Methods for generating insulin-secreting pancreatic islet-like cells

Abstract

Various embodiments of the present invention relate generally to stem cell biology, developmental biology, and regenerative medicine. More particularly, various embodiments relate to methods, compositions, culture systems, and kits for directing the differentiation of human pluripotent stem cells, including human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), into pancreatic lineage cells such as functional islet-like cells. In further embodiments, the invention relates to populations of pancreatic lineage cells produced by the disclosed methods, as well as their use in disease modelling, drug screening, and cell replacement therapies for metabolic disorders such as diabetes.

Description

METHODS FOR GENERATING INSULIN-SECRETING PANCREATIC ISLET-LIKE CELLSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of Singapore Patent Application No.10202500144Q filed 17 January 2025, the content of which being hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] Various embodiments of the present invention relate generally to stem cell biology, developmental biology, and regenerative medicine. More particularly, various embodiments relate to methods, compositions, culture systems, and kits for directing the differentiation of human pluripotent stem cells, including human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), into pancreatic lineage cells such as functional islet-like cells. In further embodiments, the invention relates to populations of pancreatic lineage cells produced by the disclosed methods, as well as their use in disease modelling, drug screening, and cell replacement therapies for metabolic disorders such as diabetes.BACKGROUND

[0003] Diabetes is a chronic disease that affects over 560 million people worldwide. More than half of the world’s population of diabetes patients reside in Asia. In particular, patients with Type 1 diabetes or severe forms of diabetes (Type 2 diabetes or other rare monogenic forms of diabetes) that require intensive insulin therapy harbour debilitating disease that affects their quality of life. An alternative treatment strategy based on regenerative medicine provides a more curative approach, through replacement of the insulin-secreting pancreatic islet cells to restore glucose control in diabetes patients.

[0004] HiPSCs have tremendous potential to be used as the starting source material for deriving many different cell types for the purpose of cell replacement therapy and regenerative medicine. Specifically, hiPSC-derived pancreatic islet cells may be developed for autologous or allogeneic cell replacement therapy for diabetes in addition to disease modelling and drug screening studies. For this to be possible, hiPSC-derived islet cells need to be generated off-the-shelf, on-demand, with consistency and positive demonstration of insulin secretion function. Various earlier research protocols deriving hiPSC-islet cells have several limitations such as lack of reproducibility, scalability, functionality, and only found to work with 2-3 cell lines.

[0005] Batch-to-batch variability arises from epigenetic differences between hiPSC lines, higher passage numbers leading to genomic instability, and complex multi-step differentiation processes that result in heterogeneous cell populations with off-target cells. These issues restrict protocols to functioning reliably with only a few specific cell lines, undermining their potential for off-the-shelf, on-demand production.

[0006] Therefore, there is still a need in the art for improved differentiating methods for generating functional islet-like cells, which may be used for treating metabolic disorders, such as diabetes.SUMMARY

[0007] The present invention satisfies the aforementioned need in the art by providing the methods, cells, compositions, and kits described herein.

[0008] In one aspect, there is provided in vitro method for generating insulin-secreting pancreatic isletlike cells from a human induced pluripotent stem cells (hiPSCs) or human Embryonic Stem Cells (hESCs) derived pancreatic progenitor 1 cells, comprising: providing the pancreatic progenitor 1 cells; differentiating the pancreatic progenitor 1 cells in vitro in a Stage 4 medium under conditions and for a period of time sufficient to obtain pancreatic progenitor 2 cells, wherein the Stage 4 medium comprises a BMP type I receptor inhibitor; differentiating the pancreatic progenitor 2 cells in vitro in a first Stage 5 medium and a second Stage 5 medium under conditions for a first period of time sufficient and a second period of time sufficient, respectively, to obtain pancreatic endocrine progenitor cells, wherein each, the first Stage 5 medium and the second Stage 5 medium, comprises the BMP type I receptor inhibitor; and differentiating the pancreatic endocrine progenitor cells in vitro in a first Stage 6 medium, a second Stage 6 medium and a third Stage 6 medium under conditions for a first period of time sufficient, a second period of time sufficient, and a third period of time sufficient, respectively, to obtain insulinsecreting pancreatic islet-like cells, wherein the first Stage 6 medium comprises glucose, ZnSC , and N-acetyl cysteine (NAC) and ISX9.

[0009] In various embodiments, the BMP type I receptor inhibitor is LDN193189.

[0010] In various embodiments, the Stage 4 medium further comprises a bovine serum albumin (BSA), an activin and a GSK30 inhibitor, the first Stage 5 medium further comprises the BSA and a fibroblast growth factor (FGF), the second Stage 5 medium comprises the BSA, a retinoid, a Hedgehog pathway antagonist, a y-secretase inhibitor, a TGF-p receptor inhibitor, a thyroid hormone, an epidermal growth factor (EGF) family ligand, and LDN193189, the first Stage 6 medium further comprises the BSA, the FGF, a retinoid, a Hedgehog pathway antagonist, and a protein kinase C (PKC) activator, the second Stage 6 medium comprises the TGF-p receptor inhibitor, the thyroid hormone, and NAC, and the third Stage 6 medium comprises the TGF-p receptor inhibitor, the thyroid hormone, NAC and an Axl inhibitor.

[0011] In various embodiments, the BSA is fatty-acid-free bovine serum albumin (FAF BSA), the activin is Activin A, the GSK3P inhibitor is CHIR99021 , the FGF is fibroblast growth factor 7 (FGF7), the retinoid is retinoic acid (RA), the Hedgehog pathway antagonist is SANT1 , the y-secretase inhibitor is XXI, the TGF-p receptor inhibitor is ALK5ill, the thyroid hormone is triiodothyronine (T3), the epidermal growth factor (EGF) family ligand is Betacellulin, the PKC activator is phorbol 12,13-d i butyrate (PDBu), the Axl inhibitor is R428, or a combination thereof.

[0012] In various embodiments, the pancreatic progenitor 1 cells are provided by the following steps of, differentiating hiPSCs or hESCs in vitro in a Stage 1 medium under conditions and for a period of time sufficient to obtain definitive endoderm cells, wherein the Stage 1 medium comprises FAF BSA, Activin A and CHIR99021 ; differentiating the definitive endoderm cells in vitro in a Stage 2 medium under conditions and for a period of time sufficient to obtain primitive gut tube cells, wherein the Stage 2 medium comprises FAF BSA and FGF7; and differentiating the primitive gut tube cells in vitro in a Stage 3 medium under conditions and for a period of time sufficient to obtain pancreatic progenitor 1 cells, wherein the Stage 3 medium comprises FAF BSA, FGF7, RA, SANT1 , phorbol 12,13-dibutyrate (PDBu), and LDN193189.

[0013] In another aspect there is provided an in vitro method for generating insulin-secreting pancreatic islet-like cells from human induced pluripotent stem cells (hiPSCs) or human Embryonic Stem Cells (hESCs), comprising: (1) differentiating hiPSCs or hESCs in vitro in a Stage 1 medium under conditions and for a period of time sufficient to obtain definitive endoderm cells, wherein the Stage 1 medium comprises fatty-acid-free BSA (FAF BSA), Activin A and CHIR99021 ; (2) differentiating the cells obtained from stage (1) in vitro in a Stage 2 medium under conditions and for a period of time sufficient to obtain primitive gut tube cells, wherein the Stage 2 medium comprises FAF BSA and fibroblast growth factor 7 (FGF7); (3) differentiating the cells obtained from stage (2) in vitro in a Stage 3 medium under conditions and for a period of time sufficient to obtain pancreatic progenitor 1 cells, wherein the Stage 3 medium FAF BSA, FGF7, retinoic acid (RA), SANT1 , phorbol 12,13-dibutyrate (PDBu), and LDN193189; (4) differentiating the cells obtained from stage (3) in vitro in a Stage 4 medium under conditions and for a period of time sufficient to obtain pancreatic progenitor 2 cells, wherein the Stage 4 medium comprises FAF BSA, FGF7, RA, SANT1 , LDN193189 and Nicotinamide; (5) differentiating the cells obtained from stage (4) in vitro in a first Stage 5 medium a second Stage 5 medium under conditions and for a first period of time sufficient and a second period of time sufficient, respectively, to obtain pancreatic endocrine progenitor cells, wherein the first Stage 5 medium comprises FAF BSA, RA, SANT1 , XXI, ALK5i II , T3, Betacellulin, and LDN193189; wherein the second Stage 5 medium comprises RA, XXI, ALK5HI, triiodothyronine (T3), Betacellulin, LDN193189 and ISX9; and (6) differentiating the cells obtained from stage (5) in vitro in a first Stage 6 medium and a second Stage 6 medium under conditions for a first period of time sufficient and a second period of time sufficient, respectively, to obtain islet-like cells, wherein the first Stage 6 medium comprises FAF BSA, glucose, ZnSO4, ALK5ill , T3, N-acetyl cysteine (NAC), and ISX9 or an equivalent factor, wherein the second Stage 6 medium comprises ALK5ill, T3, NAC and R428.

[0014] In various embodiments, all stages of the method are performed in a suspension culture system.

[0015] In various embodiments, the method further comprises, before Stage 1 , an additional step of dissociating the hiPSCs or hESCs into single cells and culturing them as spheroids in an in vitro suspension culture system.

[0016] In various embodiments, the single cells are incubated at a density of 0.6 -1.2 x 106cells per ml_ for 1 -5 days under orbital rotation at a speed of 70-90 rpm.

[0017] In various embodiments, the Stage 1 medium comprises about 1-2 % FAF BSA, about 100 ng / ml Activin A, and about 1 -3 pM CHIR99021, preferably the CHIR99021 is at a concentration of about 1 pM, and the Stage 1 period of time is about 1 day.

[0018] In various embodiments, the Stage (1 ) further comprises a subsequent Stage (1 ) differentiating step comprising replacing the Stage 1 medium with a second Stage 1 medium comprising about 100 ng / ml Activin A, wherein the subsequent differentiating step is for a period of time of about 2 days.

[0019] In various embodiments, the Stage 2 medium comprises about 2 % FAF BSA and about 50 ng / ml FGF7, and the Stage 2 period of time is about 3 days.

[0020] In various embodiments, the Stage 3 medium comprises about 2% FAF BSA, about 50 ng / ml FGF7, about 2 pM RA, about 0.25 pM SANT1, about 0.5 pM PDBu, and 0.1-0.2 pM LDN193189, and the Stage 3 period of time is about 2 days.

[0021] In various embodiments, the Stage 4 medium comprises about 2% FAF BSA, about 50 ng / ml FGF7, about 0.1 pM RA, about 0.25 pM SANT1 , 0.1-0.2 pM LDN193189 and about 10 mM Nicotinamide, wherein the Stage 4 period of time is about 4-6 days, preferably about 5 days.

[0022] In various embodiments, the first Stage 5 medium comprises about 2% FAF BSA, about 0.1 pM RA, about 0.25 pM SANT1 , about 1 pM XXI, about 10 pM ALK5il I , about 1 pM T3, about 20 ng / ml Betacellulin, and about 0.1-0.2 pM LDN193189, wherein the Stage 5 period of time is about 4 days.

[0023] In various embodiments, Stage (5) further comprises a subsequent Stage (5) differentiating step comprising replacing the first Stage 5 medium with the second Stage 5 medium comprising about 25 nM RA, about 1pM XXI, about 10pM ALK5HI, about 1pM T3, about 20 ng / ml Betacellulin, about 0.1-0.2 pM LDN193189 and about 0-10 pM ISX9 or an equivalent factor, wherein the second period of time of about 3 days, preferably the equivalent factor is selected from the group consisting of CHIR99021 , 6-bromoindirubin-3'-oxime (BIO), lithium chloride (LiCI), T3, Alk5 inhibitors, PKA / cAMP agonists and epigenetic modulators.

[0024] In various embodiments, the Stage 6 first medium comprises about 2% FAF BSA, about 20mM glucose, about 10pM ZnSC , about 10pM ALK5HI, about 1pM T3, about 1mM NAC, and about 0-1 OpM ISX9, wherein the first period of time is about 2-3 days.

[0025] In various embodiments, Stage (6) further comprises a subsequent 2ndStage (6) differentiating step comprising replacing the first Stage 6 medium with the second Stage 6 medium comprising about 10pM ALK5HI, about 1pM T3, and about 1mM NAC, wherein the second period of time is about 2-4 days.

[0026] In various embodiments, Stage (6) further comprises a subsequent 3rdStage (6) differentiating step comprising replacing the second Stage 6 medium with a third Stage 6 medium comprising about 10pM ALK5III, about 1pM T3, about 1 mM NAC, about 0.5pM R428, wherein the third period of time is about 10-20 days, preferably about 12 days.

[0027] In various embodiments, LDN193189 is used continuously from Stage 3 to Stage 5 to inhibit BMP signalling, thereby enhancing pancreatic lineage commitment and pancreatic progenitor development.

[0028] In various embodiments, the insulin-secreting pancreatic islet-like cells exhibit glucose-stimulated insulin secretion (GSIS) in response to glucose at a concentration of 15-25.5 mM, and KCI stimulation.

[0029] In various embodiments, the insulin-secreting pancreatic islet-like cells exhibit decreased insulin secretion in response to a low glucose concentration of 0-3 mM.

[0030] In various embodiments, the hiPSCs are Asian donor-derived hiPSCs selected from the group consisting of i70b, i173b, is104, 43e5 and is043.

[0031] In another aspect, there is provided a population of insulin-secreting pancreatic islet-like cells generated using the in vitro method disclosed herein, wherein the islet-like cells express islet cell markers selected from the group comprising C-peptide, PDX1 , NKX6.1 , and glucagon.

[0032] In another aspect, there is provided a composition comprising the population of insulinsecreting pancreatic islet-like cells disclosed herein.

[0033] In another aspect, there is provided a method of treating a metabolic disorder in a subject in need thereof, comprising administering the composition disclosed herein to the subject, wherein the metabolic disorder is selected from the group consisting of diabetes, hyperglycemia, pre-diabetes, diabetes induced by pancreatectomy, monogenic forms of diabetes, and neonatal diabetes.

[0034] In another aspect, there is provided a use of the composition disclosed herein in the manufacture of a medicament for treating a metabolic disorder in a subject in need thereof, wherein the metabolic disorder is selected from the group consisting of diabetes, hyperglycemia, pre-diabetes, diabetes induced by pancreatectomy, monogenic forms of diabetes, and neonatal diabetes.

[0035] In various embodiments, the subject is human.

[0036] In various embodiments, the insulin-secreting pancreatic islet-like cells are administered via a route selected from the group consisting of portal vein injection, subcutaneous implantation, intramuscular injection, and delivery via a medical device.

[0037] In various embodiments, the subject is a mouse.

[0038] In various embodiments, the insulin-secreting pancreatic islet-like cells are administered via transplantation to the kidney.

[0039] In another aspect, there is provided a kit for use in generating insulin-secreting pancreatic isletlike cells, comprising one or more of the Stage (1 )-(6) medium defined in the method disclosed herein, preferably the kit comprises the Stage 4 medium, the Stage 5 medium, and the Stage 6 medium, and optionally the Stage 1 medium, the Stage 2 medium, and the Stage 3 medium.

[0040] In another aspect, there is provided a composition comprising: a population of cells, wherein at least 70% cells are pancreatic endocrine progenitor cells that express NEUROG3 (NGN3); and a medium comprising effective amounts of glucose, ZnSC , N-acetyl cysteine (NAG) and ISX9 for differentiating the endocrine progenitor cells into insulin-secreting pancreatic islet-like cells.

[0041] In various embodiments, the medium comprises about 20mM glucose, about 10pM ZnSCu, about 1 mM N-acetyl cysteine (NAC), and about 0-1 OpM ISX9.

[0042] In various embodiments, the medium further comprises a bovine serum albumin (BSA), a TGF-p receptor inhibitor, and a thyroid hormone.

[0043] In various embodiments, the BSA is fatty-acid-free bovine serum albumin (FAF BSA), the TGF- receptor inhibitor is ALK5III, and the thyroid hormone is triiodothyronine (T3).

[0044] In various embodiments, the medium comprises about 2% fatty-acid-free bovine serum albumin (FAF BSA), about 10pM ALK5ill, and about 1pM triiodothyronine (T3).

[0045] In another aspect, there is provided a composition comprising: a population of cells, wherein at least 70% cells are pancreatic progenitor 2 cells that express PDX1 and NKX6.1 ; and a medium comprising an effective amount of BMP type I receptor inhibitor for differentiating the pancreatic progenitor 2 cells into endocrine progenitor cells.

[0046] In various embodiments, the effective amount of BMP type I receptor inhibitor is about 0.1 -0.2 pM of LDN193189.

[0047] In various embodiments, the medium further comprises a bovine serum albumin (BSA), a retinoid, a Shh signalling pathway antagonist, a y-secretase inhibitor, a TGF-p receptor inhibitor, a thyroid hormone, and an epidermal growth factor (EGF) family ligand.

[0048] In various embodiments, the BSA is fatty-acid-free bovine serum albumin (FAF BSA), the retinoid is retinoic acid (RA), the Shh signalling pathway antagonist is SANT1 , the y-secretase inhibitor is XXI, the TGF-f> receptor inhibitor ALK5i 11 , the thyroid hormone is triiodothyronine (T3), and the EGF family ligand is Betacellulin.

[0049] In various embodiments, the medium comprises about 2% a fatty-acid-free bovine serum albumin (FAF BSA), about 0.1 pM retinoic acid (RA), about 0.25 pM SANT1 , about 1 pM XXI, about 10 pM ALK5ill, about 1 pM triiodothyronine (T3), and about 20 ng / ml Betacellulin.

[0050] In another aspect, there is provided a composition comprising:a population of cells, wherein at least 70% cells are pancreatic progenitor 1 cells that express PDX1 , FOXA2 and HNF1 P; and a medium comprising an effective amount of BMP type I receptor inhibitor for differentiating the pancreatic progenitor 1 cells into pancreatic progenitor 2 cells.

[0051] In various embodiments, the effective amount of BMP type I receptor inhibitor is 0.1 - 0.2 pM of LDN193189.

[0052] In various embodiments, the medium further comprises a bovine serum albumin (BSA), a fibroblast growth factor (FGF), a retinoid, a Shh signalling pathway antagonist, and a nicotinamide metabolic cofactor.

[0053] In various embodiments, the BSA is fatty-acid-free bovine serum albumin (FAF BSA), the FGF is fibroblast growth factor 7, the retinoid is retinoic acid (RA), the Shh signalling pathway antagonist is SANT1 , and the nicotinamide metabolic cofactor is Nicotinamide.

[0054] In various embodiments, the medium comprises about 2% a fatty-acid-free bovine serum albumin (FAF BSA), about 50 ng / ml FGF7, about 0.1 pM retinoic acid (RA), about 0.25 pM SANT1, and about 10 mM Nicotinamide.

[0055] In another aspect, there is provided a composition comprising:a population of cells, wherein at least 70% cells are insulin-secreting pancreatic islet-like cells that express INS, PCSK1 , PCSK2, MAFA, PDX1 , and NKX6.1 ; and a medium comprising a TGF-p receptor inhibitor, a thyroid hormone, a redox-modulating agent, and an Axl inhibitor, wherein the insulin-secreting pancreatic islet-like cells have at 100% more insulin content, normalized to total genomic DNA, relative to primary human islets.

[0056] In various embodiments, the TGF-p receptor inhibitor is ALK5HI, the thyroid hormone is triiodothyronine (T3), the redox-modulating agent is N-acetyl cysteine (NAC), and the Axl inhibitor is R428.

[0057] In various embodiments, the medium comprises 10 pM ALK5III, 1 pM T3, 1 mM N-acetyl cysteine (NAC), and 0.5 pM R428.

[0058] In another aspect, there is provided a composition comprising: a population of cells, wherein at least 70% cells are insulin-secreting pancreatic islet-like cells that express INS, PCSK1 , PCSK2, MAFA, PDX1 , and NKX6.1 ; and a medium comprising a TGF-p receptor inhibitor, a thyroid hormone, a redoxmodulating agent, and an Axl inhibitor, wherein the insulin-secreting pancreatic islet-like cells exhibit about 8-30 fold increased insulin secretion in response to glucose at a concentration of 15-25.5 mM.

[0059] In various embodiments, the TGF-p receptor inhibitor is ALK5III, the thyroid hormone is triiodothyronine (T3), the redox-modulating agent is N-acetyl cysteine (NAC), and the Axl inhibitor is R428.

[0060] In various embodiments, the medium comprises 10 pM ALK5III, 1 pM T3, 1 mM N-acetyl cysteine (NAC), and 0.5 pM R428.

[0061] In another aspect, there is provided a pharmaceutical composition a population of cells and a pharmaceutically acceptable excipient, wherein at least 70% cells are insulin-secreting pancreatic isletlike cells that express INS, PCSK1 , PCSK2, MAFA, PDX1 , and NKX6.1 , wherein: the insulin-secreting pancreatic islet-like cells have at least 50% more insulin content, normalized to total genomic DNA, relative to primary human islets, the insulin-secreting pancreatic islet-like cells exhibit a biphasic insulin secretion in response to glucose stimulation and KCI stimulation, the insulin-secreting pancreatic isletlike cells exhibit about 8-30 fold increased insulin secretion when contacted with a high glucose concentration in a range of 15-25.5 mM, and the insulin-secreting pancreatic islet-like cells exhibit decreased insulin secretion when contacted with a low glucose concentration in a range of 0-3 mM following contacting with the high glucose concentration.

[0062] In another aspect, there is provided a pharmaceutical composition comprising a population of cells and a pharmaceutically acceptable excipient, wherein at least 70% cells are insulin-secreting pancreatic islet-like cells that express INS, PCSK1 , PCSK2, MAFA, PDX1 , and NKX6.1 , wherein the insulin-secreting pancreatic islet-like cells have at least 100% more insulin content, normalized to total genomic DNA, relative to primary human islets.

[0063] In various embodiments, the population of cells comprise C-peptide positive cells, glucagon positive cells and somatostatin positive cells.

[0064] In various embodiments, the insulin-secreting pancreatic islet-like cells are generated from human induced pluripotent stem cells (hiPSCs).

[0065] In various embodiments, the islet-like cells express islet cell markers selected from the group comprising C-peptide, PDX1 , NKX6.1 , and glucagon.

[0066] In various embodiments, the hiPSCs are Asian donor-derived hiPSCs selected from the group consisting of i70b, i173b, is104, 43e5 and is043.

[0067] In another aspect, there is provided a method of treating a metabolic disorder in a subject in need thereof, comprising administering an effective amount of the pharmaceutical composition disclosed herein to the subject, wherein the metabolic disorder is selected from the group consisting of type 1 diabetes mellitus (T1 DM), type 2 diabetes mellitus (T2DM), hyperglycemia, pre-diabetes, diabetes induced by surgical pancreatectomy, monogenic diabetes, and neonatal diabetes.

[0068] In various embodiments, the pharmaceutical composition is administered by portal vein infusion, subcutaneous implantation, intra-muscular implantation, or intra-omental implantation.

[0069] In another aspect, there is provided a use of the pharmaceutical composition disclosed herein in the manufacture of a medicament for treating a metabolic disorder in a subject in need thereof, wherein the metabolic disorder is selected from the group consisting of type 1 diabetes mellitus (T 1 DM), type 2 diabetes mellitus (T2DM), hyperglycemia, pre-diabetes, diabetes induced by surgical pancreatectomy, monogenic diabetes, and neonatal diabetes.

[0070] It is understood that all embodiments disclosed herein in relation to one aspect of the invention are similarly applicable to all other aspects of the invention.BRIEF DESCRIPTION OF DRAWINGS

[0071] Various embodiments will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[0072] FIG. 1 shows a schematic illustration of the protocol and stages for generating pancreatic isletlike clusters (hiPSC-islets) from human induced pluripotent stem cells (hiPSCs). The top series of illustrations schematically depicts the sequential transition of cell aggregates through staged differentiation phases. The lower series of microscopy images corresponds to each stage, demonstrating characteristic morphological changes, including formation of dense three-dimensional aggregates, increasing cellular compaction, and emergence of islet-like architecture. Scale bars of100pm indicate relative size at each differentiation step. The series collectively represents an exemplary workflow for producing functional, insulin-secreting hiPSC-derived islet-like cells.

[0073] FIG.2A-2B show a dynamic human insulin secretion profile of hiPSC-derived islets in response to high glucose (16.7 mM) and KCI (30 mM): FIG.2A shows insulin concentration (pg / L) measured over time during exposure to low glucose (2.8 mM), high glucose (16.7 mM), a return to low glucose, followed by potassium chloride (KCI, 30 mM) and a final low-glucose phase. The data demonstrate stimulusdependent increases in secreted insulin, including a robust secretion peak in response to high glucose and a secondary response upon KCI stimulation; and FIG. 2B shows the corresponding fold-change in insulin secretion by hiPSC-derived islet-like clusters across the same sequential stimulation paradigm shown in FIG. 2A. Fold-change values are plotted over time relative to the baseline low-glucose condition (2.8 mM). The trace reveals marked increases in insulin secretion upon high-glucose challenge (16.7 mM) and an additional pronounced response following KCI-induced depolarisation, with secretion returning to basal levels during low-glucose washout phases.

