Method for preparing endoderm stem cells and pancreatic islets derived therefrom

Abstract

The present disclosure provides methods of producing a population of endodermal stem cells (EnSC), a population of EnSC-derived PP cells, a population of EnSC-derived EP cells, or regenerative pancreatic islet tissue (E-islets). The disclosure also relates to EnSC, EnSC-derived PP cells, EnSC-derived EP cells or regenerated pancreatic islet tissue obtained using the method, and therapeutic uses thereof.

Description

TECHNICAL FIELD

[0001] The present application relates generally to methods for preparing endodermal stem cells (EnSCs), progenitor EnSC-derived pancreatic progenitor (PP) cells, EnSC-derived endocrine progenitor (EP) cells, and EnSC-derived pancreatic islets (regenerative islet tissue (E-islets)), and the therapeutic use of EnSCs, EnSC-derived PP cells, EnSC-derived EP cells, and regenerative islet tissue in the treatment of diseases or conditions associated with impaired islet function. BACKGROUND

[0002] Human pluripotent stem cells (hPSCs) are capable of differentiating into a variety of functional cells or tissues, such as pancreatic progenitor cells (PPs) and islet tissue. Such cells have been shown to survive, function, and reverse hyperglycemia in animal models of diabetes. See, e.g., Pagliuca, F.W. et al., Cell 159, 428-439 (2014); Rezania, A. et al., Nat. Biotechnol. 32, 1121-1133 (2014); and Sneddon, J.B. et al., Cell Stem Cell 22, 810-823 (2018). Additionally, recent clinical trials have shown that hPSC-derived pancreatic endodermal cells can further mature into diet-responsive beta-like cells and secrete insulin when implanted subcutaneously in patients with type 1 diabetes, although the amount of secretion was insufficient to achieve independence from exogenous insulin. See, e.g., Ramzy, A. et al., Cell Stem Cell 28, 2047-2061 (2021); and Shapiro, A.M.J. et al., Cell Rep. Med. 2, 100466 (2021).

[0003] Nonetheless, the clinical application of hPSC-derived cells is negatively impacted by the complex differentiation procedures and the risk that any undifferentiated cells remaining in the system can form teratomas in vivo. Additionally, laboratory research and cell-based therapies using hPSCs are hindered by their limited ability to generate pure populations of differentiated cell types in vitro. Such limitations will affect the reproducibility and reliability of experiments, and the safety and efficacy of potential therapeutic applications.

[0004] Accordingly, there is a need to develop improved methods for generating high purity non-tumorigenic intermediate stem cell types for safer and more effective therapeutic applications, particularly for the treatment of diseases associated with impaired islet function, such as diabetes. SUMMARY

[0005] It is an object of the present application to provide a method for generating a population of endodermal stem cells, comprising: a) providing a population of pluripotent stem cells; b) culturing the population of pluripotent stem cells in a first culture medium comprising a Nodal signaling agonist and a WNT signaling agonist; c) culturing the cells in a second culture medium comprising a Nodal signaling agonist and fibroblast growth factor (FGF); d) culturing the cells in a third culture medium comprising a factor belonging to the TGF-β superfamily, FGF, hepatocyte growth factor (HGF), and VEGF; and e) culturing the cells in a fourth culture medium comprising a WNT signaling agonist, a TGF-β inhibitor, and epidermal growth factor (EGF); This generates a population of endoderm stem cells.

[0006] In another embodiment, the present disclosure provides a method for producing a pancreatic endocrine cell population, comprising: a) providing an endoderm stem cell population; b) culturing the endoderm stem cell population in the presence of a BMP inhibitor, a Nodal signaling agonist, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and nicotinamide to generate a pancreatic progenitor (PP) cell population; c) culturing the PP cell population in the presence of a BMP inhibitor, a TGF-β inhibitor, and a γ-secretase inhibitor to generate an endocrine progenitor (EP) cell population; and d) culturing the EP cell population in the presence of the factors involved in step c and T3 and nicotinamide; This gives rise to the pancreatic endocrine cell population.

[0007] In another embodiment, the present disclosure provides a method for treating a disease or condition associated with impaired islet function in a subject in need thereof, comprising: administering to the subject an effective amount of pancreatic endocrine cells (or regenerated islet tissue) derived from an endoderm stem cell population, thereby treating the disease or condition associated with impaired islet function in a subject in need thereof.

[0008] In another embodiment, the present disclosure provides a population of EnSCs produced according to the methods provided herein.

[0009] In another embodiment, the present disclosure provides a pancreatic endocrine cell population or regenerated pancreatic islet tissue produced according to the methods provided herein.

[0010] In another embodiment, the present disclosure provides a pharmaceutical composition comprising an EnSC population, an EnSC-derived PP cell population, an EnSC-derived EP cell population, a pancreatic endocrine cell population, or a regenerated pancreatic islet tissue produced according to the methods provided herein.

[0011] In another embodiment, the present disclosure provides a use of an endoderm stem cell population for the manufacture of a regenerative pancreatic islet tissue for treating a disease or condition associated with impaired pancreatic islet function in a subject in need thereof.

[0012] In another embodiment, the present disclosure provides a use of a regenerative pancreatic islet tissue for the manufacture of a medicament for treating a disease or condition associated with impaired pancreatic islet function in a subject in need thereof.

[0013] In another embodiment, the present disclosure provides a kit for generating a population of EnSCs from a population of pluripotent stem cells, wherein the kit comprises a first set of factors, a second set of factors, a third set of factors, and a fourth set of factors, wherein the first set of factors comprises a Nodal signaling agonist and a WNT signaling agonist, the second set of factors comprises a Nodal signaling agonist and a fibroblast growth factor (FGF), the third set of factors comprises a factor belonging to the TGF-beta superfamily, a FGF, a hepatocyte growth factor (HGF), and a VEGF, and the fourth set of factors comprises a WNT signaling agonist, a TGF-beta inhibitor, and an epidermal growth factor (EGF).

[0014] In another embodiment, the present disclosure provides a kit for generating a population of pancreatic endocrine cells from a population of EnSCs, wherein the kit comprises a fifth set of factors, a sixth set of factors, and a seventh set of factors, wherein the fifth set of factors comprises a BMP inhibitor, a Nodal signaling agonist, a FGF10, an EGF, a SANT1, a retinoic acid, an ascorbic acid, and / or a nicotinamide, the sixth set of factors comprises a BMP inhibitor, a TGF-beta inhibitor, and / or a gamma-secretase inhibitor, and the seventh set of factors comprises a T3 and / or a nicotinamide.

[0015] In another embodiment, the present disclosure provides a kit for generating a population of pancreatic endocrine cells from a population of pluripotent stem cells, wherein the kit comprises a first set of factors to a seventh set of factors, wherein the first set of factors comprises a Nodal signaling agonist and a WNT signaling agonist, the second set of factors comprises a Nodal signaling agonist and a fibroblast growth factor (FGF), the third set of factors comprises a factor belonging to the TGF-beta superfamily, a FGF, a hepatocyte growth factor (HGF), and a VEGF, the fourth set of factors comprises a WNT signaling agonist, a TGF-beta inhibitor, and an epidermal growth factor (EGF), the fifth set of factors comprises a BMP inhibitor, a Nodal signaling agonist, a FGF10, an EGF, a SANT1, a retinoic acid, an ascorbic acid, and a nicotinamide, the sixth set of factors comprises a BMP inhibitor, a TGF-beta inhibitor, and a gamma-secretase inhibitor, and the seventh set of factors comprises a T3 and a nicotinamide.

[0016] In another embodiment, the present disclosure provides a kit for generating a pancreatic endocrine cell population from a PP cell population, wherein the kit comprises a set of factors comprising a BMP inhibitor, a TGF-β inhibitor, and a γ-secretase inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Quality control of EnSCs is shown. (a) EnSC morphology. (b) FACS results, showing the proportion of SOX17+ / FOXA1+ cells in EnSCs. (c) Karyotyping results of the WB20 EnSC cell line. (d) EnSC growth curve. (e) EnSC qualification criteria and pathogen testing results. (f) Circle plots of genomic variation from WGS analysis of parental PBMCs (left) and EnSCs (line WB20) (right). From the outside to the inside, each of the nine circles represents an aspect of genomic variation. Circle 1: chromosome; Circle 2: density map of single nucleotide variations (SNVs); Circle 3: density map of Indel insertions; Circle 4: density map of Indel deletions; Circle 5: density map of mutation sites occurring in coding regions; Circle 6: density map of mutation sites occurring in non-coding regions; Circle 7: position map of copy number variations (CNVs), with red and blue columns indicating increases and decreases in copy number, respectively; Circle 8: position map of structural variations (SVs), with orange and green columns indicating deletions and insertions, respectively; Circle 9: type association map of SVs, with blue, red and green lines indicating inversions, interchromosomal translocations and intrachromosomal translocations, respectively.

[0018] Figure 2 Quality control of intermediate pancreatic differentiation stages of EnSCs is shown. (a) and (b) Morphology and cellular composition of differentiation cultures at the pancreatic progenitor (PP) stage. (a) Representative phase-contrast images of EnSC-derived PP cells. (b) FACS data showing the proportion of NKX6-1+ / PDX1+ PP cells at this stage. (c) and (d) Morphology and cellular composition of differentiation cultures at the endocrine progenitor (EP) stage. (c) Representative phase-contrast images of EnSC-derived EP cells. (d) FACS data showing the proportion of NKX6-1+ / PDX1+ and CHGA- / NKX6-1+ PP cells that differentiate toward CHGA+ endocrine progenitors at the early endocrine progenitor stage (EP day 3), and the proportion of CHGA+ endocrine progenitors and emerging endocrine (C-peptide+ or glucagon+) cells at the late endocrine progenitor stage (EP day 8). Scale bar, 100 μm.

[0019] Figure 3Quality control of the regenerated islet tissue is shown. (a) Morphology of the regenerated islet tissue (scale bar, 100 μm). (b) Results from FACS analysis of pancreatic endocrine cells in the regenerated islet tissue, where the chromogranin A+ population represents the pan-endocrine compartment, the C-peptide+ glucagon- population represents beta cells, the glucagon+ population represents alpha cells, and the somatostatin+ population represents delta cells. (c) Immunofluorescence staining of the regenerated islet tissue with C-peptide (CPEP) positive beta cells, glucagon (GCG) positive alpha cells, and somatostatin (SST) positive delta cells (scale bar, 50 μm). (d) Expression levels of indicated genes in the regenerated islet tissue and adult islets, as measured by quantitative RT-PCR (qRT-PCR), where PDX1, NKX6.1, and insulin (INS) are expressed by adult beta cells, and glucagon (GCG) and somatostatin (SST) are typically expressed by adult alpha and delta cells, respectively. (e) and (f) Clusters and gene expression in subpopulations of the regenerated islet tissue, as revealed by single-cell transcriptome analysis (scRNA seq, 10x Genomics). (e) is a tSNE cluster plot of the scRNA seq, showing various subpopulations of the regenerated islet tissue (excludes 2721 cells with low UMI from 15244 sequenced cells). (f) shows expression of representative genes for cell types (indicated by boxes with blue dashed lines) in the regenerated islet tissue. (g) C-peptide secretion from human (primary) islets and the regenerated islet tissue in response to low and high glucose stimulation under static conditions (GSIS). (h) FACS data revealing that non-target liver lineages (alpha fetoprotein / AFP+ or albumin / ALB+ cells) are not detectable in the regenerated islet tissue. (i) Criteria and results of pathogen testing of the regenerated islet tissue.

[0020] Figure 4Preclinical studies and clinical outcomes of autologous regenerative islet tissue transplantation in T2D patients. (a) Brief flow of the procedure showing the main procedures involved in the generation and quality control of autologous regenerative islet tissue and the post-surgical evaluation of safety and efficacy of regenerative islet tissue transplantation. (b)-(d) Regenerative islet tissue reverses hyperglycemia in immunocompromised (SCID Beige) mice with STZ-induced diabetes. (b) Schematic representation of regenerative islet tissue transplantation under the kidney capsule in diabetic mice. (c) Fasting glycemic kinetics in diabetic mice (blue line represents the sham-treated group, which shows persistent hyperglycemia and death within 2 months, and red line represents the regenerative islet tissue-transplanted group, which indicates that hyperglycemia is reversed within one month and the curative effect disappears after the transplanted organ is removed by nephrectomy). (d) Human C-peptide secretion in STZ-induced diabetic mice at 90 and 180 days after regenerative islet tissue transplantation, both after fasting and 30 minutes after intraperitoneal glucose injection. (e)-(g) Immunogenicity of regenerative islet tissue in humanized mice. (e) Schematic representation of patient-specific regenerative islet tissue transplantation under the kidney capsule in diabetic mice (NCG hIL 15, non-obese diabetic mice with genetically embedded human IL15, suffering from severe combined immunodeficiency and interleukin 2 receptor gamma deficiency) humanized with patient and volunteer PBMCs. (f) Fasting glycemic kinetics of STZ-induced diabetic humanized mice (blue line represents the control group in which patient regenerative islet tissue was transplanted into three diabetic mice humanized with volunteer PBMCs, and red line represents the group in which patient regenerative islet tissue was transplanted into three diabetic mice humanized with patient PBMCs). (g) Human C-peptide secretion in humanized diabetic mice at 7 and 14 days after regenerative islet tissue transplantation, both after fasting and 30 minutes after intraperitoneal glucose injection ([U.D.]: not detectable). (h) Clinical outcomes of time in strict target range (TITR), time in range (TIR), and hemoglobin AlC (HbAlc) and insulin (degludec) dose during 116 weeks. Changes in degludec dose (yellow line) are shown on the left y-axis. Proportion of time in strict target range (TITR, 3.9-7.8 mM) (green line) and time in range (TIR, 3.9-10.0 mM) (blue line) and serum hemoglobin AlC content (red line) are shown on the right y-axis. (i) Continuous interstitial glucose fluctuations derived from CGM measurements at weeks 52 and 105 compared to pre-surgery. Green horizontal lines at 3.9 and 7.8 mM divide the healthy target glucose range.Brown (pre-surgery) / green (week 52) / navy (week 105) line, medium brown (pre-surgery) / medium green (week 52) / medium blue (week 105) area, and light brown (pre-surgery) / light green (week 52) / light blue (week 105) area represent median (50%), 25-75% range, and 5-95% range, respectively. (j)-(l) Results of the Mixed Meal Tolerance Test (MMTT) performed by monitoring serum levels of fasting and meal-stimulated circulating glucose (j), C-peptide (k), and insulin (l) to assess islet function.

[0021] Figure 5 Regenerative islet tissue is shown to improve diabetes in STZ-induced diabetic monkeys. (a) Schematic representation of the portal vein implantation of regenerative islet tissue in STZ-induced diabetic monkeys. Two diabetic monkeys were transplanted with 6000 (monkey 1) or 30000 (monkey 2) regenerative islet tissue, respectively. Monkey 1 was used to test the feasibility of the portal vein injection of regenerative islet tissue without digital subtraction angiography (DSA), while monkey 2 was used to assess the short-term safety and efficacy of regenerative islet tissue. Tail tip blood glucose was measured before morning and afternoon feeding. Exogenous insulin treatment doses were determined according to pre-prandial blood glucose in animals. (b) Daily pre-prandial blood glucose levels (left) and insulin administration doses (right) during the 28-day period before and after surgery (Pre: pre-surgery; Post: post-surgery; A.M.: before breakfast; P.M.: before dinner). (c) and (d) Results of the 8-time point intravenous glucose tolerance test (IVGTT) in STZ-induced diabetic monkeys transplanted with regenerative islet tissue. Regularity of blood glucose (c) and human C-peptide (d) was monitored by IVGTT before diabetes model establishment / STZ treatment (before diabetes model establishment), 2 weeks before transplantation (Tx 2 weeks before), and 2, 3, and 4 weeks after transplantation (Tx 2, 3, and 4 weeks after).

[0022] Figure 6Characterization of humanized mice. (a) and (b) Characterization of NCG-hIL15 mice humanized with patient (a) or volunteer (b) derived PBMCs by the presence of hCD45+ human cells in peripheral blood. (a) Proportion of live cells (by SSC and FSC), human-derived (hCD45+) and mouse-derived (mCD45+) blood cells in three patient humanized mice. (b) Proportion of live cells, human-derived and mouse-derived blood cells in three volunteer humanized mice. (c) Immunofluorescence staining of grafts collected under the kidney capsule of patient humanized mice for the presence of human beta cells (C-peptide+ and NKX6-1+), alpha cells (glucagon+) and delta cells (somatostatin+); C-peptide (CPEP, red), glucagon (GCG, green), somatostatin (SST, purple) and NKX6-1 (cyan); the area delimited by the white dotted line indicates mouse kidney tissue; scale bar, 50 pm.

