TW202536168A - Methods for preparing endoderm stem cells and islets derived therefrom - Google Patents

Abstract

The present disclosure provides methods of generating a population of endoderm stem cells (EnSCs), a population of EnSC-derived PP cells, a population of EnSC-derived EP cells, or E-islets. The present disclosure also relates to the EnSCs, EnSC-derived PP cells, EnSC-derived EP cells, or E-islets obtained using the methods and therapeutic uses thereof.

Description

Method for preparing endoderm stem cells and islet tissue derived therefrom.

This application generally relates to methods for preparing endoderm stem cells (EnSC), EnSC-derived pancreatic precursor (PP) cells, EnSC-derived endocrine precursor (EP) cells, and EnSC-derived islet tissue (regenerated islet tissue (E-islets)), and the therapeutic use of EnSC, EnSC-derived PP cells, EnSC-derived EP cells, and regenerated islet tissue in the treatment of diseases or conditions related to impaired islet function.

Human pluripotent stem cells (hPSCs) can differentiate into various functional cells or tissues, such as pancreatic precursor cells (PPs) and islet tissue. These cells have been shown to survive, function, and reverse hyperglycemia in animal models of diabetes. See, for example, Pagliuca, FW et al. , Cell 159, 428-439 (2014); Rezania, A. et al. , Nat. Biotechnol. 32, 1121-1133 (2014) ; and Sneddon, JB et al. , Cell Stem Cell 22, 810-823 (2018) . Furthermore, recent clinical trials have demonstrated that when subcutaneously implanted in patients with type 1 diabetes, hPSC-derived pancreatic endodermal cells can further mature into diet-responsive β-like cells and secrete insulin, although the secretion amount is insufficient to achieve exogenous insulin independence. See, for example, Ramzy, A. et al. , Cell Stem Cell 28, 2047-2061 (2021) ; and Shapiro, AMJ et al. , Cell Rep. Med. 2, 100466 (2021) .

Nevertheless, the clinical application of hPSC-derived cells is negatively impacted by the complex differentiation process and the risk that any undifferentiated cells remaining in the system may form teratomas in vivo. Furthermore, the use of hPSCs in laboratory studies and cell-based therapies is hampered by their limited ability to generate pure differentiated cell populations in vitro. These limitations will affect the reproducibility and reliability of experiments, as well as the safety and efficacy of potential therapeutic applications.

Therefore, there is a need to develop improved methods for generating high-purity non-tumor-forming intermediate stem cell types for safer and more effective therapeutic applications, especially for treating diseases associated with impaired pancreatic function (such as diabetes).

One objective of this application is to provide a method for generating an endoderm stem cell population, comprising: a) providing a pluripotent stem cell population; b) culturing the pluripotent stem cell population in a first culture medium containing Nodal signaling agonist and WNT signaling agonist; c) culturing the cells in a second culture medium containing Nodal signaling agonist and fibroblast growth factor (FGF); d) culturing the cells in a third culture medium containing factors belonging to the TGF-β superfamily, FGF, hepatocyte growth factor (HGF), and VEGF; and e) The cells were cultured in a fourth medium containing a WNT signaling agonist, a TGF-β inhibitor, and epidermal growth factor (EGF) to generate an endoderm stem cell population.

In another embodiment, this disclosure provides a method for generating 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 niacinamide to generate a pancreatic precursor (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 precursor (EP) cell population; and d) The EP cell population was cultured in the presence of the factors involved in step c, as well as T3 and niacinamide, thereby generating a pancreatic endocrine cell population.

In another embodiment, this disclosure provides a method for treating a disease or condition associated with impaired pancreatic function in an individual of need, comprising: administering to the individual 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 pancreatic function in the individual of need.

In another instance, this disclosure provides an EnSC population generated according to the method provided herein.

In another embodiment, this disclosure provides a population of pancreatic endocrine cells or regenerated islet tissue produced according to the methods provided herein.

In another embodiment, this 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 regenerated islet tissue generated according to the methods provided herein.

In another embodiment, this disclosure provides the use of an endoderm stem cell population for the manufacture of regenerated islet tissue for the treatment of diseases or symptoms associated with impaired islet function in individuals in need.

In another embodiment, this disclosure provides the use of regenerated islet tissue in the manufacture of a medicament for treating diseases or symptoms associated with impaired islet function in individuals in need.

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

In another embodiment, this disclosure provides a kit for generating pancreatic endocrine cell populations from EnSC populations, 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 comprises a BMP inhibitor, a Nodal signaling agonist, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and/or niacinamide, the sixth group of factors comprises a BMP inhibitor, a TGF-β inhibitor, and/or a γ-secretase inhibitor, and the seventh group of factors comprises T3 and/or niacinamide.

In another embodiment, this disclosure provides a kit for generating pancreatic endocrine cell populations from pluripotent stem cell populations, wherein the kit comprises factors in groups one through seven, wherein the first group comprises Nodal signaling agonists and WNT signaling agonists, the second group comprises Nodal signaling agonists and fibroblast growth factor (FGF), and the third group comprises factors belonging to the TGF-β superfamily, FGF, and hepatocyte growth factor. The fourth group of factors includes HGF and VEGF, WNT signaling promoters, TGF-β inhibitors and epidermal growth factor (EGF), the fifth group of factors includes BMP inhibitors, Nodal signaling promoters, FGF10, EGF, SANT1, retinoic acid, ascorbic acid and niacinamide, the sixth group of factors includes BMP inhibitors, TGF-β inhibitors and γ-secretase inhibitors, and the seventh group of factors includes T3 and niacinamide.

In another embodiment, this disclosure provides a kit for generating pancreatic endocrine cell populations from PP cell populations, wherein the kit contains a set of factors including a BMP inhibitor, a TGF-β inhibitor, and a γ-secretase inhibitor.

The following description of this disclosure is intended only to illustrate various embodiments of this disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, variations, and modifications can be made without departing from the scope of this disclosure, and it should be understood that such equivalent embodiments are included herein. All references cited herein (including disclosures, patents, and patent applications) are incorporated herein by reference in their entirety.

I. General definition

Unless the context clearly indicates otherwise, the singular terms “a,” “an,” and “the” include the plural. For example, reference to “(a) cell” means one or more cells, and reference to “(the) method” includes reference to equivalent steps and methods disclosed herein and/or known to those skilled in the art. Similarly, unless the context clearly indicates otherwise, the word “or” is intended to include “and.” Although methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, suitable methods and materials are described below.

As used herein, the term “about” or “approximately” means within 20% of the given value or range, preferably within 10%, and even better within 5%.

As used in this article, the term "cell" refers to an individual cell, a cell line, or a culture derived from such cells.

As used in this article, the term "Nodal signaling enhancer" refers to substances that stimulate or enhance the Nodal signaling pathway. The Nodal signaling pathway plays a crucial role in pattern formation and differentiation during the early stages of chordate development, particularly before and during gastrulation. This pathway is essential for establishing the body axis and for the formation of the mesoderm and endoderm.

As used herein, the term "Notch signaling pathway" refers to a pathway that plays a crucial role in regulating cell fate, cell proliferation, and cell death during development. This pathway is unique in that it primarily involves interactions between adjacent cells because the ligands that activate Notch receptors are mostly transmembrane proteins. Inhibition of the Notch signaling pathway can be achieved by substances that disrupt the function of any protein within the pathway or prevent functional interactions between proteins in two pathways. Exemplary inhibitors of the Notch signaling pathway include (but are not limited to) γ-secretase inhibitors and RBPJ inhibitor-1. See, for example, Hurtado, C et al ., Disruption of NOTCH signaling by a small molecule inhibitor of the transcription factor RBPJ. Sci Rep 9, 10811 (2019) .

As used in this article, the term "WNT signaling enhancer" 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 key role in many physiological processes, including stem cell behavior, cell polarity, and tissue development.

As used in this article, the term "fibroblast growth factor" or "FGF" refers to members of the FGF family, which is a diverse group of secreted proteins that transmit signals through receptor tyrosine kinase and regulate various cellular processes.

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 regulating many aspects of physiological development and tissue stability. Members of the TGF-β family include secretory peptides that play key roles in embryogenesis and adult tissue maintenance, but also promote the development of various diseases. BMPs, or bone morphogenetic proteins, constitute a subset of the signaling molecules within the TGF-β superfamily. Specifically, "BMP4" refers to the protein encoded by the human BMP4 gene, which is a member of the BMP family and thus part of the broader TGF-β superfamily.

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, for example, by at least 10% or more, such as 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, such as its ability to reduce the amount and/or activity of TGF-β or its receptors, can be determined, for example, by measuring the amount and/or activity of the expression product of TGF-β or its receptors. In some embodiments, the inhibitor may be an inhibitory nucleic acid; an aptamer; an antibody or a binding fragment thereof; or a small molecule.

As used herein, the term "γ-secretase inhibitor" refers to an agent that can reduce the expression and/or activity of γ-secretase, for example, by at least 10% or more, such as 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, such as its ability to reduce the amount and/or activity of γ-secretase, can be determined, for example, by measuring the amount and/or activity of the expression product of γ-secretase. In some embodiments, the inhibitor may be an inhibitory nucleic acid; an aptamer; an antibody or a binding fragment thereof; or a small molecule. γ-secretase is a protease complex that cleaves single-pass transmembrane proteins within a transmembrane domain. It is a main membrane protein and belongs to the class of intramembrane proteases.

As used herein, the term "BMP inhibitor" refers to an agent that can reduce the expression and/or activity of BMP, for example, by at least 10% or more, such as 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, such as its ability to reduce the amount and/or activity of BMP, can be determined, for example, by measuring the amount and/or activity of the BMP expression product. In some embodiments, the inhibitor may be an inhibitory nucleic acid; an aptamer; an antibody or a binding fragment thereof; or a small molecule.