[0074] FIG.3 shows dynamic glucose-stimulated insulin-secretion (GSIS) profiles of stem cell-derived islet spheroids (SC-islets) generated from multiple hiPSC lines (including i70b, 1173b and 43e5). Insulin secretion, normalised to basal secretion at 2.8 mM glucose, is plotted over time during sequential perifusion. Each pattern and line represents an independent differentiation batch or hiPSC line, demonstrating the temporal kinetics and magnitude of insulin release under each stimulus. Each pattern and line corresponds to an independent in vitro differentiation set-up paired with a dynamic GSIS experiment.

[0075] FIG. 4 shows total insulin content normalised to total genomic DNA for primary human islets and for stem cell-derived islets (SC-islets) generated from multiple hiPSC lines (43e5, i70b, i173b) as well as a reference hESC line (H9). Bars represent mean insulin content, with individual data points shown that have been normalised to genomic DNA content for each preparation. The SC-islets display insulin content values within a range comparable to those measured in cadaveric human islets. Each pattern corresponds to islets from different sources, human islets, hiPSC-derived islets, or hESC-derived islets, paired with total insulin content data.

[0076] FIG. 5A-5F shows fluorescence microscopy images displaying the expression of key islet markers with top images being 10x magnification and the bottom image being 20x magnification: FIG.5A C-Peptide (processed form of insulin in green) and beta cell transcription factor PDX1 ; FIG. 5B insulin (in green) and beta cell transcription factor NKX6.1 ; FIG.5C C-Peptide (processed form of insulin in green) and glucagon (alpha cell hormone in red) in the hiPSC-derived islet spheroids; FIG.5D shows representative flow cytometric data depicting the expression of beta cell marker insulin (INS) alongside the alpha cell marker glucagon (GCG), and other beta cell markers PDX1 and NKX6.1 , from two independent differentiations; FIG. 5E shows UMAP visualization of single-cell RNA sequencing data from day 35 hiPSC-derived islets reveals distinct clustering of major islet and associated cell types,including acinar, alpha (a), beta ( ), bihormonal (a / P), delta (5), ductal, enterochromaffin, endothelial, epsilon, mesenchymal-like cells and progenitor-like unassigned cell types; and FIG. 5F shows UMAP projections of single-cell RNA sequencing data from day 35 hiPSC-derived islets and expression of key marker genes used to annotate cell populations. INS identifies cells, GCG a cells, SST 6 cells, PPY y (PP) cells, PRSS1 pancreatic acinar cells and KRT19 ductal cells. Each dot represents a single cell, colored by relative gene expression level (light to dark), overlaid on the same UMAP embedding.

[0077] FIG.6A shows blood glucose profiles obtained during an intraperitoneal glucose tolerance test (IPGTT) performed in streptozotocin (STZ)-induced diabetic mice, diabetic mice transplanted with hiPSC-derived islet spheroids (SC-islets), and healthy control mice. Blood glucose concentrations (mmol / L) were measured over a 120-minute period following glucose injection. Healthy control mice exhibit rapid glucose clearance, whereas untreated STZ-diabetic mice show sustained hyperglycaemia. Mice transplanted with SC-islets display an intermediate response, with enhanced glucose lowering compared to untreated STZ mice; and FIG.6B shows the area under the curve (AUC) quantification for glucose levels during IPGTT in the same three groups: healthy control mice, STZ-diabetic mice, and SC-islet-transplanted mice. The AUC measurements show markedly hyperglycaemic levels in STZ-diabetic mice, whereas SC-islet-transplanted mice exhibit significantly reduced AUC values, approaching those of healthy controls, suggesting that hyperglycaemia is more quickly resolved in SC-islet-transplanted mice.

[0078] FIG. 7A shows serum concentrations of human insulin measured in STZ-diabetic mice transplanted with SC-islets during an intraperitoneal glucose tolerance test (IPGTT); FIG. 7B and 7C shows serum concentrations of human c-peptide measured in STZ-diabetic mice transplanted with SC-islets during an IPGTT at 12 weeks and 20 weeks post-transplantation; and FIG. 7D shows weekly serum C-peptide concentrations measured in non-fasted STZ-diabetic mice transplanted with SC-islets over a monitoring period of approximately two months post-transplantation. Each pattern represents human insulin concentration measured during in vivo GSIS in diabetic mice transplanted with hiPSC-derived islets.

[0079] FIG. 8 shows fluorescence microscopy images of explanted SC-islet grafts retrieved eight weeks after transplantation into STZ-diabetic mice. The grafts were stained for human insulin (INS) or C-peptide (C-PEP), |3-cell transcription factors PDX1 or NKX6.1 , glucagon (GCG), and nuclear marker DAPI. The upper panels show lower-magnification images, and the corresponding lower panels show higher-magnification views of the highlighted regions. The images demonstrate the presence of insulin / C-peptide-positive p-like cells, p-cell transcription factor-positive cells, and glucagon-positive a-like cells within the explanted grafts.DEFINITIONS

[0080] As used herein, pluripotent stem cells (PSCs) refer to undifferentiated cells defined by their ability, at the single cell level, to both self-renew and differentiate. Stem cells may produce progenycells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells may be characterized by their ability to differentiate into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm, and ectoderm).

[0081] As used herein, human induced pluripotent stem cells (hiPSCs) refer to pluripotent stem cells generated by reprogramming differentiated or somatic human cells to an embryonic-stem-cell-like state through the ectopic expression of defined reprogramming factors, such as OCT4, SOX2, KLF4, and c-MYC, or functional equivalents, variants, or subsets thereof. hiPSCs exhibit the defining properties of pluripotent stem cells, including long-term self-renewal and the ability to differentiate into cell types of all three germ layers (endoderm, mesoderm, and ectoderm). hiPSCs typically display ESC-like morphology and express hallmark pluripotency markers such as OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81 , SSEA-3, and SSEA-4, together with reduced or absent expression of lineage-specific differentiation markers. hiPSCs further exhibit epigenetic features characteristic of a pluripotent state, including reactivation of the pluripotency network, X-chromosome reactivation in female lines, and global transcriptional profiles resembling those of hESCs. When assessed in vivo, hiPSCs retain the capacity to form teratomas comprising derivatives of all three germ layers, confirming their pluripotent identity.

[0082] As used herein, human embryonic stem cells (hESCs) refer to pluripotent stem cells derived from the inner cell mass of a human blastocyst-stage embryo. hESCs are characterised by their ability to self-renew indefinitely in vitro under appropriate culture conditions while maintaining an undifferentiated state and by their inherent capacity to differentiate into functional cell types representative of the endoderm, mesoderm, and ectoderm germ layers. hESCs typically express a characteristic set of pluripotency-associated markers, including OCT4, SOX2, NANOG, TRA-1-60, TRA-1-81 , SSEA-3, and SSEA-4, and exhibit morphological, epigenetic, and transcriptomic features consistent with a pluripotent identity. hESCs also demonstrate the ability to form teratomas containing multiple differentiated tissue types when introduced into permissive in vivo environments, thereby functionally validating their pluripotent state.

[0083] As used herein, the term “differentiation” refers to the process by which an unspecialized (“uncommitted”) or less specialised cell acquires the features of a specialised cell, for example, a p islet cell. A differentiated cell is one that has taken on a more specialised (“committed”) position within the lineage of a cell. The term “committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.

[0084] As used herein, the term “growth factor” refers to a polypeptide, cytokine, or extracellular signalling ligand that binds to a cognate cell-surface receptor to regulate cell proliferation, survival, patterning, or lineage specification. Growth factors may include members of the TGF-p superfamily,fibroblast growth factors (FGFs), epidermal growth factor (EGF) family ligands, insulin-like growth factors, hepatocyte growth factors, or other developmentally relevant signalling proteins. Non-limiting examples of growth factors useful in the present differentiation methods include Activin A, a TGF-p superfamily ligand functioning as an Activin / Nodal pathway agonist for definitive endoderm induction; FGF7 (Keratinocyte Growth Factor, KGF), a fibroblast growth factor that promotes foregut and early pancreatic lineage specification; and Betacellulin, an EGF-family ligand that enhances endocrine progenitor formation and p-cell maturation. Additional representative growth factors that may be employed include other TGF-p family ligands (e.g., TGF-pi , BMP proteins, Nodal, GDF proteins), other FGFs (e.g., FGF2, FGF7, FGF10), EGF or HB-EGF, IGF-I or IGF-II, Wnt ligands (e.g., Wnt3a), neuregulins, VEGF, PDGF, and HGF. As used herein, the term “TGF-p family ligand” encompasses any member of the Transforming Growth Factor-p superfamily of secreted polypeptides, including, without limitation, TGF-p isoforms (e.g., TGF-p1 , TGF-P2, TGF-P3), activins (e.g., Activin A, Activin B, Activin AB), inhibins (e.g., Inhibin A, Inhibin B), nodal and nodal-related proteins (e.g., Nodal, GDF1 , GDF3), bone morphogenetic proteins (e.g., BMP2, BMP4, BMP5, BMP6, BMP7, BMP8a / b, BMP9, BMP10, BMP11 / GDF11 , BMP12 / GDF7, BMP13 / GDF6, BMP14 / GDF5, BMP15), growth and differentiation factors (e.g., GDF8 / myostatin, GDF9, GDF10), anti-Mullerian hormone (AMH / MIS), and Lefty proteins (Leftyl , Lefty2), or functional equivalents or variants thereof.

[0085] As used herein, the term “small-molecule differentiation agent” refers to a synthetic or naturally occurring organic compound, typically having a molecular weight below approximately 1 kDa, that modulates an intracellular signalling pathway, transcriptional program, or cellular metabolic state to direct lineage specification, patterning, or maturation. Such compounds include agonists or inhibitors of Wnt, Hedgehog, Notch, TGF-p, BMP, PKC, tyrosine kinase, redox, or metabolic pathways, among others. Small-molecule differentiation agents further encompass retinoids such as all-trans retinoic acid (RA), which act through RAR / RXR receptors to regulate developmental gene expression, as well as metabolic cofactors such as nicotinamide, which influence cellular metabolism and support endocrine lineage commitment.

[0086] As used herein, the term “Wnt pathway activator” refers to any polypeptide ligand, recombinant protein, or small-molecule compound capable of activating or potentiating canonical Wnt / p-catenin signalling. Non-limiting examples include recombinant Wnt ligands (e.g., Wnt3a, Wnt1 , Wnt2, Wnt7a, Wnt8a, Wnt10b), R-spondin family proteins (R-spondin 1-4), GSK3P inhibitors (e.g., CHIR99021 , BIO, LiCI, SB-216763, SB-415286, tideglusib), tankyrase inhibitors (e.g., XAV939, IWR-1 , JW55, G007-LK), and other p-catenin-stabilizing small molecules such as ISX9, Purvalanol A, QS11 , and LY2090314, or functional analogues or derivatives thereof. The Wnt pathway activator comprises a small-molecule NEUROD1 enhancer, such as ISX9, that promotes endocrine lineage specification and maturation.

[0087] As used herein, a “protein kinase C activator” refers to any compound that activates one or more PKC isoforms to modulate intracellular signalling and differentiation. Suitable PKC activators include phorbol esters such as phorbol 12, 13-dibutyrate (PDBu), phorbol 12-myristate 13-acetate(PMA), phorbol 12, 13-dibutyrate, and ingenol derivatives. Additional examples include diacylglycerol (DAG) analogues such as OAG (oleoyl -acetyl-g lycerol) or synthetic PKC agonists.

[0088] As used herein, the term “metabolic cofactor” refers to any molecule that participates in or regulates cellular metabolic pathways, redox balance, NAD+metabolism, or energy homeostasis. In certain embodiments, the metabolic cofactor comprises nicotinamide, a vitamin B3 derivative used to promote endocrine lineage progression. Other suitable metabolic cofactors include nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), NAD+precursors, pyruvate, coenzyme A derivatives, glutathione, and vitamin B derivatives (e.g., riboflavin or thiamine).

[0089] As used herein, the term “retinoid” refers to natural or synthetic derivatives of vitamin A that activate RAR or RXR receptors and regulate developmental gene expression. Suitable retinoids include all-trans retinoic acid (RA), 9-cis-retinoic acid, 13-cis-retinoic acid, retinaldehyde, and synthetic retinoid analogues such as bexarotene, adapalene, tazarotene, TTNPB, and other RAR / RXR agonists.

[0090] As used herein, a “Hedgehog pathway antagonist” refers to any compound that inhibits Hedgehog signalling, including inhibitors of Smoothened (SMO), Patched, or downstream Gli transcription factors. Suitable antagonists include SANT1 , KAAD-cyclopamine, vismodegib, sonidegib, glasdegib, cyclopamine, GANT61 , and other SMO or Gli inhibitors.

[0091] As used herein, the term “Notch pathway inhibitor” refers to any agent that inhibits Notch receptor activation, ligand binding, y-secretase activity, or downstream transcriptional responses. Suitable inhibitors include y-secretase inhibitors such as Compound XXI (XXI), DAPT, LY411575, RO4929097, MK-0752, and antibodies targeting Notch receptors (Notch1-4) or ligands (e.g., Delta-like or Jagged family proteins).

[0092] As used herein, a “BMP pathway inhibitor” refers to any molecule that inhibits BMP receptor kinases (ALK2, ALK3, ALK6), BMP ligands, or downstream SMAD1 / 5 / 8 phosphorylation. Suitable inhibitors include LDN193189, dorsomorphin, DMH1 , K02288, ML347, Noggin, Gremlin, or soluble BMP receptor fragments that act as ligand traps.

[0093] As used herein, a “TGF-p receptor inhibitor” refers to any compound that inhibits signalling through TGF-p type I or type II receptors, including ALK4, ALK5, and ALK7. Suitable inhibitors include ALK5III, SB-431542, SB-505124, A-83-01 , LY2157299 (galunisertib), SD-208, and other selective ALK5 antagonists or kinase inhibitors that block SMAD2 / 3 signalling.

[0094] As used herein, the term “thyroid hormone” refers to naturally occurring or synthetic derivatives of thyroxine (T4) or triiodothyronine (T3) that regulate metabolic and developmental pathways. Suitable thyroid hormones include triiodothyronine (T3), levothyroxine (T4), liothyronine, and synthetic thyroid receptor agonists such as GC-1 (sobetirome) or KB2115.

[0095] As used herein, a “redox-modulating agent” refers to any compound that regulates oxidative stress, ROS levels, antioxidant pathways, or cellular redox status. Suitable redox-modulating agents include N-acetyl cysteine (NAG), glutathione, lipoic acid, ascorbic acid (vitamin C), edaravone, thiol antioxidants, Trolox, and mitochondrial-targeted antioxidants such as MitoQ.

[0096] As used herein, the term “receptor tyrosine kinase inhibitor” refers to any molecule that inhibits receptor tyrosine kinase (RTK) signalling, including inhibitors of AXL, MER, TYRO3, or related kinases. In certain embodiments, the RTK inhibitor comprises an AXL inhibitor, such as R428 (bemcentinib). Additional AXL inhibitors include BGB324, foretinib, cabozantinib, bosutinib, UNC2025, and other TAM-family kinase inhibitors.

[0097] As used herein, the term “nutrient supplement” refers to any metabolically utilizable molecule added to a culture medium to support cellular energy production, biosynthetic activity, or metabolic maturation. Nutrient supplements include, without limitation, carbohydrates such as glucose (e.g., D-glucose), galactose, or mannose; metabolic intermediates such as pyruvate; amino acids; and other carbon sources that contribute to ATP generation or metabolic flux. In certain embodiments, the nutrient supplement comprises glucose, which supports oxidative metabolism and promotes functional maturation of endocrine lineage cells, including islet-like cells. Nutrient supplements may be used individually or in combination to optimise metabolic conditions for differentiation or maturation.

[0098] As used herein, the term “ionic supplement” refers to any inorganic ion or salt added to a culture medium to support ionic balance, membrane potential, enzymatic cofactor activity, hormone processing, or structural stability of differentiated cells. Ionic supplements include, without limitation, zinc salts such as zinc sulfate (ZnS04), zinc chloride, zinc acetate, or zinc citrate, as well as other metal ions including magnesium chloride, calcium chloride, potassium chloride, and phosphate or bicarbonate salts. In certain embodiments, the ionic supplement comprises zinc sulfate, which contributes to insulin maturation, crystallisation, and storage within p-like cells. Ionic supplements may be included individually or in combination to maintain optimal ionic homeostasis during endocrine differentiation.

[0099] As used herein, the term “markers” may refer to nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest so that they can be used to indicate a certain state (such as a developmental stage), a characteristic property and / or identity of the cell. In this context, differential expression means an increased level for a positive marker and a decreased level for a negative marker as compared to an undifferentiated cell or a cell at another stage of differentiation. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.

[0100] As used herein, a cell that is “positive” or “+” for a specific marker, may refer to the specific marker being beyond detection limit and sufficiently significant in the cell. A proper detection limit can be determined by one skilled in the art depending on the testing method. In particular, positive by flow cytometry ( “FC” ) is usually greater than about 2%. Positive by polymerase chain reaction cytometry ( “PCR” ) is usually less than or equal to about 35 cycles (Cts) .

[0101] As used herein, “suspension culture” refers to a culture of cells, single cells or clusters, suspended in medium rather than adhering to a surface in contrast to adherent culture, such as planar culture. One or more culturing stages of the present method can comprise suspension culture or planar culture. The culture stages are described in more detail in the following section and examples.

[0102] As used herein, “cells expressing markers characteristic of the definitive endoderm” refers broadly to cells that exhibit one or more molecular markers associated with definitive endoderm identity, irrespective of whether the entire population has fully committed to that lineage. “Definitive endoderm cells” refer to cells that have undergone definitive endoderm specification and display a characteristic combination of lineage-defining markers, such as co-expression of SOX17 and FOXA2 with loss of pluripotency markers.

[0103] As used herein, “cells expressing markers characteristic of the primitive gut tube” refers broadly to cells that exhibit one or more molecular markers associated with early foregut or primitive gut tube identity, irrespective of whether the entire population has fully committed to that lineage. “Primitive gut tube cells” referto cells that have undergone primitive gut tube specification and display a characteristic combination of lineage-defining markers, such as expression of HNF1p and HNF4a in combination with continued FOXA2 expression and decreased CXCR4 levels relative to definitive endoderm. Accordingly, cells expressing markers characteristic of the primitive gut tube, or primitive gut tube cells, may be obtained at Stage 2.

[0104] As used herein, “cells expressing markers characteristic of pancreatic progenitor 1 cells” refers broadly to cells that exhibit one or more molecular markers associated with early pancreatic progenitor specification, irrespective of whether the entire population has fully committed to that lineage. “Pancreatic progenitor 1 cells” refers to cells that have undergone early pancreatic specification and display a characteristic combination of markers, such as expression of PDX1 together with continued FOXA2 and HNFip and low-to-moderate expression of SOX9 or TBX3. Accordingly, cells expressing markers characteristic of pancreatic progenitor 1 cells, or pancreatic progenitor 1 cells, may be obtained at Stage 3.

[0105] As used herein, “cells expressing markers characteristic of pancreatic progenitor 2 cells” refers broadly to cells that express one or more markers associated with a more advanced pancreatic progenitor state, regardless of whether the population is fully homogeneous. “Pancreatic progenitor 2 cells” refers to cells that exhibit a lineage-advanced marker profile, such as co-expression of PDX1 andNKX6.1 , optionally in combination with elevated SOX9 or HNF1 and reduced expression of early endodermal markers such as CXCR4. Accordingly, cells expressing markers characteristic of pancreatic progenitor 2 cells, or pancreatic progenitor 2 cells, may be obtained at Stage 4.

[0106] As used herein, “cells expressing markers characteristic of pancreatic endocrine progenitor cells” refers broadly to cells that express one or more markers indicative of endocrine lineage commitment, even if the population contains a mixture of states. “Pancreatic endocrine progenitor cells” refers to cells that have undergone endocrine lineage specification and display a characteristic combination of markers, including expression of NEUROG3 (NGN3) as a defining transcription factor, optionally together with PAX4, PAX6, NKX2.2, or ISL1. Accordingly, cells expressing markers characteristic of pancreatic endocrine progenitor cells, or pancreatic endocrine progenitor cells, may be obtained at Stage 5.

[0107] As used herein, “cells expressing markers characteristic of functional islet-like cells” refers broadly to cells that express one or more markers associated with mature or maturing pancreatic endocrine function, including insulin production, glucose responsiveness, or hormone secretion. “Functional islet-like cells” refer to cells that exhibit a mature endocrine phenotype and express a characteristic combination of markers such as INS (insulin), PCSK1 / 2 (prohormone convertases), MAFA, PDX1 , and NKX6.1 , optionally with reduced or absent expression of progenitor-associated genes (e.g., NGN3). Such cells may additionally secrete insulin or C-peptide in response to glucose stimulation. Accordingly, cells expressing markers characteristic of functional islet-like cells, or functional islet-like cells, may be obtained at Stage 6. “hPSC-islets” in this context refers to islets including C-peptide positive cells, glucagon positive cells and somatostatin positive cells, which were derived from human pluripotent stem cells, by the present method

[0108] As used herein, the term “treat” , “treating” or “treatment” means the control, reversal or cure of a disease or condition, or one or more symptoms or complications thereof in a subject, including alleviation of a symptom or complication, delay in progression of a disease or condition, or complete cure of a disease or condition. In some embodiments, the term “treat” , “treating” or “treatment” includes those for prophylactic purpose which are administered before the onset or development of a disease or condition, or one or more symptoms or complications thereof. An effective treatment can be determined by measuring physiologic parameters, observing morphology or by any means known in the art or developed in the future for the same purpose.

[0109] As used herein, the term “diabetes” refers to a syndrome that can be characterized by disordered metabolism resulting in abnormally high blood glucose levels (hyperglycemia) . The two most common forms of diabetes are due to either a diminished production of insulin (in Type 1) , or diminished response by the body to insulin (in Type 2 and gestational) . Type 1 diabetes (Type 1 diabetes, Type I diabetes mellitus (T1 DM) , Insulin dependent diabetes mellitus (IDDM) , juvenile diabetes) is a disease that results in the permanent destruction of insulin-producing beta cells of thepancreas. Type 2 diabetes (non-insulin-dependent diabetes mellitus (NIDDM) , or adult-onset diabetes) is a metabolic disorder that is primarily characterized by insulin resistance (diminished response by the body to insulin) , relative insulin deficiency, and hyperglycemia. Complications associated with diabetes include, but are not limited to hypoglycaemia, ketoacidosis, or nonketotic hyperosmolar coma, cardiovascular disease, renal failure, retinal damage, nerve damage, and microvascular damage. In some embodiments, a mammal is pre-diabetic, which can be characterized, for example, as having elevated fasting blood glucose or elevated post-prandial blood glucose.

[0110] As used herein, a “therapeutically effective amount” refers to an amount of islet-like cells sufficient to confer a measurable therapeutic benefit, including reduction of blood glucose levels, improvement of glucose tolerance, restoration of insulin production, or reduction of exogenous insulin requirements.DETAILED DESCRIPTION

[0111] The following detailed description refers to, by way of illustration, specific details and embodiments in which the invention may be practised. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. Other embodiments may be utilised and structural and logical changes may be made without departing from the scope of the invention. Embodiments described below in the context of the methods are analogously valid for the respective cells, cell populations, compositions, uses, kits, and vice versa. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0112] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprises" means "includes." In case of conflict, the present specification, including explanations of terms, will prevail. “About”, as used herein in connection with numerical values refers to the referenced numerical value ±10% or ±5%.

[0113] The present inventors designed a multi-stage directed differentiation protocol to differentiate hiPSCs and hESCs into islet-like cells (hiPSC-islets) that are capable of glucose-stimulated insulin secretion (GSIS) in vitro using a suspension culture system. The islet-like cells comprise of insulinsecreting beta cells as well as other endocrine cell types. In particular, the method described herein is capable of differentiating hiPSCs or hESCs into early, intermediate and mature developmental stages that mimic specific stages of human pancreatic beta cell development. The differentiation results in islet-like cells that comprise of insulin-secreting beta cells as well as other endocrine cell types such as glucagon-expressing alpha cells. The method described herein yield 60-80% of insulin-positive cells, as determined by flow cytometry. The method described herein also results in achieving high peakinsulin secretion (e.g., 8-30-fold). Further, the method described herein also yields a higher amount of insulin, when normalized to total gDNA, relative to human primary islets. As such, the methods described herein advantageously improve cost-efficiencies for making insulin using a suspension culture system, which ultimately reduces cost per dose.

[0114] Accordingly, in one aspect, provided herein is a method of generating insulin-secreting pancreatic islet-like cells, also referred to herein as stem cell-derived islets.

[0115] In various embodiments, the method comprises a staged differentiation protocol, or series of differentiation steps, that sequentially directs stem cells through definitive endoderm, primitive gut tube, early pancreatic progenitor, late pancreatic progenitor, endocrine progenitor, and islet-like cell fates. The staged approach recapitulates developmental signalling events using defined media comprising combinations of growth factors, small molecules, hormones, and signalling pathway modulators.

