- 23 of differentiation from hPSCs.

[0053] In some embodiments, the FGFR agonist is FGF2, FGF9, or FGF4, preferably FGF2.

[0054] In an aspect there is provided a method for generating proglucagon+ cells from a human pluripotent stem cell-derived population of PDX1 + progenitors, the method comprising providing a population of human pluripotent stem cell-derived cells identified as PDX1+ progenitors for differentiation into proglucagon+ cells; and endocrine differentiating the population of progenitors in the presence of a Bone Morphogenetic Protein Receptor (BMPR) agonist.

[0055] In some embodiments, the PDX1+ progenitors are NKX6-1|OW.

[0056] In some embodiments, the BMPR agonist is BMP4.

[0057] In some embodiments, the proglucagon+ cells are islet-like alpha cells.

[0058] In an aspect there is provided a method for generating ARX expressing cells from a human pluripotent stem cell-derived population of PDX1 +progenitors, the method comprising providing a population of human pluripotent stem cell-derived cells identified as PDX1+ progenitors for differentiation into proglucagon+ cells; and endocrine differentiating the population of progenitors in the presence of a Bone Morphogenetic Protein Receptor (BMPR) agonist. In some embodiments, the BMPR agonist is added for between day 13-16 or day 13- 23 of differentiation from hPSCs, or alternatively between days 0-3, 0-10 or 3-10 of endocrine differentiation of pancreatic progenitors.

[0059] In an aspect there is provided the population of enterochromaffin cells produced by the method described herein.

[0060] In an aspect there is provided the population of islet-like beta and / or delta cells produced by the method described herein.

[0061] In an aspect there is provided the population of proglucagon+ cells or ARX expressing cells produced by the method described herein.

[0062] In an aspect there is provided a use of the population described herein for cell therapy in a subject in need thereof.

[0063] The term "subject" or “patient” as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans.

[0064] As used herein, “cell therapy” may refer to any therapy in which viable cells are injected, grafted or implanted into a patient in order to effectuate a medicinal effect. For example, by transplanting insulin producing cells into a patient as a treatment option for diabetes.

[0065] The term “treatment” as used herein as applied to a subject, refers to an approach aimed at obtaining beneficial or desired results, including clinical results and includes medical procedures and applications including for example pharmaceutical interventions, surgery, radiotherapy and naturopathic interventions as well as test treatments for treating diabetes. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, remission from a disease state, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0066] In an aspect there is provided a use of the population described herein for the screening of therapeutic agents. In an aspect there is provided a use of the population described herein to investigate the effect of one or more therapeutic agents, diagnostic agents and / or conditions on said population or said population’s development or differentiation.

[0067] As used herein, “pharmaceutically acceptable carrier1' means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include osmotically active agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent.

[0068] As used herein, “therapeutically effective amount' refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the pharmacological agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects.

[0069] The advantages of the present invention are further illustrated by the following examples. The examples and their particular details set forth herein are presented for illustration only and should not be construed as a limitation on the claims of the present invention.

[0070] EXAMPLES

[0071] Methods and Materials

[0072] Human pluripotent stem cell differentiation

[0073] A method of producing PDX1+ endodermal cells from a pluripotent stem cell population, the method comprising one or more of steps comprising contacting a pluripotent stem cell population with a combination of a nodal agonist, optionally ActA and a wnt signaling agonist, optionally, Wnt3a or CIHR 99021 , and a FGF agonist, optionally bFGF to produce definitive endoderm-like cells; contacting the definitive endoderm cells with a FGF agonist, optionally FGF10, and optionally wnt signaling agonist, optionally Wnt3a and / or a noggin component, optionally Dorsomorphin, to produce early foregut cells; contacting the early foregut cells with a BMP inhibitor component, optionally Noggin, Retinoic acid (RA) or RA analog, and optionally cyclopamine-KAAD (Cyc), a FGF agonist, optionally FGF10 to provide a PDX1 + endodermal population; and contacting the PDX1+ endodermal cell population with an EGF component and a tankyrase inhibitor and optionally Nicotinamide, and optionally a BMP inhibitor component, optionally Noggin and optionally a FGF agonist, optionally FGF10 in an amount sufficient to induce the differentiation of at least a portion of the endodermal cell population into PDX1+ / NKX6-1+ pancreatic progenitor cells typically at day 13 of differentiation.

[0074] Differentiation of PDX1+ progenitor cells into endocrine cells is performed by, for example, methods previously disclosed1 715-18(Table 1), whereby additionally contacting the cells with a MEK inhibitor optionally PD0325901 and optionally a BMP inhibitor optionally LDN193189 (LDN) is used to generate endocrine cell population expressing SLC18A1. PD0325901 can be substituted for example with FGF inhibitors such as FIIN1 -hydrochloride, PD173074 and ERK inhibitors such as SCH772984, LDN 193189 can be substituted for example with other noggin components such as chordin Dorsomorphin and BMPRs.