As used in this article, the term "Activin A" refers to a member of the TGF-β superfamily that is structurally similar to TGF-β 1 and conducts signals through the common SMAD2/3 (mothers against decapentaplegic) homologues 2 and 3.

As used in this article, the term "CHIR99021" refers to the GSK-3a and GSK-3b inhibitors with CAS number 252917-06-9.

As used in this article, the term "bFGF" is used interchangeably with the term "FGF2" and refers to the growth factor and signal transduction protein encoded by the FGF2 gene.

As used in this article, the term "Wnt3A" refers to the protein encoded by the WNT3A gene in the human body.

As used in this article, the term "A83-01" refers to the TGF-β type I receptor inhibitor with CAS number 909910-43-6.

As used in this article, the term "Rspondin1" refers to the secretory protein in the human body encoded by the RSPO1 gene.

As used in this article, the term "LDN-193189" refers to the selective BMP signal transduction inhibitor with CAS number 1062368-24-4.

As used in this article, the term "TPPB" refers to protein kinase C activator with CAS number 497259-23-1.

As used in this article, the term "Noggin" refers to a protein involved in the development of many body tissues, including nerve tissue, muscles, and bones. In humans, noggin is encoded by the NOG gene.

As used in this article, the term "GSI-XX" refers to the γ-secretase inhibitor with CAS number 209984-56-5.

As used in this article, the term "epidermal growth factor" or "EGF" refers to a protein that stimulates cell growth and differentiation by binding to its receptor (epidermal growth factor receptor (EGFR)). The term "TGF-α" refers to a protein encoded by the TGFA gene in the human body. TGF-α is a member of the epidermal growth factor (EGF) family.

As used in this article, the term "hepatocyte growth factor" or "HGF" refers to a mitogen produced by stromal cells that stimulates the proliferation, motility, morphogenesis, and angiogenesis of epithelial cells in various organs. It exerts its effect through tyrosine phosphorylation of the receptor c-Met. HGF plays an important role in organ development, the self-repair of injured tissues, and the protection of epithelial and non-epithelial organs through anti-apoptotic and anti-inflammatory signaling. See, for example, 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 .

As used herein, the term "vascular endothelial growth factor" or "VEGF" refers to a growth factor with significant pro-angiogenic activity, exerting pro-proliferative and anti-apoptotic effects on endothelial cells. It enhances vascular permeability, promotes cell migration, and actively regulates both normal and pathological angiogenesis. The human VEGF family includes several members, including VEGF-A (with different isoforms), VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, placental 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 primarily found on vascular and lymphoid endothelial cells. VEGFR-2 exhibits 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, for example, 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 .

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

As used in this article, the term "islets" refers to the smaller clusters of specialized cells in the pancreas, also known as Langerhans islets. These islets are responsible for secreting hormones that regulate blood glucose levels. Islets contain different types of cells, including B cells (β cells) that secrete insulin, A cells (α cells) that secrete glucagon, D cells (δ cells) that secrete somatostatin, and pancreatic polypeptide-secreting cells that secrete pancreatic polypeptides. Islets function as the endocrine component of the pancreas and play a crucial role in maintaining blood glucose levels.

As used herein, the term "regenerated islet tissue" refers to a cluster of pancreatic endocrine cells obtained by optimized differentiation culture of EnSC using the methods described in this disclosure. These regenerated islet tissues possess morphology, endocrine cell composition, gene phenotype, and/or function similar to natural islets.

As used herein, the terms "treatment," "treat," or "treating" in relation to a condition refer to the management, elimination, reduction, or improvement of a condition and/or its associated symptoms. While not excluding the possibility that treatment of a condition does not necessarily eliminate the condition or its associated symptoms completely, as used herein, the term "treatment" may include "preventive treatment," which is administered before the development of any symptoms or manifestations of a condition to reduce or prevent the occurrence or recurrence of the condition in individuals who are not currently ill but are at risk, or whose condition is prone to recurrence, or who have a risk of relapse or are prone to recurrence, or to reduce the likelihood of relapse of a previously controlled condition. Within the meaning of this invention, "treatment" also includes relapse prevention or a preventative phase, as well as the treatment of acute or chronic symptoms and/or functional impairments. Treatment can target symptoms, such as by suppressing them. Treatment can be effective in short, medium, or long-term, such as in maintenance therapy.

As used in this article, “auto” cells refer to any cells derived from the same individual into which they are later reintroduced.

As used in this article, "allogeneic" cells refer to any cells derived from different individuals of the same species.

As used herein, the term "effective dose" refers to the amount of a compound or cell sufficient to achieve therapeutic effect on a disease when administered to a patient. An effective dose can be an amount that effectively achieves prevention and/or an amount that effectively achieves avoidance. An effective dose can be an amount that effectively achieves relief, an amount that effectively achieves the following objectives: prevention of the occurrence of symptoms, reduction of the occurrence and severity of symptoms, elimination of the occurrence of symptoms, slowing the development of symptoms, preventing the development of symptoms, and/or achieving prevention and avoidance of symptoms. "Effective dose" can vary depending on the disease and its severity, as well as the patient's age, weight, medical history, sensitivities, and pre-existing conditions. For the purposes of this invention, the term "effective dose" and "therapeutic effective dose" are synonymous.

As used herein, the term "individual" is not limited to a specific species or sample type. For example, the term "individual" can refer to a patient, and usually to a human patient. However, this term is not limited to humans and therefore encompasses a wide range of mammal species, such as non-human veterinary mammals, such as dogs, cats, rabbits, pigs, rodents, horses, or monkeys.

II. Endoderm stem cells

In one embodiment, this disclosure provides a method for generating an endoderm stem cell (EnSC) population, comprising: a) providing a pluripotent stem cell population; b) culturing the pluripotent stem cell population in a first culture medium containing Nodal signaling agonist and WNT signaling agonist; c) in a medium containing Nodal signaling agonist and fibroblast growth factor (FGF). The cells are cultured in a second medium; d) the cells are cultured in a third medium containing factors belonging to the TGF-β superfamily, FGF, hepatocyte growth factor (HGF) and VEGF; and e) the cells are cultured in a fourth medium containing a WNT signaling agonist, a TGF-β inhibitor and epidermal growth factor (EGF); thereby generating an endoderm stem cell population.

In some implementations, the culture time for step b is 1 to 2 days. In some implementations, the culture time for step b is 1 day. In some implementations, the culture time for step c is 2 to 6 days. In some implementations, the culture time for step c is 4 days. In some implementations, the culture time for step d is 2 to 6 days. In some implementations, the culture time for step d is 4 days.

In some embodiments, steps b to d are performed at less than 10% _O2 , such as less than 8 _% , less than 6 _% , less than 4 _% , or less than 2% _{O2. In some embodiments, steps b to d are performed at 5% O2 .}

In some embodiments, the method further includes washing the cells obtained from step b before starting step c, washing the cells obtained from step c before starting step d, and washing the cells obtained from step d before starting step e. In some embodiments, there is no washing step in steps b through e.

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 endoderm stem cell population express SOX17, CDX2, SOX9, GATA6, and/or FOXA1.

The expression of the marker can be detected by any method known in this technique, including (but not limited to) fluorescently activated cell sorting (FACS), Western blotting, mRNA amplification-based methods (such as PCR, isothermal amplification, etc., which may include reverse transcription and can be applied to detect expression from single cells or multiple cells), Northern blotting, immunostaining, etc. Furthermore, the expression of such markers can be inferred from the expression of reporter structures controlled by genetic elements (such as fluorescent proteins, whose expression can be detected visually; antibiotic resistance genes, whose expression can be detected by cell survival rates in the presence of antibiotics, etc.), such as promoters of a aforementioned marker or fragments thereof.

II-1. Pluripotent stem cells

In some embodiments, the pluripotent stem cell population is autologous or allogeneic. In some embodiments, the pluripotent stem cells are selected from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).

As used herein, the terms "embryonic stem cell" and "ESC" are used interchangeably and refer to the pluripotent stem cells of the inner cell mass of the blastocyst (see U.S. Patents 5,843,780 and 6,200,806, which are incorporated herein by reference). Distinguishing features of embryonic stem cells define their phenotype. Therefore, a cell possesses one or more distinctive features of embryonic stem cells that distinguish it from other cells; it then exhibits the phenotype of an embryonic stem cell. Illustrative distinguishing features of embryonic stem cells include (but are not limited to) gene expression profiles, proliferative capacity, differentiation capacity, karyotype, responsiveness to specific culture conditions, and similar features.

As used herein, the terms "induced pluripotent stem cells" and "iPSCs" are used interchangeably and refer to pluripotent cells artificially derived from self-differentiated somatic cells (i.e., non-pluripotent cells) (e.g., through complete or partial inversion induction). Pluripotent cells can differentiate into cells of all three germ layers.

The ectoderm, mesoderm, and endoderm are the three germ layers that form during embryonic development. The mesoderm is the middle layer, the ectoderm is the outer layer, and the endoderm is the inner layer. The ectoderm forms the surface ectoderm, neural crests, and neural tube. The surface ectoderm develops into the epidermis, hair, nails, lens of the eye, sebaceous glands, cornea, tooth enamel, and the epithelium of the mouth and nose. The neural crests of the ectoderm develop into the peripheral nervous system, adrenal medulla, melanocytes, facial cartilage, and dentin of teeth. The neural tubes of the ectoderm develop into the brain, spinal cord, posterior pituitary gland, motor neurons, and retina. The mesoderm forms the stroma, mesothelium, non-epithelial blood cells, and coelomic cells, which constitute muscles (smooth and striated muscles), bones, cartilage, connective tissue, adipose tissue, the circulatory system, the lymphatic system, the dermis, the urogenital system, the plasma membrane, and the notochord. The endoderm forms the pharynx, esophagus, stomach, small intestine, colon, liver, pancreas, bladder, the epithelial portions of the trachea and bronchi, lungs, thyroid gland, and parathyroid gland.