[0116] In various embodiments, the method is an in vitro method.

[0117] In various embodiments, each differentiation step comprises contacting (i.e. incubating) the cells with a stage-specific culture medium under conditions and for a period of time sufficient to induce transition to the desired lineage. Thus, differentiating the cells may comprise exposing cells, or spheroid, to the corresponding stage-specific medium(s) comprising one or more growth factors, small-molecule differentiation agents, nutrient and / or ionic supplements as described herein, and incubating the cells under suitable temperature, and culture conditions for a defined duration sufficient to obtain cells expressing markers characteristic of the definitive endoderm, primitive gut tube cells, pancreatic progenitor 1 cells, pancreatic progenitor 2 cells, pancreatic endocrine progenitor cells, or functional isletlike cells, respectively. In various embodiments, each differentiation stage may further comprise one or more medium changes, washing steps to reduce carry-over of factors from the previous stage, and optional supplementation or renewal of one or more agents during the culture period, so as to maintain the desired signalling environment and promote efficient progression to the targeted cell population.

[0118] In various embodiments, the islet-like cells comprise or consist essentially of p-like cells, which are cells that display key features of pancreatic p-cells, including expression of p-cell-associated transcription factors and hormones such as insulin (INS), NKX6.1 , PDX1 , MAFA, and PCSK1 / 2, and the capacity to process proinsulin and secrete insulin or C-peptide in response to glucose stimulation, p-like cells may further exhibit functional properties characteristic of mature p-cells, including glucose-stimulated insulin secretion (GSIS), zinc-dependent insulin crystallisation, appropriate redox and metabolic responses. In various embodiments, the islet-like cell population may additionally contain non-p endocrine cell types (e.g., a-, 5-, or PP-like cells), but is enriched for or predominantly composed of p-like cells that collectively confer physiological insulin secretory responses comparable to native human islets.

[0119] In various embodiments, the hiPSCs are Asian donor-derived hiPSCs selected from the group consisting of i70b, 1173b, is104, 43e5 and is043.

[0120] The differentiation of hiPSCs or hESCs into functional islet-like cells in vitro cell cultures, may comprises a number of consecutive multiple-stages. In particular, in this step-wise differentiation, “Stage 1” or “Step (1) ” may refer to the first step in the differentiation process, the differentiation of hiPSCs or hESCs into definitive endoderm cells. “Stage 2” or “Step (2) ” refers to the second step, the differentiation of the definitive endoderm cells into primitive gut tube cells. “Stage 3” or “Step (3) ’’ refers to the third step, differentiation of primitive gut tube cells into progenitor 1 cells. “Stage 4” or “Step (4) ” refers to the fourth step, differentiation of progenitor 1 cells into progenitor 2 cells. “Stage 5” or “Step (5) ” refers to the fifth step, differentiation of progenitor 2 cells into pancreatic endocrine progenitor cells. “Stage 6” or “Step (6) ” refers to the sixth step, the differentiation of pancreatic endocrine progenitor cells into islet-like cells.

[0121] In various embodiments, the method may comprise the following series of stages (or steps), differentiating hiPSCs or hESCs to obtain definitive endoderm cells or cells expressing markers characteristic of the definitive endoderm;differentiating the cells obtained from stage (1) to obtain primitive gut tube cells, or cells expressing markers characteristic of primitive gut tube cells;differentiating the cells obtained from stage (2) to obtain pancreatic progenitor 1 cells, or cells expressing markers characteristic of pancreatic progenitor 1 cells;differentiating the cells obtained from stage (3) to obtain pancreatic progenitor 2 cells, or cells expressing markers characteristic of pancreatic progenitor 2 cells;differentiating the cells obtained from stage (4) to obtain pancreatic endocrine progenitor cells, or cells expressing markers characteristic of pancreatic endocrine progenitor cells; differentiating the cells obtained from stage (5) to obtain functional islet-like cells, or cells expressing markers characteristic of functional islet-like cells.

[0122] In various embodiments, the entire differentiation method and stages are performed in a suspension culture system. In various embodiments, the method disclosed herein uses stage-specific media formulations such that each stage is performed in one or more culture medium. Thus, the method may comprise the following series of stages (or steps) performed in a series of culture media,(1 ) differentiating hiPSCs or hESCs in a first culture medium under conditions and for a period of time sufficient to obtain definitive endoderm cells or cells expressing markers characteristic of the definitive endoderm;(2) differentiating the cells obtained from stage (1 ) in a second culture medium under conditions and for a period of time sufficient to obtain primitive gut tube cells, or cells expressing markers characteristic of primitive gut tube cells;(3) differentiating the cells obtained from stage (2) in a third culture medium under conditions and for a period of time sufficient to obtain pancreatic progenitor 1 cells, or cells expressing markers characteristic of pancreatic progenitor 1 cells;(4) differentiating the cells obtained from stage (3) in a fourth culture medium under conditions and for a period of time sufficient to obtain pancreatic progenitor 2 cells, or cells expressing markers characteristic of pancreatic progenitor 2 cells;(5) differentiating the cells obtained from stage (4) in a fifth culture medium under conditions and for a period of time sufficient to obtain pancreatic endocrine progenitor cells, or cells expressing markers characteristic of pancreatic endocrine progenitor cells;(6) differentiating the cells obtained from stage (5) in a sixth culture medium under conditions and for a period of time sufficient to obtain functional islet-like cells, or cells expressing markers characteristic of functional islet-like cells.

[0123] It will be appreciated that the mediums used for each of the stages may also be termed as stage 1 culture medium, stage 2 culture medium, stage 3 culture medium, stage 4 culture medium, stage 5 culture medium, and stage 6 culture medium, corresponding respectively to the first culture medium, second culture medium, third culture medium, fourth culture medium, fifth culture medium, and sixth culture medium.

[0124] In various embodiments, the method may begin with providing a population of cells that are already committed to a defined intermediate lineage stage, rather than starting from undifferentiated hiPSCs or hESCs. Thus, in various embodiments, the method comprises providing a population of definitive endoderm cells derived from hiPSCs or hESCs, and subjecting the definitive endoderm cells to the corresponding differentiation stage (i.e. stage 2) and subsequent differentiation stages as described herein to obtain functional islet-like cells.

[0125] In various embodiments, the method may comprise providing definitive endoderm cells, primitive gut tube cells, pancreatic progenitor 1 (PP1 ) cells, pancreatic progenitor 2 (PP2) cells, or pancreatic endocrine progenitor (EP) cells and carrying out only the downstream stages appropriate for maturation into functional islet-like cells.

[0126] Accordingly, the method for generating insulin-secreting pancreatic islet-like cells may comprise providing definitive endoderm cells, primitive gut tube cells or pancreatic progenitor 1 cells, followed by performing differentiation stages of (2)-(6), (3)-(6), or (4)-(6), respectively. For example, the method may comprise providing pancreatic progenitor 1 , followed by performing stage (4)-(6) differentiation steps as disclosed herein, whereby the initial differentiation stages (1)-(3) are not essential in generating functional islet-like cells from the provided PP1 cells.

[0127] In various embodiments, the method comprises providing a population of pancreatic progenitor 1 (PP1) cells derived from hiPSCs or hESCs, and subjecting the PP1 cells to the corresponding differentiation stage (i.e. stage 4) and subsequent differentiation stages as described herein to obtain islet-like cells.

[0128] In various embodiments, the mediums used for the stages may comprise a basal medium. The term “basal medium” as described herein refers to a composition providing the nutrients such as amino acids, vitamins, carbohydrates and salts which support the survival and growth of cells. In various embodiments, supplements are added to the basal medium to provide cells with compounds which are essential for their growth and / or differentiation. Exemplary basal medium suitable for culturing mammalian stem cells is known in the art, including but not limited to Dulbecco’s Modified Eagle Media (DMEM) or DMEM-derived media (e.g. DMEM basic, DMEM / F12, Knockout-DMEM (KO-DMEM), MCDB131 , RPMI 1640, CMRL1066 or the like). In various embodiments, the basal medium may comprise MCDB131 or CMRL1066.

[0129] To induce differentiation of cells, one or more differentiating agents may be included in the medium depending on the culture stage. The differentiating agent as described herein refers to any agent that facilitates the development from hiPSC or hESC, or other pancreatic cell lineages (i.e. PP1 ) towards functional islet-like cells. A differentiating agent can function by improving or increasing the generation or growth of a desired cell type, and / or inhibiting or decreasing the generation or growth of one or more undesired cell types, by known or unknown mechanisms. FIG. 1 shows a specific nonlimiting, embodiment of a preferred combination of differentiating agents that maybe used in each stage of the method of the present invention.

[0130] In various embodiments, the one or more differentiating agents may comprise growth factors and small-molecule differentiation agents, including any polypeptide ligand or organic compound capable of activating or inhibiting a signalling pathway relevant to the induction, patterning, or maturation of stem-cell-derived lineages. In various embodiments, the media used in each stage may comprise one or more (i) small molecule differentiating agents, (ii) growth factors, (iii) nutrient supplements, (iv) ionic supplements, and combinations thereof.

[0131] In various embodiments, the basal medium may comprise B27 supplements, FBS (fetal calf serum), BSA (bovine serum albumin), glucose, sodium bicarbonate, vitamin C, insulin-transferrin-selenium (ITS-X), penicillin / streptomycin, non-essential amino acids (NEAA), sodium pyruvate, Zinc Sulphate, and / or GlutaMAX. In various embodiments, the basal medium of any stage in the method disclosed herein may comprise BSA. In various embodiments, the basal medium of all stages in the method disclosed herein comprises BSA. The BSA encompasses any suitable form of albumin derived from bovine serum or produced recombinantly, including, but not limited to, fatty-acid-free BSA, Fraction-V BSA, low-endotoxin BSA, cell-culture-grade BSA, protease-free BSA, immunoglobulin-depleted BSA, or recombinant BSA or human serum albumin analogues thereof. In various embodiments, the BSA may be added to the medium in an amount of 0.01 -10%, preferably 0.5-5%, more preferably 0.5-3%, most preferably 1 -2%. As used herein, the percentage of BSA refers to a weight / volume (w / v) concentration, meaning grams of BSA per 100 mL of culture medium (e.g., 1% = 10 mg / mL; 2% = 20 mg / mL).

[0132] In various embodiments, the BSA may be a fatty-acid-free BSA (FAF BSA). In various embodiments, the FAF BSA is in an amount of about 1-2%. In various embodiments, the FAF BSA is in an amount of about 2%.

[0133] One skilled in the art would also understand that the use of a certain type of product is not mandatory in the present application. Any basal medium or nutritional supplements can be substituted with a functional alternative.

[0134] In various embodiments, each staged differentiation occurs in a complete suspension culture system for spheroids (i.e. 3D clusters of cells). Accordingly, the cells after each differentiation step may be three-dimensional (3D) aggregates of cells formed under suspension or low-adhesion culture conditions, in which cells self-organise into compact, spherical clusters.

[0135] In the context of the present differentiation method, spheroids may comprise aggregates of human pluripotent stem cells (hiPSCs or hESCs), or lineage-committed derivatives thereof, that are maintained in a 3D configuration to promote uniform exposure to agents and enhance cell-cell and cellmatrix signalling during differentiation. Spheroid culture supports improved viability, developmental patterning, and maturation compared with two-dimensional monolayer culture, and enables the generation of pancreatic progenitors and functional islet-like cells with structural and physiological properties more closely resembling native islets. In various embodiments, spheroids maybe formed by seeding pluripotent stem cells at a defined density under orbital shaking, stirred-suspension, or nonadherent culture conditions, and are maintained throughout one or more stages of differentiation as described herein.

[0136] In various embodiments, the method disclosed herein may include an initial step prior to stage 1 that prepares and provides the hiPSCs or hESCs to be differentiated and incubated in stage 1. In various embodiments, the method comprises a step, before stage 1 , of providing the hiPSCs or hESCs. In various embodiments, the providing step may comprise dissociating the hiPSCs or hESCs into single cells and culturing them as spheroids in a suspension culture system. The hiPSCs or hESCs may be dissociated into single cells and seeded into low-adhesion or bioreactor culture vessels to form the spheroids. In various embodiments, the single cells are incubated at a density of 0.6-1 .2 x 106cells per ml_ for 1-5 days under orbital rotation at a speed of 70-90 rpm. This will allow aggregation and establishment of stable spheroid structures suitable for differentiation in stage 1 .

[0137] In various embodiments, the dissociation of hiPSCs or hESCs into single cells and seeding a density may be carried out in a pluripotent stem cell maintenance (for example, mTeSR or an equivalent medium). The total cell number may range from 3-4 x 106cells per well of a 6-well plate. Cells may be cultured for 1 -2 days as spheroids.

[0138] In various embodiments, Stage 1 differentiation maybe initiated using pre-formed spheroids of hiPSCs or hESCs that have been cultured in a pluripotent stem cell maintenance medium (for example, mTeSR or an equivalent medium) under three-dimensional suspension conditions under orbital rotation at a speed of 70-90 rpm. Following spheroid formation, the maintenance medium may be removed and replaced with a Stage 1 differentiation medium.

[0139] In various embodiments, the method disclosed herein comprises stepwise differentiation into early (e.g., definitive endoderm and primitive gut tube), intermediate (e.g., pancreatic progenitors and endocrine progenitors) and mature developmental stages that mimic specific stages of human beta cell development, thereby generating glucose-responsive, insulin-secreting pancreatic islet-like cells from hiPSC or hESCs spheroids, or cells that are already committed to a defined intermediate lineage stage (e.g., PP1), in a complete suspension culture system.o Stage 1

[0140] In stage 1 , the provided hiPSCs or hESCs are incubated in a stage 1 (first) culture medium under suitable conditions to initiate differentiation of hiPSCs or hESCs to definitive endoderm cells.

[0141] In various embodiments, the stage 1 culture medium comprises a basal medium supplemented with one or more differentiating agents selected from growth factors and Wnt pathway activators.

[0142] In various embodiments, the stage 1 culture medium comprises a basal medium supplemented with BSA, preferably FAF BSA. In various embodiments, the FAF BSA is in an amount of about 1-2%, preferably 1%.

[0143] In various embodiments, the stage 1 culture medium may comprise one or more TGF-p family ligands. In various embodiments, the one or more TGF-0 family ligand may be in an amount of about 10-200 ng / ml, preferably about 50-150 ng / ml, more preferably about 80-120 ng / ml. In various embodiments, the one or more TGF-p family ligand may be in an amount of about 100 ng / ml.

[0144] In various embodiments, the stage 1 culture medium may comprise Activin A. In various embodiments, the Activin A may be in an amount of about 80-120 ng / ml, preferably 90-100 ng / ml, more preferably about 100 ng / ml.

[0145] In various embodiments, the stage 1 culture medium may comprise one or more Wnt pathway activators, preferably small-molecule Wnt activators. In various embodiments, the one or more Wnt pathway activators may be in a concentration of about 0.1 -10 pM, 0.5-5 pM, 0.5-3pM, or 1-3 pM. In various embodiments, the one or more Wnt pathway activators may be in a concentration of about 1 pM.

[0146] In various embodiments, the stage 1 culture medium may comprise CHIR99021. In various embodiments, the CHIR99021 may be in a concentration of about 0.5-5 pM, preferably 0.5-2 pM, more preferably about 1 pM.

[0147] In various embodiments, the stage 1 culture medium comprises a basal medium supplemented with one or more TGF-p family ligands and one or more Wnt pathway activators. In various embodiments, the stage 1 culture medium comprises a basal medium supplemented with FAF BSA, a TGF-p family ligand and a Wnt pathway activator.

[0148] In various embodiments, the stage 1 culture medium comprises FAF BSA in an amount of about 1-2%, Activin A in an amount of about 90-110 ng / ml, and CHIR99021 at a concentration of about 1-3 pM. In various embodiments, the stage 1 culture medium comprises FAF BSA in an amount of about 1%, Activin A in an amount of about 100 ng / ml, and CHIR99021 at a concentration of about 1 pM.

[0149] In various embodiments, the stage 1 differentiation in the stage 1 culture medium may be carried out for a period of about 12 hours to 2 days, preferably about 1 day.

[0150] In various embodiments, the stage 1 culture medium may be further supplemented by adding a TGF-p family ligand, at one or more time points during the period of the stage 1 differentiation. In various embodiments, the stage 1 culture medium may be further supplemented by adding Activin A at one or more time points during the period of the stage 1 differentiation.

[0151] In various embodiments, stage 1 may further comprise an additional culture step where the stage 1 culture medium (1a) is exchanged for a new stage 1 culture medium (1b) in a subsequent differentiation in stage 1 that follows the initial differentiation stage 1 . The stage 1 culture medium (1a) may be termed as the first stage 1 culture medium and the new stage 1 culture medium (1 ) may be termed as the second stage 1 culture medium, for ease of distinguishing the two.

[0152] In various embodiments, the exchanged second stage 1 culture medium (1b) may comprise one or more TGF-p family ligands in an amount of about 10-200 ng / ml, preferably about 50-150 ng / ml, more preferably about 80-120 ng / ml. In various embodiments, the one or more TGF-p family ligand may be in an amount of about 100 ng / ml in the second stage 1 culture medium (1b).

[0153] In various embodiments, the exchanged second stage 1 culture medium (1b) may comprise FAF BSA in an amount of about 1-2% and Activin A in an amount of about 80-120 ng / ml, preferably about 100 ng / ml, in the complete or substantial absence of the Wnt pathway activator.

[0154] In various embodiments, the additional culture step of the hiPSC or hESC spheroids in this exchanged second stage 1 culture medium (1b) may be carried out for a period of about 1-3 days, preferably about 2 days.

[0155] In various embodiments, stage (1) further comprises a subsequent differentiating step comprising replacing the Stage 1 medium with a second Stage 1 medium comprising about 100 ng / ml Activin A, wherein the subsequent differentiating step is for a period of about 2 days.

[0156] Accordingly, in various embodiments, stage 1a may be carried out for a period of about 1 day and stage 1b may be carried out for a period of about 2 days, such that stage 1 in total maybe performed for a period of about 3 days.

[0157] In various embodiments, the culture conditions of the incubation of the hiPSC or hESC cells may be standard mammalian cell culture conditions known in the art, for example at about 37 °C in a humidified incubator with about 5% CO2, optionally with gentle agitation (e.g., orbital shaking at about 60-80 rpm or an equivalent suspension culture system) to maintain the spheroids in suspension and to promote uniform exposure to the medium components.

[0158] In various embodiments, the culturing stage 1 comprises incubating the hiPSC or hESC cells in the stage 1 culture medium for a period of time, as outlined above, under suitable temperature, gas, and agitation conditions, with a single medium change performed, for example, from 1a to 1 b. Under these conditions, the hiPSC or hESC spheroids are exposed to high levels of Activin / Nodal signalling, optionally in combination with transient Wnt pathway activation, to drive differentiation toward a definitive endoderm cell lineage.

[0159] During or following Stage 1 differentiation, the resulting cell population may comprise or consist essentially of cells exhibiting molecular and phenotypic characteristics of definitive endoderm. In various embodiments, such definitive endoderm cells may express one or more lineage-specific transcription factors, surface markers, or signalling molecules associated with endoderm specification. Non-limiting examples of definitive endoderm markers include SOX17, FOXA1 , FOXA2, GATA4, GATA6, CXCR4, and EPCAM, which may be detected at the protein or transcript level. Additional markers such as HNFip, HNF4a, and CER1 may also be upregulated in response to Activin / Nodal pathway stimulation. In various embodiments, definitive endoderm identity may be confirmed by co-expression of SOX17 and FOXA2, optionally in combination with high surface expression of CXCR4 and downregulation or loss of pluripotency markers such as OCT4 and NANOG. The proportion of cells expressing one or more of these markers may be used as an indicator of differentiation efficiency or progression into the primitive gut tube lineage.

[0160] Accordingly, cells expressing markers characteristic of the definitive endoderm, or definitive endoderm cells, may be obtained at stage 1 .

[0161] Upon completion of Stage 1 , the differentiated cells are advanced into Stage 2 by removing the Stage 1 medium and replacing it with the Stage 2 differentiation medium (i.e. second culture medium).In various embodiments, medium exchange may be performed by allowing the cells to settle or by gently collecting them by low-speed centrifugation, followed by aspiration of the spent medium and optional washing with basal medium to minimise carry-over of stage 1 components to stage 2.o Stage 2

[0162] In stage 2, the cells obtained from stage 1 are incubated in a stage 2 (second) culture medium under suitable conditions to initiate differentiation to primitive gut tube cells.

[0163] In various embodiments, the stage 2 culture medium comprises a basal medium supplemented with one or more growth factors.

[0164] In various embodiments, the stage 2 culture medium comprises a basal medium supplemented with one or more of glucose, sodium bicarbonate, vitamin C, insulin-transferrin selenium (ITS-X), penicillin-streptomycin and glutamax. In various embodiments, the stage 2 culture medium comprises a basal medium supplemented with BSA, preferably FAF BSA. In various embodiments, the FAF BSA is in an amount of about 1 -2%, preferably 2%.

[0165] In various embodiments, the stage 2 culture medium may comprise one or more fibroblast growth factors (FGFs), preferably FGF7. In various embodiments, the one or more FGFs may be in an amount of about 10-100 ng / ml, preferably about 20-80 ng / ml, more preferably about 30-70 ng / ml. In various embodiments, the one or more FGFs may be in an amount of about 50 ng / ml.

[0166] In various embodiments, the stage 2 culture medium comprises a basal medium supplemented with one or more FGFs. In various embodiments, the stage 2 culture medium comprises a basal medium supplemented with FAF BSA, and a FGF7.

[0167] In various embodiments, the stage 2 culture medium comprises FAF BSA in an amount of about 1-2%, and FGF7 in an amount of about 30-70 ng / ml. In various embodiments, the stage 2 culture medium comprises FAF BSA in an amount of about 2%, and FGF7 in an amount of about 50 ng / ml.

[0168] In various embodiments, the stage 2 differentiation in the stage 2 culture medium may be carried out for a period of about 1 -4 days, preferably about 3 days.

[0169] In various embodiments, the stage 1 cells are incubated under standard culture conditions to promote patterning and differentiation toward a primitive gut tube fate. In various embodiments, the culture conditions of stage 2 may be standard mammalian cell culture conditions known in the art, for example at about 37 °C in a humidified incubator with about 5% CO2, optionally with gentle agitation (e.g., orbital shaking at about 60-80 rpm or an equivalent suspension culture system) to maintain the cells in suspension and to promote uniform exposure to the medium components.

[0170] In various embodiments, primitive gut tube cells obtained during or after Stage 2 differentiation are characterised by molecular signatures indicative of early foregut specification. Primitive gut tube cells may express one or more markers associated with anterior definitive endoderm and early pancreatic endoderm formation, including, for example, HNF1 , HNF4a, FOXA1 , FOXA2, SOX17, TBX3, and PDX1 at low or emerging levels. In certain embodiments, primitive gut tube identity may be confirmed by the maintenance or moderate expression of FOXA2 from definitive endoderm into primitive gut tube and foregut, in combination with upregulation of early foregut markers such as HNF1 or HNF4a, and by the gradual reduction of CXCR4 expression relative to Stage 1. The presence and proportion of cells expressing one or more of these markers may be used as an indicator of successful transition from definitive endoderm into the primitive gut tube lineage.

[0171] Accordingly, cells expressing markers characteristic of the primitive gut tube, or primitive gut tube cells, may be obtained at stage 2.

[0172] Upon completion of Stage 2, the differentiated cells are advanced into Stage 3 by removing the Stage 2 medium and replacing it with the Stage 3 differentiation medium ( .e., the third culture medium). In various embodiments, medium exchange may be performed by allowing the cells to settle by gravity or by gently collecting them by low-speed centrifugation, followed by aspiration of the spent Stage 2 medium. The cells may optionally be washed once or more with the basal medium to minimise carryover of Stage 2 factors to stage 3.o Stage 3

[0173] In stage 3, the cells obtained from stage 2 are incubated in a stage 3 (third) culture medium under suitable conditions to initiate differentiation to pancreatic progenitor 1 (PP1) cells. In various embodiments, the stage 3 culture medium comprises a basal medium supplemented with one or more growth factors and one or more small-molecule differentiating agents.

[0174] In various embodiments, the stage 3 culture medium comprises a basal medium supplemented with one or more of glucose, sodium bicarbonate, vitamin C, insulin-transferrin selenium (ITS-X), penicillin-streptomycin and glutamax. In various embodiments, the stage 3 culture medium comprises a basal medium supplemented with BSA, preferably FAF BSA. In various embodiments, the FAF BSA is in an amount of about 1 -2%, preferably about 2%.

[0175] In various embodiments, the stage 3 culture medium may comprise one or more fibroblast growth factors (FGFs), preferably FGF7. In various embodiments, the one or more FGFs may be in an amount of about 10-100 ng / ml, preferably about 20-80 ng / ml, more preferably about 30-70 ng / ml. In various embodiments, the one or more FGFs may be in an amount of about 50 ng / ml.