[0023] Figure 7 Clinical assessments and results showing glycemic control. (a) Schematic representation of the follow-up time points for routine and diabetes-specific clinical assessments, and treatments received by the patient during the entire follow-up period. Endocrine function and diabetes-specific parameters were tested by mixed meal tolerance test (MMTT) at baseline and at 4, 8, 12, 16, 20, 24, 36 and 48 weeks and thereafter at the indicated time points. Glycemic control was measured in the patient using a 24-hour real-time continuous glucose monitoring (CGM) system. Baseline and follow-up CGM glucose values were measured during the first 52 weeks and thereafter during the indicated periods, and the average duration of CGM device wear was at least 3 days. The primary conditioning regimen included antihyperglycemic drugs and immunosuppressant therapy. Antidiabetic therapy included metformin (0.75 g bid, tapered off at week 44 and discontinued at week 56) and acarbose (50 mg tid, tapered off at week 44 and discontinued at week 48). Insulin analogue degludec was administered since 2021 (20 U once daily at bedtime), but was discontinued immediately after the transplant of the regenerative pancreatic tissue and resumed since week 2 and tapered off, and stopped at the end of week 11. Graft versus host disease was treated with mycophenolate mofetil (0.5 g bid since kidney transplant) and tacrolimus (1 to 3 mg bid orally administered after kidney transplant, dose depending on serum FK506 concentration). The asterisk indicates the follow-up time points for clinical assessments, and the delta indicates CGMS monitoring phases. The checkered pattern indicates phases of tapering. (b) Results derived from the mixed meal tolerance test (MMTT) in 5 points (0, 30, 60, 120, 180 minutes) of intravenous glucose (Glu) and C-peptide (C-Pep) in the patient humanized mice at baseline (week 0) and at week 4, 8, 12, 16, 20, 24, 36 and 48. The dotted line indicates the mean value of the patient at baseline. Figure 4 (j)) C-peptide ( Figure 4(k)) and insulin Figure 4 (l)) Area under the curve (AUC) of values. (c) Continuous glucose monitoring during MMTT (0 to 240 minutes) measured by CGM device at 5-minute intervals at each follow-up time point. Horizontal dashed lines at 3.9 and 10.0 mM divide the target glucose range. Fold change in AUC (right panel) is the area under the curve at each follow-up time point normalized to the AUC at week 2 (2W). (d) Postprandial glucose excursions (mean glucose excursions, MAGE), the gold standard of glycemic variability, is represented by the mean glucose values (in 95% range) at 1.5 hours before and 2 hours after each meal at baseline, week 52, and week 105. Green horizontal lines at 3.9 and 7.8 mM divide the target glucose excursion in healthy subjects.

[0024] Figure 8 Continuous glucose monitoring is shown at each follow-up time point. (a)-(l) Continuous glucose monitoring (CGM) traces pre-surgery (a) and at each follow-up time point (b)-(l). For each follow-up time point, GCM data were collected for 72 consecutive hours. Median (navy line), 25-75% (mid-blue area), and 5-95% (light blue area) ranges are shown, with green horizontal lines dividing the target glucose range in diabetic patients (3.9 to 10.0 mM). Postprandial glucose excursions (MAGE) calculated from mean glucose values (in 95% range) are shown in red on each panel.

[0025] Figure 9 Teratoma formation analysis is shown.

[0026] Figure 10 Quality control acceptance criteria for EnSCs and regenerative islet tissue are shown.

[0027] Figure 11 Primer list is shown.

[0028] Figure 12 Antibody list is shown.

[0029] Figure 13 Key laboratory values before and after transplantation are shown.

[0030] Figure 14 Primary follow-up objectives are shown.

[0031] Figure 15 Exploratory objectives are shown. DETAILED DESCRIPTION

[0032] The following description of the present disclosure merely exemplifies various implementations of the present disclosure. Accordingly, the particular modifications discussed are not to be taken in a limiting sense as the scope of the present disclosure encompasses various equivalents, variations, and modifications of the concepts disclosed herein, and it is understood that such equivalent implementations are within the scope of the present disclosure. All references cited herein, including publications, patents, and patent applications, are hereby incorporated by reference in their entirety.

[0033] I. General Definitions

[0034] The singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. For example, reference to "a cell" refers to one or more cells, and reference to "the method" includes reference to equivalent steps and methods disclosed herein and / or known to one of ordinary skill in the art, and so forth. Similarly, "or" is intended to mean "and / or" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below.

[0035] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0036] As used herein, the term "cell" refers to an individual cell, a cell line, or a culture derived from such a cell, as used herein.

[0037] As used herein, the term "Nodal signaling agonist" refers to an agent that stimulates or enhances the Nodal signaling pathway. The Nodal signaling pathway plays a key role in patterning and differentiation during early stages of chordate development, especially prior to and during gastrulation. This pathway is required for establishing the body axes and for the formation of mesoderm and endoderm.

[0038] As used herein, the term "Notch signaling pathway" refers to a pathway that plays a critical role in regulating cell fate, cell proliferation, and cell death during development. This pathway is unique in that it primarily involves interactions between neighboring cells, as the ligands that activate the Notch receptor are mostly transmembrane proteins. Inhibition of the Notch signaling pathway can be achieved by a substance that disrupts the function of any protein within the pathway or prevents functional interactions between two pathway proteins. Exemplary inhibitors of the Notch signaling pathway include, but are not limited to, gamma-secretase inhibitors, RBPJ inhibitor-1. See, e.g., Hurtado, C, et al. Disruption of NOTCH signaling by a small molecule inhibitor of the transcription factor RBPJ. Sci Rep 9, 10811 (2019).

[0039] As used herein, the term "WNT signaling agonist" refers to a substance that stimulates or enhances the WNT signaling pathway. The WNT signaling pathway is a fundamental intercellular communication mechanism that plays a critical role in many physiological processes, including stem cell behavior, cell polarity, and tissue development.

[0040] As used herein, the term "fibroblast growth factor" or "FGF" refers to a member from the FGF family, which is a diverse set of secreted proteins that signal through receptor tyrosine kinases and regulate a variety of cellular processes.

[0041] As used herein, the term "TGF-β superfamily" refers to a superfamily of factors involved in the TGF-β signaling pathway. The TGF-β signaling pathway is a conserved mechanism that regulates many manifestations of physiological development and tissue homeostasis. TGF-β family members include secreted polypeptides that play a critical role in embryogenesis and adult tissue maintenance, but also contribute to the development of various diseases. BMPs or bone morphogenetic proteins constitute a subset of signaling molecules within the TGF-β superfamily. In particular, "BMP4" refers to a protein encoded by the BMP4 gene in humans, which is a member of the BMP family and thus part of the broader TGF-β superfamily.

[0042] As used herein, the term "TGF-β inhibitor" refers to an agent that can reduce the expression and / or activity of TGF-β or its receptors, e.g., by at least 10% or more, e.g., by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more. The efficacy of an inhibitor, e.g., its ability to reduce the amount and / or activity of TGF-β or its receptors, can be determined, e.g., by measuring the amount of an expression product of TGF-β or its receptors and / or its activity. In some embodiments, the inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody or binding fragment thereof; or a small molecule.

[0043] As used herein, the term "γ-secretase inhibitor" refers to an agent that can reduce the expression and / or activity of γ-secretase, e.g., by at least 10% or more, e.g., by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more. The efficacy of an inhibitor, e.g., its ability to reduce the amount and / or activity of γ-secretase, can be determined, e.g., by measuring the amount of an expression product of γ-secretase and / or its activity. In some embodiments, the inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody or binding fragment thereof; or a small molecule. γ-secretase is a proteinase complex that cleaves single-pass transmembrane proteins within the transmembrane domain. It is a membrane-bound protein and belongs to the category of intramembrane proteases.

[0044] As used herein, the term "BMP inhibitor" refers to an agent that can reduce the expression and / or activity of BMP, e.g., by at least 10% or more, e.g., by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more. The efficacy of an inhibitor, e.g., its ability to reduce the amount and / or activity of BMP, can be determined, e.g., by measuring the amount of an expression product of BMP and / or its activity. In some embodiments, the inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody or binding fragment thereof; or a small molecule.

[0045] As used herein, the term "Activin A" refers to a member of the TGF-β superfamily that is structurally similar to TGF-β1 and signals through the common SMAD2 / 3 (mothers against decapentaplegic homologs 2 and 3) pathway.

[0046] As used herein, the term "CHIR99021" is a GSK-3a and GSK-3b inhibitor with CAS Number 252917-06-9.

[0047] As used herein, the term "bFGF" is used interchangeably with the term "FGF2" and refers to a growth factor and signaling protein encoded by the FGF2 gene.

[0048] As used herein, the term "Wnt3A" refers to a protein encoded by the WNT3A gene in humans.

[0049] As used herein, the term "A83-01" is a TGF-beta type I receptor inhibitor with CAS number 909910-43-6.

[0050] As used herein, the term "Rspondin 1" refers to a secreted protein encoded by the RSPO1 gene in humans.

[0051] As used herein, the term "LDN-193189" is a selective BMP signaling inhibitor with CAS number 1062368-24-4.

[0052] As used herein, the term "TPPB" is a protein kinase C activator with CAS number 497259-23-1.

[0053] As used herein, the term "Noggin" refers to a protein involved in the development of many body tissues, including neural tissue, muscle, and bone. In humans, noggin is encoded by the NOG gene.

[0054] As used herein, the term "GSI-XX" is a gamma-secretase inhibitor with CAS number 209984-56-5.

[0055] As used herein, the term "epidermal growth factor" or "EGF" is a protein that stimulates cell growth and differentiation by binding to its receptor, the epidermal growth factor receptor (EGFR). The term "TGF-a" refers to a protein encoded by the TGFA gene in humans. TGF-a is a member of the epidermal growth factor (EGF) family.

[0056] As used herein, the term "hepatocyte growth factor" or "HGF" refers to a mitogen produced by stromal cells that stimulates epithelial cell proliferation, motility, morphogenesis, and angiogenesis in various organs. It acts through tyrosine phosphorylation of the receptor c-Met. HGF plays an important role in organ development, self-repair of wounded tissues, and protection of epithelial and non-epithelial organs via anti-apoptotic and anti-inflammatory signals. See, e.g., Nakamura T, et al. The discovery of hepatocyte growth factor (HGF) and its significance for cell biology, life sciences and clinical medicine. Proc Jpn Acad Ser B Phys Biol Sci. 2010; 86(6):588-610.

[0057] As used herein, the term "vascular endothelial growth factor" or "VEGF" refers to a growth factor with significant pro-angiogenic activity that exerts mitogenic and anti-apoptotic effects on endothelial cells. It enhances vascular permeability, promotes cell migration, and actively promotes the regulation of normal and pathological angiogenic processes. The VEGF family in humans includes several members, including VEGF-A (with different isoforms), VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, placenta growth factor (PlGF), and endocrine gland-derived vascular endothelial growth factor (EG-VEGF). VEGF binds to tyrosine kinase cell receptors (VEGFRs), such as VEGFR-1, VEGFR-2, and VEGFR-3, which are mainly expressed on vascular and lymphatic endothelial cells. VEGFR-2 has the strongest pro-angiogenic activity. VEGF and its receptors are also expressed on non-endothelial cells. Anti-VEGF and anti-VEGFR therapies are currently considered key to blocking angiogenesis in cancer and other pathological processes. See, e.g., Melincovici CS, et al. Vascular endothelial growth factor (VEGF)-key factor in normal and pathological angiogenesis. Rom J Morphol Embryol. 2018; 59(2):455-467.

[0058] As used herein, the term "endoderm stem cell" or "EnSC" refers to a specific type of cell derived from pluripotent stem cells that is unique to the germ layer and is non-tumorigenic. See, e.g., Cheng, X., et al. Cell Stem Cell 10, 371-384 (2012).

[0059] As used herein, the term "islet" refers to a small cluster of specialized cells in the pancreas, also known as islets of Langerhans. Such islets are responsible for secreting hormones that regulate blood glucose levels. Islets contain different types of cells, including B cells (beta cells) that secrete insulin, A cells (alpha cells) that secrete glucagon, D cells (delta cells) that secrete somatostatin, and pancreatic polypeptide secreting cells that secrete pancreatic polypeptide. Islets serve as the endocrine portion of the pancreas and play a key role in maintaining blood glucose levels.

[0060] As used herein, the term "regenerative islet tissue" refers to a cluster of pancreatic endocrine cells obtained using the methods described in the present disclosure through optimized differentiation culture of EnSCs. Such regenerative islet tissue has similar morphology, endocrine cell composition, gene phenotype, and / or functionality as native islets.

[0061] As used herein, the term "treatment," "treat," or "treating" with respect to a disorder means managing, eliminating, reducing, or ameliorating the disorder and / or its associated symptoms. Although not precluded, treating a disorder does not require complete elimination of the disorder or its associated symptoms. As used herein, the term "treatment" can include "prophylactic treatment," which is administered before any symptoms or manifestations of a disorder develop, to lessen or prevent the onset of the disorder, or to lessen or prevent the recurrence of the disorder in subjects who are not ill but who are at risk, or who are susceptible to, or who has a history of, the disorder. Within the meaning of the present invention, "treatment" also includes prevention of recurrence or prevention stage, to and treatment of acute or chronic signs, symptoms, and / or dysfunction. Treatment can target symptoms, for example, to suppress symptoms. Treatment can act over a short period, an intermediate period, or can be long-term treatment, such as in the case of maintenance therapy.

[0062] As used herein, "autologous" cells refer to any cells derived from the same subject into which they are later reintroduced.

[0063] As used herein, "allogeneic" cells refer to any cells derived from a different subject of the same species.

[0064] As used herein, the term "effective amount" refers generally to the amount of a compound or cell that, when administered to a patient for treatment of a disease, is sufficient to effect treatment for the disease. The effective amount can be an amount effective to achieve prophylaxis and / or an amount effective to achieve prevention. The effective amount can be an amount effective to achieve alleviation, an amount effective to achieve the following: prevention of the occurrence of a sign / symptom, reduction in the severity of the occurrence of a sign / symptom, elimination of the occurrence of a sign / symptom, slowing of the progression of the occurrence of a sign / symptom, prevention of the progression of the occurrence of a sign / symptom, and / or achievement of prophylaxis of the occurrence of a sign / symptom. The "effective amount" can vary depending on the disease and its severity, and the age, weight, medical history, sensitivity, and pre-existing conditions of the patient being treated. For the purposes of this disclosure, the term "effective amount" is synonymous with "therapeutically effective amount."

[0065] As used herein, the term "subject" is not limited to a particular species or sample type. For example, the term "subject" can refer to a patient, and typically refers to a human patient. However, this term is not limited to humans, and thus encompasses a variety of mammalian species, such as non-human veterinary mammals, such as dogs, cats, rabbits, pigs, rodents, horses, or monkeys.

[0066] II. Endodermal Stem Cells

[0067] In one embodiment, the disclosure provides a method for producing a population of endodermal stem cells (EnSCs), comprising: a) providing a population of pluripotent stem cells; b) culturing the population of pluripotent stem cells in a first medium comprising a Nodal signaling agonist and a WNT signaling agonist; c) culturing the cells in a second medium comprising a Nodal signaling agonist and a fibroblast growth factor (FGF); d) culturing the cells in a third medium comprising a factor belonging to the TGF-β superfamily, a FGF, a hepatocyte growth factor (HGF), and a VEGF; and e) culturing the cells in a fourth medium comprising a WNT signaling agonist, a TGF-β inhibitor, and an epidermal growth factor (EGF); thereby producing a population of endodermal stem cells.

[0068] In some embodiments, the culturing period of step b is 1 to 2 days. In some embodiments, the culturing period of step b is 1 day. In some embodiments, the culturing period of step c is 2 to 6 days. In some embodiments, the culturing period of step c is 4 days. In some embodiments, the culturing period of step d is 2 to 6 days. In some embodiments, the culturing period of step d is 4 days.

[0069] In some embodiments, steps b-d are performed under less than 10% O2, such as less than 8% O2, less than 6% O2, less than 4% O2, or less than 2% O2. In some embodiments, steps b-d are performed under 5% O2.