Inducible pluripotent stem cells (iPSCs) can be generated using methods known in this art. For example, the iPSCs provided by the methods disclosed herein can be generated from peripheral blood mononuclear cells (PBMCs). In some embodiments, PBMCs are obtained from human individuals who will undergo EnSC-derived therapy. In some embodiments, iPSCs are generated by culturing PBMCs in the presence of human SCF, FLT-3, IL-3, and IL-6, followed by transduction of PBMCs with four reprogramming factors: OCT4, SOX2, KLF4, and L-MYC.

II-2. Used for manufacturing EnSC Culture medium

The primary culture medium used in steps b through e of the method provided herein is not particularly limited. Any culture medium may be used as the primary culture medium, as long as it enables the production of endoderm stem cells. In some embodiments, the primary culture medium is serum-free and/or matrix-free. In some implementations, the basic culture medium is selected from the following group: mTeSR ^™ 1, TeSR ^™ -AOF, Essential 8 ^™ , ^NutriStem® hPSC XF medium (Sartorius), RPMI/B27 ^™ , SFD-based medium, MCDB131, Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (DMEM/F12 medium), StemPro ^™ 34-SFM, RPMI-1640, Iscove's Modified Dulbecco's Medium (IMDM), Ham's F12, and CMRL-1066.

Regarding culture temperature, culture at 35.0°C or higher has been shown to promote cell differentiation. Culture temperature is one that does not damage cells, such as preferably 35.0°C to 42.0°C, or even better 36.0°C to 40.0°C, or even more preferably 37.0°C to 39.0°C.

First culture medium

In some embodiments, the first culture medium contains a Nodal signaling activator and a WNT signaling activator. Exemplary Nodal signaling activators include (but are not limited to) activin A, Nodal, and GDF-8. In some embodiments, the Nodal signaling activator is activin A.

In some implementations, 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.

Exemplary WNT signaling enhancers include (but are not limited to) CHIR99021, Wnt3A, Wnt3a-AFM, R-Spondin-1, and BIO (6-bromoindorubin-3'-oxime), SKL2001, BML-284, CP21R7, and SB 216763. In some embodiments, the WNT signaling enhancer is CHIR99021 or Wnt3A.

In some embodiments, the concentration of the WNT signal transduction agonist is in the range of about 0.2 μM to about 4 μM, such as 0.4 μM, 0.6 μM, 0.8 μM, 1 μM, 1.2 μM, 1.4 μM, 1.6 μM, 1.8 μM, 2.0 μM, 2.2 μM, 2.4 μM, 2.6 μM, 2.8 μM, 3.0 μM, 3.2 μM, 3.4 μM, 3.6 μM, 3.8 μM or 4.0 μM.

In some embodiments, the first culture medium contains activator A and CHIR99021. In some embodiments, the first culture medium contains a culture medium supplemented with activator A and CHIR99021.

Second culture medium

In some embodiments, the second culture medium contains a Nodal signaling agonist and 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 some embodiments, the FGF is bFGF.

In some implementations, the Nodal signaling activator is activator A.

In some implementations, 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.

In some implementations, the concentration of 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.

In some embodiments, the second culture medium contains activator A and bFGF. In some embodiments, the second culture medium contains a culture medium supplemented with activator A and bFGF.

Those skilled in this art will understand that other necessary supplements as described can be added to the culture medium. In some embodiments, the second culture medium further comprises VEGF, ascorbic acid, and/or glutamax.

Third culture medium

In some implementations, the third culture medium contains factors belonging to the transforming growth factor-β (TGF-β) superfamily, FGF, hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF).

Illustrative factors belonging to the TGF-β superfamily include (but are not limited to) BMP4, BMP2, and BMP7. In some implementations, the factor belonging to the TGF-β superfamily is BMP4.

In some implementations, the concentration of factors 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.

In some embodiments, the FGF is bFGF. In some 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.

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.

In embodiments, 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.

In some embodiments, the third culture medium contains BMP4, bFGF, HGF, and VEGF. In some embodiments, the third culture medium contains a culture medium supplemented with BMP4, bFGF, HGF, and VEGF.

Those skilled in this art will understand that other necessary supplements as described can be added to the culture medium. In some embodiments, the third culture medium further comprises TGF-α and/or dexamethasone.

Fourth culture medium

In some implementations, the fourth culture medium contains a WNT signaling agonist, a TGF-β inhibitor, and epidermal growth factor (EGF). Examples of 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 some implementations, the TGF-β inhibitor is A83-01.

In some implementations, the WNT signaling activator is Wnt3A.

In some embodiments, the concentration of the WNT signal transduction agonist is in the range of about 0.2 μM to about 4 μM, such as 0.4 μM, 0.6 μM, 0.8 μM, 1 μM, 1.2 μM, 1.4 μM, 1.6 μM, 1.8 μM, 2.0 μM, 2.2 μM, 2.4 μM, 2.6 μM, 2.8 μM, 3.0 μM, 3.2 μM, 3.4 μM, 3.6 μM, 3.8 μM or 4.0 μM.

In some implementations, 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, and 2.0 mM.

In some implementations, 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.

In some embodiments, the fourth culture medium does not contain FGF2 and/or CHIR99021.

In some embodiments, the fourth culture medium comprises Wnt3A, A83-01, and EGF. In some embodiments, the fourth culture medium comprises a culture medium supplemented with Wnt3A, A83-01, and EGF (e.g., MCDB131).

Those skilled in the art will understand that other necessary supplements as described can be added to the culture medium. In some embodiments, the fourth culture medium further comprises Rspondin1, ascorbic acid, and/or glutamic acid.

In some embodiments, the method for generating an endoderm stem cell population includes: a) providing an induced pluripotent stem cell population; b) culturing the cells in a first medium containing activin A and CHIR99021; c) culturing the cells in a second medium containing activin A and bFGF; d) culturing the cells in a third medium containing BMP4, bFGF, HGF, and VEGF; and e) culturing the cells in a fourth medium containing Wnt3A, A83-01, and EGF; thereby generating an endoderm stem cell population.

In some embodiments, methods for generating endoderm stem cell populations include: a) providing an induced pluripotent stem cell population; b) culturing cells in a first medium containing approximately 50 ng/mL to approximately 150 ng/mL activator A and approximately 1 μM to 3 μM CHIR99021; c) culturing cells in a second medium containing approximately 50 ng/mL to approximately 150 ng/mL activator A and approximately 4 ng/mL to approximately 6 ng/mL bFGF; d) culturing cells in a second medium containing 40 ng/mL to approximately 60 ng/mL BMP4, 5 ng/mL to approximately 15 ng/mL bFGF, 15 ng/mL to approximately 35 ng/mL HGF, and 5 ng/mL to approximately 15 ng/mL HGF. Cells were cultured in a third medium containing 10 ng/mL of VEGF; and e) cells were cultured in a fourth medium containing about 0.5 μM to about 1.5 μM of Wnt3A, about 0.1 mM to about 1.0 mM of A83-01 and 10 ng/mL to about 30 ng/mL of EGF; thereby generating an endoderm stem cell population.

In some embodiments, methods for generating endoderm stem cell populations include: a) providing an induced pluripotent stem cell population; b) culturing the cells for 12 to 48 hours (e.g., 24 hours) in a first medium containing about 50 ng/mL to about 150 ng/mL activator A and about 1 μM to 3 μM CHIR99021; c) in a medium containing about 50 ng/mL to about 150 ng/mL activator 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 μM CHIR99021. d) Culture cells in a second medium containing approximately 40 ng/mL to approximately 60 ng/mL of BMP4, approximately 5 ng/mL to approximately 15 ng/mL of bFGF, approximately 15 ng/mL to approximately 35 ng/mL of HGF, approximately 5 ng/mL to approximately 15 ng/mL of VEGF, approximately 10 ng/mL to approximately 30 ng/mL of TGF-α, and approximately 20 ng/mL to approximately 60 ng/mL of dexamethasone for approximately 2 to 6 days (e.g., 4 days); and e) Culture cells in a third medium containing 0.5 μM to approximately 1.5 μM of Wnt3A, approximately 0.1 mM to approximately 1.0 mM of A83-01, and approximately 10 ng/mL to approximately 30 ng/mL of dexamethasone. Cells were cultured in a fourth medium containing ng/mL EGF, approximately 20 ng/mL to approximately 80 ng/mL Rspondin1, approximately 0.1 mM to 1.0 mM ascorbic acid, and approximately 1 mM to approximately 3 mM glutamic acid to produce an endoderm stem cell (EnSC) population.

In some implementation methods, such as collecting EnSCs every 3 to 4 days and dissociating them into single cells for subsequent proliferation, secondary proliferation, or differentiation.

In some implementations, EnSCs (e.g., generation 20) are selected for quality control testing, including morphology, survival, purity, sterility, karyotype, and whole-genome sequencing. Clinically acceptable EnSCs should, for example, be free of known cancer-related mutations, have the lowest total mutation load compared to PBMCs derived from EnSCs, and have a minimal likelihood of tumorigenesis. Clinically acceptable EnSCs may be frozen and stored for future use.

III. Pancreatic endocrine cells

In another embodiment, this disclosure provides a method for generating 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/or niacinamide. The process involves: a) generating a pancreatic precursor (PP) cell population; b) culturing the PP cell population in the presence of a BMP inhibitor, a TGF-β inhibitor, and/or a γ-secretase inhibitor to generate an endocrine precursor (EP) cell population; and c) culturing the EP cell population in the presence of T3 and niacinamide; thereby generating a pancreatic endocrine cell population. In some embodiments, the PP cell population in step c refers to a uniform cluster of cells formed from the PP cell population.