[0075] Alternatively, differentiation of PDX1+ progenitor cells into endocrine cells is performed by, for example, methods previously disclosed1 7 15'18(Table 1), whereby additionally contacting the cells with a FGF agonist optionally FGF2 is used to generate a pancreatic endocrine cell population expressing CPEPTIDE, NKX6-1 and containing few SLC18A1 expressing cells.

[0076] Alternatively, differentiation of PDX1+ progenitor cells into endocrine cells is performed by, for example, methods previously disclosed1 7 15'18. (Table 1), whereby additionally contacting the cells with a BMP agonist optionally BMP4 is used to generate a pancreatic endocrine cell population expressing ARX and GLUCAGON and containing few SLC18A1 expressing cells.

[0077] Table 1 : Media used days 13-22.

[0078] Legend: v / v (volume / volume); *lsoxazole can be used instead of Latrunculin A.

[0079] Differentiation of endocrine cells into islet-like cells is performed by, for example, the methods previously disclosed (Balboa). Final stages for example can include contacting the population to be differentiated (e.g. day 23 differentiated cells) with media containing components described in Table 2.

[0080] Table 2: Media used in final stage (days 23 onwards, media changed every 3 days) In vitro glucose-stimulated insulin secretion assay Under direct visualization using a low-power stereomicroscope, 20-30 hPSC-islets were individually selected and transferred to one well of a bottom 96-well plate. 3 sets of hPSC-islets were analyzed per condition. All subsequent steps were performed using a base KRB media (129 mM NaCI, 4.8 mM KCI, 2.5 mM CaCI2, 1.2 mM MgCI2, 1.2 mM KH2PO4, 10 mM HEPES, 5 mM NaHCO3) supplemented with 0.1% fatty acid- free bovine serum albumin (BSA) buffered to a pH of 7.4. Cells were first rested in a low glucose media (2.8 mM) for 30 minutes to 1 hour. Cells were then spun down at 1 ,000 RPM for 1 minute, supernatant was carefully removed, fresh “low glucose” (2.8 mM) media was added to each well. After 1 hour, cells were again spun down at 1 ,000 RPM for 1 minute, supernatant was carefully collected, and fresh “high-glucose” (16.7 mM) was added to each well. After 1 hour, cells spun down at 1 ,000 RPM for 1 minute, supernatant was carefully collected, and cells were rested in fresh low-glucose (2.8 mM) media for 30 minutes. Cells were then exposed to “KCI” media containing potassium chloride (30 mM) and high-glucose (16.7 mM) for 1 hour, after which supernatant was collected. For the low-glucose, high-glucose and KCI steps, human insulin content was quantified using the Ultra-Sensitive low-range HTRF kit (Cisbio #62IN2PEG) using the PHERAstar FS plate reader (BMG Labtech)

[0081] Flow cytometry

[0082] For profiling of hPSC-islets, cells were incubated with TrypLE Express with DNase at 10 pL / mL (1%) for 7-10 minutes at 37°C, then gently dissociated with a P1000 Pipetman. An equal volume of staining buffer containing 10% heat-inactivated Gibco FBS (ThermoFisher Scientific #12483-020) and 90% PBS without Calcium and Magnesium (Corning #21-040), supplemented with DNase (100 pL / mL) was added to stop the dissociation reaction.

[0083] Dissociated hPSC-islet cells in suspension culture were filtered through a 35 micrometer cell strainer, centrifuged, and the supernatant was discarded. All centrifuge steps were done at 930G for 2 minutes, and all antibody staining was performed in U- bottom 96-well plates. Cells were analyzed on a BD LSRFortessa flow cytometer. For multicolor experiments, compensation beads were used with fluophore-conjugated antibodies to calculate compensation parameters. Experimental data was analyzed using FlowJo Software (BD). Staining details ample gating strategies for specific experiments are provided below. Intracellular flow cytometry of hPSC-islets: Cells were incubated with Zombie Violet Fixable Viability dye (diluted at 1 pL / mL in PBS without Calcium and Magnesium) for 20 minutes at room temperature. Cells were centrifuged, and then resuspended with Cytofix / Cytoperm (BD #555722) solution for 20 minutes at room temperature. Cells were centrifuged and resuspended in primary antibody solution (table 2.10) for at least 1 hour at room temperature (most antibodies) or overnight at 4°C (any solution containing anti-human NKX6.1 primary antibody). Cells were washed once, resuspended in secondary antibody solution (table 2.11), and left to incubate for 20 minutes at room temperature. Cells were then washed once and resuspended in staining buffer for analysis. All washes and antibody dilutions were performed with Perm / Wash solution (10% BD Perm / Wash 10X stock solution, 90% PBS without Calcium and Magnesium). Due to cross-reactivity of donkey anti-mouse secondary antibodies against rat primary antibodies, solutions containing rat primary antibodies were always added after incubations with mouse primary antibodies and anti-mouse secondary antibodies were completed.

[0084] Table 2.10: Primary antibodies used in flow cytometry

[0085] Legend: FC, flow cytometry.