[0070] In certain embodiments, the method further comprises washing the cells obtained from step b prior to starting step c, washing the cells obtained from step c prior to starting step d, and washing the cells obtained from step d prior to starting step e. In certain embodiments, there are no washing steps in steps b through e.

[0071] In some embodiments, at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%) of the population of endoderm stem cells express SOX17, CDX2, SOX9, GATA6, and / or FOXA1.

[0072] Expression of a marker can be detected by any method known in the art, including, but not limited to, fluorescence-activated cell sorting (FACS), Western Blotting, methods based on amplification of mRNA (e.g., PCR, isothermal amplification, etc., which can include reverse transcription and can be applied to detect expression from a single cell or multiple cells), Northern blotting, immunostaining, and the like. In addition, expression of a marker can be inferred from expression of a reporter construct under the control of a genetic element that confers cell type-specific expression, such as a promoter for one of the foregoing markers or fragments thereof, such as a fluorescent protein, whose expression can be detected visually; an antibiotic resistance gene, whose expression can be detected by cell survival in the presence of the antibiotic; and the like.

[0073] II-1. Pluripotent Stem Cells

[0074] In certain embodiments, the population of pluripotent stem cells is autologous or allogeneic. In certain embodiments, the pluripotent stem cells are selected from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).

[0075] As used herein, the terms "embryonic stem cell" and "ESC" are used interchangeably and refer to a pluripotent stem cell of the inner cell mass of a blastocyst embryo (see U.S. Patent Nos. 5,843,780; 6,200,806, which are incorporated herein by reference). Distinctive features of an embryonic stem cell define the phenotype of an embryonic stem cell. Thus, a cell has the phenotype of an embryonic stem cell if it has one or more unique features of an embryonic stem cell such that the cell can be distinguished from other cells. Exemplary distinctive embryonic stem cell features include, but are not limited to, gene expression profile, proliferative capacity, differentiative capacity, karyotype, responsiveness to particular culture conditions, and the like.

[0076] As used herein, the terms "induced pluripotent stem cell" and "iPSC" are used interchangeably and refer to a pluripotent cell artificially derived (e.g., by complete or partial reversion induction) from a differentiated somatic cell (i.e., from a non-pluripotent cell). A pluripotent cell can differentiate into cells of all three germ layers of a developing embryo.

[0077] The ectoderm, mesoderm, and endoderm are the three germ layers formed during embryonic development, with the mesoderm as the middle layer, the ectoderm as the outer layer, and the endoderm as the inner layer. The ectoderm forms the surface ectoderm, neural crest, and neural tube, with the surface ectoderm developing into the epidermis, hair, nails, lens of the eye, sebaceous glands, cornea, enamel of the teeth, epithelium of the mouth and nose; the neural crest of the ectoderm developing into the peripheral nervous system, adrenal medulla, melanocytes, facial cartilage, and dentin of the teeth; and the neural tube of the ectoderm developing into the brain, spinal cord, posterior pituitary, motor neurons, and retina. The mesoderm forms the mesenchyme, mesothelium, non-epithelial blood cells, and coelomic cells, which make up the muscle (smooth and striated), bone, cartilage, connective tissue, adipose tissue, circulatory system, lymphatic system, dermis, urogenital system, serosal membranes, and notochord. The endoderm forms the epithelial portion of the pharynx, esophagus, stomach, small intestine, colon, liver, pancreas, urinary bladder, trachea and bronchi, lungs, thyroid, and parathyroid.

[0078] Induced pluripotent stem cells (iPSCs) can be generated using methods known in the art. For example, the iPSCs provided in the methods of the present disclosure can be generated from peripheral blood mononuclear cells (PBMCs). In certain embodiments, the PBMCs are obtained from a human subject who will undergo EnSC derivation therapy. In certain embodiments, the iPSCs are generated by culturing PBMCs in the presence of human SCF, FLT-3, IL-3, and IL-6, followed by transduction of the PBMCs with four reprogramming factors, OCT4, SOX2, KLF4, and L-MYC.

[0079] II-2. Media for preparing EnSCs

[0080] The basal medium used for culturing in steps b to e of the methods provided herein is not particularly limited. Any medium can be used as the basal medium, as long as it enables the generation of endoderm stem cells. In certain embodiments, the basal medium is serum-free and / or matrix-free. In certain embodiments, the basal medium is selected from the group consisting of mTeSR TM 1. TeSR TM - AOF, Essential 8 TM 、 hPSC XF Medium (Sartorius), RPMI / B27 TM, SFD-based medium, MCDB 131, Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 (DMEM / F12 medium), StemPro TM 34-SFM, RPMI-1640, Iscove's Modified Dulbecco's Medium (IMDM), Ham's F12, and CMRL-1066.

[0081] For the culture temperature, culturing at a temperature of 35.0°C or greater is confirmed to promote cell differentiation. The culture temperature is a temperature that does not destroy the cells, such as preferably 35.0°C to 42.0°C, or more preferably 36.0°C to 40.0°C, or still more preferably 37.0°C to 39.0°C.

[0082] The first medium

[0083] In certain embodiments, the first medium comprises a Nodal signaling agonist and a WNT signaling agonist. Exemplary Nodal signaling agonists include, but are not limited to, Activin A, Nodal, and GDF-8. In certain embodiments, the Nodal signaling agonist is Activin A.

[0084] In certain embodiments, the concentration of the Nodal signaling agonist is in the range of about 20 ng / mL to about 200 ng / mL, such as 40 ng / mL, 60 ng / mL, 80 ng / mL, 100 ng / mL, 120 ng / mL, 140 ng / mL, 160 ng / mL, 180 ng / mL, or 200 ng / mL.

[0085] Exemplary WNT signaling agonists include, but are not limited to, CHIR99021, Wnt3A, Wnt3a-AFM, R-Spondin-1, and BIO (6-bromoindirubin-3'-oxime), SKL2001, BML-284, CP21R7, and SB 216763. In certain embodiments, the WNT signaling agonist is CHIR99021 or Wnt3A.

[0086] In certain embodiments, the concentration of the WNT signaling agonist is in the range of about 0.2 μΜ to about 4 μΜ, such as 0.4 μΜ, 0.6 μΜ, 0.8 μΜ, 1 μΜ, 1.2 μΜ, 1.4 μΜ, 1.6 μΜ, 1.8 μΜ, 2.0 μΜ, 2.2 μΜ, 2.4 μΜ, 2.6 μΜ, 2.8 μΜ, 3.0 μΜ, 3.2 μΜ, 3.4 μΜ, 3.6 μΜ, 3.8 μΜ, or 4.0 μΜ.

[0087] In certain embodiments, the first medium comprises Activin A and CHIR99021. In certain embodiments, the first medium comprises a medium supplemented with Activin A and CHIR99021.

[0088] Second medium

[0089] In certain embodiments, the second medium comprises a Nodal signaling agonist and a fibroblast growth factor (FGF). Exemplary FGFs include, but are not limited to, basic FGF (bFGF), FGF4, and chimeric fibroblast growth factor (FGFC), FGF1, FGF10, and FGF7. In certain embodiments, the FGF is bFGF.

[0090] In certain embodiments, the Nodal signaling agonist is Activin A.

[0091] In certain embodiments, the concentration of the Nodal signaling agonist is in the range of about 20 ng / mL to about 200 ng / mL, such as 40 ng / mL, 60 ng / mL, 80 ng / mL, 100 ng / mL, 120 ng / mL, 140 ng / mL, 160 ng / mL, 180 ng / mL, or 200 ng / mL.

[0092] In certain embodiments, the concentration of the FGF is in the range of about 1 ng / mL to 10 ng / mL, such as 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, or 10 ng / mL.

[0093] In certain embodiments, the second medium comprises Activin A and bFGF. In certain embodiments, the second medium comprises a medium supplemented with Activin A and bFGF.

[0094] One of skill in the art will appreciate that other necessary described supplements can be added to the medium. In certain embodiments, the second medium further comprises VEGF, ascorbic acid, and / or glutaMAX.

[0095] Third medium

[0096] In certain embodiments, the third culture medium comprises a factor belonging to the transforming growth factor-beta (TGF-β) superfamily, FGF, hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF).

[0097] Exemplary factors belonging to the TGF-β superfamily include, but are not limited to, BMP4, BMP2, and BMP7. In certain embodiments, the factor belonging to the TGF-β superfamily is BMP4.

[0098] In certain embodiments, the concentration of the factor belonging to the TGF-β superfamily is in the range of about 20 ng / mL to about 200 ng / mL, such as 40 ng / mL, 50 ng / mL, 60 ng / mL, 80 ng / mL, 100 ng / mL, 120 ng / mL, 140 ng / mL, 160 ng / mL, 180 ng / mL, or 200 ng / mL.

[0099] In certain embodiments, the FGF is bFGF. In certain embodiments, the concentration of FGF is in the range of about 1 ng / mL to 20 ng / mL, such as 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, or 19 ng / mL.

[0100] In certain embodiments, the concentration of VEGF is in the range of about 1 ng / mL to 20 ng / mL, such as 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, or 19 ng / mL.

[0101] In an embodiment, the concentration of HGF is in the range of about 1 ng / mL to 40 ng / mL, such as 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, or 39 ng / mL.

[0102] In certain embodiments, the third culture medium comprises BMP4, bFGF, HGF, and VEGF. In certain embodiments, the third culture medium comprises culture medium supplemented with BMP4, bFGF, HGF, and VEGF.

[0103] Those skilled in the art will appreciate that other necessary supplements described above may be added to the culture medium. In certain embodiments, the third culture medium further comprises TGF-α and / or dexamethasone.

[0104] Fourth culture medium

[0105] In certain embodiments, the fourth culture medium includes a WNT signaling agonist, a TGF-β inhibitor, and epidermal growth factor (EGF). Exemplary TGF-β inhibitors include, but are not limited to, A83-01, SB431542, ALK5 inhibitors, LDN-193189, Galunisertib (LY2157299), LY2109761, SB525334, SB505124, GW788388, LY364947, RepSox, LDN-193189, K02288, LDN-214117, SD-208, Vactosertib (TEW-7197), ML347, LDN-212854, DMH1, Pirfenidone, Alantolactone, SIS3, Hesperetin, and Dorsomorphin. In certain embodiments, the TGF-β inhibitor is A83-01.

[0106] In certain embodiments, the WNT signaling agonist is Wnt3A.

[0107] In certain embodiments, the concentration of the WNT signaling agonist is in the range of about 0.2 mM to about 4 mM, such as 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, 1.2 mM, 1.4 mM, 1.6 mM, 1.8 mM, 2.0 mM, 2.2 mM, 2.4 mM, 2.6 mM, 2.8 mM, 3.0 mM, 3.2 mM, 3.4 mM, 3.6 mM, 3.8 mM, or 4.0 mM.

[0108] In certain embodiments, the concentration of the TGF-b inhibitor is in the range of about 0.1 mM to about 2.0 mM, such as 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM.

[0109] In certain embodiments, the concentration of the EGF is in the range of about 2 ng / mL to about 40 ng / mL, such as about 4 ng / mL, about 6 ng / mL, about 8 ng / mL, about 10 ng / mL, about 12 ng / mL, about 14 ng / mL, about 16 ng / mL, about 18 ng / mL, about 20 ng / mL, about 22 ng / mL, about 24 ng / mL, about 26 ng / mL, about 28 ng / mL, about 30 ng / mL, about 32 ng / mL, about 34 ng / mL, about 36 ng / mL, about 38 ng / mL, or about 40 ng / mL.

[0110] In certain embodiments, the fourth medium does not include FGF2 and / or CHIR99021. In certain embodiments, the fourth medium does not include FGF.

[0111] In certain embodiments, the fourth medium includes Wnt3A, A83-01, and EGF. In certain embodiments, the fourth medium includes a medium (e.g., MCDB131) supplemented with Wnt3A, A83-01, and EGF.

[0112] One of skill in the art will appreciate that other necessary described supplements can be added to the medium. In certain embodiments, the fourth medium further includes RSPONDIN1, ascorbic acid, and / or glutamine.

[0113] In certain embodiments, the method for producing a population of endoderm stem cells comprises: a) providing a population of induced pluripotent stem cells; b) culturing the cells in a first medium comprising Activin A and CHIR99021; c) culturing the cells in a second medium comprising Activin A and bFGF; d) culturing the cells in a third medium comprising BMP4, bFGF, HGF, and VEGF; and e) culturing the cells in a fourth medium comprising Wnt3A, A83-01, and EGF; thereby producing a population of endoderm stem cells.

[0114] In certain embodiments, the method for producing a population of endoderm stem cells comprises: a) providing a population of induced pluripotent stem cells; b) culturing the cells in a first medium comprising about 50 ng / mL to about 150 ng / mL Activin A and about 1 μΜ to 3 μΜ CHIR99021; c) culturing the cells in a second medium comprising about 50 ng / mL to about 150 ng / mL Activin A and about 4 ng / mL to about 6 ng / mL bFGF; d) culturing the cells in a third medium comprising 40 ng / mL to about 60 ng / mL BMP4, 5 ng / mL to about 15 ng / mL bFGF, 15 ng / mL to about 35 ng / mL HGF, and 5 ng / mL to about 15 ng / mL VEGF; and e) culturing the cells in a fourth medium comprising about 0.5 μΜ to about 1.5 μΜ Wnt3A, about 0.1 mM to about 1.0 mM A83-01, and 10 ng / mL to about 30 ng / mL EGF; thereby producing a population of endoderm stem cells.

[0115] In certain embodiments, a method for producing an endodermal stem cell population comprises: a) providing a population of induced pluripotent stem cells; b) culturing the cells in a first medium comprising about 50 ng / mL to about 150 ng / mL Activin A and about 1 mM to 3 mM CHIR99021 for 12 hours to 48 hours (e.g., 24 hours); c) culturing the cells in a second medium comprising about 50 ng / mL to about 150 ng / mL Activin A, about 4 ng / mL to about 6 ng / mL bFGF, about 5 ng / mL to about 15 ng / mL VEGF, about 0.1 mM to 1.0 mM ascorbic acid, and about 1 mM to about 3 mM glutaMAX for about 2 to 6 days (e.g., 4 days); d) culturing the cells in a third medium comprising about 40 ng / mL to about 60 ng / mL BMP4, about 5 ng / mL to about 15 ng / mL bFGF, about 15 ng / mL to about 35 ng / mL HGF, about 5 ng / mL to about 15 ng / mL VEGF, about 10 ng / mL to about 30 ng / mL TGF-a, and about 20 ng / mL to about 60 ng / mL dexamethasone for 2 to 6 days (e.g., 4 days); and e) culturing the cells in a fourth medium comprising 0.5 mM to about 1.5 mM Wnt3A, about 0.1 mM to about 1.0 mM A83-01, 10 ng / mL to about 30 ng / mL EGF, about 20 ng / mL to about 80 ng / mL RSPONDIN1, about 0.1 mM to 1.0 mM ascorbic acid, and about 1 mM to about 3 mM glutamine; thereby producing an endodermal stem cell (EnSC) population.

[0116] In certain embodiments, EnSCs are collected and dissociated into single cells for subsequent expansion, subcloning, or differentiation, e.g., every 3 to 4 days.

[0117] In certain embodiments, EnSCs (e.g., passage 20) are selected for quality control testing, including morphology, viability, purity, sterility, karyotype, and whole genome sequencing. Clinically acceptable EnSCs require, e.g., EnSCs that are free of known cancer-associated mutations, have a minimal total mutational burden compared to the PBMCs from which the EnSCs were derived, and have a minimal likelihood of tumor formation. Clinically acceptable EnSCs can be frozen and stored for future use.

[0118] III. Pancreatic Endocrine Cells

[0119] In another embodiment, the present disclosure provides a method for producing a population of pancreatic endocrine cells, comprising: a) providing a population of endoderm stem cells; b) culturing the population of endoderm stem cells in the presence of a BMP inhibitor, an agonist of Nodal signaling, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and / or nicotinamide to produce a population of pancreatic progenitor (PP) cells; c) culturing the population of PP cells in the presence of a BMP inhibitor, a TGF-β inhibitor, and / or a γ-secretase inhibitor to produce a population of endocrine progenitor (EP) cells; and d) culturing the population of EP cells in the presence of T3 and nicotinamide; thereby producing a population of pancreatic endocrine cells. In certain embodiments, the population of PP cells in step c refers to a uniform cell cluster formed from the population of PP cells.