[0176] In various embodiments, the stage 3 culture medium may comprise one or more small-molecule differentiating agents, wherein the small-molecule differentiating agents may comprise a retinoid, aHedgehog signalling pathway antagonist, a protein kinase C activator, a BMP pathway inhibitor, or combinations thereof.

[0177] In various embodiments, the stage 3 culture medium further comprises one or more retinoids, preferably retinoic acid (RA). In various embodiments, the one or more retinoids may be at a concentration of about 0.1-5 pM, preferably about 1-3 pM, more preferably about 2 pM.

[0178] In various embodiments, the stage 3 culture medium may comprise one or more Hedgehog signalling pathway antagonists (r.e., Shh signalling pathway antagonist), preferably SANT1 . In various embodiments, the one or more Hedgehog pathway antagonists may be at a concentration of about 0.05-1 pM, preferably about 0.1 -0.5 pM, more preferably about 0.25 pM.

[0179] In various embodiments, the stage 3 culture medium may comprise one or more protein kinase C (PKC) activators, preferably PDBu. In various embodiments, the one or more PKC activators may be at a concentration of about 0.1-1 pM, preferably about 0.25-0.75 pM, more preferably about 0.5 pM.

[0180] In various embodiments, the stage 3 culture medium may comprise one or more BMP pathway inhibitors, preferably LDN193189. In various embodiments, the one or more BMP pathway inhibitors may be at a concentration of about 0.05-0.5 pM, preferably about 0.1 -0.3 pM, more preferably about 0.1 -0.2 pM.

[0181] In various embodiments, the stage 3 culture medium comprises a basal medium supplemented with one or more FGFs and one or more small-molecule differentiation agents selected from retinoids, Hedgehog pathway antagonists, PKC activators, and BMP pathway inhibitors. In various embodiments, the stage 3 culture medium comprises BSA, an FGF, a retinoid, a Hedgehog pathway antagonist, a PKC activator, and a BMP pathway inhibitor.

[0182] In various embodiments, the stage 3 culture medium comprises a basal medium supplemented with FAF BSA, FGF7, RA, SANT1 , PDBu, and LDN193189. In various embodiments, the stage 3 culture medium comprises FAF BSA in an amount of about 1-2%, and FGF7 in an amount of about 30-70 ng / ml, together with RA at a concentration of about 1-3 pM, SANT1 at a concentration of about 0.1 -0.5 pM, PDBu at a concentration of about 0.25-0.75 pM, and LDN193189 at a concentration of about 0.1 -0.2 pM. In various embodiments, the stage 3 culture medium comprises FAF BSA in an amount of about 2%, FGF7 in an amount of about 50 ng / ml, RA at a concentration of about 2 pM, SANT1 at a concentration of about 0.25 pM, PDBu at a concentration of about 0.5 pM, and LDN193189 at a concentration of about 0.1 -0.2 pM.

[0183] In various embodiments, the stage 3 differentiation in the stage 3 culture medium may be carried out for a period of about 1 -3 days, preferably about 2 days.

[0184] In various embodiments, the cells in stage 3 are incubated under standard culture conditions to promote patterning and differentiation toward a pancreatic progenitor 1 fate. In various embodiments, the culture conditions of stage 3 may be standard mammalian cell culture conditions known in the art, for example at about 37 °C in a humidified incubator with about 5% CO2, optionally with gentle agitation (e.g., orbital shaking at about 60-80 rpm or an equivalent suspension culture system) to maintain the cells in suspension and to promote uniform exposure to the medium components.

[0185] In various embodiments, pancreatic progenitor 1 (PP1 ) cells obtained during or after Stage 3 differentiation are characterized by molecular signatures indicative of early pancreatic progenitor specification. Pancreatic progenitor 1 cells may express one or more markers associated with early pancreatic endoderm, including, for example, PDX1 together with continued expression of FOXA2 and HNF1 p, and low-to-moderate expression of SOX9 or TBX3. This stage follows definitive endoderm and primitive gut tube (PGT), where SOX17 / CXCR4 are high, transitioning to PDX1 / NKX6.1 enrichment. In certain embodiments, pancreatic progenitor 1 identity may be confirmed by the emergence or upregulation of PDX1 in combination with FOXA2 and / or HNF1p, and by a reduction in CXCR4 expression relative to primitive gut tube cells. The presence and proportion of cells expressing one or more of these markers may be used as an indicator of successful transition from the primitive gut tube into the pancreatic progenitor 1 lineage.

[0186] Accordingly, cells expressing markers characteristic of pancreatic progenitor 1 cells, or pancreatic progenitor 1 cells, may be obtained at Stage 3.

[0187] Upon completion of Stage 3, the differentiated cells are advanced into Stage 4 by removing the Stage 3 medium and replacing it with the Stage 4 differentiation medium ( / .e., the fourth culture medium). In various embodiments, medium exchange may be performed by allowing the cells to settle by gravity or by gently collecting them by low-speed centrifugation, followed by aspiration of the spent Stage 3 medium. The cells may optionally be washed once or more with the basal medium to minimise carry-over of Stage 3 factors into Stage 4.o Stage 4

[0188] In stage 4, the cells obtained from stage 3 are incubated in a stage 4 (fourth) culture medium under suitable conditions to initiate differentiation to pancreatic progenitor 2 (PP2) cells. In various embodiments, the stage 4 culture medium comprises a basal medium supplemented with one or more growth factors and one or more small-molecule differentiating agents.

[0189] In various embodiments, the stage 4 culture medium comprises a basal medium supplemented with BSA, preferably FAF BSA. In various embodiments, the FAF BSA is in an amount of about 1 -2%, preferably about 2%. The media is supplemented with glucose, sodium bicarbonate, vitamin C, insulin-transferrin selenium (ITS-X), penicillin-streptomycin and glutamax.

[0190] In various embodiments, the stage 4 culture medium may comprise one or more fibroblast growth factors (FGFs), preferably fibroblast growth factor 7 (FGF7). In various embodiments, the one or more FGFs may be in an amount of about 10-100 ng / ml, preferably about 20-80 ng / ml, more preferably about 30-70 ng / ml. In various embodiments, the one or more FGFs may be in an amount of about 50 ng / ml.

[0191] In various embodiments, the stage 4 culture medium may comprise one or more small-molecule differentiating agents, wherein such agents may comprise a retinoid, a Hedgehog signalling pathway antagonist, a BMP pathway inhibitor, and a metabolic cofactor, and combinations thereof. In various embodiments, the stage 4 culture medium may comprise a BMP type I receptor inhibitor, and optionally a retinoid, a Hedgehog signalling pathway antagonist, and a metabolic cofactor.

[0192] In various embodiments, the stage 4 culture medium comprises one or more retinoids, preferably retinoic acid (RA). In various embodiments, the one or more retinoids may be at a concentration of about 0.01 -1 pM, preferably about 0.05-0.2 pM, more preferably about 0.1 pM.

[0193] In various embodiments, the stage 4 culture medium may comprise one or more Hedgehog signalling pathway antagonists, preferably SANT 1. In various embodiments, the one or more Hedgehog pathway antagonists may be at a concentration of about 0.051 pM, preferably about 0.10.5 pM, more preferably about 0.25 pM.

[0194] In various embodiments, the stage 4 culture medium may comprise one or more BMP pathway inhibitors, preferably LDN193189. In various embodiments, the one or more BMP pathway inhibitors may be at a concentration of about 0.05-0.5 pM, preferably about 0.1 -0.3 pM, more preferably about 0.1 -0.2 pM.

[0195] In various embodiments, the stage 4 culture medium comprises one or more metabolic cofactors, preferably nicotinamide. In various embodiments, the nicotinamide may be present at a concentration of about 1 -50 mM, preferably about 5-20 mM, more preferably about 10 mM.

[0196] In various embodiments, the stage 4 culture medium comprises a basal medium supplemented with one or more FGFs and one or more small-molecule differentiation agents selected from retinoids, Hedgehog pathway antagonists, BMP pathway inhibitors, and metabolic cofactors. In various embodiments, the stage 4 culture medium comprises BSA, an FGF, a retinoid, a Hedgehog pathway antagonist, a BMP pathway inhibitor, and a metabolic cofactor.

[0197] In various embodiments, the stage 4 culture medium comprises a basal medium supplemented with FAF BSA, FGF7, RA, SANT1 , LDN193189, and nicotinamide. In various embodiments, the stage 4 culture medium comprises FAF BSA in an amount of about 1 -2%, and FGF7 in an amount of about 30-70 ng / ml, together with RA at a concentration of about 0.05-0.2 pM, SANT1 at a concentration ofabout 0.1 -0.5 pM, LDN193189 at a concentration of about 0.1 -0.2 pM, and nicotinamide at a concentration of about 5-20 mM. In various embodiments, the stage 4 culture medium comprises FAF BSA in an amount of about 2%, FGF7 in an amount of about 50 ng / ml, RA at a concentration of about 0.1 pM, SANT1 at a concentration of about 0.25 pM, LDN193189 at a concentration of about 0.1 -0.2 pM, and nicotinamide at a concentration of about 10 mM.

[0198] In various embodiments, the stage 4 differentiation in the stage 4 culture medium may be carried out for a period of about 4-6 days, preferably about 5 days.

[0199] In various embodiments, the cells are incubated under standard culture conditions to promote patterning and differentiation toward a pancreatic progenitor 2 (PP2) fate. In various embodiments, the culture conditions of stage 4 may be standard mammalian cell culture conditions known in the art, for example at about 37 °C in a humidified incubator with about 5% CO2, optionally with gentle agitation (e.g., orbital shaking at about 60-80 rpm or an equivalent suspension culture system) to maintain the cells in suspension and to promote uniform exposure to the medium components.

[0200] In various embodiments, PP2 cells obtained during or after Stage 4 differentiation are characterised by molecular signatures indicative of an advanced pancreatic progenitor state. PP2 cells may express one or more markers associated with pancreatic lineage maturation, including, for example, co-expression of PDX1 and NKX6.1, optionally together with elevated SOX9 or HNF1p and reduced expression of primitive or early endodermal markers such as CXCR4. In certain embodiments, PP2 identity may be confirmed by the robust emergence of PDX1+NKX6.1+cells, optionally in combination with high SOX9 expression. The presence and proportion of cells expressing one or more of these markers may be used as an indicator of successful transition from PP1 to PP2.

[0201] In various embodiments, PP2 cells or cells expressing markers characteristic of PP2 cells are obtained following completion of Stage 4. In various embodiments, the PP2 cells are capable of being differentiated into hiPSC-derived functional islet-like cells. In various embodiments, the hiPSC-derived functional islet-like cells demonstrate a rapid first-phase insulin secretion upon transition from low glucose concentration (e.g., about 2-3 mM, preferably about 2.8 mM) to high glucose concentration (e.g., about 15-25 mM, preferably about 16.7 mM). In various embodiments, the insulin secretion peak is at least six-fold, at least eight-fold, at least ten-fold, at least twelve-fold, at least fifteen-fold, at least eighteen-fold, at least twenty-fold, at least twenty-five-fold, at least thirty-fold or more for the hiPSC-derived functional islet-like cells relative to undifferentiated parent hiPSC. In various embodiments, the hiPSC-derived functional islet-like cells demonstrate a second phase insulin secretion in response to KCI-induced depolarisation following stimulation with high glucose concentration before returning to base level. In various embodiments, the second phase insulin secretion peak is at least six-fold, at least eight-fold, at least ten-fold, at least twelve-fold, at least fifteen-fold, at least eighteen-fold, at least twenty-fold, or more for the hiPSC-derived functional islet-like cells relative to undifferentiated parent hiPSC.

[0202] Accordingly, cells expressing markers characteristic of PP2 cells, or PP2 cells, maybe obtained at Stage 4.

[0203] Upon completion of Stage 4, the differentiated cells are advanced into Stage 5 by removing the Stage 4 medium and replacing it with the Stage 5 differentiation medium (i.e., the fifth culture medium). In various embodiments, medium exchange may be performed by allowing the cells to settle by gravity or by gently collecting them by low-speed centrifugation, followed by aspiration of the spent Stage 4 medium. The cells may optionally be washed once or more with the basal medium to minimize carryover of Stage 4 factors into Stage 5.o Stage 5

[0204] In stage 5, the cells obtained from stage 4 are incubated in a stage 5 (fifth) culture medium under suitable conditions to initiate differentiation to pancreatic endocrine progenitor cells. In various embodiments, the stage 5 culture medium comprises a basal medium supplemented with one or more growth factors and one or more small-molecule differentiating agents.

[0205] In various embodiments, the stage 5 culture medium comprises a basal medium supplemented with BSA, preferably FAF BSA. In various embodiments, the FAF BSA is in an amount of about 1 -2%, preferably about 2%. The media is supplemented with glucose, sodium bicarbonate, vitamin C, insulin-transferrin selenium (ITS-X), penicillin-streptomycin, heparin and glutamax.

[0206] In various embodiments, the stage 5 culture medium may comprise one or more growth factors, preferably one or more EGF-family ligands. In various embodiments, the one or more growth factors may comprise betacellulin. In various embodiments, the betacellulin may be in an amount of about 5-100 ng / ml, preferably about 10-40 ng / ml, more preferably about 20 ng / ml.

[0207] In various embodiments, the stage 5 culture medium may comprise one or more small-molecule differentiating agents, wherein such small molecules may comprise one or more retinoids, Hedgehog pathway antagonists, Notch pathway inhibitors, TGF-p receptor inhibitors, BMP pathway inhibitors, Wnt pathway activators, thyroid hormones, or combinations thereof. In various embodiments, the stage 5 culture medium may comprise a BMP pathway inhibitor, and optionally one or more retinoids, Hedgehog pathway antagonists, Notch pathway inhibitors, TGF-p receptor inhibitors, Wnt pathway activators, thyroid hormones, or combinations thereof.

[0208] In various embodiments, the stage 5 culture medium may comprise one or more retinoids, preferably retinoic acid (RA). In various embodiments, the one or more retinoids may be at a concentration of about 0.01 -1 pM, preferably about 0.05-0.2 pM, more preferably about 0.1 pM.

[0209] In various embodiments, the stage 5 culture medium may comprise one or more Hedgehog signalling pathway antagonists, preferably SANT 1. In various embodiments, the one or more Hedgehog pathway antagonists may be at a concentration of about 0.05-1 pM, preferably about 0.1 -0.5 pM, more preferably about 0.25 pM.

[0210] In various embodiments, the stage 5 culture medium may comprise one or more Notch pathway inhibitors, preferably a y-secretase inhibitor such as Compound XXI (XXI). In various embodiments, the one or more Notch pathway inhibitors may be at a concentration of about 0.1 -5 pM, preferably about 0.5-2 pM, more preferably about 1 pM.

[0211] In various embodiments, the stage 5 culture medium may comprise one or more TGF-p receptor inhibitors, preferably ALK5ill. In various embodiments, the one or more TGF-p receptor inhibitors may be at a concentration of about 1 -20 pM, preferably about 5-15 pM, more preferably about 10 pM.

[0212] In various embodiments, the stage 5 culture medium may comprise one or more BMP pathway inhibitors, preferably LDN193189. In various embodiments, the one or more BMP pathway inhibitors may be at a concentration of about 0.05-0.5 pM, preferably about 0.1 -0.3 pM, more preferably about 0.1 -0.2 pM.

[0213] In various embodiments, the stage 5 culture medium may comprise one or more thyroid hormones, preferably triiodothyronine (T3). In various embodiments, the T3 may be at a concentration of about 0.1-5 pM, preferably about 0.5-2 pM, more preferably about 1 pM.

[0214] In various embodiments, the stage 5 culture medium comprises BSA, a growth factor, a retinoid, a Hedgehog pathway antagonist, a Notch pathway inhibitor, a TGF-p receptor inhibitor, a BMP pathway inhibitor, a Wnt pathway activator, and a thyroid hormone.

[0215] In various embodiments, the stage 5 culture medium comprises a basal medium supplemented with FAF BSA, RA, SANT1 , Compound XXI, ALK5III, T3, betacellulin, and LDN193189. In various embodiments, the fifth culture medium comprises FAF BSA in an amount of about 1 -2%, betacellulin in an amount of about 10-40 ng / ml, together with RA at a concentration of about 0.05-0.2 pM, SANT1 at a concentration of about 0.1 -0.5 pM, Compound XXI at a concentration of about 0.5-2 pM, ALK5ill at a concentration of about 5-15 pM, T3 at a concentration of about 0.5-2 pM, and LDN193189 at a concentration of about 0.1 -0.2 pM. In various embodiments, the stage 5 culture medium comprises FAF BSA in an amount of about 2%, betacellulin in an amount of about 20 ng / ml, RA at a concentration of about 0.1 pM, SANT1 at a concentration of about 0.25 pM, Compound XXI at a concentration of about 1 pM, ALK5ill at a concentration of about 10 pM, T3 at a concentration of about 1 pM, and LDN193189 at a concentration of about 0.1 -0.2 pM.

[0216] In various embodiments, the Stage 5 differentiation in the stage 5 culture medium may be carried out for a period of about 3-5 days, preferably about 4 days.

[0217] In various embodiments, the stage 5 culture medium may further supplemented by adding a retinoid; a Hedgehog pathway antagonist; a Notch pathway inhibitor; a TGF-p receptor inhibitor; a thyroid hormone; an EGF-family growth factor; a BMP pathway inhibitor; and a Wnt pathway activator, at one or more time points during the period of the stage 5 differentiation, or after the period (i.e. 3-5 days) of the stage 5 differentiation. In various embodiments, the stage 5 culture medium may further supplemented by adding RA, SANT1 , Compound XXI, ALK5ill, T3, betacellulin, LDN193189 and ISX9 or equivalent factors.

[0218] In various embodiments, stage 5 may further comprise an additional culture step where the stage 5 culture medium (5a) is exchanged for a new stage 5 culture medium (5b) in a subsequent differentiation step in stage 5 that follows the initial differentiation stage 5. The stage 5 culture medium (5a) may be termed as the first stage 5 culture medium and the new stage 5 culture medium (5b) may be termed as the second stage 5 culture medium, for ease of distinguishing the two.

[0219] In various embodiments, the second stage 5 culture medium comprises one or more small molecules selected from a retinoid, a Hedgehog pathway antagonist, a Notch pathway inhibitor, a TGF-P receptor inhibitor, a BMP pathway inhibitor, a thyroid hormone, a Wnt pathway activator, or combinations thereof.

[0220] In various embodiments, the second stage 5 culture medium comprises one or more smallmolecule differentiation agents and one or more growth factors selected from: a retinoid; a Hedgehog pathway antagonist; a Notch pathway inhibitor; a TGF-p receptor inhibitor; a thyroid hormone; an EGF-family growth factor; a BMP pathway inhibitor; and a Wnt pathway activator.

[0221] In various embodiments, the retinoid comprises a retinoic acid (RA), which may be present at a concentration of about 0.005-0.1 pM, preferably about 0.01-0.05 pM, more preferably about 25 nM.

[0222] In various embodiments, the Hedgehog pathway antagonist comprises a Smoothened inhibitor such as SANT 1 , which may be present at a concentration of about 0.1 -0.5 pM, preferably about 0.25 pM.

[0223] In various embodiments, the Notch pathway inhibitor comprises a y-secretase inhibitor such as Compound XXI (XXI), which may be present at a concentration of about 1 pM.

[0224] In various embodiments, the TGF- receptor inhibitor comprises a selective ALK5 inhibitorsuch as ALK5HI, which may be present at a concentration of about 10 pM.

[0225] In various embodiments, the thyroid hormone comprises triiodothyronine (T3), which may be present at a concentration of about 1 pM.

[0226] In various embodiments, the EGF-family growth factor comprises betacellulin, which may be present at an amount of about 10-40 ng / ml, preferably about 20 ng / ml.

[0227] In various embodiments, the BMP pathway inhibitor comprises a type I BMP receptor inhibitor such as LDN193189, which may be present at a concentration of about 0.1 -0.2 pM.

[0228] In various embodiments, the Wnt pathway activator comprises a small-molecule Wnt pathway activator such as Isoxazole (ISX9) or an equivalent factor, which may be present at a concentration of about 0-20 pM, preferably about 0-10 pM, more preferably about 5-10 pM. In various embodiments, the equivalent factor may be selected from the group consisting of CHIR99021 , 6-bromoindirubin-3'-oxime (BIO), lithium chloride (LiCI) T3, Alk5 inhibitors, PKA / cAMP agonists and epigenetic modulators.

[0229] In various embodiments, the second stage 5 culture medium comprises FAF BSA, RA, SANT 1 , Compound XXI, ALK5III, T3, betacellulin, LDN193189 and ISX9 or an equivalent factor.

[0230] In various embodiments, the second stage 5 culture medium comprises: RA at a concentration of about 0.005-0.1 pM, preferably about 0.01-0.05 pM, more preferably about 25 nM; SANT1 at a concentration of about 0.1 -0.5 pM, preferably about 0.25 pM; Compound XXI at a concentration of about 1 pM; ALK5ill at a concentration of about 10 pM; T3 at a concentration of about 1 pM; betacellulin at an amount of about 10-40 ng / ml, preferably about 20 ng / ml; LDN193189 at a concentration of about 0.1 -0.2 pM; and a Wnt pathway activator such as ISX9 at a concentration of about 0-20 pM, preferably about 0-10 pM, more preferably about 5-10 pM depending on the desired endocrine commitment.

[0231] In various embodiments, Stage 5 may comprise a 1stculture period using the first stage 5 culture medium, followed by a subsequent 2ndculture period using the second stage 5 culture medium. In various embodiments, the Stage 5 differentiation in the fifth culture medium(s) may be carried out for a total period of about 5-10 days, comprising the 1stculture period in the first stage 5 culture medium of about 3-5 days, preferably about 4 days, followed by the 2ndculture period in the second stage 5 culture medium of about 2-4 days, preferably about 3 days.

[0232] In various embodiments, the cells are incubated under standard culture conditions to promote differentiation toward a pancreatic endocrine progenitor fate. In various embodiments, the culture conditions of Stage 5 may be standard mammalian cell culture conditions known in the art, for example at about 37 °C in a humidified incubator with about 5% CO2, optionally with gentle agitation (e.g., orbital shaking at about 60-80 rpm or an equivalent suspension culture system) to maintain the cells in suspension and ensure uniform exposure to the medium components.

[0233] In various embodiments, the culturing stage 5 comprises incubating the PP2 cells in the stage 5 culture medium(s) for a period of time, as outlined above, under suitable temperature, gas, and agitation conditions. In various embodiments, Stage 5 comprises a first culture period in which the cells are exposed to a defined combination of growth factors and small-molecule differentiation agents, followed by a second culture period in which additional or renewed small-molecule differentiation agents are introduced to reinforce endocrine lineage specification. Under these conditions, the spheroid cultures are exposed to signalling environments that modulate Notch, Hedgehog, TGF- , BMP, Wnt, and thyroid hormone pathways, thereby promoting the transition from PP2 cells to pancreatic endocrine progenitor cells.

[0234] In various embodiments, pancreatic endocrine progenitor (EP) cells obtained during or after Stage 5 differentiation are characterised by molecular signatures indicative of endocrine lineage specification. Pancreatic endocrine progenitor cells may express one or more endocrine-associated markers, including, for example, NEUROG3 (NGN3) as a defining transcription factor, optionally together with PAX4, PAX6, NKX2.2, and ISL1 . In certain embodiments, pancreatic endocrine progenitor identity may be confirmed by robust induction of NGN3 expression. The presence and proportion of cells expressing one or more of these markers may be used as an indicator of successful transition from the PP2 lineage to the pancreatic EP lineage.

[0235] Accordingly, cells expressing markers characteristic of pancreatic endocrine progenitor cells may be obtained at Stage 5.

[0236] In various embodiments, EP cells or cells expressing markers characteristic of EP cells are obtained following completion of Stage 5. In various embodiments, the EP cells are capable of being differentiated into hiPSC-derived functional islet-like cells, wherein the hiPSC-derived functional isletlike cells demonstrate a rapid first-phase insulin secretion upon transition from low glucose concentration (e.g., about 2-3 mM, preferably about 2.8 mM) to high glucose concentration (e.g., about 15-25 mM, preferably about 16.7 mM). In various embodiments, the insulin secretion peak is at least six-fold, at least eight-fold, at least ten-fold, at least twelve-fold, at least fifteen-fold, at least eighteenfold, at least twenty-fold, at least twenty-five-fold, at least thirty-fold or more for the hiPSC-derived functional islet-like cells relative to undifferentiated parent hiPSC. In various embodiments, the hiPSC-derived functional islet-like cells demonstrate a second phase insulin secretion in response to KCI-induced depolarisation following stimulation with high glucose concentration before returning to base level. In various embodiments, the second phase insulin secretion peak is at least six-fold, at least eightfold, at least ten-fold, at least twelve-fold, at least fifteen-fold, at least eighteen-fold, at least twenty-fold, or more for the hiPSC-derived functional islet-like cells relative to undifferentiated parent hiPSC.