[0086] Table 2.11 : Secondary antibodies used in intracellular flow cytometry

[0087] Legend: FC, flow cytometry.

[0088] Statistical analysis and illustrations

[0089] Graphpad Prism (v 9.1.4) was used for statistical analysis and data visualization. Statistical tests employed are indicated in the figure captions, Experimental schematics were designed using BioRender (www.biorender.com').

[0090] Results and Discussion

[0091] Composition of endocrine induction media influences both degree of endocrine commitment and hormone lineage allocation

[0092] To study endocrine lineage specification, we added various permutations of small molecules to early endocrine induction media (days 13-16) and characterized the degree of endocrine commitment (CHGA expression) and hormone lineage specification in each case, including NKX6-1 +CPEP+ beta-like cells, ARX+ alpha- lineage cells, and SLC18A1+ EC-like cells. To ensure we did not miss effects related to altered differentiation kinetics, we sampled each condition on day 23 and again on day 29 of differentiation (Fig. 1 b). The compounds we chose to evaluate included the MEK1 / 2 inhibitor PD03 alongside tankyrase inhibitor WIKI4, and LatA, as these two compounds had been recently described to enhance endocrine differentiation of pancreatic progenitors15 18. These three compounds were added alone or in combination to a base protocol including ATRA, Thyroid hormone (T3), and inhibitors of the notch, ROCK and ALK5 pathways, as these had previously been found to be indispensable for endocrine differentiation of hPSC-derived pancreatic progenitors1 17(Fig. 1 B).

[0093] We first looked at general changes in endocrine differentiation between days 23 and 29 to evaluate the ideal time point for hPSC-islet characterization. Considerable increases in most endocrine lineages were observed over this period, with a mean absolute increase in CHGA+ endocrine-committed cells of 10.9% across all conditions and replicates, and a similar increase in the islet-specific endocrine marker ISL1 (Fig. 1 c,f). These changes appeared to be mostly the result of beta-like cells differentiation (NKX6-1+CPEP+), as the frequency of ARX+ and SLC18A1+ cells changed little between these time points (Fig. 1c-e,g-j). Overall, these suggested that hPSC-islet differentiation was not complete by day 23, and we thus focused most of our analyses on the later time point.

[0094] Looking at the effects of individual factors, we next observed a general trend towards greater CHGA expression as more compounds were included (Fig. 1f). This was mirrored by an increase in the frequency of NKX6.1+CPEP+ co-expressing cells, while the proportion of ARX+ (putative early alpha-like cells) remained relatively unchanged (Fig. 1e,g,h). Strikingly, a large increase in SLC18A1+ occurred only when LatA and PD03 were used in combination (Fig. 1d,i). To determine if we could improve SLC18A1+ cell differentiation, we undertook a second set of experiments to characterize EC lineage specification when PD03 was added at various time points between days 13 and 23 to a base media including LatA and WIKI4 (Fig. 2a). Under these conditions, the frequency of EC-like cells was highest when PD03 was included through endocrine differentiation (days 13-22, Fig. 2b-c). These experiments confirmed that PD03 treatment could strongly bias the differentiation of endocrine progenitors towards the EC lineage. In an additional set of experiments, we further observed that this effect was not specific to PD03, but generalizable to small molecule inhibitors of MEK1 / 2-related pathways, including ERK1 / 2 (SCH772984), FGFR1 / 3 (PD173074), and to a more limited extent, EGFR (Gefitinib) (Fig. 2d). These findings generally support the notion that cell-extrinsic factors can modulate differentiation of progenitors to favour specific endocrine lineages.

[0095] We next sought to better understand the phenotype of EC-like cells. In some experiments, we observed that the frequencies of SLC18A1+ and NKX6.1+CPEP+ cells added up to >100%, suggesting overlap in expression of these markers (Fig. 1g,i). Sub-gating on SLC18A1+ cells generated across all LatA / WIKI4 / PD03 permutations of the endocrine media revealed that nearly half of EC-like cells coexpressed the beta-like cell markers NKX6-1 and CPEP (Fig. 3a-c). Intriguingly, although the overall frequency of EC-like cells remained relatively unchanged between day 23 and 29, the frequency of EC-like cells co-expressing beta-like cell markers decreased in this interval. Further analysis of these markers revealed that this change was primarily due to a decrease in the proportion of SLC18A1+ cells co-expressing CPEP, while the frequency of NKX6-1 expression in this population remained stable at over 80% (Fig 3b-c). Because ISL1 is expressed in all primary pancreatic islet cells4849as well as most intestinal enteroendocrine cells, but is specifically excluded from primary intestinal EC cells50, we assessed its expression within the EC-like cells obtained in our protocols. While the majority of EC-like cells lacked ISL1 expression, minority of cells that retained its expression was observed even until day 29 (Fig. 3d). In summary, we observed that SLC18A1+ EC-like cells generally lack ISL1 but express NKX6-1 and often co-express CPEP, particularly at early stages of differentiation. Given that the total frequency of SLC18A1+ cells remained stable over time while the frequency of triple-positive SLC18A1+NKX6-1+CPEP+ cells decreased, we speculate that these triple positive cells represent an intermediate population that will resolve to single positive SLC18A1+ EC-like cells or die in these culture conditions.