[0120] In certain embodiments, the culturing period of step b is 2 to 6 days. In certain embodiments, the culturing period of step c is 3 to 8 days. In certain embodiments, the culturing period of step d is 7 to 21 days.

[0121] III-1. Endoderm stem cells as starting cells

[0122] In certain embodiments, the EnSCs are produced by the methods described under the section "II. Endoderm stem cells".

[0123] In certain embodiments, the clinically acceptable EnSCs have one or more of the following characteristics: 1) have typical epithelial morphology with clear cell boundaries and a diameter of 3-5 μm as detected by microscopy (e.g., phase contrast observation); 2) at least 90% of the cells are FOXA1 positive as measured by flow cytometric analysis; 3) have an intact karyotype as analyzed by karyotype testing; 4) contain no known cancer-associated mutations and have a minimal total mutational burden compared to the original PBMCs as detected by whole genome sequencing; 5) have at least 90% viable cells prior to freezing and at least 60% viable cells after thawing as detected by flow cytometric analysis; 6) are mycoplasma undetectable, sterile, and virus negative as analyzed by pathogen testing; 7) no teratoma is found as analyzed by teratoma formation testing.

[0124] III-2. Production of pancreatic progenitor (PP) cells

[0125] To induce pancreatic endoderm and subsequently pancreatic progenitor (PP) cells, EnSCs are treated with a cocktail containing a plurality of factors in a basal medium (e.g., MCDB). In certain embodiments, a population of pancreatic progenitor (PP) cells is generated by culturing a population of endodermal stem cells in the presence of a BMP inhibitor, an agonist of Nodal signaling, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and / or nicotinamide. Exemplary BMP inhibitors include, but are not limited to, Noggin, Dorsomorphin, and LDN-193189. In certain embodiments, the BMP inhibitor comprises Noggin.

[0126] In certain embodiments, the concentration of the BMP inhibitor is in the range of about 2 ng / mL to about 40 ng / mL, such as about 4 ng / mL, about 6 ng / mL, about 8 ng / mL, about 10 ng / mL, about 12 ng / mL, about 14 ng / mL, about 16 ng / mL, about 18 ng / mL, about 20 ng / mL, about 22 ng / mL, about 24 ng / mL, about 26 ng / mL, about 28 ng / mL, about 30 ng / mL, about 32 ng / mL, about 34 ng / mL, about 36 ng / mL, about 38 ng / mL, or about 40 ng / mL.

[0127] In certain embodiments, the agonist of Nodal signaling is Activin A. In certain embodiments, the concentration of the agonist of Nodal signaling is in the range of about 0.1 ng / mL to about 1.0 ng / mL, such as 0.2 ng / mL, 0.3 ng / mL, 0.4 ng / mL, 0.5 ng / mL, 0.6 ng / mL, 0.7 ng / mL, 0.8 ng / mL, 0.9 ng / mL, or 1.0 ng / mL.

[0128] In certain embodiments, the concentration of FGF10 is in the range of about 2 ng / mL to about 40 ng / mL, such as about 4 ng / mL, about 6 ng / mL, about 8 ng / mL, about 10 ng / mL, about 12 ng / mL, about 14 ng / mL, about 16 ng / mL, about 18 ng / mL, about 20 ng / mL, about 22 ng / mL, about 24 ng / mL, about 26 ng / mL, about 28 ng / mL, about 30 ng / mL, about 32 ng / mL, about 34 ng / mL, about 36 ng / mL, about 38 ng / mL, or about 40 ng / mL.

[0129] In certain embodiments, the concentration of EGF is in the range of about 2 ng / mL to about 40 ng / mL, such as about 4 ng / mL, about 6 ng / mL, about 8 ng / mL, about 10 ng / mL, about 12 ng / mL, about 14 ng / mL, about 16 ng / mL, about 18 ng / mL, about 20 ng / mL, about 22 ng / mL, about 24 ng / mL, about 26 ng / mL, about 28 ng / mL, about 30 ng / mL, about 32 ng / mL, about 34 ng / mL, about 36 ng / mL, about 38 ng / mL, or about 40 ng / mL.

[0130] In certain embodiments, the concentration of SANT1 is in the range of about 0.1 μΜ to about 1.0 μΜ, such as 0.2 μΜ, 0.3 μΜ, 0.4 μΜ, 0.5 μΜ, 0.6 μΜ, 0.7 μΜ, 0.8 μΜ, 0.9 μΜ, or 1.0 μΜ.

[0131] In certain embodiments, the concentration of retinoic acid is in the range of about 0.5 μΜ to about 4 μΜ, such as 1 μΜ, 1.5 μΜ, 2 μΜ, 2.5 μΜ, 3 μΜ, 3.5 μΜ, or 4 μΜ.

[0132] In certain embodiments, the concentration of ascorbic acid is in the range of about 0.1 mM to about 1.0 mM, such as 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, or 1.0 mM.

[0133] In certain embodiments, the concentration of nicotinamide is in the range of about 1 mM to about 20 mM, such as 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM.

[0134] In certain embodiments, a population of pancreatic progenitor (PP) cells is generated by culturing a population of endodermal stem cells in the presence of Noggin, Activin A, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and / or nicotinamide.

[0135] In certain embodiments, a population of pancreatic progenitor (PP) cells is generated by culturing a population of endodermal stem cells in the presence of Noggin, Activin A, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, nicotinamide, RSPONDIN1, LDN-193189, and / or TPPB.

[0136] In certain embodiments, a pancreatic progenitor (PP) cell population is generated by culturing a population of endoderm stem cells in the presence of Noggin, activin A, FGF10, LDN193189, RSPONDIN1, TPPB and / or EGF for 1-3 days (e.g., 2 days), followed by culturing the cells in the presence of LDN193189, FGF10, EGF, SANT1, ascorbic acid and / or retinoic acid (Sigma) for 1-3 days (e.g., 2 days), followed by culturing the cells in the presence of FGF10, EGF, SANT1, retinoic acid, nicotinamide and / or ascorbic acid for 1-3 days (e.g., 2 days).

[0137] In certain embodiments, EnSC-derived PP cells are evaluated for microbial contamination, morphology, purity, and viability. In certain embodiments, clinically acceptable PP cells have one or more of the following characteristics: 1) typical epithelial morphology with indistinct cell borders and a diameter no greater than 3 μm as detected by microscopy (e.g., phase contrast); 2) at least 60% of cells are PDX1 positive as measured by flow cytometry; 3) at least 90% of cells are viable as measured by flow cytometry; and 4) no detectable mycoplasma, sterile, virus negative, and no more than 1 EU / mL of endotoxin as analyzed by pathogen testing.

[0138] In certain embodiments, at the end of this stage, pancreatic progenitor (PP) cells are single-cell dispersed and suspended in, for example, a three-dimensional environment (e.g., AggreWell (STEMCELL), Culture plates, Nunclon TM Sphera TM 96-well plate, or The cells are then plated in spherical-well round-bottom ultra-low attachment microtiter plates to allow for uniform cell cluster formation for a certain period of time (eg, 1-5 days, such as 3 days) for further islet tissue remodeling and maturation.

[0139] III-3. Generation of endocrine progenitor (EP) cells

[0140] For endocrine progenitor (EP) induction, triple inhibition of BMP, TGF-β, and Notch signaling pathways can be manipulated for several days. In certain embodiments, the uniform cell clusters obtained using the methods described under the section "Generation of Pancreatic Progenitor (PP) Cells" are further cultured in the presence of a BMP inhibitor, a TGF-β inhibitor, and / or a γ-secretase inhibitor. In certain embodiments, the BMP inhibitor comprises Noggin. In certain embodiments, the TGF-β inhibitor is A83-01. Exemplary γ-secretase inhibitors include, but are not limited to, Compound E, GSI-XX and GSI-XXI, DAPT, LY-411575, RO4929097, and dibenzazepine (DBZ). In certain embodiments, the γ-secretase inhibitor comprises GSI-XX.

[0141] In certain embodiments, the concentration of the BMP inhibitor is in the range of about 1 ng / mL to about 40 ng / mL, such as 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 22 ng / mL, 24 ng / mL, 26 ng / mL, 28 ng / mL, 30 ng / mL, 32 ng / mL, 34 ng / mL, 36 ng / mL, 38 ng / mL, or 40 ng / mL.

[0142] In certain embodiments, the concentration of the TGF-β inhibitor is in the range of about 0.1 mM to about 2.0 mM, such as 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM.

[0143] In certain embodiments, the concentration of the γ-secretase inhibitor is in the range of about 0.5 μΜ to about 4 μΜ, such as 1 μΜ, 1.5 μΜ, 2 μΜ, 2.5 μΜ, 3 μΜ, 3.5 μΜ, or 4 μΜ.

[0144] In certain embodiments, the sixth medium comprises Noggin, A83-01, and / or GSI-XX.

[0145] In certain embodiments, the uniform cell clusters are further cultured in the presence of Noggin, A83-01, and / or GSI-XX. In certain embodiments, the uniform cell clusters are further cultured in the presence of Noggin, A83-01, GSI-XX, retinoic acid, and / or SANT1.

[0146] In certain embodiments, the EnSC-derived EP cells are evaluated for microbial contamination, morphology, purity, and viability. In certain embodiments, the clinically acceptable EP cells have one or more of the following characteristics: 1) at least 60% of the cells are PDX1 and NKX6-1 positive as measured by flow cytometric analysis techniques; 2) have at least 90% viable cells as detected by flow cytometric analysis techniques; and 3) are mycoplasma undetectable, sterile, virus negative, and have no more than 1 EU / mL endotoxin as analyzed by pathogen testing.

[0147] III-4. Generation of Regenerative Pancreatic Islet Tissue (Maturation of Endocrine Cells)

[0148] For maturation of endocrine cells, additional factors can be added to the culture medium used for generation of endocrine progenitor cells described under section "III-3. Generation of Endocrine Progenitor Cells (EP)." In certain embodiments, the additional factors are selected from the group consisting of 3,3',5-triiodo-L-thyronine (T3) and nicotinamide. In certain embodiments, the additional factors include T3 and nicotinamide.

[0149] In certain embodiments, the concentration of T3 is in the range of about 0.5 μΜ to about 4 μΜ, such as 1 μΜ, 1.5 μΜ, 2 μΜ, 2.5 μΜ, 3 μΜ, 3.5 μΜ, or 4 μΜ.

[0150] In certain embodiments, the concentration of nicotinamide is in the range of about 1 mM to about 20 mM, such as 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM.

[0151] In certain embodiments, the additional factors further include BMP4.

[0152] In certain embodiments, a method for producing a population of pancreatic endocrine cells comprises: a) providing a population of endoderm stem cells; b) culturing the population of endoderm stem cells in the presence of Noggin, Activin A, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and / or nicotinamide to produce a population of pancreatic progenitor (PP) cells; c) culturing the population of PP cells in the presence of Noggin, A83-01, and / or GSI-XX to produce a population of endocrine progenitor (EP) cells; and d) culturing the population of EP cells in the presence of T3 and / or nicotinamide; thereby producing a population of pancreatic endocrine cells.

[0153] In certain embodiments, the method for generating a pancreatic endocrine cell population comprises: a) providing an endoderm stem cell population; b) culturing the endoderm stem cell population in the presence of about 10 ng / mL to about 30 ng / mL Noggin, about 0.1 ng / mL to about 1.0 ng / mL Activin A, about 10 ng / mL to about 30 ng / mL FGF10, about 10 ng / mL to about 30 ng / mL EGF, about 100 nM to about 300 nM LDN 193189, about 10 ng / mL to about 30 ng / mL RSPONDIN1 and / or about 300 nM to about 800 nM TPPB for 1-3 days (e.g., 2 days), followed by culturing the endoderm stem cell population in the presence of about 100 nM to about 300 nM LDN 193189, about 10 ng / mL to about 30 ng / mL RSPONDIN1 and / or about 300 nM to about 800 nM TPPB. The cells are further cultured for 1-3 days (e.g., 2 days) in the presence of about 10 ng / mL to about 30 ng / mL FGF10, about 10 ng / mL to about 30 ng / mL EGF, about 0.3 μM to about 0.8 μM SANT1, about 0.3 mM to about 0.8 mM ascorbic acid, and / or about 0.2 μM to about 3 μM retinoic acid, and further subsequently cultured in the presence of about 10 ng / mL to about 60 ng / mL FGF10, about 10 ng / mL to about 30 ng / mL EGF, about 0.1 μM to about 0.4 μM SANT1, about 0.1 μM to about 0.3 μM retinoic acid, about 5 mM to about 15 mM nicotinamide, and / or about 0.3 mM to about 0.8 mM ascorbic acid. c) culturing the PP cell population in the presence of about 10 ng / mL to about 30 ng / mL Noggin, about 0.1 mM to about 1.0 mM A83-01, about 1 μM to about 3 μM GSI-XX, about 0.05 μM to about 0.2 μM retinoic acid and / or about 0.05 μM to about 0.2 μM SANT1 to produce an endocrine progenitor (EP) cell population; culturing the EP cell population in the presence of the factors involved in step c) and about 0.5 μM to about 1.5 μM T3, about 5 mM to about 15 mM nicotinamide and / or about 1 ng / mL to about 3 ng / mL BMP4; thereby producing a pancreatic endocrine cell population.

[0154] In certain embodiments, the EnSCs, EnSC-derived PP cells, EnSC-derived EP cells, or EnSC-derived mature endocrine cells described herein are capable of forming three-dimensional cell aggregates that mimic native islet architecture and promote intercellular interactions. Subsequently, additional three-dimensional cultivation of the cell aggregates yields functional islet-like structures, i.e., EnSC-derived islets, i.e., regenerated islet tissue. The resulting regenerated islet tissue is then assessed for maturity and functionality, including marker expression, endocrine cell composition, hormone secretion, and glucose responsiveness.

[0155] In certain embodiments, the regenerative islet tissue is evaluated for microbial contamination, morphology, purity, and viability, and also analyzed for endocrine cell composition, such as insulin+NKX6-1+ beta cells, glucagon+ alpha cells, somatostatin+ delta cells, and chromogranin A+ endocrine cells, by, e.g., flow cytometry techniques and single cell transcriptome analysis (scRNA-seq).

[0156] In certain embodiments, the in vitro functionality of the regenerative islet tissue can be analyzed by glucose-stimulated insulin secretion (GSIS) of human cadaveric islets, and the in vivo functionality can be assessed by renal capsule or hepatic portal vein transplantation into a streptozotocin-induced diabetic animal (e.g., mouse or monkey) model. In certain embodiments, non-target hepatocytes (HNF4A+albumin+), cholangiocytes (SOX9+CK7+), intestinal epithelial cells (CDX2+), and pancreatic duct cells (SOX9+PTF1A+PDX1+) can be estimated from, e.g., scRNA-seq data.

[0157] In certain embodiments, a clinically acceptable regenerative islet tissue has one or more of the following characteristics: 1) has dense spherical cell clusters with indistinct cell boundaries, with an average diameter of about 150 pm, as detected by microscopy (e.g., phase contrast observation); 2) has at least 85% of cells positive for PDX1 (i.e., pancreatic lineage), at least 60% of cells positive for CHGA+ (i.e., endocrine lineage), at least 40% of cells positive for C-peptide (i.e., beta cells), at least 20% of cells positive for glucagon (i.e., alpha cells), at least 15% of cells positive for somatostatin (i.e., delta cells), and less than 2% of cells positive for AFP+ (i.e., non-target liver lineage), as measured by flow cytometry techniques; 3) has at least 90% viable cells, as measured by flow cytometry techniques; 4) has at least 1-fold (e.g., 1.5-fold) change in insulin or C-peptide, as detected by static glucose-stimulated insulin (C-peptide) secretion analysis; 5) has no detectable mycoplasma, sterility, viral negativity, and no more than 1 EU / mL endotoxin, as analyzed by pathogen testing.

[0158] In certain embodiments, at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) of the regenerative islet tissue provided herein is positive for C-peptide, glucagon, PDX1, and / or NKX6-1.

[0159] C-peptide is a by-product of insulin production, which is secreted by the beta cells of the islets together with insulin. The secretion of human C-peptide represents the activity of the beta cells within the islets and their ability to produce and release insulin. Insulin is the key hormone responsible for regulating blood glucose levels, and C-peptide is often used as a marker for insulin secretion due to its co-secretion with insulin in equimolar amounts. See, e.g., Wilcox G. Insulin and insulin resistance. Clin Biochem Rev. 2005 May; 26(2): 19-39.