[0237] Upon completion of Stage 5, the differentiated cells are advanced into Stage 6 by removing the Stage 5 medium and replacing it with the Stage 6 differentiation medium ( / '.e., the sixth culture medium). In various embodiments, medium exchange may be performed by allowing the cells to settle by gravityor by gently collecting them by low-speed centrifugation, followed by aspiration of the spent Stage 5 medium. The cells may optionally be washed once or more with the basal medium to minimize carryover of Stage 5 factors into Stage 6.o Stage 6

[0238] In stage 6, the cells obtained from stage 5 are incubated in a stage 6 (sixth) culture medium under suitable conditions to initiate differentiation toward functional islet-like cells. In various embodiments, the sixth culture medium comprises a basal medium supplemented with one or more small-molecule differentiating agents and one or more nutrient and ionic supplements.

[0239] In various embodiments, the stage 6 culture medium comprises a basal medium supplemented with BSA, preferably FAF BSA. In various embodiments, the FAF BSA is in an amount of about 1 -2%, preferably about 2%. The media is supplemented with glucose, sodium bicarbonate, vitamin C, insulin-transferrin selenium (ITS-X), penicillin-streptomycin, heparin and glutamax.

[0240] In various embodiments, the stage 6 culture medium comprises one or more nutrients and ionic supplements, preferably glucose and zinc salts. In various embodiments, the glucose may be present at a concentration of about 5-40 mM, preferably about 10-25 mM, more preferably about 20 mM. In various embodiments, the zinc supplement may comprise zinc sulfate (ZnS04), which may be present at a concentration of about 1-50 pM, preferably about 5-20 pM, more preferably about 10 pM.

[0241] In various embodiments, the stage 6 culture medium may comprise one or more small-molecule differentiating agents selected from: (i) a TGF-p receptor inhibitor; (ii) a thyroid hormone; (iii) a redoxmodulating agent; (iv) a Wnt pathway activator; or combinations thereof.

[0242] In various embodiments, the TGF-f> receptor inhibitor comprises a selective ALK5 inhibitorsuch as ALK5il I, which may be present at a concentration of about 5-20 pM, preferably about 10 pM.

[0243] In various embodiments, the thyroid hormone comprises triiodothyronine (T3), which may be present at a concentration of about 0.5-2 pM, preferably about 1 pM.

[0244] In various embodiments, the redox-modulating agent comprises N-acetyl cysteine (NAG), which may be present at a concentration of about 0.5-5 mM, preferably about 1-2 mM, more preferably about 1 mM.

[0245] In various embodiments, the Wnt pathway activator comprises a small-molecule Wnt / p-catenin activator such as ISX9, which may be present at a concentration of about 1 -20 pM, preferably about 1 -10 pM, more preferably about 10 pM.

[0246] In various embodiments, the stage 6 culture medium may comprise BSA, a nutrient, an ionic supplement, a TGF-p receptor inhibitor, a thyroid hormone, a redox-modulating agent, and a Wnt pathway activator. In various embodiments, the stage 6 culture medium may comprise glucose, ZnSCM, and N-acetyl cysteine (NAC) and ISX9.

[0247] In various embodiments, the stage 6 culture medium may comprise FAF BSA, glucose, ZnS04, ALK5i II , T3, NAC, and ISX9. In various embodiments, the stage 6 culture medium comprises FAF BSA in an amount of about 1 -2%, glucose at a concentration of about 10-25 mM, ZnSO4at a concentration of about 5-20 pM, ALK5i 11 at a concentration of about 10 pM, T3 at a concentration of about 1 pM, NAC at a concentration of about 1 mM, and ISX9 at a concentration of about 0-10 pM. In various embodiments, the stage 6 culture medium comprises FAF BSA in an amount of about 2%, glucose at a concentration of about 20 mM, ZnS04at a concentration of about 10 pM, ALK5III at a concentration of about 10 pM, T3 at a concentration of about 1 pM, NAC at a concentration of about 1 mM, and ISX9 at a concentration of about 0-10 pM.

[0248] In various embodiments, the Stage 6 differentiation in the stage 6 culture medium may be carried out for a period of about 1 -3 days, preferably about 2 days.

[0249] In various embodiments, the stage 6 culture medium may be further supplemented by adding a TGF-p receptor inhibitor, a thyroid hormone, a redox-modulating agent, and a receptor tyrosine kinase inhibitor, preferably an AXL inhibitor. In various embodiments, the stage 6 culture medium may be further supplemented by adding ALK5ill, T3, NAC, and / or R428 at one or more time points during, or after, the period of the stage 6 differentiation.

[0250] In various embodiments, stage 6 may further comprise one or more additional culture steps where the stage 6 culture medium (6a) is exchanged for a second stage 6 culture medium (6b) in a subsequent differentiation in stage 6 that follows the initial differentiation stage 6. The stage 6 culture medium (6a) may be termed as the first stage 6 culture medium and the newly exchanged stage 6 culture medium (6b) may be termed as the second stage 6 culture medium, for ease of distinguishing the media in this stage.

[0251] In various embodiments, the second stage 6 culture medium comprises one or more signalling pathway modulators selected from: a TGF- receptor inhibitor; a thyroid hormone; and a redoxmodulating agent.

[0252] In various embodiments, the TGF- receptor inhibitor comprises a selective ALK5 inhibitorsuch as ALKSill, which may be present at a concentration of about 5-20 pM, preferably about 10 pM. In various embodiments, the thyroid hormone comprises triiodothyronine (T3), which may be present at a concentration of about 0.5-2 pM, preferably about 1 pM. In various embodiments, the redox-modulatingagent comprises N-acetyl cysteine (NAC), which may be present at a concentration of about 0.5-5 mM, preferably about 1 mM.

[0253] In various embodiments, the second stage 6 culture medium comprises ALK5ill at a concentration of about 10 pM; T3 ata concentration of about 1 pM; and NAC at a concentration of about 1 mM.

[0254] In various embodiments, the second stage 6 culture medium may comprise BSA, a TGF-p receptor inhibitor, a thyroid hormone, and a redox-modulating agent. In various embodiments, the second stage 6 culture medium may comprise FAF BSA, ALK5ill, T3, and NAC. In various embodiments, the second stage 6 culture medium comprises FAF BSA in an amount of about 1 -2%, ALK5ill at a concentration of about 10 pM, T3 at a concentration of about 1 pM, and NAC at a concentration of about 1 mM.

[0255] In various embodiments, Stage 6 may comprise a 1stculture period using the first stage 6 culture medium, followed by a subsequent 2ndculture period using the second stage 6 culture medium. In various embodiments, the 2ndstage 6 culture period may about 1 -3 days, preferably 2 days. In various embodiments, the Stage 6 differentiation may be carried out for a total period of about 2-6 days, preferably about 4 days, comprising the 1stculture period followed by the 2ndculture period.

[0256] In various embodiments, stage 6 may further comprise a further additional culture step where the second stage 6 culture medium (6b) is exchanged for a third stage 6 culture mediums (6c) in a subsequent differentiation in stage 6 that follows the differentiation stage 6 using the first and second stage 6 culture mediums. The newly exchanged stage 6 culture medium (6c) may be termed as the third stage 6 culture medium, for ease of distinguishing the media in this stage.

[0257] In various embodiments, the third stage 6 culture medium may comprise a TGF-p receptor inhibitor, a thyroid hormone, a redox-modulating agent, and a receptor tyrosine kinase inhibitor, preferably an AXL inhibitor.

[0258] In various embodiments, the TGF-p receptor inhibitor comprises a selective ALK5 inhibitorsuch as ALKSill, which may be present at a concentration of about 5-20 pM, preferably about 10 pM. In various embodiments, the thyroid hormone comprises triiodothyronine (T3), which may be present at a concentration of about 0.5-2 pM, preferably about 1 pM. In various embodiments, the redox-modulating agent comprises N-acetyl cysteine (NAC), which may be present at a concentration of about 0.5-5 mM, preferably about 1 mM. In various embodiments, the receptor tyrosine kinase inhibitor comprises an AXL kinase inhibitor such as R428 (bemcentinib). In various embodiments, the receptor tyrosine kinase inhibitor may be present at a concentration of about 0.1-2 pM, preferably about 0.25-1 pM, more preferably about 0.5 pM. In various embodiments, R428 may be present at a concentration of about 0.1-2 pM, preferably about 0.25-1 pM, more preferably about 0.5 pM.

[0259] Accordingly, the third stage 6 culture medium differs from the second stage 6 culture medium, in that it further comprises the receptor tyrosine kinase inhibitor, such as the AXL kinase inhibitor R428.

[0260] In various embodiments, the third stage 6 culture medium may comprise BSA, aTGF-p receptor inhibitor, a thyroid hormone, a redox-modulating agent, and a receptor tyrosine kinase inhibitor. In various embodiments, the third stage 6 culture medium may comprise FAF BSA, ALK5ill, T3, NAC and R428. In various embodiments, the third stage 6 culture medium comprises FAF BSA in an amount of about 1 -2%, ALK5il I at a concentration of about 10 pM, T3 at a concentration of about 1 pM, NAC at a concentration of about 1 mM, and R428 at a concentration of about 0.5 pM. The media is supplemented with glucose, sodium bicarbonate, vitamin C, insulin-transferrin selenium (ITS-X), penicillinstreptomycin, heparin and glutamax.

[0261] In various embodiments, Stage 6 may comprise a 1stculture period using the first stage 6 culture medium, followed by a subsequent 2ndculture period using the second stage 6 culture medium, and a 3rdculture period using the third stage 6 culture medium. In various embodiments, the 3rdstage 6 culture period may about 10-20 days, preferably 11-18 days. In various embodiments, the Stage 6 differentiation in the stage 6 culture mediums may be carried out for a total period of about 15-30 days, comprising the 1s’ culture period of about 1 -3 days, preferably about 2 days, followed by the 2ndculture period of about 2-6 days, preferably about 2 days, followed by the 3rdculture period of about 10-20 days, preferably about 12 days.

[0262] In various embodiments, the cells are incubated under standard mammalian cell culture conditions to promote differentiation toward a functional islet-like cell fate. In various embodiments, the culture conditions of Stage 6 may comprise incubation at about 37 °C in a humidified incubator with about 5% CO2, optionally with gentle agitation (e.g., orbital shaking at about 60-80 rpm or an equivalent suspension culture system) to maintain the cells in suspension and ensure uniform exposure to the medium components.

[0263] In various embodiments, the Stage 6 culturing step comprises incubating the cells obtained from Stage 5 in the stage 6 culture medium(s) for a period of time, as outlined above, under suitable temperature, gas, and optional agitation conditions. In various embodiments, Stage 6 comprises sequential culture periods in which the cells are first exposed to a defined combination of nutrient and ionic supplements together with one or more small-molecule differentiating agents, followed by subsequent periods in which selected agents are withdrawn or replaced to promote further maturation. Under these conditions, the spheroid cultures experience signalling environments that modulate TGF-P, thyroid hormone, redox, and receptor tyrosine kinase pathways while supporting metabolic maturation through defined glucose and zinc supplementation. These combined cues promote the transition from pancreatic endocrine progenitor cells to functional islet-like cells exhibiting glucoseresponsive insulin secretion. In various embodiments, such exposure results in upregulation of p-cell-associated transcriptional and functional markers, endocrine hormone processing, and acquisition of physiological properties characteristic of mature islet-like cells.

[0264] In various embodiments, functional islet-like cells obtained during or after Stage 6 differentiation are characterised by molecular and functional signatures indicative of mature pancreatic endocrine identity. Functional islet-like cells may express one or more markers associated with p-cell maturation, including, for example, INS (insulin), MAFA, NKX6.1 , PDX1 , PCSK1 , PCSK2, IAPP, and GLUT2. In certain embodiments, functional islet-like identity may be confirmed by glucose-stimulated insulin secretion or expression of mature p-cell transcription factors such as MAFA. The presence and proportion of cells expressing one or more of these markers may be used as an indicator of successful transition from endocrine progenitors into functional islet-like cells. In various embodiments, the isletlike cells express core (3-cell markers (insulin, C-peptide), 0-cell transcription factors (PDX1 , NKX6.1), oc-cell marker (glucagon), 8-cell marker (somatostatin) and s-cell marker (ghrelin).

[0265] In various embodiments, hiPSC-derived functional islet-like cells or cells expressing markers characteristic of functional islet-like cells cells are obtained following completion of Stage 6. In various embodiments, the hiPSC-derived functional islet-like cells demonstrate a rapid first-phase insulin secretion upon transition from low glucose concentration (e.g., about 2-3 mM, preferably about 2.8 mM) to high glucose concentration (e. ., about 15-25 mM, preferably about 16.7 mM). In various embodiments, the insulin secretion peak is at least six-fold, at least eight-fold, at least ten-fold, at least twelve-fold, at least fifteen-fold, at least eighteen-fold, at least twenty-fold, at least twenty-five-fold, at least thirty-fold or more for the hiPSC-derived functional islet-like cells relative to undifferentiated parent hiPSC. In various embodiments, the hiPSC-derived functional islet-like cells demonstrate a second phase insulin secretion in response to KCI-induced depolarisation following stimulation with high glucose concentration before returning to base level. In various embodiments, the second phase insulin secretion peak is at least six-fold, at least eight-fold, at least ten-fold, at least twelve-fold, at least fifteenfold, at least eighteen-fold, at least twenty-fold, or more for the hiPSC-derived functional islet-like cells relative to undifferentiated parent hiPSC.

[0266] Upon completion of Stage 6, the differentiated cells may be harvested, further matured, cryopreserved, encapsulated, or used in downstream applications such as transplantation, drug testing, genetic analysis, or functional assays.

[0267] In various embodiments, the method disclosed herein comprises an initial step of providing a population of pancreatic progenitor 1 (PP1) cells derived from hiPSCs or hESCs, and subjecting the PP1 cells to the stage 4, stage 5 and stage 6 differentiation steps. In this regard, the method may comprise, providing the pancreatic progenitor 1 cells;differentiating the pancreatic progenitor 1 cells in vitro in a Stage 4 medium under conditions and for a period of time sufficient to obtain pancreatic progenitor 2 cells, wherein the Stage 4 medium comprises a BMP type I receptor inhibitor;differentiating the pancreatic progenitor 2 cells in vitro in a first Stage 5 medium and a second Stage 5 medium under conditions for a first period of time sufficient and a second period of time sufficient, respectively, to obtain pancreatic endocrine progenitor cells, wherein each, the first Stage 5 medium and the second Stage 5 medium, comprises the BMP type I receptor inhibitor; and differentiating the pancreatic endocrine progenitor cells in vitro in a first Stage 6 medium, a second Stage 6 medium and a third Stage 6 medium under conditions for a first period of time sufficient, a second period of time sufficient, and a third period of time sufficient, respectively, to obtain insulinsecreting pancreatic islet-like cells, wherein the first Stage 6 medium comprises glucose, ZnSCu, and N-acetyl cysteine (NAG) and ISX9.

[0268] In various embodiments, the BMP type I receptor inhibitor is LDN193189. In various embodiments, the Stage 4 medium further comprises comprising a bovine serum albumin, an activin and a GSK3P inhibitor. In various embodiments, the first Stage 5 medium further comprises the BSA and fibroblast growth factor (FGF). In various embodiments, the second Stage 5 medium comprises the BSA, a retinoid, a Hedgehog pathway antagonist, a y-secretase inhibitor, a TGF-p receptor inhibitor, a thyroid hormone, an epidermal growth factor (EGF) family ligand, and LDN193189. In various embodiments, the first Stage 6 medium further comprises the BSA, the FGF, a retinoid, a Hedgehog pathway antagonist, and a protein kinase G (PKG) activator. In various embodiments, the second Stage 6 medium comprises the TGF-p receptor inhibitor, the thyroid hormone, and NAG. In various embodiments, the third Stage 6 medium comprises the TGF-p receptor inhibitor, the thyroid hormone, NAC and an Axl inhibitor. In various embodiments, the BSA is fatty-acid-free bovine serum albumin (FAF BSA), the activin is Activin A, the GSK3P inhibitor is CHIR99021 , the FGF is fibroblast growth factor 7 (FGF7), the retinoid is retinoic acid (RA), the Hedgehog pathway antagonist is SANT1 , the y-secretase inhibitor is XXI, the TGF-p receptor inhibitor is ALK5i 11 , the thyroid hormone is triiodothyronine (T3), the epidermal growth factor (EGF) family ligand is Betacellulin, the PKG activator is phorbol 12,13-dibutyrate (PDBu), the Axl inhibitor is R428, or a combination thereof.

[0269] In various embodiments, the method may comprise:providing PP1 cells,(4) differentiating the PP1 cells in vitro in a stage 4 culture medium to obtain cells expressing markers characteristic of PP2 cells, wherein the stage 4 culture medium comprises FAF BSA, an FGF, a retinoid, a Hedgehog pathway antagonist, a BMP pathway inhibitor, and a metabolic cofactor, and wherein the stage 4 differentiation is carried out for a period of about 4-6 days, preferably about 5 days;(5) differentiating the cells obtained from stage (4) in vitro in a stage 5 culture medium to obtain cells expressing markers characteristic of pancreatic EP cells, wherein the stage 5 culture medium comprises FAF BSA, a growth factor, a retinoid, a Hedgehog pathway antagonist, a Notch pathway inhibitor, a TGF-p receptor inhibitor, a BMP pathway inhibitor, a Wnt pathway activator, and a thyroid hormone, and wherein the stage 5 differentiation is carried out for a period of about 3-5 days, preferably about 4 days,optionally, the stage 5 culture medium is exchanged for a second stage 5 culture medium comprising FAF BSA a growth factor, a retinoid, a Hedgehog pathway antagonist, a Notch pathway inhibitor, a TGF-p receptor inhibitor, a BMP pathway inhibitor, a Wnt pathway activator, and a thyroid hormone, in a subsequent stage 5 differentiation, wherein the subsequent differentiating is carried out under suitable conditions for a period of about 2-4 days, preferably about 3 days:(6) differentiating the cells obtained from stage (5) in vitro in a stage 6 culture medium to obtain cells expressing markers characteristic of functional islet-like cells, wherein the stage 6 culture medium comprises FAF BSA, a nutrient, an ionic supplement, a TGF-p receptor inhibitor, a thyroid hormone, a redox-modulating agent, and a Wnt pathway activator, and wherein the stage 5 differentiation is carried out for a period of about 3-5 days, preferably about 4 days,optionally, the stage 6 culture medium is exchanged for a second stage 6 culture medium comprising FAF BSA, a TGF-p receptor inhibitor, a thyroid hormone, and a redox-modulating agent, in a subsequent stage 6 differentiation, wherein the subsequent differentiating step is carried out under suitable conditions for a period of about 1-3 days, preferably 2 days, andoptionally, the second stage 6 culture medium is exchanged for a third stage 6 culture medium comprising FAF BSA, a TGF-p receptor inhibitor, a thyroid hormone, a redox-modulating agent and a receptor tyrosine kinase inhibitor, in a third stage 6 differentiation, wherein the third differentiating step is carried out under suitable conditions for a period of about 10-20 days.

[0270] In various embodiments, the method may comprise the steps of,providing PP1 cells,differentiating the PP1 cells in a stage 4 culture medium to obtain cells expressing markers characteristic of PP2 cells, wherein the stage 4 culture medium comprises FAF BSA, FGF7, RA, SANT1 , LDN193189, and nicotinamide, and wherein the stage 4 differentiation is carried out for a period of about 5 days;differentiating the cells obtained from stage (4) in a stage 5 culture medium to obtain cells expressing markers characteristic of pancreatic endocrine progenitor cells, wherein the stage 5 culture medium comprises FAF BSA, RA, SANT1 , Compound XXI, ALK5ill, T3, betacellulin, and LDN193189, and wherein the stage 5 differentiation is carried out for a period of about 4 days,optionally, the stage 5 culture medium is exchanged for a second stage 5 culture medium comprising FAF BSA, RA, SANT1 , Compound XXI, ALK5ill, T3, betacellulin, LDN193189 and ISX9 or an equivalent factor, in a subsequent stage 5 differentiation, wherein the subsequent differentiating step is carried out under suitable conditions for a period of about 3 days;differentiating the cells obtained from stage (5) in a stage 6 culture medium to obtain cells expressing markers characteristic of functional islet-like cells, wherein the stage 6 culture medium comprises FAF BSA, glucose, ZnS04, ALK5ill, T3, NAC, and ISX9, and wherein the stage 5 differentiation is carried out for a period of about 2 days,optionally, the stage 6 culture medium is exchanged for a second stage 6 culture medium comprising FAF BSA, ALK5ill, T3, and NAC, in a subsequent stage 6 differentiation, wherein the subsequent differentiating step is carried out under suitable conditions for a period of about 2 days, andoptionally, the second stage 6 culture medium is exchanged for a third stage 6 culture medium comprising FAF BSA, ALK5III , T3, NAC and R428, in a third stage 6 differentiation, wherein the third differentiating step is carried out under suitable conditions for a period of about 10-20 days.

[0271] In various embodiments, LDN193189 is used continuously from Stage 3 to Stage 5 to inhibit BMP signalling, thereby enhancing pancreatic lineage commitment and pancreatic progenitor development.

[0272] In various embodiments, the method may comprise the steps of,(1) differentiating hiPSCs or hESCs in a stage 1 culture medium to obtain cells expressing markers characteristic of the definitive endoderm, wherein the stage 1 culture medium comprises BSA, a TGF-p family ligand and a Wnt pathway activator, and wherein the differentiating is carried out under suitable conditions for a period of about 12 hours to 2 days, preferably about 1 day,optionally, the stage 1 culture medium is exchanged for a second stage 1 culture medium comprising BSA and a TGF-p family ligand, in a subsequent stage 1 differentiation, wherein the subsequent differentiating is carried out under suitable conditions for a period of about 1-3 days, preferably about 2 days;(2) differentiating the cells obtained from stage (1) in a stage 2 culture medium to obtain cells expressing markers characteristic of primitive gut tube cells, wherein the stage 2 culture medium comprises FAF BSA, and a FGF, and wherein the stage 2 differentiation is carried out for a period of about 1 -4 days, preferably about 3 days;(3) differentiating the cells obtained from stage (2) in a stage 3 culture medium to obtain cells expressing markers characteristic of pancreatic progenitor 1 cells, wherein the stage 3 culture medium comprises FAF BSA, an FGF, a retinoid, a Hedgehog pathway antagonist, a PKC activator, and a BMP pathway inhibitor, and wherein the stage 3 differentiation is carried out for a period of about 1-3 days, preferably about 2 days;(4) differentiating the cells obtained from stage (3) in a stage 4 culture medium to obtain cells expressing markers characteristic of pancreatic progenitor 2 cells, wherein the stage 4 culture medium comprises FAF BSA, an FGF, a retinoid, a Hedgehog pathway antagonist, a BMP pathway inhibitor, and NAD+ precursor and modulator, and wherein the stage 4 differentiation is carried out for a period of about 4-6 days, preferably about 5 days;(5) differentiating the cells obtained from stage (4) in a stage 5 culture medium to obtain cells expressing markers characteristic of pancreatic endocrine progenitor cells, wherein the stage 5 culture medium comprises FAF BSA, a growth factor, a retinoid, a Hedgehog pathway antagonist, a Notch pathway inhibitor, a TGF-p receptor inhibitor, a BMP pathway inhibitor, a Wnt pathway activator, and a thyroid hormone, and wherein the stage 5 differentiation is carried out for a period of about 3-5 days, preferably about 4 days,optionally, the stage 5 culture medium is exchanged for a second stage 5 culture medium comprising FAF BSA a growth factor, a retinoid, a Hedgehog pathway antagonist, a Notch pathway inhibitor, a TGF-p receptor inhibitor, a BMP pathway inhibitor, a Wnt pathway activator, and a thyroidhormone, in a subsequent stage 5 differentiation, wherein the subsequent differentiating step is carried out under suitable conditions for a period of about 2-4 days, preferably about 3 days;(6) differentiating the cells obtained from stage (5) in a stage 6 culture medium to obtain cells expressing markers characteristic of functional islet-like cells, wherein the stage 6 culture medium comprises FAF BSA, a nutrient, an ionic supplement, a TGF-p receptor inhibitor, a thyroid hormone, a redox-modulating agent, and a Wnt pathway activator, and wherein the stage 5 differentiation is carried out for a period of about 3-5 days, preferably about 4 days,optionally, the stage 6 culture medium is exchanged for a second stage 6 culture medium comprising FAF BSA, a TGF-p receptor inhibitor, a thyroid hormone, and a redox-modulating agent, in a subsequent stage 6 differentiation, wherein the subsequent differentiating step is carried out under suitable conditions for a period of about 1-3 days, preferably 2 days, andoptionally, the second stage 6 culture medium is exchanged for a third stage 6 culture medium comprising FAF BSA, a TGF-p receptor inhibitor, a thyroid hormone, a redox-modulating agent and a receptor tyrosine kinase inhibitor, in a third stage 6 differentiation, wherein the third differentiating step is carried out under suitable conditions for a period of about 10-20 days.