[0096] FGF2 and BMP4 suppress SLC18A1 and promote ISL1 expression

[0097] The previous experiments demonstrated that day 13 hPSC-PPs exhibit some degree of plasticity in their ability to commit to various endocrine cell types, particularly the EC-like and beta-like lineages. After finding greater EC-like commitment upon treatment with PD03, which is known for its MEK1 / 2-inhibiting activity51we wondered whether treating cells with MEK1 / 2 agonists might instead inhibit the emergence of these cells. Two candidate MEK / ERK-activating ligands from different molecular families were thus tested for their ability to suppress EC lineage differentiation: FGF2, which had recently been found to increase ERK1 / 2 phosphorylation in hPSCs undergoing pancreatic differentiation52; and Betacellulin (BTC), which has been used in multiple pancreatic endocrine differentiation protocols3 1753and which has also been shown to interact with the MEK-ERK signaling pathway54. We treated day 13 pancreatic progenitors with each ligand for short (days 13-16) or prolonged (days 13- 23) durations (Fig. 4a) and first observed no significant impact on endocrine commitment, with nearly all cells in all treatment conditions expressing NKX2.2+, which is broadly produced in islet cells55(Fig. 4b). In contrast, all FGF2 / BTC conditions reduced the proportion of SLC18A1+ EC-like cells (Fig. 4c, e) and simultaneously increased the frequency of ISL1+ cells (Fig. 4d,e), though this was statistically and quantitatively most significant for prolonged FGF2 treatment. Analysis of islet endocrine lineages revealed that neither BTC nor FGF2 treatment affected the frequency of SLC18A1-NKX6.1+CPEP+ beta-like cells (Fig. 4f), and instead generally increased the proportion of the ARX+ cells, which is characteristically expressed by cells of the alpha lineage5657(Fig. 4g). We also observed that prolonged FGF2 treatment was specifically associated with an increase in both CPEP-GCG-SST+ cells (Fig. 4h) as well as an increase in SST+ cells co-expressing the delta cell transcription factor HHEX58(Fig. 4i-j). These findings suggest that that MEK1 / 2-associated ligands can toggle early endocrine progenitors away from the SLC18A1+ EC-like fate towards the ISL1+ islet-like lineages, with a potential ligand-specific effect of FGF2 for the induction of delta-like cells.

[0098] In an attempt to identify other pathways that toggle between the EC-like and islet-like endocrine lineages, we next re-evaluated the BMP pathway. We compared the effects of the canonical BMP ligand, BMP4, with the effects the endogenous BMP antagonist NOGGIN (NOG) and the small molecule BMP receptor inhibitor LDN-193189 (LDN). Cells were treated with each of these compounds from days 13 to 16 (early), days 16 to 23 (late), or days 13-23 (prolonged) (Fig. 5a). Neither BMP4 nor BMP antagonists induced any meaningful changes in endocrine commitment (NKX2.2 expression, Fig. 5b). In contrast, BMP4 stimulation significantly decreased SLC18A1 expression (Fig. 5c, e). This was accompanied by increased ISL1 expression, particularly with late and prolonged treatments (Fig. 5c, e). As with FGF2 and BTC stimulation, the shift from SLC18A1 expression to ISL1 expression was not accompanied by any increase in the frequency of SLC18A1-NKX6.1+CPEP+ beta-like cells (Fig. 5i,j). Instead, early and prolonged BMP4 treatments were associated with large increases in the alpha-lineage marker ARX (Fig. 5f,h) and decreases in beta-like cells (Fig. 5i,j), while late treatment had a non-significant impact on both lineages. We also observed a significant increase in CPEP-GCG-SST+ cells with late and prolonged BMP4 stimulation (Fig. 5g), though this was quantitatively less than that observed with prolonged FGF2 treatment. We observed no significant effect with NOG treatment on any of the lineages characterized, while the more potent but potentially less-specific LDN59led to small but significant increases in SLC18A1 expression (Fig. 5c) , accompanied by a small decrease in the frequency of beta-like cells (Fig. 5i). The limited effect of these BMP antagonists could suggest that this pathway exhibits a very low degree of endogenous activation in our differentiation system.

[0099] As we had now identified several factors that could promote the EC lineage, we sought to determine whether we could develop a protocol for the generation of hPSC-islets highly enriched with EC-like cells. To do this, we used a base protocol containing DBZ, REPSOX (Rezania Pagliuca), WIKI4 (Sharon), ISX9 (liu, DU), and added PD03 and LDN, alone or in combination (Fig. 6a). By day 26, dramatic upregulation of SLC18A1 expression was observed in cells treated simultaneously with both compounds, reaching as high as 80% in some experiments (Fig. 6b and c). accompanied by a decrease in ISL1+ (Fig. 6d) and SLC18A1-NKX6.1+CPEP+ cells (Fig. 6e). Intriguingly, no significant difference in the frequency of ARX+ cells was observed between the conditions (Fig. 6f), suggesting that these treatments may toggle preferentially between the beta-like and EC-like fates. Taken together, these findings demonstrate how rational combinations of small molecules and pathway modulators can be used to guide hPSC-PPs towards specific endocrine lineages.