[0160] Glucagon serves as a marker for the islets due to its specific secretion from the alpha-cells within the islets and its key role in regulating blood glucose levels, especially during the fasting state. This hormone counteracts the effects of insulin, promoting gluconeogenesis and glycogenolysis in the liver, thereby increasing blood glucose levels.

[0161] PDX1, which is referred to as pancreas duodenal homeobox 1, is a marker for the islets due to its important role in pancreatic development and beta-cell function. As a homeodomain transcription factor, PDX1 regulates the expression of genes that are essential for pancreatic development and maintenance of pancreatic cell identity.

[0162] NKX6-1 is a pancreas-specific marker that is expressed in pancreatic progenitor (PP) cells, endocrine progenitor (EP) cells, and a subset of certain pancreatic endocrine cells.

[0163] III-5. Media for preparing pancreatic endocrine cells

[0164] Any cell culture system known in the art can be used in the present disclosure. In certain embodiments, adherent culture systems are used in the methods of producing a population of pancreatic endocrine cells. The term "adherent culture" refers to a cell culture system in which cells are cultured on a solid surface, which can in turn be coated with a matrix. The cells can or can not be tightly adhered to the solid surface or matrix. Matrices for adherent culture can further include any one or combination of, e.g., polystyrene, polyester, polycarbonate, poly(N-isopropylacrylamide), polyornithine, laminin, polylysine, purified collagen, gelatin, cellulose, extracellular matrix, fibronectin, tenacin, vitronectin, polyglycolic acid (PGA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), Matrigel, hydroxyapatite, and amniotic membrane.

[0165] In certain embodiments, suspension culture can be used in the methods of producing a population of pancreatic endocrine cells. The term "suspension culture" as used herein refers to culturing cells such that the cells do not adhere to a solid support or culture vessel. To transfer cells to a suspension culture, the cells are removed from the culture receptacle, for example, by a cell scraper and transferred to a sterile low attachment dish that does not allow the cells to adhere to the surface of the dish, which contains the culture medium. Thus, the cells are cultured in suspension without adhering to the substrate or bottom of the culture dish.

[0166] A culture medium suitable for culturing cells is any medium suitable for growing a certain cell type in a culture dish. Culture media include, for example, MCDB, Ham's F10 (Sigma), Ham's F12 medium, Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM) (Sigma), IMDM medium, Medium 199, Eagle's Minimal Essential Medium (EMEM), aMEM medium, CMRL 1066, DMEM / F12, and mixtures thereof. Any of these media can be supplemented, as necessary, with salts (such as sodium chloride, calcium, magnesium, and phospho salts), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present in the final concentration range of micromolar), glucose or equivalent energy source, albumin, insulin, transferrin, selenium, fatty acids, 2-mercaptoethanol, thiomeritol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antioxidants, pyruvate, cytokines, and, where necessary, analogs thereof. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, are those previously used for culturing the cells, and will be apparent to the ordinarily skilled artisan.

[0167] IV. Pharmaceutical compositions and therapeutic uses

[0168] IV-1. Pharmaceutical compositions

[0169] In another embodiment, the present disclosure provides a population of EnSCs produced according to the methods provided herein, e.g., under the section "II. Endodermal Stem Cells."

[0170] In another embodiment, the present disclosure provides a population of PP cells, EP cells, and pancreatic endocrine cells produced according to the methods provided herein, e.g., under the section "III. Pancreatic Endocrine Cells."

[0171] In another embodiment, the present disclosure provides a pharmaceutical composition comprising a population of EnSCs, a population of PP cells, a population of EP cells, or a population of pancreatic endocrine cells provided herein, and a pharmaceutically acceptable medium.

[0172] The term "pharmaceutically acceptable" indicates that the one or more specified carriers, vehicles, diluents, excipients, and / or salts are generally chemically and / or physically compatible with the other ingredients constituting a formulation and physiologically acceptable to the recipient thereof. Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, a pharmaceutically acceptable liquid, gel or solid carrier, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffering agents, antioxidants, anesthetics, suspending / dispersing agents, chelating agents or chelators, diluents, adjuvants, excipients, or nontoxic auxiliary substances, other components known in the art, or various combinations thereof.

[0173] The compositions described herein can also include components that aid in implantation. The compositions described herein can be pyrogen-free or substantially pyrogen-free and free of pathogens, including bacterial contaminants, mold contaminants, and viruses.

[0174] In certain embodiments, the pharmaceutical composition can further include an immunosuppressant or an immunotolerizing agent.

[0175] IV-2. Therapeutic Uses

[0176] In another embodiment, the present disclosure is directed to the therapeutic use of an EnSC, an EnSC-derived PP cell, an EnSC-derived EP cell, an EnSC-derived pancreatic endocrine cell, or a regenerative islet tissue provided herein.

[0177] In one embodiment, the present disclosure provides a method of treating a disease or condition associated with impaired islet function in a subject in need thereof, comprising: administering to the subject an effective amount of a pancreatic endocrine cell derived from a population of EnSCs or a regenerative islet tissue, thereby treating a disease or condition associated with impaired islet function in a subject in need thereof. In certain embodiments, the population of EnSCs is generated according to the methods provided herein, e.g., under the section "II. Endodermal Stem Cells." In certain embodiments, the population of pluripotent stem cells is autologous or allogeneic.

[0178] A "disease or condition associated with impaired islet function" includes any disease or condition that can be treated by administering pancreatic endocrine cells or regenerative islet tissue, including diseases in which the subject has a reduced number or death, reduced density, or otherwise become dysfunctional, of pancreatic endocrine cells. As used herein, the term "impaired islet function" encompasses not only a reduction in optimal function of the islets of Langerhans in the pancreas, resulting in a reduced production or secretion of insulin and glucagon, but also partial or complete failure of islet function. This broad range of dysfunction can result in abnormal blood glucose levels, potentially leading to diabetes or other metabolic disorders. In the case of complete failure of function, the body loses the ability to effectively regulate blood glucose, which can lead to severe hyperglycemia and related health complications if left untreated.

[0179] In certain embodiments, the disease or condition associated with impaired islet function is diabetes. Diabetes is a chronic metabolic disease that occurs when the body cannot produce enough insulin or cannot effectively use the insulin it produces. Insulin is a hormone that plays a key role in regulating blood glucose levels. Diabetes is a leading cause of blindness, kidney failure, heart disease, stroke, and lower limb amputation.

[0180] In certain embodiments, the disease or condition associated with impaired islet function is Type 1 diabetes (T1D), Type 2 diabetes (T2D), Type 3c diabetes, or maturity onset diabetes of the young (MODY).

[0181] Type 1 diabetes (T1D), also known as juvenile diabetes, is an autoimmune disease in which the body's immune system attacks and destroys the insulin-producing cells in the pancreas.

[0182] Type 2 diabetes (T2D) typically begins with insulin resistance in peripheral tissues and continues with progressive loss of islet function due to reduction in beta-cell mass or dedifferentiation of beta cells under progressively worsening pathologic conditions. See, e.g., Talchai, C. et al., Cell 150, 1223-1234 (2012); and Chatterjee, S. et al., Lancet 389, 2239-2251 (2017). More than 30% of T2D patients eventually rely on exogenous insulin therapy. Cadaveric islet transplantation is a safe and effective treatment for insulin-dependent diabetes. See, e.g., Shapiro, A.M. et al., N. Engl. J. Med. 355, 1318-1330 (2006); and Marfil-Garza, B.A. et al., Lancet Diabetes Endocrinol. 10, 519-532 (2022). Notably, improved metabolic control after islet transplantation is associated with better kidney allograft function and long-term survival. See, e.g., Lablanche, S. et al., Lancet Diabetes Endocrinol. 6, 527-537 (2018); and Markmann, J.F. et al., Am. J. Transplant. 21, 1477-1492 (2021). However, the use of islet transplantation is severely hampered by the severe shortage of healthy donor organs and the complex isolation procedure.

[0183] 3c Type 3c diabetes is a type of diabetes that is secondary to exocrine pancreatic disease. It is also known as pancreatogenic or pan-creatogenous diabetes. The most common cause of 3c type diabetes is chronic pancreatitis, which is inflammation of the pancreas that causes damage to both exocrine and endocrine tissue. Other causes include pancreatic duct adenocarcinoma, hemochromatosis, cystic fibrosis, and previous pancreatic surgery. The pathogenesis of 3c type diabetes is not fully understood, but it is believed to be related to functional beta-cell loss in the pancreas due to underlying pancreatic disease. 3c type diabetes is less common than other forms of diabetes, but it is still relatively prevalent, especially in patients with chronic pancreatitis or pancreatic duct adenocarcinoma. Diagnosis of 3c type diabetes can be challenging because its symptoms are similar to those of other forms of diabetes, and the disease can be masked by the presence of other pancreatic conditions. See, e.g., Hart PA et al., Lancet Gastroenterol Hepatol. 2016 Nov;1(3):226-237.

[0184] Maturity onset diabetes of the young (MODY) is a monogenic form of diabetes characterized by early onset of diabetes. It is caused by a single pathogenic genetic variant that leads to abnormal function or developmental changes in insulin-secreting pancreatic beta cells. A common feature of MODY is early onset of diabetes, which usually occurs during childhood, adolescence, or young adulthood. Patients with MODY often have a strong family history of diabetes, and the condition is usually inherited within the family. Symptoms of MODY are similar to those of type 2 diabetes, including hyperglycemia and insulin resistance. Diagnosis of MODY can be challenging, as it is often confused with type 1 or type 2 diabetes. See, e.g., Bonnefond, A. et al., Nat Rev Dis Primers 9, 12 (2023).

[0185] The route of administration can include any suitable means, including, but not limited to, transcutaneous hepatic infusion, portal vein infusion, subcapsular kidney implantation, rectus abdominis injection, subcutaneous implantation, mesenteric injection, retroperitoneal injection, hepatic artery injection, iliac fossa injection, and the like. In some embodiments, the particular mode of administration selected will depend on the particular treatment, the patient’s disease condition or pathology, the nature or route of administration of other pharmaceutical or therapeutic agents administered to the subject, and the like.

[0186] In certain embodiments, the subject is a human.

[0187] In certain embodiments, the effective amount is about 0.5 to 3.0 million islet equivalent (IEQ) units. The IEQ quantity is a standard estimate of islet volume, where one IEQ equals a single spherical islet of 150 pm in diameter. See, e.g., Lembert N et al., Cell Transplant. 2003; 12(1): 33-41.

[0188] In certain embodiments, the method further comprises administering an immunosuppressant to the subject in need. When an immunosuppressant is used, it can be administered systemically or locally, and it can be administered prior to, concurrently with, or after administration of the regenerative pancreatic islet tissue.

[0189] In another embodiment, the disclosure provides use of a population of endoderm stem cells for the manufacture of regenerative pancreatic islet tissue for treating a disease or condition associated with impaired pancreatic islet function in a subject in need thereof.

[0190] In certain embodiments, the manufacture comprises the steps described herein, e.g., as described under the section “III. Pancreatic endocrine cells”.

[0191] In another embodiment, the present disclosure provides the first human tissue replacement therapy for treating a disease or condition associated with impaired islet function, such as diabetes, e.g., type 1 diabetes (T1D), type 2 diabetes (T2D). In certain embodiments, the therapy provided herein provides several significant advantages over existing options.

[0192] First, unlike traditional cadaveric islet transplantation, the therapy provided utilizes the patient's own cells (such as PBMCs) to generate functional islets. This not only improves tolerability but also provides a more readily available source compared to donor cadaveric islet transplantation.

[0193] Second, compared to hPSC-derived islets currently in clinical trials, the endoderm stem cells (EnSCs) used to generate functional islets in the present disclosure are non-tumor-forming in vivo and more suitable as progenitors for efficient mass production of islets. Their endoderm-specific properties and closer developmental relationship with the pancreatic lineage make them a superior option as islet progenitors compared to hPSCs (e.g., hiPSCs).

[0194] Third, while other trials have involved patients with T1D, the therapy disclosed herein can expand its indications to T2D, while allowing for the evaluation of the engraftment and functionality of EnSC-derived islets (regenerated islet tissue) without autoimmune interference. Furthermore, this therapy has the potential to treat advanced disease, including advanced T2D.

[0195] The above advantages can be achieved by the novel methods of the present disclosure for generating EnSCs and EnSC-derived islets (regenerating islet tissue) for therapy.

[0196] V. Kit

[0197] In one embodiment, the present disclosure provides a kit for generating an endoderm stem cell population from a pluripotent stem cell population, wherein the kit comprises a first set of factors, a second set of factors, a third set of factors, and a fourth set of factors, wherein the first set of factors comprises a Nodal signaling agonist and a WNT signaling agonist, the second set of factors comprises a Nodal signaling agonist and a fibroblast growth factor (FGF), the third set of factors comprises factors belonging to the TGF-β superfamily, FGF, hepatocyte growth factor (HGF), and VEGF, and the fourth set of factors comprises a WNT signaling agonist, a TGF-β inhibitor, and epidermal growth factor (EGF).

[0198] In certain embodiments, the fourth group of factors does not include FGF2 and / or Chir99021. In certain embodiments, the fourth culture medium does not include FGF.

[0199] In another embodiment, the present disclosure provides a kit for producing a population of pancreatic endocrine cells from a population of endoderm stem cells, wherein the kit comprises a fifth set of factors, a sixth set of factors, and a seventh set of factors, wherein the fifth set of factors comprises a BMP inhibitor, an agonist of Nodal signaling, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and / or nicotinamide, the sixth set of factors comprises a BMP inhibitor, a TGF-β inhibitor, and / or a γ-secretase inhibitor, and the seventh set of factors comprises T3 and / or nicotinamide.

[0200] In another embodiment, the present disclosure provides a kit for producing a population of pancreatic endocrine cells from a population of pluripotent stem cells, wherein the kit comprises a first set of factors through a seventh set of factors, wherein the first set of factors comprises an agonist of Nodal signaling and an agonist of WNT signaling, the second set of factors comprises an agonist of Nodal signaling and a fibroblast growth factor (FGF), the third set of factors comprises a factor belonging to the TGF-β superfamily, an FGF, a hepatocyte growth factor (HGF), and VEGF, the fourth set of factors comprises an agonist of WNT signaling, a TGF-β inhibitor, and an epidermal growth factor (EGF), the fifth set of factors comprises a BMP inhibitor, an agonist of Nodal signaling, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and nicotinamide, the sixth set of factors comprises a BMP inhibitor, a TGF-β inhibitor, and a γ-secretase inhibitor, and the seventh set of factors comprises T3 and nicotinamide.

[0201] In certain embodiments, the fourth set of factors does not comprise FGF2 and / or Chir99021. In certain embodiments, the fourth medium does not comprise FGF.

[0202] In another embodiment, the present disclosure provides a kit for producing a population of pancreatic endocrine cells from a population of PP cells, wherein the kit comprises a set of factors comprising a BMP inhibitor, a TGF-β inhibitor, and a γ-secretase inhibitor.

[0203] All publications and patents cited in this specification are hereby incorporated by reference in their entirety. Example

[0204] Example 1. Production of induced pluripotent stem cells (iPSCs) from human peripheral blood mononuclear cells (PBMCs)

[0205] This example demonstrates the production of induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) of a patient with impaired insulin secretion in T2D.

[0206] PBMCs were collected and tested for microorganisms, including bacteria, fungi, mycoplasma, HIV, HAV, HBV, HCV, HTLV, EBV, HCMV, and TP, to ensure their safety for subsequent use and subsequently used to generate iPSC cell lines under GMP conditions by the Sendai virus reprogramming system. PBMCs from patients were isolated from whole blood using a Ficoll gradient (WB as donor code). Whole blood was sampled in EDTA tubes. The blood was diluted with Dulbecco's Phosphate-Buffered Saline (DPBS) and poured onto Ficoll's solution and centrifuged at 400 g for 30 minutes at room temperature. The PBMC layer was collected and washed with DPBS. The PBMCs were frozen or continued for iPSC generation.