[0273] In various embodiments, the method may comprise the steps of,(1) differentiating hiPSCs or hESGs in a stage 1 culture medium to obtain cells expressing markers characteristic of the definitive endoderm, wherein the stage 1 culture medium comprises FAF BSA, Activin A and CHIR99021 , and wherein the differentiating is carried out under suitable conditions for a period of about 1 day,optionally, the stage 1 culture medium is exchanged for a second stage 1 culture medium comprising FAF BSA and Activin A, in a subsequent stage 1 differentiation, wherein the subsequent differentiating is carried out under suitable conditions for a period of about 2 days:(2) differentiating the cells obtained from stage (1) in a stage 2 culture medium to obtain cells expressing markers characteristic of primitive gut tube cells, wherein the stage 2 culture medium comprises FAF BSA, and FGF7, and wherein the stage 2 differentiation is carried out for a period of about 3 days;(3) differentiating the cells obtained from stage (2) in a stage 3 culture medium to obtain cells expressing markers characteristic of pancreatic progenitor 1 cells, wherein the stage 3 culture medium comprises FAF BSA, FGF7, RA, SANT1 , PDBu, and LDN193189, and wherein the stage 3 differentiation is carried out for a period of about 2 days;(4) differentiating the cells obtained from stage (3) in a stage 4 culture medium to obtain cells expressing markers characteristic of pancreatic progenitor 2 cells, wherein the stage 4 culture medium comprises FAF BSA, FGF7, RA, SANT1 , LDN193189, and nicotinamide, and wherein the stage 4 differentiation is carried out for a period of about 5 days;(5) differentiating the cells obtained from stage (4) in a stage 5 culture medium to obtain cells expressing markers characteristic of pancreatic endocrine progenitor cells, wherein the stage 5 culture medium comprises FAF BSA, RA, SANT1 , Compound XXI, ALK5HI, T3, betacellulin, and LDN193189, and wherein the stage 5 differentiation is carried out for a period of about 3 days,optionally, the stage 5 culture medium is exchanged for a second stage 5 culture medium comprising FAF BSA, RA, SANT1 , Compound XXI, ALK5HI, T3, betacellulin, LDN193189 and ISX9, in a subsequent stage 5 differentiation, wherein the subsequent differentiating step is carried out under suitable conditions for a period of about 4 days;(6) differentiating the cells obtained from stage (4) in a stage 6 culture medium to obtain cells expressing markers characteristic of functional islet-like cells, wherein the stage 6 culture medium comprises FAF BSA, glucose, ZnS04, ALK5ill, T3, NAC, and ISX9, and wherein the stage 5 differentiation is carried out for a period of about 4 days,optionally, the stage 6 culture medium is exchanged for a second stage 6 culture medium comprising FAF BSA, ALK5ill, T3, and NAC, in a subsequent stage 6 differentiation, wherein the subsequent differentiating step is carried out under suitable conditions for a period of about 2 days, and optionally, the second stage 6 culture medium is exchanged for a third stage 6 culture medium comprising FAF BSA, ALK5ill , T3, NAC and R428, in a third stage 6 differentiation, wherein the third differentiating step is carried out under suitable conditions for a period of about 10-20 days.❖ Kits for use in the method

[0274] In another aspect, there is also provided a kit for use in performing the method disclosed herein, in particular generating insulin-secreting pancreatic islet-like cells.

[0275] In various embodiments, the kit may comprise one or more of the culture media described herein in relation to the stages 1-6 of the method, reagents, or compositions suitable for performing the multi-stage differentiation of hiPSCs or hESCs or pancreatic lineage cells as described herein.

[0276] In various embodiments, the kit comprises two or more stage-specific media or medium components packaged separately for sequential use in Stages 1 -6 of the differentiation method.

[0277] In various embodiments, the kit comprises: (i) a Stage 1 medium: (ii) a Stage 2 medium; (ill) a Stage 3 medium; (iv) a Stage 4 medium; (v) a Stage 5 medium; and (vi) a Stage 6 medium, each formulated for inducing differentiation through the corresponding lineage stage.

[0278] In various embodiments, the kit further comprises printed or electronic instructions describing the sequence of medium additions, concentrations of components, recommended incubation times, and culture conditions for each stage of differentiation (Stages 1 -6). In various embodiments, the instructions specify how to perform medium exchanges, washing steps, and supplementation during multi-phase stages such as Stages 5 and 6.

[0279] In various embodiments, the kit comprises any combination of media, supplements, growth factors, small-molecule differentiating agents, or instructions described herein, packaged together forperforming one or more stages of the differentiation method, or for performing the complete multi-stage differentiation protocol from pluripotent stem cells to functional islet-like cells.❖ Pancreatic Lineage Cells, and Compositions

[0280] It is also contemplated that another aspect of the present invention may relate to the pancreatic lineage cells obtained from the methods disclosed herein. All embodiments disclosed above in relation to the methods similarly apply to the pancreatic lineage cells obtained from said methods and compositions containing the same, and vice versa.

[0281] In various embodiments, there is provided definitive endoderm cells, primitive gut tube cells, pancreatic progenitor 1 cells, pancreatic progenitor 2 cells, pancreatic endocrine progenitor cells or islet-like cells obtained from the respective stages of the method disclosed herein.

[0282] In various embodiments, there is provided islet-like cells obtained from the methods disclosed herein. The islet-like cells may be defined as being functional islet-like cells that secrete insulin or C-peptide in response to glucose stimulation, that is, insulin-secreting pancreatic islet-like cells.

[0283] In various embodiments, the insulin-secreting pancreatic islet-like cells express INS, PCSK1 , PCSK2, MAFA, PDX1 , and NKX6.1. In various embodiments, the insulin-secreting pancreatic islet-like cells have at least 30% more, at least 50% more, at least 80% more, at least 100% more, at least 150% more, or more insulin content, normalized to total genomic DNA, relative to primary human islets.

[0284] In various embodiments, the insulin-secreting pancreatic islet-like cells exhibit about 8-30 fold insulin secretion. In various embodiments, the insulin-secreting pancreatic islet-like cells exhibit about 8-30 fold insulin secretion in response to glucose at a concentration of 15-25.5 mM. In various embodiments, the insulin-secreting pancreatic islet-like cells or cells expressing markers characteristic of insulin-secreting pancreatic islet-like cells are maintained / cultured in a culture media comprising ALKSill, T3, NAC, and Axl inhibitor. In various embodiments, the composition further comprises a culture media comprising ALK5III, T3, NAC, and Axl inhibitor (e.g., R428). In various embodiments, the culture media comprises 10 pM ALK5HI, 1 pM T3, 1 mM NAC, and 0.5 pM R428.

[0285] In various embodiments, the islet-like cells express one or more markers associated with mature pancreatic endocrine identity. Such markers may include p-cell-associated genes such as INS (insulin), C-peptide, PCSK1 and PCSK2 (prohormone convertases), MAFA, PDX1 , and NKX6.1 , optionally together with additional endocrine markers including IAPP, GLUT2, and UCN3. In various embodiments, the islet-like cells may further comprise non-p-cell endocrine cell types expressing markers such as glucagon (a-cells), somatostatin (6-cells), ghrelin (e-cell) or pancreatic polypeptide (PP-cells). In various embodiments, the islet-like cells exhibit reduced or absent expression of progenitor-associated markers such as NGN3. Such cells may additionally demonstrate functionalcharacteristics of mature pancreatic endocrine cells, including glucose-stimulated insulin or C-peptide secretion.

[0286] In various embodiments, the islet-like cells obtained by the methods described herein are highly spherical and have a smooth surface compared to human islets that are more irregular and fragmented in shape. In various embodiments, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, or more of the total islet-like cells are spherical. In various embodiments, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, or more of the total islet-like cells have circularity value of at least 0.6, at least 0.7, at least 0.8 or more. In various embodiments, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, or more of the total islet-like cells have smooth surface. In various embodiments, vascularized islet-like cells can be prepared by culturing islet-like cells with pro-vascularising cell types.

[0287] In various embodiments, the islet-like cells produced by the methods described herein can survive ex vivo in a culture media for at least one week, at least two weeks, at least three weeks or more without significant loss of functional performance (e.g., insulin-producing capacity). In various embodiments, the culture media comprises ALK5III, T3, NAC, Axl inhibitor (e.g., R428 (bemcentinib)). In various embodiments, the culture media comprises 10 pM ALK5III, 1 pM T3, 1 mM NAC, 0.5 pM R428. In various embodiments, the islet-like cells can mature both, in vitro and in vivo.

[0288] In various embodiments, the islet-like cells produced by the methods described herein function like endogenous islets of Langerhans. In various embodiments, the islet spheroids show colocalization of the p-cell transcription factor PDX1 with C-peptide indicates, which indicates the generation of insulinproducing p-like cells that express essential lineage-defining transcriptional regulators. In various embodiments, the islet spheroids show insulin expression together with the p-cell maturation marker NKX6.1 , which is associated with glucose-responsive insulin secretion. In various embodiments, at least some portion of the islet spheroids are glucagon-expressing a-like cells, which are localised alongside C-peptide-positive cells. In various embodiments, islet spheroids comprising p-like and a-like cells demonstrate that the differentiation protocol supports multilineage islet endocrine development, yielding spheroids with a cellular composition similar to primary human islets. In various embodiments, the islet-like cells express markers PDX1 , NKX6.1 , and glucagon, and function to secrete C-peptides (a byproduct of insulin processing). In various embodiments, the islet-like cells express markers PDX1 , NKX6.1 , and glucagon, and functions to secrete C-peptides (a byproduct of insulin processing).

[0289] In various embodiments, the islet-like cells produce and express insulin, are glucoseresponsive and capable of glucose-stimulated insulin secretion (GSIS) in vitro and can secrete insulin / C-peptide in an in vivo environment. In various embodiments, the GSIS responses resemble the GSIS responses of endogenous mature islet cells. In various embodiments, the islet-like cells exhibit GSIS responses to at least one glucose challenge. In various embodiments, the islet-like cells exhibit GSIS responses to at least two sequential glucose challenges. In various embodiments, the islet-likecells exhibit GSIS responses to at least three sequential glucose challenges. In various embodiments, the GSIS responses resemble the GSIS response of endogenous human islets to multiple glucose challenges. In various embodiments, the GSIS response is observed immediately upon transplanting the islet-like cells into a human or animal. In various embodiments, the GSIS response is observed within approximately 24 hours of transplanting the islet-like cells into a human or animal. In various embodiments, the GSIS response is observed within approximately one week of transplanting the isletlike cells into a human or animal. In various embodiments, the GSIS response is observed within approximately two weeks of transplanting the islet-like cells into a human or animal. In various embodiments, the GSIS response is observed at least week twelve following transplanting the islet-like cells into a human or animal. In various embodiments, the GSIS response is observed at least twelve weeks following transplanting the islet-like cells into a human or animal. In various embodiments, the GSIS response is observed at least twenty weeks following transplanting the islet-like cells into a human or animal.

[0290] In various embodiments, the islet-like cells obtained by the methods described herein exhibit functional performance and insulin-producing capacity comparable to or better than those of primary human islets. In various embodiments, the functional islet-like cells exhibit biphasic insulin secretion, first-phase glucose responsiveness and KCI-induced insulin release. In various embodiments, the functional islet-like cells demonstrate a rapid first-phase insulin secretion upon transition / exposure to high glucose concentration. A robust and high peak during the first-phase insulin secretion allows for a quick response to high glucose stimulation. For example, the robust and high peak during the first-phase insulin secretion is expected to trigger more effective insulin action from the peripheral tissues in vivo (e.g., rapid suppression of hepatic glucose production). In various embodiments, the functional islet-like cells can achieve 8 to >30-fold (e.g., at least six-fold, at least eight-fold, at least ten-fold, at least twelve-fold, at least fifteen-fold, at least eighteen-fold, at least twenty-fold, at least twenty five-fold, at least thirty-fold or more) increase in peak insulin secretion in response to high glucose stimulation (e.g., 15-25 mM) relative to undifferentiated parent hiPSC, followed by a reduced steady second phase secretion, and dynamic response to high and low glucose concentrations. In various embodiments, the second phase secretion results in an insulin secretion peak that is at least six-fold, at least eight-fold, at least ten-fold, at least twelve-fold, at least fifteen-fold, at least eighteen-fold, at least twenty-fold, or more for the functional islet-like cells relative to undifferentiated parent hiPSC. In various embodiments, the functional islet-like cells exhibit glucose-stimulated insulin secretion (GSIS) in response to glucose at a concentration of 15-25.5 mM, and KCI stimulation.

[0291] In various embodiments, the functional islet-like cells exhibit decreased insulin secretion in response to low glucose concentration of 0-3 mM.

[0292] In various embodiments, the functional islet-like cells produced by the methods described herein have high insulin content, normalized to total genomic DNA, relative to primary human islets. In various embodiments, the functional islet-like cells have at least 30% more, at least 50% more, at least80% more, at least 100% more, at least 150% more, or more insulin content, normalized to total genomic DNA, relative to primary human islets. In various embodiments, the functional islet-like cells have least 1.5 times insulin content, normalized to total genomic DNA, relative to primary human islets. In various embodiments, the functional islet-like cells have at least two times insulin content, normalized to total genomic DNA, relative to primary human islets. In various embodiments, functional islet-like cells exhibit insulin-producing capacity higher than the primary human islets. In various embodiments, a smaller number of functional islet-like cells is required relative to the primary human islets for generating a comparable amount of insulin in vivo. In various embodiments, the ratio of the number of functional islet-like cells and the primary human islets required for generating comparable amounts of insulin in vivo is <0.8, <0.7, <0.6, <0.5, <0.4, or lesser.

[0293] In various embodiments, the islet-like cells produced by the methods described herein secretes C-peptide at fasting states (time 0) that is comparable with human islets in vivo. In various embodiments, the islet-like cells, following IPGTT stimulation, results in at least 1 .5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, or more increase in C-peptide secretion relative to base level prior to IPGTT stimulation.

[0294] It is also contemplated that another aspect of the present invention may relate to a population of any one of the pancreatic lineage cells obtained from the methods disclosed herein. Such a population may comprise a substantially homogeneous or heterogeneous mixture of pancreatic cell types exhibiting molecular and functional characteristics of said cell types.

[0295] It is also contemplated that another aspect of the present invention may relate to a composition comprising the population of pancreatic lineage cells obtained from the methods disclosed herein.

[0296] In various embodiments, the composition may comprise a population of pancreatic progenitor 1 cells, pancreatic progenitor 2 cells, endocrine progenitor cells, or insulin-secreting pancreatic isletlike cells. These cells may be obtained from the respective stage of differentiation in the methods disclosed herein.

[0297] In various embodiments, the composition may comprise a population of cells, wherein at least 70%, at least 80%, at least 90%, at least 95% or more cells are pancreatic progenitor 1 cells, pancreatic progenitor 2 cells, endocrine progenitor cells, or insulin-secreting pancreatic islet-like cells.

[0298] In various embodiments, the composition may comprise a population of cells, wherein at least 70%, at least 80%, at least 90%, at least 95% or more cells express markers characteristic of pancreatic progenitor 1 cells, and a medium comprising an effective amount of BMP type I receptor inhibitor for differentiating the pancreatic progenitor 1 cells into pancreatic progenitor 2 cells. In various embodiments, pancreatic progenitor 1 cells express PDX1 , FOXA2 and HNF1p. In various embodiments, the effective amount of BMP type I receptor inhibitor is 0.1 -0.2 pM of LDN193189. In various embodiments, the medium further comprises FAF BSA, FGF7, RA, SANT1 , and Nicotinamide.In various embodiments, the medium further comprises about 2% FAF BSA, about 50 ng / ml FGF7, about 0.1 pM RA, about 0.25 pM SANT1 , and about 10 mM Nicotinamide.

[0299] In various embodiments, the composition may comprise a population of cells, wherein at least 70%, at least 80%, at least 90%, at least 95% or more cells are pancreatic progenitor 2 cells, and a medium comprising an effective amount of BMP type I receptor inhibitor for differentiating the pancreatic progenitor 2 cells into pancreatic endocrine progenitor cells. In various embodiments, the pancreatic progenitor 2 cells express PDX1 and NKX6.1. In various embodiments, the effective amount of BMP type I receptor inhibitor is 0.1 -0.2 pM of LDN193189. In various embodiments, the medium further comprises FAF BSA, RA, SANT1 , XXI, ALK5ill, T3, and Betacellulin. In various embodiments, the medium further comprises about 2% FAF BSA, about 0.1 pM RA, about 0.25 pM SANT1 , about 1 pM XXI, about 10 pM ALK5ill, about 1 pM T3, and about 20 ng / ml Betacellulin.

[0300] In various embodiments, the composition may comprise a population of cells, wherein at least 70%, at least 80%, at least 90%, at least 95% or more cells are pancreatic endocrine progenitor cells, and a medium comprising effective amounts of glucose, ZnSC , NAC and ISX9 for differentiating the endocrine progenitor cells into insulin-secreting pancreatic islet-like cells. In various embodiments, the pancreatic endocrine progenitor cells express NEUROG3 (NGN3). In various embodiments, the medium comprises about 20mM glucose, about 10pM ZnSC , about 1mM NAC, and about 0-1 OpM ISX9. In various embodiments, the medium further comprises FAF BSA, ALK5i II , and T3. In various embodiments, the medium further comprises about 2% FAF BSA, about 10pM ALK5ill, and about 1pM T3.

[0301] In various embodiments, the composition may comprise a population of the islet-like cells disclosed herein. In various embodiments, the composition may comprise a substantially purified population of islet-like cells, or a mixed population of endocrine lineage cells comprising one or more p-like, ct-like, 6-like, PP-like, and / or e-like cells. In various embodiments, the composition comprises cells expressing one or more markers characteristic of functional islet-like cells, including INS, C-peptide, NKX6.1 , MAFA, PDX1 , IAPP, PCSK1 / 2, GLUT2, or UCN3, and optionally glucagon, somatostatin, grehlin or pancreatic polypeptides.

[0302] In various embodiments, the composition comprises any formulation containing or supporting cells generated according to the methods disclosed herein, including liquids, gels, hydrogels, scaffolds, cell suspensions, encapsulated cell formats, pharmaceutical formulations, cryopreserved stocks, or implantable devices.

[0303] In various embodiments, the composition is a pharmaceutical composition comprising the isletlike cells and a pharmaceutically acceptable excipient (e.g., pharmaceutically acceptable carrier, diluent, buffer, stabiliser, cryoprotectant, or excipient) suitable for administration to a subject. In variousembodiments, the pharmaceutically acceptable carrier may comprise saline, phosphate-buffered saline (PBS), Ringer’s solution, PlasmaLyte, human serum albumin (HSA), or combinations thereof.

[0304] In various embodiments, the pharmaceutical composition may further comprise one or more viscosity modulators, osmoprotectants, antioxidants, anti-apoptotic factors, extracellular matrix (ECM) proteins, or cell survival-promoting agents, such as nicotinamide, N-acetyl cysteine (NAC), laminins, vitronectin, collagen derivatives, or ROCK inhibitors. In various embodiments, the pharmaceutical composition is formulated to maintain viability, functional integrity, and insulin secretory capacity of the islet-like cells prior to, during, or after administration.

[0305] In various embodiments, the composition may further comprise the insulin-secreting pancreatic islet-like cells encapsulated within, embedded in, or adhered to a biomaterial, hydrogel, scaffold, or immunoprotective device. Examples include alginate hydrogels, PEG-based hydrogels, fibrin matrices, collagen matrices, hyaluronic acid hydrogels, or synthetic polymeric capsules.

[0306] In various embodiments, the insulin-secreting pancreatic islet-like cells may be contained within a macroencapsulation device, microcapsule, semi-permeable membrane, or retrievable implantable device configured to permit diffusion of oxygen, nutrients, glucose, and insulin or other secreted hormones while limiting immune cell infiltration.

[0307] In various embodiments, the composition may be formulated for transplantation into a subject in need thereof, optionally for treatment of diabetes mellitus, impaired glucose tolerance, or endocrine insufficiency. In such embodiments, the composition may be prepared in a form suitable for implantation into sites such as the liver (via portal infusion), subcutaneous tissue, omentum, intramuscular space, renal subcapsular space, or other transplantation sites known in the art.

[0308] In various embodiments, the composition comprises a therapeutically effective amount of insulin-secreting pancreatic islet-like cells sufficient to improve glycaemic control, restore insulin production, or provide glucose-stimulated insulin secretion upon administration to the subject.

[0309] In various embodiments, the composition may be a cryopreserved composition comprising islet-like cells suspended in a cryopreservation solution, such as a medium containing dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, trehalose, sucrose, HSA, or combinations thereof, optionally supplemented with antioxidants or metabolic protectants. The cryopreserved composition may be thawed and formulated immediately prior to administration or further differentiation.

[0310] In various embodiments, the composition may comprise the insulin-secreting pancreatic isletlike cells in combination with one or more additional cell types, including endothelial cells, mesenchymal stromal cells, pancreatic ductal cells, or immune-modifying regulatory cells, optionally to enhance engraftment, vascularisation, or function following transplantation.

[0311] In various embodiments, the composition further comprises a biocompatible matrix, such as Matrigel™, laminin-rich ECM, vitronectin-coated microcarriers, or polysaccharide-based scaffolds to support survival, mechanical integrity, and functional maturation of the islet-like cells.

[0312] In various embodiments, the composition comprises islet-like spheroids or aggregates, optionally in combination with microcarriers, ECM proteins, or hydrogel encapsulation to facilitate handling and implantation.❖ Methods of Use

[0313] All embodiments disclosed above in relation to the pancreatic lineage cells, and compositions similarly apply to their methods of use and vice versa.

[0314] In various embodiments, the present invention provides the use of the functional islet-like cells, or a population or composition comprising said islet-like cells, for any therapeutic, prophylactic, diagnostic, or research purpose. Such uses include, but are not limited to, restoring, replacing, supplementing, or modelling the function of pancreatic islets, or providing insulin-producing endocrine activity.

[0315] In various embodiments, the islet-like cells generated by the method described herein may be used to treat, alleviate, stabilise, or reverse a disease or condition associated with, resulting from, or characterised by dysfunction, loss, or deficiency of endogenous islets of Langerhans.

[0316] In various embodiments, the islet-like cells, populations, or compositions disclosed herein may be formulated with a pharmaceutically acceptable excipient (e.g., pharmaceutically acceptable carrier, excipient, buffer, or delivery matrix) for use in vivo. In various embodiments, the islet-like cells may be provided as spheroids, clusters, aggregates, or encapsulated units suitable for transplantation, infusion, or implantation into a subject. In various embodiments, the compositions may be delivered via a medical device, encapsulation, or macrodevice configured to sustain endocrine function while modulating host immune exposure.

[0317] In various embodiments, the islet-like cells, populations, or compositions may be used in non-therapeutic methods, including disease modelling, drug screening, toxicity testing, assay development, or personalised medicine. Such in vitro uses may employ the islet-like cells to evaluate p-cel I function, glucose responsiveness, endocrine gene regulation, or the effects of test compounds on insulin secretion or endocrine viability. In various embodiments, the islet-like cells may be used for modelling monogenic diabetes, autoimmune diabetes, or metabolic stress pathways in vitro. In other embodiments, the cells are used for screening small molecules, biologies, or genetic perturbations that affect p-cell survival, insulin secretion, or endocrine differentiation.

[0318] In one aspect, there is provided a method of treating a metabolic disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the islet-like cells, populations, or compositions disclosed herein. In various embodiments, a therapeutically effective amount of the islet-like cells required is smaller for transplant to achieve an insulin level in vivo relative to undifferentiated hiPSCs.

[0319] In various embodiments, the composition is a pharmaceutical composition disclosed herein.

[0320] In various embodiments, the pharmaceutical composition comprises a population of cells and a pharmaceutically acceptable excipient, wherein at least 70% cells express markers characteristic of insulin-secreting pancreatic islet-like cells, wherein the insulin-secreting pancreatic islet-like cells exhibit a biphasic insulin secretion in response to glucose stimulation and KCI stimulation. In various embodiments, the insulin-secreting pancreatic islet-like cells exhibit about 8-30 fold increased insulin secretion when contacted with a high glucose concentration (e.g. 15-25.5 mM). In various embodiments, the insulin-secreting pancreatic islet-like cells exhibit decreased insulin secretion when contacted with a low glucose concentrations (e.g. 0-3 mM) following contacting with the high glucose concentration. In various embodiments, the islet-like cells express islet cell markers selected from the group comprising C-peptide, PDX1 , NKX6.1 , and glucagon. In various embodiments, the hiPSCs are Asian donor-derived hiPSCs selected from the group consisting of i70b, i173b, is104, and is043.

[0321] In various embodiments, provided herein are methods of treating a metabolic disorder in a subject in need thereof. In various embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising a population of cells and a pharmaceutically acceptable excipient. In various embodiments, at least 70%, at least 80%, at least 90%, at least 95%, or more cells express markers characteristic of insulin-secreting pancreatic islet-like cells. In various embodiments, the methods comprise administering pharmaceutical compositions described herein by portal vein infusion, subcutaneous implantation, intra-muscular implantation, or intra-omental implantation. The metabolic disorder maybe any condition characterised by impaired insulin production, p-cell loss, glucose dysregulation, or endocrine deficiency. In various embodiments, the metabolic disorder is selected from the group consisting of: type 1 diabetes mellitus (T1 DM), type 2 diabetes mellitus (T2DM), hyperglycemia, pre-diabetes, diabetes induced by surgical pancreatectomy, monogenic diabetes (including maturity-onset diabetes of the young (MODY)), and neonatal diabetes. Also, provided herein are use of the pharmaceutical compositions described herein in the manufacture of a medicament for treating a metabolic disorder in a subject in need thereof.