[0100] Pancreatic progenitors are inherently biased towards specific endocrine lineages

[0101] Equipped with a better understanding of factors that guide hPSC-PPs towards specific islet and non-islet endocrine lineages, we revisited the impact of early endodermal patterning on endocrine lineage allocation. We directed DE-like cells towards the pancreatic lineage generating pancreatic progenitor cells expressing variable levels of NKX6-1 (Fig. 7a) . As expected, significant differences in NKX6-1 and SOX9 expression were observed at day 13 (Fig. 7b-e), though the majority of cells in the suppressed protocol maintained SOX9 despite lacking NKX6-1 (Fig. 7 b,c, e). Conversely, the suppressed protocol contained the highest number of precocious NKX2-2+ endocrine cells at this stage, in contrast to <5% in the NKX6-1 -enhanced protocol (Fig. 7c, f). Following the differentiation of DE-like cells towards progenitors with the desired NKX6-1 phenotypes, each of these progenitors were then patterned towards the alpha (using LatA with BMP4 to promote ARX expression), beta (using ISX9 with FGF2 / BTC to suppress the EC lineage and LDN to suppress the alpha lineage), and EC (using ISX9 with LDN and PD03 as elaborated above) lineages (Fig. 8). We first examined the alpha cell lineage, and observed that alpha patterning generated higher levels of ARX compared to beta and EC patterning. In contrast, for any given endocrine patterning protocol, all progenitors generated similar levels ARX+ cells (Fig. 8b, c). Interestingly, the frequency of GCG+ cells induced with alpha patterning appeared to be associated with lower progenitor NKX6-1 expression, with NKX6-1 -suppressed progenitors generating the highest frequency of ARX+GCG+ alpha-like cells (Fig. 8b, d), while NKX6-1 -enhanced progenitors produced fewer ARX+GCG+ and ARX+CPEP+ cells (Fig. 8e). Because ARX is also important for PPY-expressing gamma cell differentiation, we also profiled the frequency of PPY+ cells in a subset of experiments. Contrary to what was observed with glucagon expression, alpha patterning induced the highest frequency of ARX+PPY+ gamma-like cells from NKX6- 1 -enhanced progenitors, and this also occurred to a lesser extent with beta-islet and EC patterning (Fig. 8f). Thus, alpha-patterning successfully skewed progenitors towards the ARX lineages, but within the ARX+ population, the balance between alpha vs. gamma appeared to be primarily influenced by earlier progenitor patterning.

[0102] We next examined beta lineage patterning. In contrast to the initial protocol containing the EC-biasing factor PD03, the efficiency of the new beta lineage protocol differed considerably when applied to the different progenitors. Indeed, the greatest degree of SLC18A1-NKX6-1+CPEP+ differentiation was achieved with the NKX6-1 -enhanced progenitors (Fig. 8b, g). However, the beta lineage endocrine patterning protocol generally produced greater absolute numbers of beta-like cells per well compared to other endocrine patterning protocols, regardless of which progenitors it was used on (Fig. 8k). Surprisingly, this also applied to the NKX6-1 -suppressed progenitors (Fig. 8k). Although NKX6-1 -suppressed progenitors generated a lower frequency of betalike cells, these progenitors nonetheless retained a substantial ability to generate betalike cells (Fig. 8b, h, k). In support of this, NKX6-1 -suppressed progenitors undergoing beta lineage patterning generated on average 574,000 SLC18A1-NKX6-1+CPEP+ beta-like cells per well at day 26 (standard deviation of 225,000), which considerably exceeded the average of 112,000 NKX6-1+ progenitor cells per well at day 13 (standard deviation of 27,000)(Fig. 8g, I). This was unlikely to be the result of proliferation, as endocrine differentiation requires exit from the cell cycle61 65, and this process was initiated immediately after confirming their near-absence of NKX6-1 expression (day 13). Thus, NKX6-1 -deficient progenitors likely retain some limited ability to upregulate NKX6-1 and generate beta-like cells in vitro. In addition, NKX6-1- suppressed progenitors demonstrated a surprisingly enhanced capacity for differentiation into delta-like (SST+HHEX+) cells, greater than that of other progenitors (Fig. 8h). Intriguingly, beta and delta cell differentiation are both dependent on expression of the homeobox PAX466, underlying a potentially shared developmental program early in their specification. Thus, we postulate that our attempt at beta lineage patterning instead induced differentiation of PAX4+ islet lineages, with the balance between beta- and delta-like cells being influenced by progenitor-inherent features established prior to endocrine differentiation.