[0207] The Sendai virus reprogramming kit (Invitrogen, GMP grade) containing four reprogramming factors (OCT4, SOX2, KLF4, L-MYC) was used for iPSC generation. Two million PBMCs were first plated in a StemPro TM -34SFM (Gibco) SP34 in the presence of human SCF, FLT-3, IL-3 and IL-6 for 7 days. On the day of transduction, PBMCs were washed and counted, and the appropriate viral vector volume was calculated based on cell count and viral titer. PBMCs and viral vectors were mixed for transduction. Two days after transduction, cells were plated on culture dishes and gradually transferred to mTeSR1 culture medium over the next 7 days. iPSC colonies were selected and transferred to individual culture dishes within approximately 2 to 3 weeks and maintained in a 37°C incubator with a 5% CO2, 5% O2, 90% N2 environment.

[0208] Ten iPSC lines at passage 10 were tested for their in vitro differentiation potential (data not shown), and two of them (designated WB20 and WB34) were selected for further characterization and establishment of EnSC cell lines under GMP conditions.

[0209] Example 2. Generation of EnSCs from human iPSCs

[0210] This example demonstrates the generation of EnSCs from human iPSCs (hiPSCs).

[0211] 2.1 Human endoderm stem cell resources

[0212] Human endoderm stem cell lines were generated from patient-specific hiPSCs and maintained under serum-free and matrix-free conditions. hiPSCs were generated by the experiments described in Example 1 above.

[0213] 2.2 Generation of EnSCs from hiPSCs

[0214] EnSC cell lines were established from patient-specific hiPSC cell lines WB20 and WB34 and maintained in a 37°C incubator with 5% CO2, 5% O2, 90% N2 environment. Endoderm cells were differentiated from hiPSCs by dual activation of Nodal and WNT signaling pathways with Activin A (100 ng / mL) and CHIR99021 (2 mM) for 24 hours and subsequently cultured in the presence of bFGF (5 ng / mL), Activin A (100 ng / mL), VEGF (10 ng / mL), ascorbic acid (0.5 mM, Wako), and glutaMAX (2 mM, Invitrogen) for 4 days, followed by bFGF (10 ng / ml), TGF-a (20 ng / mL), VEGF (10 ng / mL), BMP4 (50 ng / mL), HGF (25 ng / mL), dexamethasone (40 ng / mL, Sigma) for 4 days. Thereafter, EnSCs were established by re-plating the aforementioned treated endoderm cells at 1 x 105cells / mL in MCDB131 supplemented with Wnt3A (1 mM), RSPONDIN1 (50 ng / mL), EGF (20 ng / mL), A83-01 (0.5 mM), ascorbic acid (0.5 mM, Wako), and glutamine (2 mM, Corning). 6

[0215] EnSCs were collected and dissociated into single cells for subsequent expansion, subcloning, or differentiation every 3 to 4 days. Typically, EnSCs at passage 20 were selected for quality control testing, including morphology, viability, purity, sterility, karyotype, and whole genome sequencing using well-known techniques such as flow cytometry and microscopic morphological examination.

[0216] As shown in Figure 1 and Figures 10 to 11 , the generated EnSCs were subjected to quality control under the criteria shown in Figure 10 . Specifically, whole genome sequencing (WGS) was performed on the EnSC cell lines to confirm the absence of newly arising cancer- or diabetes-related mutations not detected in the original collection of PBMCs Figure 1 . The tumor-forming potential of the EnSCs was further tested in immunocompromised mice (SCID Beige) for 6 months and it was confirmed that the generated EnSCs did not form teratomas, while pluripotent human pluripotent stem cells (hPSCs) exhibited a 100% teratoma formation percentage Figure 9 .

[0217] ​In general, EnSCs generated using the methods described herein have the following characteristics: 1) have typical epithelial morphology with clear cell boundaries and 3-5 pm in diameter as detected by microscopy (e.g., phase contrast observation); 2) at least 90% of cells are FOXA1 positive as measured by flow cytometric analysis; 3) have intact karyotype as analyzed by karyotyping; 4) contain no known cancer-associated mutations and have the lowest total mutational burden compared to the original PBMCs as detected by whole genome sequencing; 5) have at least 90% viable cells prior to freezing and at least 60% viable cells after thawing as detected by flow cytometric analysis; 6) are mycoplasma undetectable, sterile, and virus negative as analyzed by pathogen testing; 7) no teratoma is found as analyzed by teratoma formation test. Detailed description of quality control experiments of EnSCs and results thereof are provided in Example 4 below.

[0218] WB20 EnSC cell line was finally selected as a clinical grade cell line because it contains no known cancer-associated mutations and has the lowest total mutational burden compared to the patient PBMCs.

[0219] Example 3. Generation of regenerative pancreatic islet tissue from EnSCs

[0220] This example is about generation of regenerative pancreatic islet tissue from EnSCs under GMP conditions through two intermediate stages.

[0221] 3.1 Scalable differentiation of EnSCs to regenerative pancreatic islet tissue

[0222] EnSCs from Example 2 above were thawed and expanded in T225 flasks at a starting concentration of 2 x 10 6 cells / flask. Sterility was tested prior to the start of differentiation.

[0223] To induce pancreatic endoderm (stage 1), EnSCs were treated for 2 days in MCDB (Gibco) with a mix containing LDN 193189 (Stemgent) (200 nM), Noggin (R&D) (20 ng / mL), Activin A (R&D) (0.5 ng / mL), FGF10 (R&D) (20 ng / mL), RSPONDIN1 (R&D) (20 ng / mL), EGF (R&D) (20 ng / mL) and TPPB (Calbiochem) (500 nM); during the 2-4 days of induction, cells were further differentiated in MCDB supplemented with LDN 193189 (Stemgent) (200 nM), FGF10 (20 ng / mL), EGF (20 ng / mL), SANT1 (Tocris) (0.5 μΜ), Ascorbic acid (WAKO) (0.5 mM) and Retinoic acid (Sigma) (2 μΜ); during the 4-6 days of differentiation, cells were cultured in the presence of FGF10 (50 ng / mL), EGF (20 ng / mL), SANT1 (0.3 μΜ), Retinoic acid (0.2 μΜ), Nicotinamide (Sigma) (10 mM) and Ascorbic acid (0.5 mM).

[0224] To form uniform cell clusters (stage 2), pancreatic progenitor (PP) cells generated at stage 1 were single cell dispersed and suspended in AggreWell (STEMCELL) to continue uniform cell cluster formation for 3 days, and then transferred to an orbital shaker (90 to 110 rpm) for further pancreatic islet tissue reorganization and maturation.

[0225] For endocrine progenitor (EP) induction (stage 3), triple inhibition of BMP, TGF-β and Notch signaling pathways was modulated in the uniform cell clusters formed at stage 2 PP cells for 10 days in the presence of Noggin (20 ng / mL), A83-01 (Stemgent) (0.5 mM), γ-secretase inhibitor XX (GSI-XX) (2 μΜ, MERCK), Retinoic acid (0.1 μΜ) and SANT1 (0.1 μΜ).

[0226] For endocrine cell maturation (stage 4, 8-10 days), T3 (Sigma) (1 μM), nicotinamide (10 mM), and BMP4 (R&D) (2 ng / mL) were added to the stage 3 formulation. MCDB was routinely supplemented with glucose (22.5 mM, Sigma), sodium bicarbonate (Sigma), and ITS-X (Invitrogen), glutaMAX (Invitrogen), and ascorbic acid (0.5 mM, Wako). All cytokines were purchased from R&D Systems and, where applicable, were of GMP grade.

[0227] Under GMP conditions, two intermediate stages (pancreatic progenitor cells / PP and endocrine progenitor cells / EP) were used to meet the dosage requirements of each patient (1.2×10 6 Three batches of regenerated islet tissue were generated at a scale of 100 islet equivalents [IEQ] / patient).

[0228] 3.2 Characterization of EnSC-derived PP cells, EP cells, and islet tissue

[0229] PP cells generated at the end of stage 1 were evaluated for microbial contamination, morphology, purity, and viability, and were Figure 2 and Figure 10 Quality control was performed according to the criteria shown in . Overall, PP cells produced using the methods described herein had the following characteristics: 1) typical epithelial morphology with unclear cell borders and a diameter of no more than 3 μm as detected by microscopy (e.g., phase contrast); 2) at least 60% of cells were PDX1 positive as measured by flow cytometry; 3) at least 90% of cells were viable as measured by flow cytometry; and 4) mycoplasma was undetectable, sterile, virus negative, and had no more than 1 EU / mL of endotoxin as analyzed by pathogen testing.

[0230] EP cells generated at the end of stage 3 were evaluated for microbial contamination, morphology, purity, and viability, and were Figure 2 and Figure 10The quality control criteria shown in the middle are used. Overall, the EP cells generated using the methods described herein have the following characteristics: 1) have dense spherical cell clusters with indistinct cell boundaries, with an average diameter of about 150 pm as detected by microscopy (e.g., phase contrast observation); 2) at least 85% of the cells are PDX1 positive (i.e., pancreatic lineage), at least 60% of the cells are CHGA+ positive (i.e., endocrine lineage), at least 40% of the cells are C-peptide positive (i.e., beta cells), at least 20% of the cells are glucagon positive (i.e., alpha cells), at least 15% of the cells are somatostatin positive (i.e., delta cells), and less than 2% of the cells are AFP+ positive (i.e., non-target liver lineage) as measured by flow cytometric analysis; 3) have at least 90% viable cells as measured by flow cytometric analysis; 4) have at least 1-fold (e.g., 1.5-fold) change in insulin or C-peptide as detected by static glucose-stimulated insulin (C-peptide) secretion analysis; 5) are mycoplasma undetectable, sterile, virus negative, and have no more than 1 EU / mL endotoxin as analyzed by pathogen testing.

[0231] Regenerative islet tissue generated using the methods described herein is evaluated for microbial contamination, morphology, purity, and viability. The endocrine cell composition of the regenerative islet tissue (insulin+NKX6-1+ beta cells; glucagon+ alpha cells; somatostatin+ delta cells; chromogranin A+ endocrine cells) is further analyzed by flow cytometric analysis and single cell transcriptome analysis (scRNA-seq). The in vitro functionality of the regenerative islet tissue is assayed by glucose-stimulated insulin secretion (GSIS) analysis as for human cadaveric islets, and the in vivo functionality is evaluated by renal capsule or hepatic portal vein transplantation into a streptozotocin-induced diabetic mouse or monkey model. Non-target hepatocytes (HNF4A+ albumin+), cholangiocytes (SOX9+ CK7+), intestinal epithelial cells (CDX2+), and pancreatic duct cells (SOX9+ PTF1A+ PDX1+) are estimated from the scRNA-seq data.

[0232] The regenerative islet tissue generated at the end of Stage 3 is evaluated for microbial contamination, morphology, purity, and viability, and is assessed for Figure 3 and Figure 10The quality control is performed according to the criteria set forth in

[0233] Detailed descriptions of quality control experiments and results for PP cells, EP cells, and regenerative islet tissue are provided in Example 4, below. In addition, functional analysis of regenerative islet tissue produced using the methods described herein is also provided in Example 4, below.

[0234] Example 4. Quality control of EnSCs, PP cells, EP cells, and regenerative islet tissue and functional analysis of regenerative islet tissue

[0235] This example describes the quality control methods and results for EnSCs, PP cells, EP cells, and regenerative islet tissue produced using the methods described herein.

[0236] 4.1 Acceptance Criteria

[0237] Acceptance criteria for EnSCs, PP cells, EP cells, and regenerative islet tissue produced using the methods described herein are summarized in Figure 10

[0238] ​4.2 RNA extraction and quantitative real-time PCR Reverse transcription and qRT-PCR reactions were performed as previously reported (Cheng et al., 2012). RNA was prepared using an RNA kit (TIANGEN) according to the manufacturer's instructions and reverse transcribed into cDNA using random hexamers and oligo (dT) primers using GoScript reverse transcriptase (Promega). qRT-PCR reactions were performed using an ABIQ6 (Life Technology) system and SYBR green master mix (Roche). Expression levels were normalized to the housekeeping gene TBP. Primer information is provided at Figure 11 middle.

[0239] 4.3 Flow cytometry analysis technology

[0240] Single cell cell samples were collected. Surface marker staining was performed in PBS (Gibco) containing 0.2% BSA (Sigma). Cells were incubated with antibodies on ice for 30 minutes. For intracellular proteins, cells were fixed with 1.6% PFA (Servicebio) at 37°C for 30 minutes and washed with permeabilization wash buffer (BioLegend). Antibodies were incubated at room temperature for 30 minutes. Finally, cells were analyzed using a flow cytometer Celesta or Fortessa (BD). For cell viability testing, live cells were labeled with calcein blue dye (Invitrogen). Cells were incubated and analyzed using a flow cytometer Celesta or Fortessa (BD). See Figure 12 Antibody information in.

[0241] 4.4 Immunofluorescence

[0242] The regenerated islet tissue was fixed with 4% PFA for 15 minutes at 4°C and permeabilized with 0.5% Triton-100 (Sigma) and then blocked. The regenerated islet tissue was washed three times with PBST (PBS containing 0.05% Tween 20) for 10 minutes at room temperature (RT) before and after each staining step, and blocked with 2% BSA for 2 hours at 4°C. The regenerated islet tissue was stained overnight with diluted primary antibodies at 4°C, and the samples were then incubated in diluted secondary antibodies at 4°C for 2 hours. All antibodies were diluted in PBS containing 2% BSA. ProlongGold antifade reagent (Invitrogen) with 4,6-diamino-2-phenylindole (DAPI) was used to contrast stain the nuclei. The regenerated islet tissue was analyzed using a confocal fluorescence microscope (Olympus FV3000). Images of the regenerated islet tissue were captured and 3D projections were performed using Olympus software. Antibody information is listed in Figure 12 middle.

[0243] 4.5 In vitro static glucose-stimulated insulin (C-peptide) secretion assay

[0244] Prior to glucose stimulation, the regenerated islet tissue prepared using the method described in Example 3 above or primary islets provided and isolated by Shanghai Changzheng Hospital were washed at 37°C and starved for 2 hours in Krebs-Ringer buffer supplemented with 2 mM glucose. For glucose stimulation, the regenerated islet tissue was treated by Krebs-Ringer buffer with low (2 mM) glucose or high (20 mM) glucose alternately. Supernatant was collected after each 30 minutes of stimulation. C-peptide was measured by Human C-peptide ELISA kit (Mercodia, 10-1141-01) according to the manufacturer's instruction.

[0245] 4.6 Mycoplasma, sterility and endotoxin test

[0246] Culture supernatant samples were sent to certified laboratory Shanghai Simple Gene Medical Laboratory for testing.

[0247] 4.7 Karyotype analysis

[0248] EnSC samples were sent to certified laboratory Shanghai Simple Gene Medical Laboratory for standard G-banding chromosome analysis.

[0249] 4.8 Whole genome sequencing

[0250] DNA preparation

[0251] DNA degradation and contamination were monitored on 0.8% agarose gel. DNA purity was checked using Spectrophotometer (IMPLEN, CA, USA). DNA concentration was measured in 3.0 Fluorometer (Life Technologies, CA, USA) using DNA analysis kit according to the manufacturer's instruction.

[0252] DNA library preparation and sequencing

[0253] NEBNext® Ultra™ II Library Prep Kit for Illumina® (NEB, MA, USA) was used for DNA library preparation according to the manufacturer's instruction. Ultra DNA Library Prep Kit (NEB, USA) was used to construct libraries for sequencing. DNA was fragmented into -200 base pair fragments. Ends of DNA fragments were subjected to end repair procedure, which included addition of single "A" base followed by adaptor ligation. Products were purified and enriched by polymerase chain reaction (PCR) to amplify library DNA. Final libraries were quantified using KAPA Library Quant Kit (KAPA Biosystems, South Africa) and Agilent 2100 Bioanalyzer. Paired-end sequencing (2 x 150 base pairs) was performed on an Illumina NovaSeq 6000 sequencer (Illumina, USA).

[0254] 4.9 Single-cell RNA sequencing and data processing

[0255] Cell capture and cDNA synthesis

[0256] The regenerated pancreatic islet tissues produced in the methods described in Examples 1 to 3 were dissociated into single cells with 0.25% trypsin and resuspended in 1 x PBS at 1 x 10 6 The cell suspension (300 to 600 live cells per microliter as determined by Count Star) was loaded onto a Chromium Single Cell Controller (10x Genomics) to generate single-cell gel beads in emulsion according to the manufacturer's protocol using the Single Cell 3' Library and Gel Bead Kit V3.1 (10x Genomics, 1000121) and Chromium Single Cell G Chip Kit (10x Genomics, 1000120). Briefly, single cells were suspended in PBS containing 0.04% BSA. About 10,000 cells were added to each channel, and the target number of recovered cells was estimated to be about 15,000 cells. The captured cells were lysed, and the released RNA was barcoded by reverse transcription in individual GEMs. Reverse transcription was performed on a S1000™ Touch Thermal Cycler (Bio Rad) at 53 °C for 45 min, followed by 85 °C for 5 min, and held at 4 °C. cDNA was generated and then amplified, and the quality was assessed using an Agilent 4200 (completed by CapitalBio Technology, Beijing).