In some implementations, the culture time for step b is 2 to 6 days. In some implementations, the culture time for step c is 3 to 8 days. In some implementations, the culture time for step d is 7 to 21 days.

III-1. Endoderm stem cells as initiating cells

In some implementations, EnSC is generated by the method described in Chapter II. Endoderm Stem Cells .

In some implementations, clinically acceptable EnSCs have one or more of the following characteristics: 1) If detected by microscopy (e.g., phase contrast observation), they have typical epithelial morphology, with clear cell boundaries and 3-5 1) Diameter of μm; 2) At least 90% of cells are FOXA1 positive as measured by flow cytometry; 3) Has a complete karyotype as analyzed by karyotype testing; 4) Does not contain known cancer-related mutations and has the lowest total mutation load compared to the original PBMCs as detected by whole genome sequencing; 5) Has at least 90% viable cells before freezing and at least 60% viable cells after thawing as detected by flow cytometry; 6) Is not detectable by pathogen testing, is sterile and virus negative; 7) No teratoma is found as analyzed by teratoma formation testing.

III-2. Pancreatic progenitor (PP) Cell production

To induce pancreatic endoderm and subsequently generate pancreatic precursor (PP) cells, EnSCs are treated with a mixture of multiple factors in a basic culture medium (e.g., MCDB). In some embodiments, the pancreatic precursor (PP) cell population is generated by culturing endoderm stem cell populations in the presence of a BMP inhibitor, a Nodal signaling agonist, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and/or niacinamide. Exemplary BMP inhibitors include (but are not limited to) Noggin, doxorubicin, and LDN-193189. In some embodiments, the BMP inhibitor includes Noggin.

In some implementations, 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.

In some embodiments, the Nodal signaling agonist is activator A. In some embodiments, the concentration of the Nodal signaling agonist 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.

In some implementations, 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.

In some implementations, 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.

In some implementations, the concentration of SANT1 is in the range of about 0.1 μM to about 1.0 μM, such as 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM or 1.0 μM.

In some implementations, the concentration of retinoids is in the range of about 0.5 μM to about 4 μM, such as 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM or 4 μM.

In some implementations, 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.

In some implementations, 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.

In some implementations, pancreatic precursor (PP) cell populations are generated by culturing endoderm stem cell populations in the presence of Noggin, activin A, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and/or niacinamide.

In some implementations, pancreatic precursor (PP) cell populations are generated by culturing endoderm stem cell populations in the presence of Noggin, activin A, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, niacinamide, Rspondin1, LDN-193189, and/or TPPB.

In some implementations, pancreatic precursor (PP) cell populations are generated by culturing endoderm stem cell populations for 1–3 days (e.g., 2 days) in the presence of Noggin, activin A, FGF10, LDN 193189, Rspondin1, TPPB, and/or EGF, followed by culturing cells for 1–3 days (e.g., 2 days) in the presence of LDN 193189, FGF10, EGF, SANT1, ascorbic acid, and/or retinoic acid (Sigma), followed by culturing cells for 1–3 days (e.g., 2 days) in the presence of FGF10, EGF, SANT1, retinoic acid, niacinamide, and/or ascorbic acid.

In some implementations, EnSC-derived PP cells are evaluated for microbial contamination, morphology, purity, and viability. In some implementations, clinically acceptable PP cells have one or more of the following characteristics: 1) if detected by microscopy (e.g., phase contrast observation), they have typical epithelial morphology, indistinct cell boundaries, and a diameter not exceeding 3 μm; 2) if measured by flow cytometry, at least 60% of the cells are PDX1 positive; 3) if detected by flow cytometry, they have at least 90% viable cells; and 4) if analyzed by pathogen testing, they are undetectable by mycobacteria, sterile, viral negative, and have endotoxin levels not exceeding 1 EU/mL.

In some implementations, at the end of this phase, pancreatic precursor (PP) cells are dispersed as single cells and suspended in, for example, a three-dimensional environment (e.g., AggreWell (STEMCELL), Corning® Elplasia® culture dish, Nunclon™ Sphera™ 96-well dish, or Corning® spherical well round-bottom ultra-low adhesion microdisc) to allow for uniform cell clustering for a sustained period of time (e.g., 1–5 days, such as 3 days) to facilitate further islet tissue reconstruction and maturation.

III-3. Endocrine precursor (EP) Cell production

For endocrine precursor cell (EP) induction, triple inhibition of the BMP, TGF-β, and Notch signaling pathways can be manipulated for several days. In some embodiments, homogeneous cell clusters obtained using the methods described in the chapter " Genesis 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 some embodiments, the BMP inhibitor includes Noggin. In some embodiments, the TGF-β inhibitor is A83-01. Exemplary γ-secretase inhibitors include (but are not limited to) compounds E, GSI-XX and GSI-XXI, DAPT, LY-411575, RO4929097 and dibenzodiazepine (DBZ). In some embodiments, γ-secretase inhibitors include GSI-XX.

In certain embodiments, the concentration of the BMP inhibitor ranges from 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.

In some implementations, 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, and 2.0 mM.

In some implementations, the concentration of the γ-secretase inhibitor is in the range of about 0.5 μM to about 4 μM, such as 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM or 4 μM.

In some implementations, the sixth culture medium contains Noggin, A83-01 and/or GSI-XX.

In some embodiments, homogeneous cell clusters are further cultured in the presence of Noggin, A83-01, and/or GSI-XX. In some embodiments, homogeneous cell clusters are further cultured in the presence of Noggin, A83-01, GSI-XX, retinoic acid, and/or SANT1.

In some implementations, EnSC-derived EP cells are evaluated for microbial contamination, morphology, purity, and viability. In some implementations, clinically acceptable EP cells have one or more of the following characteristics: 1) at least 60% of the cells are positive for PDX1 and NKX6-1 as measured by flow cytometry; 2) at least 90% of the cells are viable as detected by flow cytometry; and 3) no mycotoxins are detected, the cells are sterile, and the virus is negative as analyzed by pathogen testing, and the cells contain no more than 1 EU/mL of endotoxin.

III-4. Regenerated pancreatic islet tissue The production of ( Maturation of endocrine cells )

For the maturation of endocrine cells, additional factors may be added to the culture medium used for the production of endocrine precursor cells as described in Chapter III-3. Production of Endocrine Precursor Cells (EP) . In some embodiments, the additional factors are selected from the group consisting of 3,3',5-triiodo-L-thyroxine (T3) and niacinamide. In some embodiments, the additional factors include T3 and niacinamide.

In some implementations, the concentration of T3 is in the range of about 0.5 μM to about 4 μM, such as 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM or 4 μM.

In some implementations, 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.

In some implementations, the additional factor further includes BMP4.

In some embodiments, methods for generating pancreatic endocrine cell populations include: 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/or niacinamide to generate a pancreatic precursor (PP) cell population; c) culturing the PP cell population in the presence of Noggin, A83-01, and/or GSI-XX to generate an endocrine precursor (EP) cell population; and d) culturing the EP cell population in the presence of T3 and/or niacinamide; thereby generating a pancreatic endocrine cell population.

In some embodiments, methods for generating pancreatic endocrine cell populations include: a) providing an endoderm stem cell population; b) culturing the endoderm stem cell population for 1–3 days (e.g., 2 days) in the presence of approximately 10 ng/mL to approximately 30 ng/mL Noggin, approximately 0.1 ng/mL to approximately 1.0 ng/mL activin A, approximately 10 ng/mL to approximately 30 ng/mL FGF10, approximately 10 ng/mL to approximately 30 ng/mL EGF, approximately 100 nM to approximately 300 nM LDN 193189, approximately 10 ng/mL to approximately 30 ng/mL Rspondin1 and/or approximately 300 nM to approximately 800 nM TPPB, followed by incubation with approximately 100 nM to approximately 300 nM LDN 193189. 193189, FGF10 at approximately 10 ng/mL to approximately 30 ng/mL, EGF at approximately 10 ng/mL to approximately 30 ng/mL, SANT1 at approximately 0.3 μM to approximately 0.8 μM, ascorbic acid at approximately 0.3 mM to approximately 0.8 mM, and/or retinoic acid at approximately 0.2 μM to approximately 3 μM, were further cultured for 1–3 days (e.g., 2 days) in the presence of FGF10 at approximately 10 ng/mL to approximately 60 ng/mL, EGF at approximately 10 ng/mL to approximately 30 ng/mL, SANT1 at approximately 0.1 μM to approximately 0.4 μM, retinoic acid at approximately 0.1 μM to approximately 0.3 μM, niacin at approximately 5 mM to approximately 15 mM, and/or retinoic acid at approximately 0.3 mM to approximately 0.8 μM. a) Culture the cells in the presence of ascorbic acid at a concentration of mM for 1–3 days (e.g., 2 days) to generate a pancreatic precursor (PP) cell population; c) Culture the PP cell population in the presence of noggin at a concentration of approximately 10 ng/mL to approximately 30 ng/mL, A83-01 at a concentration of approximately 0.1 mM to approximately 1.0 mM, GSI-XX at a concentration of approximately 1 μM to approximately 3 μM, retinoic acid at a concentration of approximately 0.05 μM to approximately 0.2 μM, and/or SANT1 at a concentration of approximately 0.05 μM to approximately 0.2 μM; and d) Culture the cells in the presence of the factors involved in step c, and T3 at a concentration of approximately 0.5 μM to approximately 1.5 μM, niacin at a concentration of approximately 5 mM to approximately 15 mM, and/or SANT1 at a concentration of approximately 1 ng/mL to approximately 3 μM. EP cell populations were cultured in the presence of ng/mL BMP4, thereby generating pancreatic endocrine cell populations.