[0322] In various embodiments, the subject may be a human. In other embodiments, the subject may be a non-human mammal, including rodents, pigs, dogs, or non-human primates, for preclinical, veterinary, or translational research applications. In various embodiments, the subject may be a mouse.

[0323] In various embodiments, there is provided the use of the islet-like cells, populations, or compositions disclosed herein in the manufacture of a medicament for treating a metabolic disorder in a subject in need thereof. In various embodiments, compositions described herein comprise at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, or more islet-like cells are spherical. In various embodiments, compositions described herein comprise the islet-like cells, wherein at least 80%, at least 90%, at least 95%, or more of the islet-like cells have diameter in a range of from 100 pm to 250 pm, from 125 pm to 250 pm, from 150 pm to 250 pm, from 100 pm to 200 pm, from 125 pm to 200 pm, or from 150 pm to 200 pm. In various embodiments, compositions described herein comprise the islet-like cells, wherein at least 80%, at least 90%, at least 95%, or more of the islet-like cells have diameter in a range of from 150 pm to 250 pm. In various embodiments, compositions described herein comprise polyhormonal beta cells, pancreatic progenitor-like, mesenchymal-like cells and / or enterochromaffin cells, as determined by scRNA-seq.

[0324] In various embodiments, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the islet-like cells are polyhormonal beta cells. In various embodiments, 5-50%, 5-40%, 5-30%, 5-20%, 10-50%, 5-40%, 10-40%, 20-40%, 5-30%, 10-30%, or 5-20% of the islet-like cells are polyhormonal beta cells. In various embodiments, 10-40% of the islet-like cells are polyhormonal beta cells. In various embodiments, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 8%, at least 10%, at least 12% or at least 15% of the islet-like cells are pancreatic progenitor-like. In various embodiments, 0.5-15%, 1-15%, 5-15%, 10-15%, 0.5-10%, 1 -10%, 5-10%, 0.5-5%, or 1-5% of the isletlike cells are pancreatic progenitor-like. In various embodiments, 1-10% of the islet-like cells are pancreatic progenitor-like. In various embodiments, at least 0.1%, at least 0.5%, at least 1%, or at least 2% of the islet-like cells are mesenchymal-like cells. In various embodiments, 0.1-2%, 0.5-2%, 1-2%, 0.1-1%, 0.5-1%, 0.1 -0.5% of the islet-like cells are mesenchymal-like cells. In various embodiments, 0.1 -1 % of the islet-like cells are mesenchymal-like cells. In various embodiments, at least 0.5%, at least 1 %, at least 2%, at least 5%, or at least 10% of the islet-like cells are enterochromaffin cells. In various embodiments, 0.5-10%, 1-10%, 5-10%, 0.5-5%, 1 -5% or 0.5-1% of the islet-like cells are enterochromaffin cells. In various embodiments, 1-5% of the islet-like cells are enterochromaffin cells.

[0325] In various embodiments, the islet-like cells, populations, or compositions disclosed herein may be administered via any medically acceptable route capable of delivering viable endocrine tissue. Suitable routes of administration include, but are not limited to, portal vein infusion, subcutaneous implantation, intra-muscular implantation, intra-omental implantation, or delivery via a medical device, encapsulation, or macrodevice. In various embodiments, for animal models such as mice, the islet-like cells may be transplanted beneath the kidney capsule to assess graft function, glucose responsiveness, or insulin secretion in vivo.

[0326] In certain embodiments, the method of treatment comprises transplanting the islet-like cells via the portal vein. In other embodiments, the cells are delivered subcutaneously, optionally encapsulated in a microcapsule or macrodevice.

[0327] In various embodiments, the subject is a mouse and the islet-like cells, populations, or compositions disclosed herein are transplanted beneath the kidney capsule. In other embodiments, the subject is a human and the islet-like cells are delivered via a portal infusion or a subcutaneous implant. In various embodiments, the islet-like cells, populations, or compositions disclosed herein may be administered to the human via transplantation to the kidney.

[0328] The present invention is further illustrated by the following examples. However, it should be understood, that the invention is not limited to the exemplified embodiments.EXAMPLESMaterials and Methods

[0329] Cell culture: All mammalian cells were routinely tested to be mycoplasma-free using a MycoAlert™ PLUS mycoplasma detection kit (Lonza Bioscience, LT07-710). All mammalian cells were cultured in a 5% CO2 humidified incubator at 37 °C. hiPSC lines generated herein were cultured in TeSR™-E8™ or mTeSR-1 ™medium (StemCell Technologies, 05990 or 85850) with daily media changes. hiPSCs were passaged once weekly by colony picking or ReLeSR™ (StemCell Technologies, 05872) according to the manufacturer’s instructions. Culture plates were precoated with 0.1% gelatin in cell culture-grade water for at least 10 min and then with MEF media for at least 48 h prior to plating.

[0330] hiPSC differentiation to SC-islets: hiPSCs were dissociated using TrypLE (Thermo Fisher, 12605010) and cultured in mTeSRI (StemCell Technologies, 85850) supplemented with 10 pM of Y-27632 dihydrochloride (AbCam, ab120129). The following day, the medium was changed to mTeSR™1, and differentiation was started 24 h after plating. Directed differentiation protocol was performed using MCDB131 basal medium formulated as mentioned above.

[0331] Dynamic glucose stimulated insulin secretion (GSIS) assay: Day 35 SC-islets were gently washed three times with pre-warmed secretion assay buffer (114 mM sodium chloride, 4.7 mM potassium chloride, 1.2 mM calcium chloride, 1.2 mM potassium phosphate, 1.16 mM magnesium sulfate, 25 mM sodium bicarbonate, 0.2% fatty acid-free BSA (Proliant, 68700), 20 mM HEPES, adjusted to pH 7.3). Cells were then incubated in secretion assay buffer for 1 h before insulin secretion capacity was assessed using an automated perifusion system (Biorep). With a flow rate set at 10OpiL / min, SC-islets were subjected to 60min of perfusion with 2.5mM glucose in KRB buffer, before sequential stimulation with 2.8 mM glucose for 5min, 16.7 mM glucose for 40 min, 2.8 mM glucose for 15min, 30mM KCI for 5min, and 2.8 mM glucose for 5min. Secreted insulin was collected as flowthrough for quantification using human insulin ELISA kit.

[0332] Total insulin content extraction: At the end of dynamic GSIS, SC-islets were collected and washed twice with phosphate-buffered saline (PBS). After washing, 300|iL of acid / ethanol solution was added. The cells were incubated at 4 °C overnight before total insulin extraction was performed usinga sonication at 20% amplitude for 3 seconds. After sonication, the insulin extracts were centrifuged at 7,000rpm for 5min. The top aqueous layer containing insulin was collected and subjected to human insulin ELISA assay (Mercodia, 10-1113-10) while the bottom layer (containing cell pellet) was boiled to dryness at 80 °C on a heat block. The dried pellet was resuspended in 100| L of nuclease free water for total DNA quantification. All data involving total insulin content were normalised to total DNA.

[0333] Immunofluorescence staining of hiPSC-derived islet sections: SC-islets and SC-islets explanted from mice kidneys were fixed in 4% paraformaldehyde (WAKO, 163-20145) overnight at 4 °C and subsequently placed in 30% sucrose overnight at overnight at 4 °C. Cryo-embedding, cryo-block processing and sectioning were performed by Advanced Molecular Pathology Laboratory (AMPL, A*STAR). For immunostaining, cryosections were fixed with 4% paraformaldehyde for 10mim. Blocking and cell membrane permeabilization were performed using 5% BSAwith 0.1% triton-X (Merck Millipore, 9410) and incubated at room temperature for 1 hr. After 1 hr, the sections were incubated with primary antibodies (anti-goat NKX6.1 LifeSpan #c124275, anti-rabbit insulin CST #C2729, anti-rat PDX1 R&D AF2419, anti-rat c-peptide #GN1 D4, anti-rabbit glucagon cst#2760s) were incubated at 1 :100 overnight at 4 °C. The sections were washed with PBS twice and secondary antibodies were incubated at room temperature at 1 :500 for 1 hr. The sections were washed with PBS twice and incubated with DAPI (1 :5000) (Sigma-Aldrich D9542) for 10min. The sections were washed twice and mounting reagent was placed with a coverslip for imaging using Zeiss inverted confocal microscope, LSM700.

[0334] Generation of diabetic mice model: A vial of 50mg of STZ (Sigma S0130) was opened and dissolved in sodium citrate dihydrate buffer (pH 4.5) to make up a final concentration of 9mg / ml. The mixture is filtered through a 0.2 urn filter. Each mouse is weighed to determine the volume of STZ to be injected, as shown in the calculations below. Within 30minutes of reconstitution of STZ, 160mg / kg is injected by intra-peritoneal injection into each mouse to induce diabetes. Blood glucose is monitored by pricking the tail vein every 2 to 3 days. By the 7thday, diabetic phenotype observed by hyperglycaemia is observed.

[0335] In vivo glucose tolerance test (GTT): Mice were fasted overnight for up to 16hrs. Mice were provided with drinking water during fasting. Glucose (2 mg / g) was administered by intraperitoneal injection at 10 ul solution per gram body weight at TO. Blood collection was performed under a mechanical restrainer and the blood from the tail vein was collected at TO, T15, T20. Blood glucose measurement was performed using a glucometer during TO, T15, T30, T60, T90, T120. The blood collected was placed at room temperature for 30min to allow coagulation, before being placed on ice. All blood samples were spun down at 10,000rpm for 10min at 4 °C. The supernatant was collected andstored at -80 °C for subsequent human insulin (Mercodia, 10-1113-10) orc-peptide (Mercodia, 10-1136-01) ELISA assays performed according to the manufacturer protocol.Results and DiscussionExample 1: Protocol to differentiate hiPSCs and hESCs into functional, insulin-secreting pancreatic islet-like cells (hiPSC-islets).

[0336] The setup of differentiation involves the dissociation of hiPSCs into single cells and seeding a density of 0.6-1.2 x 106cells per ml in a suspension culture in mTeSR media. The total cell number ranges from 3- 4 x 106cells per well of a 6-well plate. Cells are cultured for 1-2 days as spheroids on an orbital shaker at 70-80 rpm.

[0337] The directed differentiation of hiPSCs to islet cells occurs in 6 stages through a series of changes in media conditions, and differentiation agents, whereby reference to “additions” refers to the exchange of media comprising the indicated supplements.Stage 7: For initiation of differentiation from hiPSCs to definitive endoderm, 1 - 2 % fatty acid-free (FAF) BSA in Stage 1 media is used with 100 ng / ml Activin A (TGF-p family) and 1-3 pM CHIR99021 (GSK3-p) for 1 day. This is followed by the addition of 100 ng / ml Activin A for 2 days.Stage 2. For differentiation to primitive gut tube, 2 % FAF BSA in Stage 2 media is used with 50 ng / ml FGF7 (also KGF).Stage 3: For differentiation to pancreatic progenitor 1 , 2 % FAF BSA in Stage 3 media is used with 50 ng / ml FGF7, 2 pM RA, 0.25 pM SANT1 (Shh signalling pathway antagonist), 0.5 pM PDBu (protein kinase C activator), 0.1 - 0.2 pM LDN193189 (BMP pathway inhibitor) for 2 days.Stage 4: For differentiation to pancreatic progenitor 2, 2 % FAF BSA in Stage 3 media is used with 50 ng / ml FGF7, 0.1 pM RA, 0.25 pM SANT1 , 0.1 - 0.2 pM LDN193189 and 10 mM Nicotinamide for 4 - 6 days.Stage 5. For differentiation to endocrine progenitor cells, 2 % FAF BSA in Stage 5 media is used with 0.1 pM RA, 0.25 pM SANT1 , 1 pM XXI (gamma-secretase inhibitor), 10 pM ALK5III (TGF-p type I receptor inhibitor), 1 pM T3 (triiodothyronine), 20 ng / ml Betacellulin, 0.1 -0.2 pM LDN193189 for 4 days. This is followed by the addition of 25 nM RA, 0.25 pM SANT1, 1 pM XXI, 10 pM ALK5ill, 1 pM T3, 20 ng / ml Betacellulin, 0.1- 0.2 pM LDN193189 and 0- 10 pM ISX9 (Wnt / beta-catenin activator) or equivalent for 3 days.Stage 6 For differentiation to islet-like cells, 2 % FAF BSA in Stage 6 media with 20 mM glucose and 10 pM ZnSO4 is used with addition of 10 pM ALK5HI, 1 pM T3, 1 mM NAC (N-acetyl cysteine), and 0 - 10 pM ISX9 for 2 days. This is followed by the addition of 10 pM ALK5HI, 1 pM T3, 1 mM NAC for 2 - 4 days. This is followed by the addition of 10 pM ALK5ill, 1 pM T3, 1 mM NAC, 0.5 pM R428 (bemcentinib, Axl inhibitor) for 10-20 days.

[0338] The protocol used to generate the data shown in FIG. 2A and 2B is described as such: Start of spheroid culture with 3.2 x 106cells in 3 ml of mTeSR media for 1 day on an orbital shaker at 70 rpm.Stage 1 : 1 % FAF BSA media with 100 ng / ml Activin A and 1 pM CHIR99021 for 1 day. Followed by Activin A 100 ng / ml for 2 days.Stage 2: 2 % FAF BSA in stage 2 media onwards, addition of 50 ng / ml FGF7 for 3 days. Stage 3: Stage 3 media with addition of 50 ng / ml FGF7, 2 pM RA, 0.25 pM SANT1, 0.5 pM PDBu, 0.2 pM LDN193189 for 2 days.Stage 4: Stage 3 media with addition of 50 ng / ml FGF7, 0.1 pM RA, 0.25 pM SANT1 , 0.2 pM LDN193189, 10 mM Nicotinamide for 5 days.Stage 5: Stage 5 media with addition of 0.1 pM RA, 0.25 pM SANT1 , 1 pM XXI, 10 pM ALK5III, 1 pM T3, 20 ng / ml Betacellulin, 0.2 pM LDN193189 for 4 days. Followed by addition of 0.1 pM RA, 0.25 pM SANT1 , 1 pM XXI, 10 pM ALK5ill, 1 pM T3, 20 ng / ml Betacellulin, 0.2 pM LDN193189, 10 pM ISX9 for 3 days.Stage 6: From day 20, Stage 6 media with addition of 10 pM ALK5ill, 1 pM T3, 1 mM NAC, 20 mM glucose, 10 pM ZnSO4 and 10 pM ISX9 for 2 days. Followed by addition of 10 pM ALK5HI, 1 pM T3, 1 mM NAC, 20 mM glucose, 10 pM ZnSO4 for 2 days. Followed by addition of 10 pM ALK5III, 1 pM T3, 1 mM NAC, 20 mM glucose, 10 pM ZnSO4, 0.5 pM R428 for 12 days. Followed by addition of 1 pM T3, 1 mM NAC, 20 mM glucose, 10 pM ZnSO4, 0.5 pM R428 for 7 days.

[0339] In this regard, the differentiation protocol disclosed herein employs an extended duration of BMP type I receptor inhibition by LDN193189 (0.1 -0.2 pM) or equivalent culture from stage 3 to stage 5 (day 7 to day 19).

[0340] In stage 3, a combination of BMP type I receptor inhibition (0.1 -0.2 pM LDN193189) with keratinocyte growth factor signalling and stimulation of osteogenesis and adipogenesis through p-catenin transduction (50 ng / ml FGF7), retinoic acid signalling (2 pM RA), sonic hedgehog inhibitor (0.25 pM SANT1), protein kinase C activation (0.5 pM PDBu) was shown to lead to the commitment of foregut into pancreatic lineage.

[0341] In stage 4, a combination of 0.1 -0.2 pM LDN193189 with 50 ng / ml FGF7, 0.1 pM RA, 0.25 pM SANT1 , nicotinamide signalling (10 mM Nicotinamide) was shown to promote pancreatic progenitor development.

[0342] In stage 5, a combination of 0.1 -0.2 pM LDN193189 with 0.1 pM RA, 0.25 pM SANT1 , gamma-secretase inhibitor (1 pM XXI), TGF-p receptor inhibitor (10 pM ALK5ill), thyroid hormone (1 pM T3) and EGF signalling (20 ng / ml Betacellulin) was shown to play a role to further promote pancreatic progenitor development.

[0343] In addition, the addition of NEUROD1 enhancer and also Wnt / beta-catenin activator (10 pM ISX9) at stage 5, in combination with 0.1 pM RA, 0.25 pM SANT1 , 1 pM XXI, 10 pM ALK5ill, 1 pM T3, 20 ng / ml Betacellulin and 0.1 -0.2 pM LDN193189 was shown to further enhance differentiation to endocrine progenitors.

[0344] In Stage 6, the media containing up to 10 pM ZnSCu and 20 mM glucose was used in addition to anti-oxidant 1 mM NAC and 0-10 pM ISX9 for 2 days. Further, the Stage 6 media containing up to 10 pM ZnSC and 20 mM glucose is used in addition to 1 mM NAC and 0.5 pM R428 Axl inhibitor for 10-20 days to promote the maturation of insulin-secreting beta cells.

[0345] As such, the differentiation of hPSC spheroids to islet cells was shown to be possible in a fully suspension culture protocol, as well as the use of Asian-derived iPSCs to generate such functional islet cells.

[0346] The improved differentiation protocol described herein has been tested in several cell lines including hESCs (H9) and hiPSCs (i70b, i173b, 43e5, is104c). Evaluation of insulin secretion function using a dynamic glucose-stimulated insulin secretion (GSIS) assay shows that the cells respond to high glucose stimulation (16.7 mM) through secretion of insulin (both first and second phase), display reduction of insulin secretion down to basal levels upon return to low glucose conditions (2.8 mM) and also respond to KCI (30 mM) stimulation.Example 2: hiPSC-Derived Islet Spheroids Are Glucose-Responsive and Secrete Insulin

[0347] Islet cells were validated using a dynamic glucose-stimulated insulin secretion (GSIS) assay in a perifusion system, to determine the functionality of the cells through its insulin secretion profile in response to basal glucose levels (2.8 mM), high glucose (16.7 mM), and KCI stimulation (positive control).

[0348] The results presented in FIG. 3 demonstrate that the hiPSC-derived islet spheroids exhibit characteristic glucose-responsive insulin-secretion behaviour, confirming the presence of functional p-like cells within the differentiated clusters. This was demonstrated from dynamic GSIS data showing SC-islets exhibited low basal insulin secretion at 2.8mM glucose, followed by a modest increase upon stimulation with 16.7mM glucose, and a robust secretory response to 30mM KCI depolarization, followed by a return toward basal levels during low-glucose washout This was akin to human islets dynamic GSIS data in FIG.4.

[0349] The reproducibility of these stimulus-dependent secretion profiles across multiple hiPSC lines (including i70b, i173b, and 43e5) further supports the functionality of the generated islet spheroids. Collectively, these findings establish that the differentiated clusters are capable of dynamic, physiologically relevant insulin secretion in response to both metabolic (glucose) and depolarising (KCI) stimuli.Example 3: hiPSC-Derived Islet Function Is Comparable With Primary Human Islets

[0350] The generated hiPSC-derived islet cells were shown to display glucose responsiveness and contain total insulin content like that observed in primary human islets from cadaveric donors.

[0351] The data presented in FIG.4demonstrate that the functional performance and insulin-producing capacity of the hiPSC-derived islet spheroids are comparable to those of primary human islets. Primary human islets display a characteristic biphasic response to glucose challenge, including a rapid first-phase spike upon transition from 2.8 mM to 16.7 mM glucose. GSIS performed on one donor preparation also exhibit a robust insulin-secretion peak in response to KCI depolarisation, confirming preserved stimulus-secretion coupling and validating the assay conditions.

[0352] When compared with these physiological human islet responses, the hiPSC-derived islets display similar dynamic GSIS patterns, including strong first-phase glucose responsiveness and KCI-induced insulin release. This demonstrates that the differentiated stem -cell-derived p-like cells closely recapitulate the behaviour of endogenous human islets under perifusion stimuli.

[0353] FIG. 4 supports this functional equivalence by comparing total insulin content normalised to genomic DNA across primary human islets and multiple SC-islet preparations generated from hiPSC lines 43e5, i70b, and i173b, as well as hESC-derived islets (H9). Across all SC-islet lines examined, the total insulin content falls within the range observed in donor-derived human islets, indicating that the stem-cell-derived spheroids not only functionally respond to glucose but also maintain physiologically relevant levels of intracellular insulin.

[0354] Collectively, these findings confirm that the hiPSC-derived islets generated using the described differentiation protocol exhibit glucose responsiveness and insulin content comparable to primary human islets, supporting their suitability for applications in disease modelling, drug testing, and cellreplacement therapies.Example 4: hiPSC-Derived Islet Spheroids Display Expression of Key Islet Cell Markers

[0355] The fluorescence imaging results in FIG. 5A-5F demonstrate that the hiPSC-derived islet spheroids contain multiple key endocrine cell types characteristic of human pancreatic islets. As shown in FIG. 5A, the co-localisation of the p-cell transcription factor PDX1 with C-peptide indicates that the differentiation protocol successfully generates insulin-producing p-like cells that express essential lineage-defining transcriptional regulators. The presence of a substantial number of PDX1 C-peptide+cells is consistent with a committed pancreatic p-cell phenotype.

[0356] FIG. 5B further supports this finding, showing widespread insulin expression together with the P-cell maturation marker NKX6.1. The detection of NKX6.17INS+double-positive cells is indicative of cells progressing toward a fully functional p-cell identity, as NKX6.1 expression is a hallmark of mature human p-cells and is required for appropriate glucose-responsive insulin secretion.

[0357] In addition to p-like cells, the results in FIG. 5C show that the islet spheroids also contain glucagon-expressing a-like cells, as indicated by clear spatial localisation of GCG-positive cellsalongside C-peptide-positive cells. This distribution mirrors the cellular heterogeneity of native human islets, which comprise mixed endocrine cell populations organised into three-dimensional clusters. The presence of both p-like and ct-like cells demonstrates that the differentiation protocol supports multilineage islet endocrine development, yielding spheroids with a cellular composition similar to primary human islets.

[0358] FIG. 5D confirms by flow cytometry that the hiPSC-derived islets comprise distinct endocrine populations, including INS+p-like cells co-expressing the p-cell transcription factors PDX1 and NKX6.1 , and GCG+a-like cells. The reproducible detection of these marker-defined populations across independent differentiations demonstrates the robustness of the differentiation process. FIG.5E shows UMAP analysis of single-cell RNA sequencing data from day-35 hiPSC-derived islets, revealing discrete clusters corresponding to major pancreatic cell types, including a, p, bihormonal, 5, y (PP), acinar, ductal, endothelial, mesenchymal-like, and progenitor-like cells, consistent with a multicellular islet-like tissue. FIG. 5F molecularly annotates these clusters, with INS marking p cells, GCG a cells, SST 6 cells, PPY y cells, PRSS1 acinar cells, and KRT19 ductal cells. The concordance between marker gene expression and UMAP clustering confirms the identity and diversity of the cell populations within the spheroids. Together, FIGS. 5D-5F demonstrate that the hiPSC-derived islet spheroids contain well-defined pancreatic endocrine and supporting cell types, including mature p-like cells co-expressing INS, PDX1 and NKX6.1 , consistent with physiologically relevant human islet-like structures.

[0359] Together, these imaging data confirm that the hiPSC-derived islet spheroids express key islet hormones and transcription factors, supporting their identity as physiologically relevant islet-like structures.Example 5: Transplantation of hiPSC-Derived Islet Spheroids into Diabetic Mice Models For In Vivo Proof-Of-Concept

[0360] Intraperitoneal glucose tolerance test (IPGTT) was used to evaluate the function of the islet cells in vivo.

[0361] 3 to 8 weeks after cell transplantation, streptozotocin (STZ)-induced diabetic mice are fasted overnight and subsequently injected with high glucose. Blood glucose levels were monitored over a 2-hour period and mouse serum may be collected to evaluate the presence of human insulin or C-peptide secreted by the iPSC-derived islets. Hyperglycemia is resolved more quickly in diabetic mice transplanted with iPSC-derived spheroids (SC-islets) than diabetic mice without any transplants (STZ only).

[0362] Specifically, the data in FIG.6A and 6B demonstrate that transplantation of hiPSC-derived islet spheroids provides functional glucose regulation in vivo in a diabetic mouse model. During IPGTT, STZ-induced diabetic mice-lacking functional endogenous p-cells-display prolonged hyperglycaemia following glucose challenge, as expected for an insulin-deficient model. In contrast, mice transplantedwith SC-islets show substantially improved glucose clearance over the 120-minute test period, indicating that the transplanted cells secrete insulin or C-peptide in response to elevated blood glucose levels.