[0257] Single-cell RNA-Seq library preparation and sequencing

[0258] Single-cell 3' library and gel bead kit v3.1 were used to construct single-cell RNA-seq libraries according to the manufacturer's instructions. Libraries were sequenced using Illumina Novaseq6000 sequencer with a sequencing depth of at least 30,000 reads per cell, utilizing paired-end 150bp (PE150) read strategy (performed by CapitalBio Technology, Beijing). During tSNE clustering analysis, 15,244 cells were sequenced and 2,721 cells with low UMI (UMI count <5000) were excluded from sequenced cells.

[0259] 4.10 Quality control results

[0260] The morphology, purity, viability, and microbial contamination of EnSC-derived pancreatic progenitor cells (PP), endocrine progenitor cells (EP), and regenerative islet tissue were demonstrated to meet the acceptance criteria Figures 1 to 3 and Figure 10 ). The regenerative islet tissue showed similar morphology Figure 3 (panel a), endocrine cell composition Figure 3 (panels b and c), gene expression profile Figure 3 (panels d-f), and in vitro functionality (glucose-stimulated insulin secretion assay, GSIS) Figure 3 (panels g) to human cadaveric islets and exhibited functional efficacy in streptozotocin (STZ)-induced diabetic mouse Figure 4 (panels b-d) and monkey Figure 5 models. No non-targeted liver or intestinal lineage was detected when examined by scRNA-seq Figure 3 (panels f) or by FACS Figure 3 (panels h). No tumor formation or cystic / ductal structures indicative of cell proliferation Figure 9 were detected in immunocompromised animals transplanted with EnSCs or regenerative islet tissue during the experiments described in Examples 5-6 below.

[0261] Example 5. Transplantation of regenerative islet tissue into streptozotocin (STZ)-induced diabetic mouse models

[0262] This example demonstrates that no tumor formation or cystic / ductal structures indicative of cell proliferation were detected in STZ-induced diabetic mouse models transplanted with EnSCs or regenerative islet tissue.

[0263] 5.1 Resources of rodent strains

[0264] SCID Beige mice were obtained from Shanghai Lingchang Biotech Co. All experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Biochemistry and Cell Biology.

[0265] NCG-hIL15 mice were obtained from GemPharmatech Co. Ltd.

[0266] All animals were male and housed in individually ventilated cages (IVC) in a specific pathogen-free (SPF) animal facility with controlled temperature and lighting (12-hour light / dark cycle).

[0267] 5.2 Teratoma formation test

[0268] SCID Beige (4-6 weeks) male mice were transplanted with 1 x 105hiPSCs or 1 x 105EnSCs intramuscularly or subcutaneously in the nuchal region. The formation of teratomas was monitored during a 6-month period. 5 7

[0269] 5.3 Transplantation of regenerative islet tissue into streptozotocin (STZ)-induced diabetic model mice

[0270] STZ was immediately dissolved in 50 mM sodium citrate buffer (pH 4.5) to a final concentration of 20 mg / mL and kept in the dark and cold before injection. Administration of STZ was completed within 5 minutes of dissolution. SCID Beige male mice (4-6 weeks) were treated with 170 mg / kg STZ (Sigma-Aldrich, S0130) by intraperitoneal injection after 4 hours of fasting. On days 5 and 8, fasting blood glucose was measured in tail blood using a handheld glucometer (Roche) to ensure hyperglycemia. Regenerative islet tissue (1000-2000 IEQ) was transplanted under the left kidney capsule of diabetic mice. Human C-peptide secretion was measured by collection of mouse serum from the orbital cavity after 16 hours of fasting and 25 minutes after intraperitoneal injection of glucose (3 g / kg, 30% solution).

[0271] As shown in Figure 9 No tumor formation or cystic / ductal structures indicative of cell proliferation were detected in immunocompromised STZ-induced diabetic model mice transplanted with EnSCs or regenerative islet tissue produced using the methods provided herein during the experimental period.

[0272] As shown in Figure 4 ​​As shown in the middle, the regenerative islet tissue produced using the methods provided herein reversed hyperglycemia in immunocompromised (SCID Beige) mice with STZ-induced diabetes in terms of glycemic kinetics and human C-peptide secretion (subplots b-d). This indicates that the regenerative islet tissue produced using the methods provided herein can act as an islet.

[0273] Example 6. Regenerative islet tissue transplantation into STZ-induced diabetic monkeys

[0274] This example demonstrates that no tumor formation or cystic / ductal structures indicative of cell proliferation were detected in STZ-induced diabetic monkeys transplanted with EnSCs or regenerative islet tissue.

[0275] 6.1 Resources for the cynomolgus monkey model

[0276] Cynomolgus monkeys were obtained from and housed at WuXi Biologics. All animals were housed in individual stainless steel cages with controlled temperature (18-26 °C), relative humidity (40-70%) and light (12 hour light / dark cycle). All animals were provided with continuous water supply and fed with regular primate chow supplemented with fresh fruits twice daily (9-11 am and 3-4 pm). All animal care and handling were performed according to the guidelines established by the IACUC at WuXi Biologics.

[0277] 6.2 Induction of diabetes in monkeys

[0278] To induce hyperglycemia, male cynomolgus monkeys (3-6 years old) were fasted overnight and treated with intravenous injection of STZ at a dose of 50 mg / kg twice with a 2-week interval. STZ was freshly dissolved in sodium citrate buffer (pH 4.5) to a final concentration of 25 mg / ml before injection. Tail tip blood glucose was measured four times daily, 2 hours before and after morning and afternoon feeding. The exogenous insulin treatment dose for the animals was determined according to preprandial blood glucose.

[0279] 6.3 Immunosuppression strategy

[0280] Immunosuppressive treatment was initiated 2 days prior to transplantation (day -2). From day -2, Sirolimus (Pfizer) (0.5 mg, daily (once daily (q.d.)), per os (p.o.)) and Mycophenolate mofetil dispersible tablets (Roche) (62.5 mg, twice daily (b.i.d.), p.o.) were administered daily. Diclofenac sodium suppositories (Hubei Qianjiang) (50 mg, per rectum (p.r.)) were used 30 minutes prior to transplantation. ATG (Genzyme) (12.5 mg, intravenously (i.v.)) was injected 1 hour prior to transplantation and 48 hours after transplantation. Etanercept (Pfizer) (25 mg, subcutaneous injection (i.h.)) was used 1 hour prior to transplantation and on days 3 and 7 after transplantation.

[0281] 6.4 Transplantation surgery

[0282] Animals were fasted for at least 4 hours and anesthetized with ketamine (Imalgene® 50 (VIRBAC)) at 3 to 5 mg / kg i.m. Heart rate, temperature, blood oxygenation and blood pressure were monitored in real time during the surgical procedure. Transplantation of the regenerative islet tissue (6000 or 30000 IEQ) was performed by B-ultrasound guided percutaneous hepatic portal vein injection. Antibiotic treatment was continued for 7 days after transplantation.

[0283] Two diabetic monkeys were transplanted with 6,000 (monkey 1) or 30,000 (monkey 2) regenerative islet tissue, respectively. Monkey 1 was used to test the feasibility of hepatic portal vein injection of regenerative islet tissue without DSA, while monkey 2 was used to assess the short-term safety and efficacy of regenerative islet tissue.

[0284] As shown in Figure 9 No tumor formation or cystic / ductal structures indicative of cell proliferation were detected in the immunocompromised STZ-induced diabetic monkeys transplanted with EnSCs or regenerative islet tissue during the experimental period.

[0285] Example 7. Transplantation of regenerative islet tissue into diabetic humanized mice

[0286] This example demonstrates the functionality of regenerative islet tissue when transplanted into diabetic humanized mice.

[0287] 7.1 Generation of humanized mice by implantation of human PBMCs

[0288] ​PBMCs from the same patient or unrelated volunteer as in Example 1 were separated from whole blood using a Ficoll gradient. 4 hours before PBMC infusion, NCG-hIL15 female mice (6 weeks) were irradiated with 250 cGy. For each mouse, 5 million PBMCs were injected into the lateral tail vein. Flow cytometry analysis was performed weekly after injection to assess the effectiveness of PBMC implantation based on the ratio of mouse CD45 / human CD45 cells in mouse blood. The percentage of human CD45 cells increased to >40% within two weeks.

[0289] like Figure 6 As shown, the proportions of live cells (by SSC and FSC), human-derived blood cells (hCD45+), and mouse blood cells (mCD45+) in the three patient-humanized mice and the three volunteer-humanized mice were comparable (sub-panels a and b), indicating that the humanized mouse model was successfully generated.

[0290] like Figure 6 As shown, regenerated islet tissue grafts collected under the renal capsule of patient-humanized mice included human β cells (C-peptide+ and NKX6-1+), α cells (glucagon+), and δ cells (somatostatin+), as demonstrated by immunofluorescence staining of C-peptide (CPEP, red), glucagon (GCG, green), somatostatin (SST, purple), and NKX6-1 (cyan), indicating that the regenerated islet tissue was successfully transplanted into the humanized mouse model.

[0291] 7.2 Diabetes Induction, Transplantation, and Assessment

[0292] After identifying the implantation of human PBMCs, humanized mice were treated with 170 mg / kg STZ (Sigma-Aldrich, S0130) by intraperitoneal injection after 4 hours of starvation. One thousand regenerated islet tissues generated from the patient's EnSCs using the method described in Example 3 were transplanted under the left kidney capsule of 1) NCG-hIL15 mice humanized with the patient's PBMCs or 2) NCG-hIL15 mice humanized with PBMCs from unrelated volunteers. Fasting blood glucose was measured every two days. Glucose-stimulated human C-peptide secretion was performed as described above. Animals were sacrificed 28 days after transplantation, and transplant survival was detected by immunofluorescence of islet markers (C-peptide, glucagon, PDX1, NKX6-1) in tissue sections.

[0293] like Figure 4 As shown, patient-specific regenerated islet tissue survived and functioned under the renal capsule of diabetic immunocompromised mice humanized with the patient's own PBMCs, but was rejected by mice humanized with PBMCs from unrelated volunteers (sub-figure area eg), indicating that autologous regenerated islet tissue may be tolerated by the patient's immune system.

[0294] As shown in Figure 8A, the fasting blood glucose levels of STZ-induced diabetic humanized mice transplanted with patient-derived islet tissue were significantly lower than those of volunteer PBMC humanized diabetic mice transplanted with patient-derived islet tissue (panel f), which indicates that autologous patient-derived islet tissue can significantly reduce the fasting blood glucose levels of diabetic subjects. Figure 4

[0295] As shown in Figure 8B, the secretion of human C-peptide in STZ-induced diabetic humanized mice transplanted with patient-derived islet tissue was significantly higher than that of volunteer PBMC humanized diabetic mice transplanted with patient-derived islet tissue (panel g) at 7 and 14 days after islet tissue transplantation, 30 minutes after fasting and intraperitoneal (i.p.) glucose bolus injection, which indicates that autologous patient-derived islet tissue can function as islets to secrete insulin. Figure 4

[0296] Example 8. Clinical study

[0297] This example is about a clinical study of autologous EnSC-differentiated islet tissue from a T2D patient with impaired insulin secretion.

[0298] 8.1 Patient information

[0299] The patient was a 59-year-old male with a 25-year history of T2D who developed end-stage diabetic nephropathy and underwent kidney transplantation in June 2017. His estimated glomerular filtration rate (eGFR) and serum creatinine (SCr) levels were maintained at 90 to 105 ml / (min·1.73 cm 2 ) and 45-72 μmol / L, respectively, indicating good survival and function of the donor organ. He received anti-rejection drugs (Tacrolimus (Astellas) 1 mg b.i.d. and Mycophenolate mofetil (Roche) 0.5 mg b.i.d.), and a 20U dose of subcutaneous insulin injection once a day at bedtime, and oral anti-diabetic drugs (Acarbose (Bayer) 50 mg t.i.d. and Metformin (Merck) 0.75 g b.i.d.) ( Figure 7 ​​Figure 8.2: Patient’s blood glucose profile before and after autologous regenerative islet tissue transplantation. (Subfigure area a) shows the process from obtaining PBMCs from the patient until the final transplantation of the regenerative islet tissue into the patient. Figure 15 Due to the important issue of insulin administration inducing hypoglycemia, the adverse effect of anti-rejection drugs on glycemic control and the adverse effect of poor glycemic control on long-term survival of the donor kidney, the patient and the research team agreed to perform an autologous regenerative islet tissue transplantation.

[0300] 8.2 Regenerative islet tissue transplantation

[0301] Figure 4 (Subfigure area a) shows the process from obtaining PBMCs from the patient until the final transplantation of the regenerative islet tissue into the patient.

[0302] According to the regulatory guidelines of the clinical islet transplantation registration (CITR), the patient was performed under local anesthetics with heparin administration, an image-guided percutaneous transhepatic islet infusion into the main portal vein circulation. The patency of the main portal vein was assessed by monitoring the portal vein pressure during islet infusion and post-infusion doppler ultrasonography. A total of 1.2 million IEQ of regenerative islet tissue produced as a single batch and passing the acceptance criteria were directly delivered without prior cryopreservation. Portal venography and portal vein pressure were monitored throughout the procedure to ensure no portal vein embolism or portal hypertension. No glucocorticoids were used at any time. After surgery, the patient was monitored overnight and allowed out of bed the next day. The patient’s compliance with the scheduled appointments was 100% (some appointments were canceled or moved to a local hospital due to the COVID-19 pandemic).

[0303] Dose design

[0304] The rationale behind the 1.2 million IEQ unit dose of the regenerative islet tissue transplant for this T2D patient is based on the following facts: 1) there are approximately 4 to 6 million IEQ of islets in a healthy person, and it is estimated that only 1 / 3 of the islets are functional under physiological conditions upon glucose stimulation, which means that about 1.5 million IEQ of islets can be sufficient for glycemic control. This is confirmed by clinical observations that transplantation of 800,000 IEQ of cadaveric islets typically makes most T1D patients independent of exogenous insulin (see references below); 2) the patient's endogenous beta cell mass is significantly reduced (estimated at least 50%) as judged by pre- and postprandial c-peptide levels; and 3) a significant amount of the regenerative islet tissue can be lost during vascularization, as the regenerative islet tissue contains only pancreatic endoderm cells, but not vascular endothelial cells, which are important for post-transplantation revascularization of cadaveric islets. The 1.2 million IEQ dose was chosen because it was surmised that 50% of the regenerative islet tissue can be lost during implantation, and the remaining 0.6 million IEQ can be sufficient to supplement the endogenous islet function.

[0305] 8.3 Mixed Meal Tolerance Test (MMTT)

[0306] After an overnight fast (> 10 hours), at 7:30 AM, the patient was asked to consume a standard mixed meal consisting of 200 g steamed buns and 50 mL water at a constant rate within 5 minutes. Blood samples were collected before (0 minute) and after ingestion at 15, 30, 60, 120, 180, and 240 minutes. No degludec was administered 24 hours prior to MMTT, and no oral anti-diabetic drugs were administered 20 hours prior to MMTT.

[0307] 8.4 Assessment of Clinical Outcome

[0308] At designated visits, the patient was weighed and reported her Clark hypoglycemia awareness score, and routine and disease-specific assessments were performed. Endocrine function and diabetes-specific parameters were tested by mixed meal tolerance test (MMTT) at baseline and at 4, 8, 12, 16, 20, and 24 weeks and every 12 weeks thereafter (subplot area a). The patient's glycemic control was measured using a 24-hour real-time glucose monitoring system (Medtronic Guardian™ Reveal® Subcutaneous Continuous Glucose Monitoring System / CGMS). Central assessment of all information from the CGMS device was performed. Baseline and follow-up CGM glucose values were measured over the first 52 weeks, and the average duration of CGM device wear was at least 3 days. Figure 7

[0309] Safety Monitoring

[0310] ​Safety endpoints included treatment-emergent adverse events (TEAEs), treatment discontinuations due to adverse events, and adjudicated adverse events. Tumor formation was monitored every three months by enhanced magnetic resonance imaging of the upper abdomen and measurement of serum cancer-related antibodies.