[0103] Finally, EC patterning successfully generated a majority of SLC18A1+ cells across all conditions (Fig. 8i), associated with a commensurate decrease in ISL1 expression (Fig. 8j). Notably, NKX6-1 -suppressed progenitors generated the highest frequency of SLC18A1+ cells (Fig. 8i) and the lowest frequency of ISL1+ cells (Fig. 8j), while the opposite trend was observed for NKX6-1 -enhanced progenitors. Indeed, NKX6-1- enhanced progenitors exhibited the greatest degree of escape from EC patterning, preferentially generating beta-like and gamma-like cells (Fig. 8f-g). These findings suggest that NKX6-1 may serve as a gatekeeper of the islet lineage, redirecting the effects of EC-patterning towards alternate NKX6-1 -associated lineages, including beta and gamma cells.

[0104] Promoting beta-like over EC-like differentiation improves the functionality of hPSC-PP- derived pancreatic endocrine cells

[0105] To understand the functional impact of reducing EC-like specification in favour of betalike cell differentiation, we assessed the functionality of NKX6-1 -enhanced progenitors that underwent beta-cell patterning (beta-enriched) using a static glucose-stimulated insulin secretion (GSIS) assay. As a control group, we included EC-patterned endocrine cells derived from control progenitors, as this method closely resembled our initial differentiation protocol, which generated hPSC-islets highly enriched in EC-like cells (Fig. 9a, b). In a static GSIS, we observed that beta-enriched endocrine cells and EC-enriched cultures exhibited similar insulin secretion upon exposure to potassium chloride (Fig. 9a). In contrast, marked differences were observed upon treatment with high glucose, as beta-enriched endocrine cells had a significant higher stimulation index compared to the EC-enriched endocrine cells (Fig. 9b). These findings suggest that a high frequency of EC-like cells may be deleterious to insulin secretion, although it remains unclear whether this is simply due to a reduced mass of functional beta-like cells, or to an inhibitory effect of EC-like cells on insulin secretion.

[0106] Discussion

[0107] A major challenge in the generation of hPSC-islets for research or therapy is the inability to influence the composition of endocrine cell types generated. As a result, efforts to generate beta-like cells are often compromised by the excessive differentiation of less desirable cell types, including alpha-like or EC-like cells. In this chapter, we describe a strategy for the directed differentiation of hPSCs towards each of the major islet cell types, including the elusive delta-islet lineage, as well as the recently-identified EC lineage. We demonstrate that soluble factors can guide endocrine differentiation of hPSC-PPs towards general endocrine programs, but also that hPSC-PPs display strong inherent biases towards specific endocrine lineages, and these two factors jointly determine the final hPSC-islet composition. Our findings are among the first to describe a system for the generation of hPSC-islets with a tunable composition of multiple defined cell types.

[0108] Although pancreatic endocrine differentiation has been extensively studied, the molecular cues guiding endocrine progenitors towards specific hormone-producing lineages remain elusive. An early study controlling the timing of endocrine commitment in the murine fetal pancreas found that the emergence of each islet cell type occurred in temporally-defined windows, suggesting that progenitors pass through “phases” of competence for each endocrine lineage68. Although this phenomenon was found to be largely mesenchyme-independent, it remains unclear whether these windows of competence are acquired in a cell-autonomous fashion (i.e. whether progenitors harbour inherent biases towards endocrine lineages that vary over time), or whether they are driven by temporal variations in paracrine signaling within the epithelium that influence hormone lineage allocation68. Supporting the notion of inherent biases, another study observed that, prior to endocrine commitment, progenitors exhibit epigenetic heterogeneity with regards to transcription factors that control the differentiation of specific endocrine lineages69. We observed that extracellular cues, notably those that modulate the BMP and FGF / BTC / MEK pathways could modulate endocrine output of hPSC-PPs. Using this knowledge, we derived specific protocols for preferentially patterning hPSC-PPs towards alpha (ARX+GCG+), beta (SLC18A1-NKX6-1+CPEP+) and EC (SLC18A1+) lineages. Although these protocols were able to effectively generate the cell types desired, other endocrine cell types not specifically targeted also appeared to favour specific methods of endocrine patterning. Thus, gamma-like cells (ARX+PPY+) emerged preferentially with alpha patterning while delta-like (HHEX+SST+) cells specifically formed under beta patterning conditions. We note that human alpha and gamma lineages appear to share a requirement for the transcription factor ARX57, while the beta and delta lineages likewise share a requirement for PAX466, and that these two transcription factors are mutually inhibitory56. Intriguingly, although gamma and delta cells were optimally induced by different methods of endocrine patterning, both preferentially emerged from NKX6-1 suppressed progenitors. We thus speculate that the alpha / gamma (“ARX”) and beta / delta (“PAX4”) lineage pairs develop under control of “shared” programs, partially under control of cell-extrinsic stimuli, which integrate with progenitor-specific features to dictate final endocrine fate.