[0363] The glucose-lowering effect observed in SC-islet-transplanted mice is further supported by the AUG analysis shown in FIG. 6B. SC-islet recipients exhibit significantly reduced glucose AUG values compared to untreated STZ mice, demonstrating a measurable restoration of glucose homeostasis. Although not fully equivalent to healthy controls, the improvement in glycaemic control confirms that the transplanted hiPSC-derived spheroids are functional in vivo and capable of responding dynamically to metabolic stimuli.

[0364] Taken together, the findings shown in FIG. 6A and 6B provide in vivo proof-of-concept that hiPSC-derived islet spheroids can engraft, survive, and exert physiologically relevant glucose-regulating activity in diabetic animals, supporting their potential use in cell-replacement therapy for diabetes.

[0365] The results presented in FIG. 7A, 7B, 7C and 7D demonstrate that hiPSC-derived islet spheroids not only engraft in vivo but also maintain functional insulin secretion capacity over extended periods following transplantation into diabetic mice. As shown in FIG. 7A, human insulin becomes detectable in the serum of transplanted STZ-diabetic mice during IPGTT, indicating that the SC-islets respond appropriately to glucose stimulation and are capable of releasing human insulin into circulation in a physiologically regulated manner. As shown in FIG. 7B and 7C, human C-peptide becomes detectable in the serum of transplanted STZ-diabetic mice during IPGTT, indicating that the SC-islets respond appropriately to glucose stimulation and are capable of releasing human insulin into circulation in a physiologically regulated manner for long-term periods of 8 weeks and 20 weeks posttransplantation. FIG. 7D further supports the long-term functionality of the transplanted islets. Weekly measurement of serum human C-peptide-a stable marker of endogenous insulin secretion-reveals persistent and sustained detection over a period of up to two months post-transplantation in non-fasted animals. These measurements confirm that the hiPSC-derived p-like cells remain viable, engrafted, and metabolically active over the full monitoring period.

[0366] Collectively, the data in FIG. 7A, 7B, 7C and 7D provide strong in vivo evidence that transplanted hiPSC-derived islet spheroids can survive, integrate, and continuously secrete human insulin and C-peptide in diabetic hosts. This sustained endocrine activity supports the utility of SC-islets as a potential cell-replacement therapy for restoring insulin production in diabetes.

[0367] The immunofluorescence data shown in FIG.8 demonstrate that hiPSC-derived islet spheroids survive long-term in vivo and retain multi-lineage endocrine identity following transplantation. After eight weeks in diabetic mice, the explanted graft tissue contains widespread insulin- or C-peptide-positive cells, indicating the continued presence of human p-like cells capable of producing insulin or itsprocessed peptide products. These insulin- or C-peptide-producing cells are observed throughout the graft and display a distribution pattern consistent with organised endocrine tissue.

[0368] In addition to insulin-producing cells, the grafts also contain cells expressing key p-cell transcription factors such as PDX1 and NKX6.1 . The co-localisation of these transcription factors with C-peptide or insulin confirms that the transplanted p-like cells maintain lineage-appropriate molecular identity during the eight-week engraftment period. This retention of p-cell transcriptional markers supports the conclusion that the SC-islets undergo stable integration and maturation in vivo rather than dedifferentiation or loss of phenotype.

[0369] Furthermore, the explanted tissue contains glucagon-positive a-like cells co-existing alongside p-like cells. This demonstrates that the transplanted spheroids preserve multi-hormonal islet composition and maintain a-cell identity within the graft microenvironment. The presence of both p- and a-like cells mirrors the cellular heterogeneity of native human islets and supports the physiological relevance of the transplanted tissue.

[0370] Collectively, the findings in FIG.8 show that hiPSC-derived islet grafts survive, retain endocrine identity, and preserve expression of p-cell (INS / C-PEP, PDX1 , NKX6.1) and a-cell (GCG) markers after eight weeks in vivo, confirming long-term stability and persistence of human islet phenotypes posttransplant.

[0371] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims.

[0372] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The methods, cells, compositions, kits and uses described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0373] The invention illustratively described herein may suitably be practiced in the absence of anyelement or elements, limitation or limitations, not specifically disclosed herein. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed 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.

[0374] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

Claims

1. CLAIMSWhat is Claimed is:

1. An in vitro method for generating insulin-secreting pancreatic islet-like cells from a human induced pluripotent stem cells (hiPSCs) or human Embryonic Stem Cells (hESCs) derived pancreatic progenitor 1 cells, comprising:providing the pancreatic progenitor 1 cells:differentiating the pancreatic progenitor 1 cells in vitro in a Stage 4 medium under conditions and for a period of time sufficient to obtain pancreatic progenitor 2 cells, wherein the Stage 4 medium comprises a BMP type I receptor inhibitor;differentiating the pancreatic progenitor 2 cells in vitro in a first Stage 5 medium and a second Stage 5 medium under conditions for a first period of time sufficient and a second period of time sufficient, respectively, to obtain pancreatic endocrine progenitor cells, wherein each, the first Stage 5 medium and the second Stage 5 medium, comprises the BMP type I receptor inhibitor; and differentiating the pancreatic endocrine progenitor cells in vitro in a first Stage 6 medium, a second Stage 6 medium and a third Stage 6 medium under conditions for a first period of time sufficient, a second period of time sufficient, and a third period of time sufficient, respectively, to obtain insulinsecreting pancreatic islet-like cells, wherein the first Stage 6 medium comprises glucose, ZnSC , and N-acetyl cysteine (NAC) and ISX9.

2. The in vitro method of claim 1 , wherein the BMP type I receptor inhibitor is LDN193189.

3. The in vitro method of claim 2, wherein:the Stage 4 medium further comprises a bovine serum albumin (BSA), an activin and a GSK3 inhibitor,the first Stage 5 medium further comprises the BSA and a fibroblast growth factor (FGF), the second Stage 5 medium comprises the BSA, a retinoid, a Hedgehog pathway antagonist, a y-secretase inhibitor, a TGF-p receptor inhibitor, a thyroid hormone, an epidermal growth factor (EGF) family ligand, and LDN193189,the first Stage 6 medium further comprises the BSA, the FGF, a retinoid, a Hedgehog pathway antagonist, and a protein kinase C (PKC) activator,the second Stage 6 medium comprises the TGF-p receptor inhibitor, the thyroid hormone, and NAC, andthe third Stage 6 medium comprises the TGF-p receptor inhibitor, the thyroid hormone, NAC and an Axl inhibitor.

4. The in vitro method of claim 3, wherein:the BSA is fatty-acid-free bovine serum albumin (FAF BSA),the activin is Activin A,the GSK3P inhibitor is CHIR99021 ,the FGF is fibroblast growth factor 7 (FGF7),the retinoid is retinoic acid (RA),the Hedgehog pathway antagonist is SANT1 ,the y-secretase inhibitor is XXI,the TGF-p receptor inhibitor is ALK5il I ,the thyroid hormone is triiodothyronine (T3),the epidermal growth factor (EGF) family ligand is Betacellulin,the PKG activator is phorbol 12,13-dibutyrate (PDBu),the Axl inhibitor is R428, ora combination thereof.

5. The in vitro method of any one of claims 1-4, wherein the pancreatic progenitor 1 cells are provided by the following steps of,differentiating hiPSCs or hESCs in vitro in a Stage 1 medium under conditions and for a period of time sufficient to obtain definitive endoderm cells, wherein the Stage 1 medium comprises FAF BSA, Activin A and CHIR99021 ;differentiating the definitive endoderm cells in vitro in a Stage 2 medium under conditions and for a period of time sufficient to obtain primitive gut tube cells, wherein the Stage 2 medium comprises FAF BSA and FGF7; anddifferentiating the primitive gut tube cells in vitro in a Stage 3 medium under conditions and for a period of time sufficient to obtain pancreatic progenitor 1 cells, wherein the Stage 3 medium comprises FAF BSA, FGF7, RA, SANT1 , phorbol 12,13-dibutyrate (PDBu), and LDN193189.

6. An in vitro method for generating insulin-secreting pancreatic islet-like cells from human induced pluripotent stem cells (hiPSCs) or human Embryonic Stem Cells (hESCs), comprising:(1) differentiating hiPSCs or hESCs in vitro in a Stage 1 medium under conditions and for a period of time sufficient to obtain definitive endoderm cells, wherein the Stage 1 medium comprises fatty-acid-free BSA (FAF BSA), Activin A and CHIR99021 ;(2) differentiating the cells obtained from stage (1 ) in vitro in a Stage 2 medium under conditions and for a period of time sufficient to obtain primitive gut tube cells, wherein the Stage 2 medium comprises FAF BSA and fibroblast growth factor 7 (FGF7);(3) differentiating the cells obtained from stage (2) in vitro in a Stage 3 medium under conditions and for a period of time sufficient to obtain pancreatic progenitor 1 cells, wherein the Stage 3 medium comprises FAF BSA, FGF7, retinoic acid (RA), SANT1 , phorbol 12,13-dibutyrate (PDBu), and LDN193189;(4) differentiating the cells obtained from stage (3) in vitro in a Stage 4 medium under conditions and for a period of time sufficient to obtain pancreatic progenitor 2 cells, wherein the Stage 4 medium comprises FAF BSA, FGF7, RA, SANT1 , LDN193189 and Nicotinamide;(5) differentiating the cells obtained from stage (4) in vitro in a first Stage 5 medium a second Stage 5 medium under conditions and for a first period of time sufficient and a second period of time sufficient, respectively, to obtain pancreatic endocrine progenitor cells, wherein the first Stage 5 medium comprises FAF BSA, RA, SANT1 , XXI, ALK5III, T3, Betacellulin, and LDN193189;wherein the second Stage 5 medium comprises RA, XXI, ALK5HI, triiodothyronine (T3), Betacellulin, LDN193189 and ISX9; and(6) differentiating the cells obtained from stage (5) in vitro in a first Stage 6 medium and a second Stage 6 medium under conditions for a first period of time sufficient and a second period of time sufficient, respectively, to obtain islet-like cells, wherein the first Stage 6 medium comprises FAF BSA, glucose, ZnSC , ALK5ill, T3, N-acetyl cysteine (NAC), and ISX9 or an equivalent factor, wherein the second Stage 6 medium comprises ALK5ill, T3, NAC and R428.

7. The in vitro method of any one of claims 1-6, wherein all stages of the method are performed in a suspension culture system.

8. The in vitro method of claim 6, further comprising, before Stage 1 , an additional step of dissociating the hiPSCs or hESCs into single cells and culturing them as spheroids in an in vitro suspension culture system.

9. The in vitro method of claim 8, wherein the single cells are incubated at a density of 0.6 -1.2 x 106cells per mL for 1 -5 days under orbital rotation at a speed of 70-90 rpm.

10. The in vitro method of claim 5 or 6, wherein the Stage 1 medium comprises about 1-2 % FAF BSA, about 100 ng / ml Activin A, and about 1-3 pM CHIR99021 , preferably the CHIR99021 is at a concentration of about 1 pM, and the Stage 1 period of time is about 1 day.11 . The in vitro method of claim 10, wherein Stage (1 ) further comprises a subsequent Stage (1 ) differentiating step comprising replacing the Stage 1 medium with a second Stage 1 medium comprising about 100 ng / ml Activin A, wherein the subsequent differentiating step is for a period of time of about 2 days.

12. The in vitro method of claim 5 or 6, wherein the Stage 2 medium comprises about 2 % FAF BSA and about 50 ng / ml FGF7, and the Stage 2 period of time is about 3 days.

13. The in vitro method of claim 5 or 6, wherein the Stage 3 medium comprises about 2% FAF BSA, about 50 ng / ml FGF7, about 2 pM RA, about 0.25 pM SANT1 , about 0.5 pM PDBu, and 0.1-0.2 pM LDN193189, and the Stage 3 period of time is about 2 days.

14. The in vitro method of any one of claims 4-13, wherein the Stage 4 medium comprises about 2% FAF BSA, about 50 ng / ml FGF7, about 0.1 pM RA, about 0.25 pM SANT1 , 0.1 -0.2 pM LDN193189and about 10 mM Nicotinamide, wherein the Stage 4 period of time is about 4-6 days, preferably about 5 days.

15. The in vitro method of any one of claims 4-14, wherein the first Stage 5 medium comprises about 2% FAF BSA, about 0.1 pM RA, about 0.25 pM SANT1 , about 1 pM XXI, about 10 pM ALK5III, about 1 pM T3, about 20 ng / ml Betacellulin, and about 0.1 -0.2 pM LDN193189, wherein the Stage 5 period of time is about 4 days.

16. The in vitro method of claim 15, wherein Stage (5) further comprises a subsequent Stage (5) differentiating step comprising replacing the first Stage 5 medium with the second Stage 5 medium comprising about 25 nM RA, about 1pM XXI, about 10pM ALK5III, about 1pM T3, about 20 ng / ml Betacellulin, about 0.1 -0.2 pM LDN193189 and about 0-10 pM ISX9 or an equivalent factor, wherein the second period of time of about 3 days, preferably the equivalent factor is selected from the group consisting of CHIR99021 , 6-bromoindirubin-3'-oxime (BIO), lithium chloride (LiCI) T3, Alk5 inhibitors, PKA / cAMP agonists and epigenetic modulators.

17. The in vitro method of any one of claims 4-16, wherein the Stage 6 first medium comprises about 2% FAF BSA, about 20mM glucose, about 10pM ZnSCU, about 10pM ALK5HI, about 1pM T3, about 1 mM NAG, and about 0-1 OpM ISX9, wherein the first period of time is about 2-3 days.

18. The in vitro method of claim 17, wherein Stage (6) further comprisesa subsequent 2ndStage (6) differentiating step comprising replacing the first Stage 6 medium with the second Stage 6 medium comprising about 10pM ALK5ill, about 1pM T3, and about 1mM NAC, wherein the second period of time is about 2-4 days.

19. The in vitro method of claim 18, wherein Stage (6) further comprisesa subsequent 3rdStage (6) differentiating step comprising replacing the second Stage 6 medium with a third Stage 6 medium comprising about 10pM ALK5ill, about 1pM T3, about 1mM NAC, about 0.5pM R428, wherein the third period of time is about 10-20 days, preferably about 12 days.

20. The in vitro method of any one of claims 4-19, wherein LDN193189 is used continuously from Stage 3 to Stage 5 to inhibit BMP signalling, thereby enhancing pancreatic lineage commitment and pancreatic progenitor development.21 . The in vitro method of any one of claims 1-20, wherein the insulin-secreting pancreatic isletlike cells exhibit glucose-stimulated insulin secretion (GSIS) in response to glucose at a concentration of 15-25.5 mM, and KCI stimulation.

22. The in vitro method of claim 21 , wherein the insulin-secreting pancreatic islet-like cells exhibit decreased insulin secretion in response to low glucose concentration of 0-3 mM.

23. The in vitro method of any one of claims 1-22, wherein the hiPSCs are Asian donor-derived hiPSCs selected from the group consisting of i70b, i173b, is104, 43e5 and is043.

24. A population of insulin-secreting pancreatic islet-like cells generated using the in vitro method of any one of claims 1 -23, wherein the islet-like cells express islet cell markers selected from the group comprising C-peptide, PDX1 , NKX6.1 , and glucagon.

25. A composition comprising the population of insulin-secreting pancreatic islet-like cells of claim 24.

26. An method of treating a metabolic disorder in a subject in need thereof, comprising administering the composition of claim 25 to the subject, wherein the metabolic disorder is selected from the group consisting of diabetes, hyperglycemia, pre-diabetes, diabetes induced by pancreatectomy, monogenic forms of diabetes, and neonatal diabetes.

27. Use of the composition of claim 25 in the manufacture of a medicament for treating a metabolic disorder in a subject in need thereof, wherein the metabolic disorder is selected from the group consisting of diabetes, hyperglycemia, pre-diabetes, diabetes induced by pancreatectomy, monogenic forms of diabetes, and neonatal diabetes.

28. The method of claim 26, or the use of claim 27, wherein the subject is human.

29. The method or the use of claim 28, wherein the insulin-secreting pancreatic islet-like cells are administered via a route selected from the group consisting of portal vein injection, subcutaneous implantation, intra-muscular injection, and delivery via a medical device.

30. The method of claim 26, or the use of claim 27, wherein the subject is mouse.31 . The method or the use of claim 30, wherein the insulin-secreting pancreatic islet-like cells are administered via transplantation to kidney.

32. A kit for use in generating insulin-secreting pancreatic islet-like cells, comprising one or more of the Stage (1 )-(6) medium defined in the method of any one of claims 1 -6, preferably the kit comprises the Stage 4 medium, the Stage 5 mediums, and the Stage 6 mediums, and optionally the Stage 1 medium, the Stage 2 medium, and the Stage 3 medium.

33. A composition comprising:a population of cells, wherein at least 70% cells are pancreatic endocrine progenitor cells that express NEUROG3 (NGN3); anda medium comprising effective amounts of glucose, ZnSC , N-acetyl cysteine (NAC) and ISX9 for differentiating the endocrine progenitor cells into insulin-secreting pancreatic islet-like cells.

34. The composition of claim 33, wherein the medium comprises about 20mM glucose, about 10pM ZnSC>4, about 1 mM N-acetyl cysteine (NAC), and about 0-1 OpM ISX9.

35. The composition of claim 33 or 34, wherein the medium further comprises a bovine serum albumin (BSA), a TGF-p receptor inhibitor, and a thyroid hormone.

36. The composition of claim 35, wherein:the BSA is fatty-acid-free bovine serum albumin (FAF BSA),the TGF-p receptor inhibitor is ALK5il I, andthe thyroid hormone is triiodothyronine (T3).

37. The composition of claim 36, wherein the medium comprises about 2% fatty-acid-free bovine serum albumin (FAF BSA), about 10pM ALK5ill, and about 1pM triiodothyronine (T3).

38. A composition comprising:a population of cells, wherein at least 70% cells are pancreatic progenitor 2 cells that express PDX1 and NKX6.1 ; anda medium comprising an effective amount of BMP type I receptor inhibitor for differentiating the pancreatic progenitor 2 cells into endocrine progenitor cells.

39. The composition of claim 38, wherein the effective amount of BMP type I receptor inhibitor is about 0.1 - 0.2 pM of LDN193189.

40. The composition of claim 38 or 39, wherein the medium further comprises a bovine serum albumin (BSA), a retinoid, a Shh signalling pathway antagonist, a y-secretase inhibitor, a TGF-p receptor inhibitor, a thyroid hormone, and an epidermal growth factor (EGF) family ligand.41 . The composition of claim 40, wherein:the BSA is fatty-acid-free bovine serum albumin (FAF BSA),the retinoid is retinoic acid (RA),the Shh signaling pathway antagonist is SANT1 ,the y-secretase inhibitor is XXI,the TGF-p receptor inhibitor ALK5il I ,the thyroid hormone is triiodothyronine (T3), andthe EGF family ligand is Betacellulin.

42. The composition of claim 41 , wherein the medium comprises about 2% a fatty-acid-free bovine serum albumin (FAF BSA), about 0.1 pM retinoic acid (RA), about 0.25 pM SANT1 , about 1 pM XXI, about 10 pM ALK5il I , about 1 pM triiodothyronine (T3), and about 20 ng / ml Betacellulin.

43. A composition comprising:a population of cells, wherein at least 70% cells are pancreatic progenitor 1 cells that express PDX1, FOXA2 and HNF1 ; anda medium comprising an effective amount of BMP type I receptor inhibitor for differentiating the pancreatic progenitor 1 cells into pancreatic progenitor 2 cells.

44. The composition of claim 43, wherein the effective amount of BMP type I receptor inhibitor is 0.1 - 0.2 pM of LDN193189.

45. The composition of claim 43 or 44, wherein the medium further comprises a bovine serum albumin (BSA), a fibroblast growth factor (FGF), a retinoid, a Shh signalling pathway antagonist, and a nicotinamide metabolic cofactor.

46. The composition of claim 45, wherein:the BSA is fatty-acid-free bovine serum albumin (FAF BSA),the FGF is fibroblast growth factor 7,the retinoid is retinoic acid (RA),the Shh signalling pathway antagonist is SANT1 , andthe nicotinamide metabolic cofactor is Nicotinamide.

47. The composition of claim 46, wherein the medium comprises about 2% a fatty-acid-free bovine serum albumin (FAF BSA), about 50 ng / ml FGF7, about 0.1 pM retinoic acid (RA), about 0.25 pM SANT 1 , and about 10 mM Nicotinamide.

48. A composition comprising:a population of cells, wherein at least 70% cells are insulin-secreting pancreatic islet-like cells that express INS, PCSK1 , PCSK2, MAFA, PDX1 , and NKX6.1 ; anda medium comprising a TGF-f> receptor inhibitor, a thyroid hormone, a redox-modulating agent, and an Axl inhibitor,wherein the insulin-secreting pancreatic islet-like cells have at 100% more insulin content, normalized to total genomic DNA, relative to primary human islets.

49. The composition of claim 48, wherein:the TGF-p receptor inhibitor is ALK5ill,the thyroid hormone is triiodothyronine (T3),the redox-modulating agent is N-acetyl cysteine (NAG), andthe Axl inhibitor is R428.

50. The composition of claim 49, wherein the medium comprises 10 pM ALK5HI, 1 pM T3, 1 mM N-acetyl cysteine (NAC), and 0.5 pM R428.51 . A composition comprising:a population of cells, wherein at least 70% cells are insulin-secreting pancreatic islet-like cells that express INS, PCSK1 , PCSK2, MAFA, PDX1 , and NKX6.1 ; anda medium comprising a TGF- receptor inhibitor, a thyroid hormone, a redox-modulating agent, and an Axl inhibitor,wherein the insulin-secreting pancreatic islet-like cells exhibit about 8-30 fold increased insulin secretion in response to glucose at a concentration of 15-25.5 mM.

52. The composition of claim 51 , wherein:the TGF-p receptor inhibitor is ALK5HI,the thyroid hormone is triiodothyronine (T3),the redox-modulating agent is N-acetyl cysteine (NAC), andthe Axl inhibitor is R428.

53. The composition of claim 52, wherein the medium comprises 10 pM ALK5ill, 1 pM T3, 1 mM N-acetyl cysteine (NAC), and 0.5 pM R428.

54. A pharmaceutical composition comprising a population of cells and a pharmaceutically acceptable excipient, wherein at least 70% cells are insulin-secreting pancreatic islet-like cells that express INS, PCSK1 , PCSK2, MAFA, PDX1 , and NKX6.1 , wherein:the insulin-secreting pancreatic islet-like cells have at least 50% more insulin content, normalized to total genomic DNA, relative to primary human islets,the insulin-secreting pancreatic islet-like cells exhibit a biphasic insulin secretion in response to glucose stimulation and KCI stimulation,the insulin-secreting pancreatic islet-like cells exhibit about 8-30 fold increased insulin secretion when contacted with a high glucose concentration in a range of 15-25.5 mM, andthe insulin-secreting pancreatic islet-like cells exhibit decreased insulin secretion when contacted with a low glucose concentration in a range of 0-3 mM following contacting with the high glucose concentration.

55. A pharmaceutical composition comprising a population of cells and a pharmaceutically acceptable excipient, wherein at least 70% cells are insulin-secreting pancreatic islet-like cells that express INS, PCSK1 , PCSK2, MAFA, PDX1 , and NKX6.1 , wherein the insulin-secreting pancreatic islet-like cells have at least 100% more insulin content, normalized to total genomic DNA, relative to primary human islets.

56. The pharmaceutical composition of claim 55, wherein the population of cells comprise C-peptide positive cells, glucagon positive cells and somatostatin positive cells.

57. The pharmaceutical composition of claim 55 or 56, wherein the insulin-secreting pancreatic islet-like cells are generated from human induced pluripotent stem cells (hiPSCs).

58. The pharmaceutical composition of claim 57, wherein the hiPSCs are Asian donor-derived hiPSCs selected from the group consisting of i70b, i173b, is104, 43e5 and is043.

59. A method of treating a metabolic disorder in a subject in need thereof, comprising administering an effective amount of the pharmaceutical composition of any one of claims 54-58 to the subject, wherein the metabolic disorder is selected from the group consisting of type 1 diabetes mellitus (T1 DM), type 2 diabetes mellitus (T2DM), hyperglycemia, pre-diabetes, diabetes induced by surgical pancreatectomy, monogenic diabetes, and neonatal diabetes.

60. The method of claim 59, wherein the pharmaceutical composition is administered by portal vein infusion, subcutaneous implantation, intra-muscular implantation, or intra-omental implantation.61 . Use of the pharmaceutical composition of any one of claims 54-58 in the manufacture of a medicament for treating a metabolic disorder in a subject in need thereof, wherein the metabolic disorder is selected from the group consisting of type 1 diabetes mellitus (T1 DM), type 2 diabetes mellitus (T2DM), hyperglycemia, pre-diabetes, diabetes induced by surgical pancreatectomy, monogenic diabetes, and neonatal diabetes.

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