[0311] Three primary clinical outcomes were monitored over the first 116 weeks (i.e., glycemic targets, reduction in exogenous insulin, and fasting and prandial stimulated circulating C-peptide / insulin levels) Figure 13 and Figure 14 A list of follow-up assessment results is provided in Table 1. Significant changes in patient glycemic control were observed as early as week 2 post-transplant, as MAGE decreased from 5.50 mmol / L to 3.60 mmol / L, and notably, TITR rapidly increased from 56.7% to 77.8% ( Figure 4 (Figure 16) and (panel h) and Figure 8 (Figure 17) and Figure 15 ) over the same period.

[0312] During the period between week 4 and week 12, a significant reduction in dynamic average glucose fluctuations was observed (from 5.50 (baseline) to 2.6 mmol / L) Figure 15 ) and a steady increase in TITR (from 81% to 90%) Figure 4 (Figure 16) and (panel h), Figure 8 (Figure 17) and Figure 15 ) over the same period. After week 32, the patient’s TITR had reached 99% and remained there Figure 4 (Figure 16) and (panel h and I), Figure 15 ) and postprandial glucose fluctuations / MAGE (the gold standard for glycemic variability) decreased from 5.50 mM (baseline) to 1.60 mM Figure 7 (Figure 17) and (panel d), Figure 8 (Figure 16) and (panel h-l), Figure 15 ) over the same period. Importantly, no episodes of hypoglycemia or severe hyperglycemia were observed during the entire follow-up period of 116 weeks post-surgery Figure 8 and Figure 15 ) over the same period. Importantly, no episodes of hypoglycemia or severe hyperglycemia were observed during the entire follow-up period of 116 weeks post-surgery

[0313] In addition, the MMTT revealed a trend towards stabilization of glycemic variability post-surgery, as evidenced by a steady fasting glucose concentration and a significant reduction in postprandial glucose concentration (maximum of 21.3 mM at baseline vs. 9.1 mM at week 105) Figure 4 (Figure 16) and (panel j), Figure 14Consistently, the area under the curve (AUC) derived from 5-point intravenous glucose values ​​decreased to 40% of baseline ( Figure 7 (sub-panel b)), which was further confirmed by the AUC of the values ​​obtained from the continuous glucose monitoring system (CGM) ( Figure 7 (Sub-panel c)). Hemoglobin A1c levels decreased from 6.6% (baseline) to 5.5% (week 85) and 4.6% (week 113) ( Figure 4 (Sub-figure area h) and Figure 16).

[0314] Notably, insulin requirements gradually decreased until complete withdrawal by the end of week 11 ( Figure 4 (sub-figure area h)), and oral antidiabetic drugs were gradually reduced from week 44 and discontinued at week 48 (acarbose) and week 56 (metformin) ( Figure 7 (Sub-image area a)).

[0315] When compared with preoperative levels, the mean postoperative fasting C-peptide level (0.68 nmol / L) increased 3-fold ( Figure 4 (sub-graph area k) and Figure 15 Notably, C-peptide ( Figure 4 (Sub-plot k)) and insulin ( Figure 4 (Sub-graph area 1)) showed a significant increase in secretion compared to the pre-operative test, which was confirmed by AUC ( Figure 7 (Sub-image area b)).

[0316] During the 116-week follow-up period, no tumor formation was detected by upper abdominal MRI or by measurement of serum tumor-associated antigen markers. Treatment-emergent adverse events (TEAEs) included: 1) transient abdominal distension and loss of appetite within 4 to 8 weeks, which resolved with methionine trichloride; and 2) reversible weight loss of <5% (80 kg to 76 kg).

[0317] Data from the first 116 weeks revealed significant improvements in glycemic control and islet function. The grafts were well tolerated, with no tumor formation or serious transplant-related adverse events. These data suggest that stem cell-derived islet tissue can rescue islet function in patients with advanced T2D.

[0318] While the present disclosure has been particularly shown and described with reference to particular embodiments, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the spirit and scope of the present disclosure as disclosed herein.

Claims

1. A method for generating an endoderm stem cell population, comprising: a) providing a population of pluripotent stem cells; b) culturing the population of pluripotent stem cells in a first culture medium comprising a Nodal signaling agonist and a WNT signaling agonist; c) culturing the cells in a second culture medium comprising a Nodal signaling agonist and fibroblast growth factor (FGF); d) culturing the cells in a third culture medium comprising a factor belonging to the TGF-β superfamily, FGF, hepatocyte growth factor (HGF), and VEGF; and e) culturing the cells in a fourth culture medium comprising a WNT signaling agonist, a TGF-β inhibitor, and epidermal growth factor (EGF); This generates a population of endoderm stem cells.

2. The method according to claim 1, wherein the fourth culture medium does not include FGF2 and / or Chir99021.

3. The method of any one of the preceding claims, wherein the Nodal signaling agonist is selected from the group consisting of activin A, Nodal, and GDF-8.

4. The method of claim 3, wherein the Nodal signaling agonist is activin A.

5. The method of any one of the preceding claims, wherein the WNT signaling agonist is selected from the group consisting of: CHIR99021, Wnt3A, Wnt3a-AFM, R-Spondin-1 and BIO (6-bromoindirubin-3'-oxime), SKL2001, BML-284, CP21R7 and SB 216763. The method of claim 5 , wherein the WNT signaling agonist is CHIR99021 or Wnt3A.

7. The method according to any one of the preceding claims, wherein the FGF is selected from the group consisting of basic FGF (bFGF), FGF4 and chimeric fibroblast growth factor (FGFC), FGF1, FGF10 and FGF7. The method according to claim 7 , wherein the FGF is bFGF.

9. The method according to any one of the preceding claims, wherein the factor belonging to the TGF-β superfamily is selected from the group consisting of: BMP4, BMP2 and BMP7.

10. The method according to claim 9, wherein the factor belonging to the TGF-β superfamily is BMP4.

11. The method of any one of the preceding claims, wherein the TGF-β inhibitor is selected from the group consisting of: A83-01, SB431542, ALK5 inhibitors, LDN-193189, Galunisertib (LY2157299), LY2109761, SB525334, SB505124, GW788388, LY364947, RepSox, LDN-193189, K02288, LDN-214117, SD-208, Vactosertib (TEW-7197), ML347, LDN-212854, DMH1, Pirfenidone, Alantolactone, SIS3, Hesperetin, and Dorsomorphin.

12. The method of claim 11, wherein the TGF-β inhibitor is A83-01.

13. The method according to any one of the preceding claims, wherein the pluripotent stem cells are selected from embryonic stem cells (ESC) and induced pluripotent stem cells.

14. A method according to any one of the preceding claims, comprising: a) providing a population of induced pluripotent stem cells; b) culturing the cells in a first culture medium comprising activin A and CHIR99021; c) culturing the cells in a second culture medium comprising activin A and bFGF; d) culturing the cells in a third culture medium comprising BMP4, bFGF, HGF, and VEGF; and e) culturing the cells in a fourth culture medium comprising Wnt3A, A83-01, and EGF; thereby generating a population of endoderm stem cells.

15. The method of any one of the preceding claims, wherein the second culture medium further comprises VEGF, ascorbic acid and / or glutaMAX.

16. The method according to any one of the preceding claims, wherein the third culture medium further comprises TGF-α and / or dexamethasone.

17. The method according to any one of the preceding claims, wherein the fourth culture medium further comprises Rspondin 1, ascorbic acid and / or glutamine.

18. The method according to any one of the preceding claims, wherein the incubation period of step b is 1 to 2 days (preferably 1 day).

19. The method according to any one of the preceding claims, wherein the culturing period of step c is 2 to 6 days (preferably 4 days).

20. The method according to any one of the preceding claims, wherein the culturing period of step d is 2 to 6 days (preferably 4 days).

21. The method of any one of the preceding claims, wherein steps b to d are performed at less than 10% O2 (preferably less than 8%, less than 6%, less than 4% or less than 2%; more preferably 5%).

22. The method of any of the preceding claims, wherein at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98% or at least 99%) of the endoderm stem cell population express SOX17, CDX2, SOX9, GATA6 and / or FOXA1.

23. The method of any one of the preceding claims, wherein the first culture medium, the second culture medium, the third culture medium, and the fourth culture medium independently contain a culture medium selected from the group consisting of mTeSR1, TeSR-AOF, Essential 8, NutriStem hPSC XF medium (Sartorius), RPMI / B27, SFD-based culture medium, MCDB131, DMEM / F12 medium, StemPro34-SFM, RPMI1640, IMDM, DMEM, Ham's F12, and CMRL1066.

24. The method according to any one of the preceding claims, wherein the first culture medium, the second culture medium, the third culture medium and the fourth culture medium are serum-free and / or matrix-free.

25. A population of endoderm stem cells produced according to the method of any preceding claim.

26. A method for producing a pancreatic endocrine cell population, comprising: a) providing an endoderm stem cell population; b) culturing the endoderm stem cell population in the presence of a BMP inhibitor, a Nodal signaling agonist, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and nicotinamide to generate a pancreatic progenitor (PP) cell population; c) culturing the PP cell population in the presence of a BMP inhibitor, a TGF-β inhibitor, and a γ-secretase inhibitor to generate an endocrine progenitor (EP) cell population; and d) culturing the EP cell population in the presence of the factors involved in step c, as well as T3 and nicotinamide; thereby producing a pancreatic endocrine cell population.

27. The method of claim 26, wherein the endoderm stem cell population comprises the endoderm stem cell population of claim 25.

28. The method of claim 26 or 27, wherein the BMP inhibitor is selected from the group consisting of Noggin, Doxormorphine, and LDN-193189.

29. The method of claim 28, wherein the BMP inhibitor comprises Noggin.

30. The method of any one of claims 26 to 29, wherein the TGF-β inhibitor is selected from the group consisting of: A83-01, SB431542, ALK5 inhibitors, LDN-193189, galactosidinib (LY2157299), LY2109761, SB525334, SB505124, GW788388, LY364947, RepSox, LDN-193189, K02288, LDN-214117, SD-208, vaquetinib (TEW-7197), ML347, LDN-212854, DMH1, pirfenidone, iliacin, SIS3, and hesperetin and doxormorphine.

31. The method of claim 30, wherein the TGF-β inhibitor comprises A83-01.

32. The method of any one of claims 26 to 31, wherein the γ-secretase inhibitor is selected from the group consisting of Compound E, GSI-XX and GSI-XXI, DAPT, LY-411575, RO4929097, and dibenzazepine (DBZ).

33. The method of any one of claims 26 to 32, wherein the Nodal signaling agonist is selected from the group consisting of activin A, Nodal, and GDF-8.

34. The method of claim 33, wherein the Nodal signaling agonist is activin A.

35. The method according to any one of claims 26 to 34, comprising: a) providing an endoderm stem cell population; b) culturing the endoderm stem cell population in the presence of Noggin, activin A, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and nicotinamide to generate a pancreatic progenitor (PP) cell population; c) culturing the PP cell population in the presence of Noggin, A83-01, and GSI-XX to generate an endocrine progenitor (EP) cell population; and d) culturing the EP cell population in the presence of T3 and nicotinamide; This gives rise to the pancreatic endocrine cell population.

36. The method according to any one of claims 26 to 35, wherein step b comprises culturing the endoderm stem cell population in the presence of Noggin, activin A, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, nicotinamide, and in the presence of at least one of Rspondin1, LDN-193189 and TPPB to produce a pancreatic progenitor (PP) cell population.

37. The method of any one of claims 26 to 36, wherein step c comprises culturing the PP cell population in the presence of Noggin, A83-01, GSI-XX, and in the presence of at least one of retinoic acid and SANT1 to produce an endocrine progenitor (EP) cell population.

38. The method of any one of claims 26 to 37, wherein step d comprises culturing the EP cell population in the presence of T3, nicotinamide, and BMP4.

39. The method according to any one of claims 26 to 38, wherein the culturing period of step b is 2 to 6 days.

40. The method according to any one of claims 26 to 39, wherein the culturing period of step c is 3 to 8 days.

41. The method according to any one of claims 26 to 40, wherein the culturing period of step d is 7 to 21 days.

42. The method according to any one of claims 26 to 41, wherein steps b to d are independently performed in a two-dimensional (2D) or three-dimensional (3D) environment.

43. A population of pancreatic endocrine cells produced according to the method of any one of claims 26 to 42.

44. A pharmaceutical composition comprising the pancreatic endocrine cell population according to any one of claims 26 to 43 and a pharmaceutically acceptable culture medium.

45. A method for treating a disease or condition associated with impaired pancreatic islet function in a subject in need thereof, comprising: administering to the subject an effective amount of pancreatic endocrine cells derived from an endoderm stem cell population (i.e., regenerating pancreatic islet tissue), Thereby treating a disease or condition associated with impaired pancreatic islet function in said subject in need thereof.

46. ​​The method of claim 45, wherein the population of endoderm stem cells is produced according to the method of any one of claims 1 to 25.

47. The method of claim 46, wherein the population of pluripotent stem cells is autologous or allogeneic.

48. The method of any one of claims 45 to 47, wherein the administering comprises portal vein infusion, subrenal capsule implantation, rectus abdominis injection, subcutaneous implantation, mesenteric injection, retroperitoneal injection, hepatic artery injection, or iliac fossa injection.

49. The method of any one of claims 45 to 48, wherein the subject is a human.

50. The method of any one of claims 45 to 48, wherein the effective amount is about 0.5 to 3.0 million islet equivalent (IEQ) units.

51. The method of any one of claims 45 to 50, wherein the disease or condition associated with impaired islet function is diabetes, such as type 1 diabetes (T1D), type 2 diabetes (T2D), type 3c diabetes, or maturity-onset diabetes of the young (MODY).

52. Use of a population of endoderm stem cells for producing regenerated pancreatic islet tissue for treating a disease or condition associated with impaired pancreatic islet function in a subject in need thereof.

53. Use according to claim 52, wherein said manufacturing comprises the steps defined in any one of claims 26 to 42.

54. A kit for generating an endoderm stem cell population from a pluripotent stem cell population, wherein the kit comprises a first set of factors, a second set of factors, a third set of factors, and a fourth set of factors, wherein The first group of factors includes Nodal signaling agonists and WNT signaling agonists, The second group of factors includes Nodal signaling agonists and fibroblast growth factor (FGF), The third group of factors includes factors belonging to the TGF-β superfamily, FGF, hepatocyte growth factor (HGF) and VEGF, and The fourth group of factors includes WNT signaling agonists, TGF-β inhibitors and epidermal growth factor (EGF).

55. The kit of claim 54, wherein the fourth group of factors does not include FGF2 and / or Chir99021.

56. A kit for generating a pancreatic endocrine cell population from an endoderm stem cell population, wherein the kit comprises a fifth group of factors, a sixth group of factors, and a seventh group of factors, wherein The fifth group of factors includes BMP inhibitors, Nodal signaling agonists, FGF10, EGF, SANT1, retinoic acid, ascorbic acid and / or nicotinamide, The sixth group of factors includes BMP inhibitors, TGF-β inhibitors and / or γ-secretase inhibitors, and The seventh group of factors includes T3 and / or nicotinamide.

57. A kit for generating a pancreatic endocrine cell population from a pluripotent stem cell population, wherein the kit comprises a first group of factors to a seventh group of factors, wherein The first group of factors includes Nodal signaling agonists and WNT signaling agonists, The second group of factors includes Nodal signaling agonists and fibroblast growth factor (FGF), The third group of factors includes factors belonging to the TGF-β superfamily, FGF, hepatocyte growth factor (HGF) and VEGF, The fourth group of factors includes WNT signaling agonists, TGF-β inhibitors and epidermal growth factor (EGF), The fifth group of factors includes BMP inhibitors, Nodal signaling agonists, FGF10, EGF, SANT1, retinoic acid, ascorbic acid and nicotinamide, The sixth group of factors includes BMP inhibitors, TGF-β inhibitors and γ-secretase inhibitors, and The seventh group of factors includes T3 and nicotinamide.

58. The kit of claim 57, wherein the fourth group of factors does not include FGF2 and / or Chir99021.

59. A kit for generating a pancreatic endocrine cell population from a PP cell population, wherein the kit comprises a set of factors comprising a BMP inhibitor, a TGF-β inhibitor, and a γ-secretase inhibitor.

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