In some embodiments, the EnSCs, EnSC-derived PP cells, EnSC-derived EP cells, or EnSC-derived mature endocrine cells described herein can form three-dimensional cell aggregates that mimic the natural islet structure and promote intercellular interactions. Subsequently, additional three-dimensional culture of the cell aggregates produces functional islet-like structures, namely EnSC-derived islet tissue, or regenerated islet tissue. The maturity and functionality of the resulting regenerated islet tissue are then evaluated, including marker expression, endocrine cell composition, hormone secretion, and glucose responsiveness.

In some implementations, regenerated islet tissue is assessed for microbial contamination, morphology, purity, and survival rate. Endocrine cell composition, such as insulin+NKX6-1+ β cells, glucagon+ α cells, somatostatin+ δ cells, and chromogranin A+ endocrine cells, is also analyzed using techniques such as flow cytometry and single-cell transcriptomic analysis (scRNA-seq).

In some implementations, the in vitro functionality of regenerated islet tissue can be analyzed as glucose-stimulated insulin secretion (GSIS) of human cadaveric islets, and in vivo functionality can be assessed by transplanting renal capsules or portal veins into streptozotocin-induced diabetic animal models (e.g., mice or monkeys). In some implementations, non-target hepatocytes (HNF4A+ albumin+), bile duct epithelial cells (SOX9+ CK7+), intestinal epithelial cells (CDX2+), and pancreatic duct cells (SOX9+ PTF1A+ PDX1+) can be estimated from, for example, scRNA-seq data.

In some implementations, clinically acceptable regenerated pancreatic islet tissue has one or more of the following characteristics: 1) as detected by microscopy (e.g., phase contrast observation), it has dense clusters of spherical cells with indistinct cell boundaries and an average diameter of about 150 mm. μm; 2) As measured by flow cytometry, 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., β cells), at least 20% of the cells are glucagon positive (i.e., α cells), at least 15% of the cells are somatostatin positive (i.e., δ cells), and less than 2% of the cells are positive for PDX1 (i.e., pancreatic lineage), CHGA+ positive (i.e., endocrine lineage), C-peptide positive (i.e., β cells), and less than 2% of the cells are positive for PDX1 (i.e., pancreatic lineage), CHGA+ positive (i.e., endocrine lineage), CHGA+ positive (i.e., somatostatin), and somatostatin positive (i.e., δ ... somatostatin positive (i.e., δ cells). 3) The cells are AFP+ positive (i.e., not the target liver lineage); 4) The cells have at least 90% viable cells as measured by flow cytometry; 5) The cells have at least a 1-fold (e.g., 1.5-fold) change in insulin or C-peptide as detected by static glucose-stimulated insulin (C-peptide) secretion analysis; 6) The cells are undetectable by pathogen testing, sterile, virus-negative, and have endotoxin levels not exceeding 1 EU/mL.

In some implementations, 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 regenerated islet tissue provided herein is positive for C-peptide, glucagon, PDX1, and/or NKX6-1.

C-peptide is a byproduct of insulin production, secreted by the β-cells of the pancreas along with insulin. The secretion of human C-peptide represents the activity of β-cells within the pancreas and their ability to produce and release insulin. Insulin is a key hormone responsible for regulating blood glucose levels, and C-peptide is commonly used as a marker of insulin secretion due to its isoproterenol co-secretion with insulin. See, for example, Wilcox G. Insulin and insulin resistance. Clin Biochem Rev. May 2005 ;26(2):19-39 .

Glucagon acts as a marker of the pancreas due to its specific secretion from α-cells within the islets of Langerhans and its crucial role in regulating blood glucose levels, especially during fasting. This hormone counteracts the effects of insulin, promoting gluconeogenesis and glycogenolysis in the liver, thereby increasing blood glucose levels.

PDX1, known as the pancreas duodenal homeobox 1, is attributed to its important role as a marker of the islets of Langerhans in pancreatic development and β-cell function. As a homologous domain transcription factor, PDX1 regulates the expression of genes crucial for pancreatic development and the maintenance of islet cell attributes.

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

III-5. Culture medium used for preparing pancreatic endocrine cells

Any cell culture system known in this art can be used in this disclosure. In some embodiments, an adherent culture system is used in a method for generating pancreatic endocrine cell populations. The term "adherent culture" refers to a cell culture system in which cells are cultured on a solid surface, which may be coated with a matrix. The cells may or may not adhere tightly to the solid surface or the matrix. Substrates used for adherent culture may further include, for example, any or a combination of polystyrene, polyester, polycarbonate, poly(N-isopropylacrylamide), polyguanine, laminin, polylysine, purified collagen, gelatin, cellulose, extracellular matrix, fibronectin, tenacin, fibronectin, polyglycolic acid (PGA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), matrix glue, hydroxyapatite, and amnion.

In some embodiments, suspension culture can be used in methods for generating pancreatic endocrine cell populations. The term "suspension culture," as used herein, refers to culturing cells in a manner that prevents them from adhering to a solid support or culture vessel. To transfer cells to a suspension culture, for example, cells are removed from a culture vessel using a cell scraper and transferred to a sterile, hypoadhesive dish that accommodates the culture medium and prevents cell adhesion to the dish surface. Thus, cells are cultured in suspension without adhering to the substrate or bottom of the culture dish.

A culture medium suitable for culturing cells is any culture medium suitable for growing a particular cell type in a culture dish. Culture media include, for example, MCDB, Hammer F10 (Sigma), Hammer F12, Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), Dürbeco Modified Eagle Medium (DMEM) (Sigma), IMDM, Medium 199, Eagle Minimal Essential Medium (EMEM), aMEM, CMRL1066, DMEM/F12, and mixtures thereof. Any of these culture media may be supplemented as needed with salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM), trace elements (defined as inorganic compounds that are typically present in the micromolar range at their final concentration), glucose or an equivalent energy source, albumin, insulin, transferrin, selenium, fatty acids, 2-hydroxyethanol, thiol glycerol, lipids, amino acids, L-glutamic acid, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antioxidants, pyruvate, intercytokines, and analogues as needed. Appropriate concentrations, including any other necessary supplements, may also be used at concentrations known to those skilled in the art. The culture conditions (such as temperature, pH, and similar conditions) are those previously used for cell culture and will be obvious to those generally skilled in the art.

IV. Pharmaceutical composition and therapeutic uses

IV-1. pharmaceutical composition

In another instance, this disclosure provides an EnSC population generated according to the methods provided herein, for example under Chapter II. Endoderm Stem Cells .

In another embodiment, this disclosure provides a population of PP cells, EP cells and pancreatic endocrine cells generated according to the methods provided herein, for example under section " III . Pancreatic Endocrine Cells ".

In another embodiment, this disclosure provides a pharmaceutical composition comprising the EnSC population, PP cell population, EP cell population or pancreatic endocrine cell population provided herein, and a pharmaceutically acceptable culture medium.

The term "pharmaceutically acceptable" indicates that one or more specified carriers, mediators, diluents, excipients, and/or salts are generally chemically and/or physically compatible with other components constituting the formulation and physiologically compatible with their acceptors. Pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous mediators, non-aqueous mediators, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspensions/dispersants, clamping or chelating agents, diluents, adjuvants, excipients, or non-toxic excipients, other components known in the art, or various combinations thereof.

The compositions described herein may also contain components that facilitate implantation. The compositions described herein may be pyrogen-free or substantially pyrogen-free and pathogen-free, wherein pathogens include bacterial contaminants, fungal contaminants and viruses.

In some embodiments, the pharmaceutical composition may further comprise an immunosuppressant or an immune tolerance reagent.

IV-2. Therapeutic uses

In another embodiment, this disclosure relates to the therapeutic use of the EnSC, EnSC-derived PP cells, EnSC-derived EP cells, EnSC-derived pancreatic endocrine cells, or regenerated islet tissue provided herein.

In one embodiment, this disclosure provides a method for treating a disease or condition associated with impaired pancreatic function in an individual of need, comprising: administering to the individual an effective amount of pancreatic endocrine cells derived from an EnSC population or regenerated islet tissue, thereby treating the disease or condition associated with impaired pancreatic function in the individual of need. In some embodiments, the EnSC population is generated according to the method provided herein, for example under Section II. Endoderm Stem Cells . In some embodiments, the pluripotent stem cell population is autologous or allogeneic.

"Diseases or conditions associated with impaired pancreatic function" include any disease or condition that can be treated by administering pancreatic endocrine cells or regenerating pancreatic tissue, including diseases in which an individual's pancreatic endocrine cells are reduced in number or die, have decreased density, or otherwise become dysfunctional. As used herein, the term "impaired pancreatic function" encompasses not only a reduction in the optimal function of the Islets of Langerhans in the pancreas, leading to decreased production or secretion of insulin and glucagon, but also partial or complete pancreatic dysfunction. This broad range of dysfunction can lead to abnormal blood glucose levels, potentially resulting in diabetes or other metabolic disorders. When this function is completely lost, the body loses its ability to effectively regulate blood sugar. If left untreated, this can lead to severe hyperglycemia and related health complications.

In some implementations, the disease or condition associated with impaired pancreatic function is diabetes. Diabetes is a chronic metabolic disorder 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 sugar levels. Diabetes is a leading cause of blindness, kidney failure, heart disease, stroke, and lower limb amputation.

In some implementations, the diseases or conditions associated with impaired pancreatic function are type 1 diabetes (T1D), type 2 diabetes (T2D), type 3c diabetes, or mature-onset diabetes mellitus (MODY).