[0109] Although EC cells are believed to share the beta / delta lineage requirement for PAX470,, we observed that EC-like cells emerged under conditions distinct from betalike and delta-like cells, preferentially from NKX6-1 -suppressed cells. Because the hPSC-derived EC lineage was uniquely characterized by decreased expression of the transcription factor ISL1 , we additionally posit that the EC cells represents a distinct program which requires both the presence of PAX4 and the absence of ISL1 (This hypothesis is depicted in Figure 10.

[0110] NKX6-1 has long been known to confer differentiation biases at multiple stages in the developing murine pancreatic epithelium. In the early fetal pancreatic primordium, NKX6-1 represses the expression of the pro-acinar transcription factor PTF1A, driving a subset of progenitors towards a bipotent duct / endocrine state42. Moreover, overexpression of NKX6-1 within this bipotent epithelium appears to generally bias these progenitors towards endocrine lineages42. Soon after, its overexpression in NGN3+ endocrine progenitors increases beta-like cell specification41. In our hPSC differentiation system, NKX6-1 appears to mark progenitors with biases towards specific endocrine lineages. Specifically, hPSC-PPs with high-grade NKX6-1 expression gave rise to higher frequencies of beta-like and gamma-like cells and resisted EC-differentiation, while progenitors lacking NKX6-1 more readily generated alpha-like, delta-like and EC-like cells. However, the final hPSC-islet composition was heavily influenced by the endocrine patterning protocol employed. Based on these findings, we tentatively propose that NKX6-1 expression prior to endocrine differentiation represents one of the progenitor-inherent features through which the alpha-gamma and beta-delta lineage pair can resolve. NKX6-1 appears to play an additional role as gatekeeper of the islet lineage, as a considerable fraction of NKX6-1 enhanced progenitors appear to resist EC specification, generating instead a variety of ISL1+ lineages (particularly beta-like and gamma-like cells). Importantly, as we did not directly manipulate NKX6-1 at the progenitor stage (e.g. through genetic knock-out or overexpression), we cannot discount the possibility that differences in the expression of this homeobox were only incidental, and other molecular differences inherent to the hPSC-PPs generated were also influential in lineage allocation.

[0111] Recently, several groups have developed methods to increase the frequencies of specific cell types. For instance, two studies have described methods for the generation of the alpha-like cells from hPSCs71 72. Another group recently identified matrix-derived factors as capable of inducing SST or CPEP induction in differentiating pancreatic progenitors73. These studies represent important contributions to our understanding of hPSC-islet differentiation in vitro, but the limited characterization of the populations generated render the interpretation of their findings challenging. Indeed, we observed that both hormones can be expressed in cells lacking transcription factors central to the identity of their canonical islet cell type (e.g. SST can be expressed in HHEX- cells, and CPEP can be expressed in NKX6-1- cells.). To build upon these studies, we relied on patterns of co-expression of multiple lineage markers gain deeper insight into the specific lineages obtained in our differentiations.. Moreover, we interrogated a wide variety of markers to discern the effects of our treatments on multiple pancreatic lineages. In doing so, we were able to confidently follow changes in islet composition throughout the various protocols tested. We believe that these findings will provide an important resource for the design of lineage-specific protocols that can be applied to a wide variety of hPSC lines.

[0112] An important focus of this work was the mechanisms underlying SLC18A1+ EC-like cell differentiation. These endeavors identified that small molecule inhibitors of BMP receptor and ERK1 / 2 signaling along with the putative agonists of these pathways could modulate the differentiation of EC-like cells. We find that EC-patterned hPSC- islets exhibit lower glucose-responsive insulin-secreting functionality than beta- lineage-patterned hPSC-islets, supporting the rationale for limiting the specification of this population in hPSC-islets intended for therapeutic purposes.

[0113] In conclusion, we present a system that efficiently generates pancreatic islet cells from hPSCs, and provide strategies to fine-tune their endocrine composition. In particular, this system permits both the enrichment and depletion of EC-like cells, which in excess appear to have deleterious effects on the insulin-secreting functionality of hPSC-islets. This study addresses an important gap in our understanding of how hPSC-PPs commit to specific endocrine lineages, and provides a framework for the rational design and optimization of hPSC-islet differentiation protocols.

[0114] Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including those in the following reference list, are incorporated by reference.

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Claims

CLAIMS:1 . A method for generating enterochromaffin cells from a human pluripotent stem cell-derived population of PDX1+ progenitors, the method comprising: providing a population of human pluripotent stem cell-derived cells identified as PDX1+ progenitors for differentiation into enterochromaffin cells; and endocrine differentiating the population of pancreatic progenitors in the presence of an inhibitor of at least one of Fibroblast Growth Factor Receptor (FGFR), MAPK and ERK Kinase (MEK), and Extracellular signal Regulated protein Kinase (ERK) pathways.