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

Type 2 diabetes (T2D) typically begins with insulin resistance in peripheral tissues and continues to gradually lose pancreatic function due to a decrease in β-cell mass or dedifferentiation of β-cells in progressively worsening pathological conditions. See, for example, 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 become dependent on exogenous insulin therapy. Dead donor islet transplantation is a safe and effective treatment for insulin-dependent diabetes mellitus. See, for example, Shapiro, AM et al ., N. Engl. J. Med. 355, 1318-1330 (2006) ; and Marfil-Garza, BA et al ., Lancet Diabetes Endocrinol. 10, 519-532 (2022) . Notably, improved metabolic control after islet transplantation is associated with better renal allogeneic transplantation function and long-term survival. See, for example, Lablanche, S. et al. , Lancet Diabetes Endocrinol. 6, 527-537 (2018) ; and Markmann, JF et al . , Am. J. Transplant. 21, 1477-1492 (2021) . However, the application of islet transplantation is severely hampered by a severe shortage of healthy donor organs and complex separation procedures.

Type 3c diabetes is a type of diabetes secondary to exocrine pancreatic disease. It is also known as pancreatogenic or pancreatogenous diabetes. The most common cause of type 3c diabetes is chronic pancreatitis, an inflammation of the pancreas that causes damage to both exocrine and endocrine tissues. Other causes include pancreatic ductal adenocarcinoma, hemochromatosis, cystic fibrosis, and previous pancreatic surgery. The pathogenesis of type 3c diabetes is not fully understood, but it is believed to be associated with loss of functional β-cells in the pancreas due to underlying pancreatic disease. Type 3c diabetes is less common than other forms of diabetes, but it is still relatively prevalent, especially in patients with chronic pancreatitis or pancreatic ductal adenocarcinoma. Diagnosing type 3c 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, for example, Hart PA et al ., Lancet Gastroenterol Hepatol. 2016 Nov ; 1(3):226-237 .

Mature-onset diabetes mellitus (MODY) is a monogenic form of diabetes characterized by early onset. It is caused by a single pathogenic gene mutation that leads to dysfunction or developmental changes in the insulin-secreting pancreatic beta cells. A common feature of MODY is its early onset, typically occurring during childhood, adolescence, or early adulthood. Individuals with MODY usually have a strong family history of diabetes, and the condition is often inherited within families. The symptoms of MODY are similar to those of type 2 diabetes, including hyperglycemia and insulin resistance. Diagnosis of MODY can be challenging because it is often confused with type 1 or type 2 diabetes. See, for example, Bonnefond, A. et al. , Nat Rev Dis Primers 9, 12 (2023) .

Routes of administration may include any suitable method, including (but not limited to) percutaneous transhepatic infusion, portal vein infusion, renal capsule transplantation, rectus abdominis injection, subcutaneous implantation, mesenteric injection, retroperitoneal injection, hepatic artery injection, iliac fossa injection, and similar methods. In some implementations, the specific mode of administration chosen will depend on the specific treatment, the patient's disease condition or symptoms, the nature of other drugs or treatments administered to the individual, or the route of administration.

In some implementations, the individual is human.

In some implementations, the effective amount is approximately 0.5 to 3.0 million islet equivalents (IEQ). IEQ is a standard estimate of islet volume, where one IEQ equals a single spherical islet with a diameter of 150 μm. See, for example, Lembert N et al ., Cell Transplant. 2003;12(1):33-41 .

In some implementations, the method further includes administering an immunosuppressant to the individual in need. When using an immunosuppressant, it can be administered systemically or locally, and it can be administered before, simultaneously with, or after the administration of regenerated pancreatic islet tissue.

In another embodiment, this disclosure provides the use of endoderm stem cell populations for the manufacture of regenerated islet tissue for the treatment of diseases or conditions associated with impaired islet function in individuals in need.

In some implementations, the manufacturing process includes the steps described herein, such as those described under Chapter III. Pancreatic Endocrine Cells .

In another embodiment, this disclosure provides a first-in-human tissue replacement therapy for the treatment of diseases or conditions associated with impaired pancreatic function, such as diabetes, including type 1 diabetes (T1D) and type 2 diabetes (T2D). In some implementations, the therapy provided herein offers several significant advantages over existing options.

First, unlike traditional cadaveric islet transplantation, this therapy 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.

Second, compared to hPSC-derived islet tissue currently undergoing clinical trials, the endoderm stem cells (EnSCs) used to generate functional islets in this disclosure are non-tumorigenic in vivo and are more suitable as precursors for efficient, large-scale islet production. Their endoderm-specific properties and closer developmental relationship to the pancreas lineage make them a superior option as islet precursors compared to hPSCs (e.g., hiPSCs).

Third, while other trials have involved patients with T1D, the therapy disclosed here can extend its indications to T2D, while allowing for the evaluation of EnSC-derived islet tissue (regenerated islet tissue) implantation and function without autoimmune interference. Furthermore, this therapy has the potential to treat advanced disease, including advanced T2D.

The aforementioned advantages can be achieved through the novel methods disclosed herein for generating EnSCs for therapy and EnSC-derived islet tissue (regenerated islet tissue).

V. Set

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

In some embodiments, the fourth factor does not contain FGF2 and/or Chir99021. In some embodiments, the fourth culture medium does not contain FGF.

In another embodiment, this disclosure provides a kit for generating pancreatic endocrine cell populations from endoderm stem cell populations, 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 comprises a BMP inhibitor, a Nodal signaling agonist, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and/or niacinamide, the sixth group of factors comprises a BMP inhibitor, a TGF-β inhibitor, and/or a γ-secretase inhibitor, and the seventh group of factors comprises T3 and/or niacinamide.

In another embodiment, this disclosure provides a kit for generating pancreatic endocrine cell populations from pluripotent stem cell populations, wherein the kit comprises factors in groups one through seven, wherein the first group comprises Nodal signaling agonists and WNT signaling agonists, the second group comprises Nodal signaling agonists and fibroblast growth factor (FGF), and the third group comprises factors belonging to the TGF-β superfamily, FGF, and hepatocyte growth factor. The fourth group of factors includes HGF and VEGF, WNT signaling promoters, TGF-β inhibitors and epidermal growth factor (EGF), the fifth group of factors includes BMP inhibitors, Nodal signaling promoters, FGF10, EGF, SANT1, retinoic acid, ascorbic acid and niacinamide, the sixth group of factors includes BMP inhibitors, TGF-β inhibitors and γ-secretase inhibitors, and the seventh group of factors includes T3 and niacinamide.

In some embodiments, the fourth factor does not contain FGF2 and/or Chir99021. In some embodiments, the fourth culture medium does not contain FGF.

In another embodiment, this disclosure provides a kit for generating pancreatic endocrine cell populations from PP cell populations, wherein the kit contains a set of factors including a BMP inhibitor, a TGF-β inhibitor, and a γ-secretase inhibitor.

All disclosures and patents cited in this specification are incorporated herein by reference in their entirety. Examples

Implementation Examples 1. From human peripheral blood mononuclear cells (PBMC) Generate induced pluripotent stem cells (iPSC)

This embodiment illustrates the generation of induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) in patients with impaired insulin secretion and type 2 diabetes mellitus (T2D).

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 for the production of iPSC cell lines under GMP conditions using the Sendai Virus Reprogramming System. Patient PBMCs were isolated from whole blood using a Ficoll gradient (WB as donor code). Whole blood was sampled using BD Vacutainer® EDTA tubes. Blood was diluted with Dulbecco's Phosphate-Buffered Saline (DPBS) and poured onto the Ficoll solution, and centrifuged at 400 g for 30 minutes at room temperature. The PBMC layer was collected and washed with DPBS. Freeze the PBMC or continue generating iPSCs.

A Sendai virus reprogramming kit (Invitrogen, GMP grade) containing four reprogramming factors (OCT4, SOX2, KLF4, L-MYC) was used for iPSC generation. According to the manufacturer's instructions, two million PBMCs were first cultured for 7 days in StemPro™-34 SFM (Gibco) SP34 in the presence of human SCF, FLT-3, IL-3, and IL-6. On the day of transduction, the PBMCs were washed and counted, and the appropriate viral vector volume was calculated based on the cell count and viral titer. The PBMCs and viral vector were then mixed for transduction. Two days after transduction, cells were plated and gradually transferred to mTeSR1 medium over the next 7 days. Select iPSC communities and transfer them to individual culture trays within approximately 2 to 3 weeks, maintaining them in an incubator at 37°C with an environment of 5% _CO2 , 5% _O2 , and 90% _N2 .

The in vitro differentiation potential of ten iPSC progeny from the 10th generation was tested (data not shown), and two of them (named WB20 and WB34) were selected for further characterization and to establish EnSC cell lines under GMP conditions.

Implementation Examples 2. From humankind iPSC Generate EnSC

This embodiment illustrates the generation of EnSC from human iPSC (hiPSC).

2.1 Cellular resources of human endoderm stem cells

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

2.2 since hiPSC Generate EnSC

EnSC cell lines were established from patient-specific hiPSC cell lines WB20 and WB34 and maintained in a 37°C incubator with an environment of 5% _CO2 , 5% _O2 , and 90% _N2 . Endoderm cells were differentiated from hiPSCs by double activation of the Nodal and WNT signaling pathways with activin A (100 ng/mL) and CHIR99021 (2 μM) for 24 hours. The cells were then cultured for 4 days in the presence of bFGF (5 ng/mL), activin A (100 ng/mL), VEGF (10 ng/mL), ascorbic acid (0.5 mM, Wako), and grutamia (2 mM, Invitrogen), followed by 4 days of culture in the presence of bFGF (10 ng/mL), TGF-α (20 ng/mL), VEGF (10 ng/mL), BMP4 (50 ng/mL), HGF (25 ng/mL), and dexamethasone (40 ng/mL, Sigma). Subsequently, EnSCs were established by re-coating the previously treated endoderm cells at a concentration of 1 × ^10⁶ cells/mL and maintained in MCDB131 supplemented with Wnt3A (1 μM), Rspondin1 (50 ng/mL), EGF (20 ng/mL), A83-01 (0.5 mM), ascorbic acid (0.5 mM, Wako), and glutamic acid (2 mM, Corning).

EnSCs are collected every 3 to 4 days and dissociated into single cells for subsequent proliferation, secondary proliferation, or differentiation. Typically, using familiar techniques such as flow cytometry and microscopic morphology examination, EnSCs from passage 20 are selected for quality control tests, including morphology, viability, purity, sterility, karyotype, and whole genome sequencing.

As shown in Figures 1 and 10-11, the generated EnSCs underwent quality control according to the criteria shown in Figure 10. Specifically, whole-genome sequencing (WGS) of the EnSC cell lines was performed to confirm the absence of any newly emerging cancer or diabetes-related mutations not detected in the original collection of PBMCs (Figure 1). The tumorigenicity of EnSCs within 6 months was further tested in immunocompromised mice (SCID Beige), and it was confirmed that the generated EnSCs did not form teratomas, while human pluripotent stem cells (hPSCs) showed a 100% teratoma formation percentage (Figure 9).

In general, EnSCs produced using the methods described herein have the following characteristics: 1) When detected by microscopy (e.g., phase contrast observation), they exhibit typical epithelial morphology with clear cell boundaries and 3-5 1) A diameter of μm; 2) At least 90% of cells are FOXA1 positive as measured by flow cytometry; 3) A complete karyotype is present as analyzed by karyotype testing; 4) No known cancer-related mutations are detected by whole genome sequencing and the lowest total mutation load compared to the original PBMCs; 5) At least 90% viable cells are present before freezing and at least 60% viable cells are present after thawing as detected by flow cytometry; 6) No mycobacteria are detected as analyzed by pathogen testing, and the sample is sterile and virus-negative; 7) No teratomas are detected as analyzed by teratoma formation testing. Detailed descriptions of EnSC's quality control experiments and their results are provided in Example 4 below.

Finally, the WB20 EnSC cell line was selected as the clinical-grade progeny line because it does not contain any known cancer-related mutations and has the lowest total mutation load compared to patient PBMCs.

Implementation Examples 3. since EnSC Generate Regenerated pancreatic islet tissue

This embodiment relates to the generation of regenerated islet tissue from EnSC under GMP conditions through two intermediate stages.

3.1 EnSC Towards Regenerated pancreatic islet tissue Expandable differentiation

The EnSC samples from Example 2 above were thawed and amplified in T225 flasks at an initial concentration of 2 × ^10⁶ samples/flask. Sterility was tested before differentiation began.

To induce pancreatic endoderm (stage 1), EnSCs were treated for 2 days in MCDB (Gibco) with a mixture 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 days 2-4 of induction, cells were supplemented with LDN 193189 (Stemgent) (200 nM), FGF10 (20 ng/mL), EGF (20 ng/mL), and SANT1 (Tocris) (0.5 nM). Cells were further differentiated in MCDB containing FGF10 (50 ng/mL), ascorbic acid (WAKO) (0.5 mM), and retinoic acid (Sigma) (2 μM); during days 4-6 of differentiation, cells were cultured in the presence of FGF10 (50 ng/mL), EGF (20 ng/mL), SANT1 (0.3 μM), retinoic acid (0.2 μM), niacinamide (Sigma) (10 mM), and ascorbic acid (0.5 mM).

To form a uniform cell cluster (stage 2), the pancreatic precursor (PP) cells produced in stage 1 were dispersed and suspended in AggreWell (STEMCELL) to continue forming a uniform cell cluster for 3 days, and then transferred to an orbital oscillator (90 to 110 rpm) for further pancreatic islet tissue reconstruction and maturation.

For endocrine precursor cell (EP) induction (stage 3), in the presence of Noggin (20 ng/mL), A83-01 (Stemgent) (0.5 mM), γ-secretase inhibitor XX (GSI-XX) (2 μM, MERCK), retinoic acid (0.1 μM), and SANT1 (0.1 μM), the homogeneous cell clusters formed by PP cells in stage 2 were concentrated and regulated by a triple inhibition of BMP, TGF-β, and Notch signaling pathways for 10 days.

For endocrine cell maturation (stage 4, days 8-10), T3 (Sigma) (1 μM), niacinamide (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), glutetamar (Invitrogen), and ascorbic acid (0.5 mM, Wako). All intercytokines were purchased from R&D Systems and, where applicable, were GMP grade.

Under GMP conditions, three batches of regenerated islet tissue were produced at a scale of 1.2 × ^10⁶ islet equivalents [IEQ]/patient through two intermediate stages (pancreatic precursor cells/PP and endocrine precursor cells/EP) to meet the dosage requirements of each patient.

3.2 Derived from EnSC Of PP Cells EP Cells and pancreatic islets organization The manifestations

The PP cells generated at the end of the first stage were evaluated for microbial contamination, morphology, purity and survival rate, and quality control was carried out according to the criteria shown in Figures 2 and 10. In general, PP cells produced using the methods described herein have the following characteristics: 1) when detected by microscopy (e.g., phase contrast observation), they have typical epithelial morphology, with indistinct cell boundaries and a diameter not exceeding 3 μm; 2) when measured by flow cytometry, at least 60% of the cells are PDX1 positive; 3) when detected by flow cytometry, they have at least 90% viable cells; and 4) when analyzed by pathogen testing, they are undetectable by mycobacteria, sterile, virus negative, and have endotoxin levels not exceeding 1 EU/mL.

The EP cells generated at the end of stage 3 were evaluated for microbial contamination, morphology, purity, and viability, and quality control was performed according to the criteria shown in Figures 2 and 10. Overall, the EP cells generated using the methods described herein have the following characteristics: 1) when detected by microscopy (e.g., phase contrast observation), they exhibit dense clusters of spherical cells with indistinct cell boundaries and an average diameter of approximately 150 μm. μm; 2) As measured by flow cytometry, 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., β cells), at least 20% of the cells are glucagon positive (i.e., α cells), at least 15% of the cells are somatostatin positive (i.e., δ cells), and less than 2% of the cells are positive for PDX1 (i.e., pancreatic lineage), CHGA+ positive (i.e., endocrine lineage), C-peptide positive (i.e., β cells), and less than 2% of the cells are positive for PDX1 (i.e., pancreatic lineage), CHGA+ positive (i.e., endocrine lineage), CHGA+ positive (i.e., somatostatin), and somatostatin positive (i.e., δ ... somatostatin positive (i.e., δ cells). 3) The cells are AFP+ positive (i.e., not the target liver lineage); 4) The cells have at least 90% viable cells as measured by flow cytometry; 5) The cells have at least a 1-fold (e.g., 1.5-fold) change in insulin or C-peptide as detected by static glucose-stimulated insulin (C-peptide) secretion analysis; 6) The cells are undetectable by pathogen testing, sterile, virus-negative, and have endotoxin levels not exceeding 1 EU/mL.

The microbial contamination, morphology, purity, and viability of regenerated islet tissue produced using the methods described herein were evaluated. The endocrine cell composition of the regenerated islet tissue (insulin+NKX6-1+ β cells; glucagon+ α cells; somatostatin+ δ cells; chromogranin A+ endocrine cells) was further analyzed using flow cytometry and single-cell transcriptomic analysis (scRNA-seq). In vitro functionality of the regenerated islet tissue was assessed using glucose-stimulated insulin secretion (GSIS) analysis of human cadaveric islets, and in vivo functionality was evaluated using renal capsule or portal vein transplantation into streptozotocin-induced diabetic mouse or monkey models. Non-target hepatocytes (HNF4A+ albumin+), bile duct epithelial cells (SOX9+ CK7+), intestinal epithelial cells (CDX2+), and pancreatic duct cells (SOX9+ PTF1A+ PDX1+) were estimated from scRNA-seq data.

The regenerated islet tissue produced at the end of stage 3 was evaluated for microbial contamination, morphology, purity, and survival rate, and quality control was performed according to the criteria shown in Figures 3 and 10. Overall, the EP cells produced using the methods described herein have the following characteristics: 1) when detected by microscopy (e.g., phase contrast observation), they exhibit dense clusters of spherical cells with indistinct cell boundaries and an average diameter of approximately 150 μm. μm; 2) If measured by flow cytometry, 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., β cells), at least 20% of the cells are glucagon positive (i.e., α cells), and at least 15% of the cells are somatostatin positive (i.e., δ cells), and Less than 2% of cells are AFP+ positive (i.e., not the target liver lineage); 3) at least 90% viable cells as measured by flow cytometry; 4) at least 1.5-fold change in insulin as detected by static glucose-stimulated insulin (C-peptide) secretion analysis; 5) no detectable mycobacteria, sterility, negative for viruses, and endotoxin levels not exceeding 1 EU/mL as analyzed by pathogen testing.

Detailed descriptions of the quality control experiments and results of PP cells, EP cells, and regenerated islet tissue are provided in Example 4 below. Additionally, functional analysis of the regenerated islet tissue produced using the methods described herein is also provided in Example 4 below.

Implementation Examples 4. EnSC , PP Cells EP Cells and Regenerated pancreatic islet tissue Quality control and Regenerated pancreatic islet tissue Functional analysis

This embodiment describes the quality control methods and results of EnSC, PP cells, EP cells, and regenerated pancreatic islet tissue produced using the methods described herein.

4.1 Qualification Criteria

The qualification criteria for EnSC, PP cells, EP cells, and regenerated islet tissue produced using the methods described herein are summarized in Figure 10.

4.2 RNA Real-time extraction and quantification PCR

Reverse transcription and qRT-PCR reactions were performed as previously reported (Cheng et al., 2012). RNA was prepared using RNA TIANGEN according to the manufacturer's instructions and reverse transcribed into cDNA using GoScript reverse transcriptase (Promega) with randomized hexamer and oligomeric (dT) primers. qRT-PCR reactions were performed using an ABI Q6 (Life Technology) system and a SYBR green master mix (Roche). Expression levels were standardized relative to housekeeping gene TBP. Primer information is provided in Figure 11.