2. The method of claim 1 , wherein the PDX1 + progenitors are NKX6-1 +.

3. The method of claim 1 or 2, wherein the inhibitor is a FGFR inhibitor.

4. The method of claim 3, wherein the FGFR inhibitor is FIIN1-hydrochloride, PD173074, Erdafitinib, pemigatinib, envatinib (E7080), ponatinib (AP24534), regorafenib (BAY 73-4506), dovitinib (TKI258), lucitanib (E3810), cediranib (AZD2171), nintedanib (BIBF 1120), brivanib (BMS-540215), LY2874455, Zoligratinib (Debio-1347), Dovitinib, (TKI258) Lactate monohydrate, FIIN-2, S49076, PRN1371 , Derazantinib(ARQ-087), ASP5878, Futibatinib (TAS-120), ODM-203, or Infigratinib (BGJ398), preferably FIIN1-hydrochloride, or PD173074, or alternatively an FGFR3 inhibitor.

5. The method of claim 1 or 2, wherein the inhibitor is a MEK inhibitor.

6. The method of claim 5, wherein the MEK inhibitor is PD0325901 , trametinib(Mekinist), cobimetinib (Cotellic), binimetinib (Mektovi), Selumetinib (AZD6244), U0126-EtOH, PD184352 (CI-1040), PD98059, TAK-733, AZD8330,Refametinib (RDEA119), or GDC-0623, preferably PD0325901 , U0126-EtOH or PD98059.

7. The method of claim 1 or 2, wherein the inhibitor is a ERK inhibitor.

8. The method of claim 7, wherein the ERK inhibitor is SCH772984, Ravoxertinib (GDC-0994), Temuterkib (LY3214996), Pluripotin (SC1), VX-11e, or Ulixertinib (BVD-523), preferably SCH772984.

9. The method of any one of claims 1-8, wherein the inhibitor is added for between day 13-22 of differentiation from hPSCs or alternatively between 0-9 days of endocrine differentiation from pancreatic progenitors.

10. The method of any one of claims 1-9, wherein the population of pancreatic progenitors is differentiated additionally in the presence of Latrunculin A.

11. The method of any one of claims 1-10, wherein the population of pancreatic progenitors is differentiated additionally in the presence of at least one of WIKI4, DBZ and Repsox.

12. The method of any one of claims 1-11 , wherein the population of pancreatic progenitors is differentiated additionally in the presence of ISX9.

13. A method for generating islet-like beta and / or delta cells from a human pluripotent stem cell-derived population of PDX1+ progenitors, the method comprising: providing a population of human pluripotent stem cell-derived cells identified as PDX1+ progenitors for differentiation into islet-like beta and / or delta cells; and endocrine differentiating the population of progenitors in the presence of a Fibroblast Growth Factor Receptor (FGFR) agonist.

14. The method of claim 13, wherein the PDX1+ progenitors are NKX6-1+.

15. The method of claim 13 or 14, for generating islet-like beta cells and the PDX1+ progenitors are NKX6-1h'9h.

16. The method of claim 13 or 14, for generating islet-like delta cells and the PDX1+ progenitors are NKX6-1|OW.

17. The method of any one of claims 13-16, wherein the FGFR agonist is added for between day 13-16 or day 13-23 of differentiation from hPSCs.

18. The method of any one of claims 13-17, wherein the FGFR agonist is FGF2, FGF9, or FGF4, preferably FGF2.

19. A method for generating proglucagon+ cells from a human pluripotent stem cell-derived population of PDX1+ progenitors, the method comprising: providing a population of human pluripotent stem cell-derived cells identified as PDX1+ progenitors for differentiation into proglucagon+ cells; and endocrine differentiating the population of progenitors in the presence of a Bone Morphogenetic Protein Receptor (BMPR) agonist.

20. The method of claim 19, wherein the PDX1+ progenitors are NKX6-1|OW.21 . The method of claim 19 or 20, wherein the BMPR agonist is BMP4.

22. The method of any one of claims 19-21 , wherein the proglucagon+ cells are islet-like alpha cells.

23. A method for generating ARX expressing cells from a human pluripotent stem cell-derived population of PDX1+ progenitors, the method comprising: providing a population of human pluripotent stem cell-derived cells identified as PDX1+ progenitors for differentiation into proglucagon+ cells; and endocrine differentiating the population of progenitors in the presence of a Bone Morphogenetic Protein Receptor (BMPR) agonist.

24. The method of any one of claims 19-23, wherein the BMPR agonist is added for between day 13-16 or day 13-23 of differentiation from hPSCs, or alternatively between days 0-3, 0-10 or 3-10 of endocrine differentiation of pancreatic progenitors.

25. The population of enterochromaffin cells produced by the method of any one of claims 1-12.

26. The population of islet-like beta and / or delta cells produced by the method of any one of claims 13-18.

27. The population of proglucagon+ cells or ARX expressing cells produced by the method of any one of claims 19-24.

28. Use of the population of any one of claims 25-27 for cell therapy in a subject in need thereof.

29. Use of the population of any one of claims 25-27 for the screening of therapeutic agents.

30. Use of the population of any one of claims 25-27 to investigate the effect of one or more therapeutic agents, diagnostic agents and / or conditions on said population or said population’s development or differentiation.

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