4.3 Flow cytometry analysis technology

Collect single-cell samples. Stain with surface markers in PBS (Gibco) containing 0.2% BSA (Sigma). Incubate cells with antibodies on ice for 30 min. For intracellular proteins, fix cells with 1.6% PFA (Servicebio) at 37°C for 30 min and wash with permeation wash buffer (BioLegend). Incubate antibodies at room temperature for 30 min. Finally, analyze cells using flow cytometry with Celesta or Fortessa (BD). For cell viability assays, label viable cells with calcium yellow-green pigment blue (Invitrogen). Incubate cells and analyze using flow cytometry with Celesta or Fortessa (BD). See Figure 12 for antibody information.

4.4 Immunofluorescence

Regenerated islet tissue was fixed with 4% PFA for 15 min at 4°C and permeabilized with 0.5% Triton-100 (Sigma), followed by inhibition. Before and after each staining step, the regenerated islet tissue was washed three times at room temperature (RT) with PBST (PBS containing 0.05% Tween 20), and then inhibited with 2% BSA at 4°C for 2 hours. The regenerated islet tissue was stained overnight at 4°C with diluted primary antibodies, and then incubated with diluted secondary antibodies at 4°C for 2 hours. All antibodies were diluted in PBS containing 2% BSA. Contrast staining of cell nuclei was performed using Prolong Gold anti-quenching reagent (Invitrogen) containing 4,6-diamino-2-phenylindole (DAPI). Regenerated islet tissue was analyzed using a confocal fluorescent microscope (Olympus FV3000). Images of the regenerated islet tissue were captured and 3D projection was performed using Olympus software. Antibody information is shown in Figure 12.

4.5 In vivo static glucose-stimulated insulin (C- peptides ) Secretion analysis

Prior to glucose stimulation, regenerated islet tissue prepared using the method described in Example 3 above, or primary islets provided and isolated by Shanghai Changzheng Hospital, was rinsed at 37°C and then ghorn for 2 hours in Krebs-Ringer buffer supplemented with 2 mM glucose. For glucose stimulation, the regenerated islet tissue was treated alternately with Krebs-Ringer buffer containing either low (2 mM) or high (20 mM) glucose. The supernatant was collected 30 minutes after each stimulation. C-peptide was measured using a human C-peptide ELISA kit (Mercodia, 10-1141-01) according to the manufacturer's instructions.

4.6 Mycotoxin, sterility and endotoxin tests

The culture supernatant samples were sent to the accredited Shanghai Simple Gene Medical Laboratory for testing.

4.7 Karyotype analysis

EnSC samples were sent to the accredited Shanghai Simple Gene Medical Laboratory for standard G-banding chromosome analysis.

4.8 Whole genome sequencing

DNA Preparation

DNA degradation and contamination were monitored on 0.8% agarose gel. DNA purity was verified using a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). DNA concentration was measured using a Qubit® DNA Analysis Kit in a Qubit® 3.0 fluorometer (Life Technologies, CA, USA).

DNA Library preparation and sequencing

According to the manufacturer's instructions, a library for sequencing was constructed using the NEB Next® Ultra DNA Library Preparation Kit for Illumina® (NEB, USA). DNA was fragmented into segments of approximately 200 base pairs. An end-repair procedure was performed on the DNA segments, including the addition of a single "A" base followed by transducer conjugation. The library DNA was amplified by purifying and enriching the product using polymerase chain reaction (PCR). The final library was quantified using the KAPA Library Quantification Kit (KAPA Biosystems, South Africa) and an Agilent 2100 bioanalyzer. Paired end sequencing (2 × 150 base pairs) was performed on an Illumina NovaSeq 6000 sequencer (Illumina, USA).

4.9 Single cell RNA Sequencing and Data Processing

Cell capture and cDNA synthesis

The regenerated islet tissue produced in the methods described in Examples 1 to 3 was dissociated into single cells with 0.25% trypsin and resuspended in 1×PBS at a concentration of 1× ^10⁶ cells/mL. According to the manufacturer's protocol, using a single-cell 3' library and gel bead kit V3.1 (10x Genomics, 1000121) and a Chromium single-cell G-chip kit (10x Genomics, 1000120), the cell suspension (300 to 600 viable cells/µL as determined by Count Star) was loaded onto a Chromium single-cell controller (10x Genomics) to generate single-cell gel beads in the emulsion. In short, single cells were suspended in PBS containing 0.04% BSA. Approximately 10,000 cells were added to each channel, and the estimated number of target cells recovered was approximately 15,000. The captured cells were lysed, and the released RNA was barcoded by reverse transcription pairs in individual GEMs. Reverse transcription was performed at 53°C for 45 minutes on an S1000™ Touch thermal cycler (Bio Rad), followed by 5 minutes at 85°C, and maintained at 4°C. cDNA was generated and subsequently amplified, and quality was evaluated using an Agilent 4200 (performed by CapitalBio Technology, Beijing).

Single cell RNA-Seq Library preparation and sequencing

According to the manufacturer, a single-cell RNA-seq library was constructed using a single-cell 3' library and a gel bead kit V3.1. The library was ultimately sequenced using an Illumina Novaseq 6000 sequencer to a depth of at least 30,000 reads per cell, employing a 150 bp (PE150) pairing end reading strategy (performed by CapitalBio Technology, Beijing). During tSNE cluster analysis, 15,244 cells were sequenced, and 2,721 cells with low UMI (UMI count < 5000) were excluded from the sequenced cells.

4.10 Quality control results

The morphology, purity, survival rate, and microbial contamination of EnSC-derived pancreatic precursor cells (PP), endocrine precursor cells (EP), and regenerated islet tissue were verified to meet the qualification criteria (Figures 1 to 3 and Figure 10). Regenerated islet tissue exhibited morphology (Fig. 3 (subsection a)) similar to that of human cadaver islets, endocrine cell composition (Fig. 3 (subsections b and c)), genotype (Fig. 3 (subsections d-f)), and in vitro functionality (glucose-stimulated insulin secretion analysis, GSIS) (Fig. 3 (subsection g)), and demonstrated functional efficacy in streptozotocin (STZ)-induced diabetic mice (Fig. 4 (subsections b-d)) and monkey (Fig. 5) models. This was confirmed by scRNA-seq (Fig. 3 (subsection f)) or FACS (Fig. 3). During the detection of (sub-region h), no non-target liver or intestinal lineages were detected. During the experiments described in Examples 5 and 6 below, no tumor formation or cystic/ductal structures indicating cell proliferation were detected in immunocompromised animals that received EnSC or regenerated islet tissue (Figure 9).

Implementation Examples 5. Regenerated pancreatic islet tissue Transplantation to Lezomycin (STZ) In induced diabetic model mice

This embodiment demonstrates that no tumor formation or cystic/ductal structures indicating cell proliferation were detected in STZ-induced diabetic mouse models transplanted with EnSC or regenerated islet tissue.

5.1 Resources of the biting animal system

SCID Beige mice were obtained from Shanghai Lingchang Biotech. All experiments were conducted in accordance with protocols approved by the Laboratory Animal Care and Use Committee of the Shanghai Institute of Biochemistry and Cell Biology.

The NCG-hIL15 mice were obtained from GemPharmatech Ltd.

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

5.2 Teratoma formation test

SCID Beige (4 to 6 weeks) male mice were implanted with 1 × ^10⁵ hiPSCs or 1 × ^10⁷ EnSCs intramuscularly or subcutaneously in the cervix. Teratoma formation was monitored during the 6-month period.

5.3 Regenerated pancreatic islet tissue Transplantation to Lezomycin (STZ) In induced diabetic model mice

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 at low temperature before injection. STZ administration was completed within 5 minutes of dissolution. After 4 hours of hunger, male SCID Beige mice were treated with intraperitoneal injection of 170 mg/kg STZ (Sigma-Aldrich, S0130) for 4 to 6 weeks. Fasting blood glucose was measured in tail blood using a Roche handheld glucometer on days 5 and 8 to confirm hyperglycemia. Regenerated islet tissue (1000 to 2000 IEQ) was transplanted under the left renal capsule of diabetic mice. The secretion of human C-peptide stimulated by glucose was measured by collecting mouse serum through the eye socket 25 minutes after fasting for 16 hours and after intraperitoneal injection of glucose (3 g/kg, 30% solution).

As shown in Figure 9, during the experiment, no tumor formation or cystic/ductal structures indicating cell proliferation were detected in immunocompromised STZ-induced diabetic model mice transplanted with EnSC or regenerated islet tissue produced using the methods provided herein.

As shown in Figure 4, in terms of glycemic dynamics and human C-peptide secretion (subplots b-d), the regenerated islet tissue produced using the methods presented herein reversed hyperglycemia in STZ-induced diabetes mellitus (SCID Beige) mice. This demonstrates that the regenerated islet tissue produced using the methods presented herein can function as islets.

Implementation Examples 6. Regenerated pancreatic islet tissue transplanted to STZ Induced diabetic monkeys

This embodiment demonstrates that no tumor formation or cystic/ductal structures indicating cell proliferation were detected in STZ-induced diabetic monkeys transplanted with EnSC or regenerated islet tissue.

6.1 Resources for crab-eating macaque models

The cynomolgus macaques were acquired from and housed at WuXi Biologics. All animals were kept in individual stainless steel cages with controlled temperature (18°C to 26°C), relative humidity (40% to 70%), and light (12-hour light/dark cycle). A continuous water supply was provided to all animals, and they were fed twice daily (9:00 AM to 11:00 AM and 3:00 PM to 4:00 PM) supplemental with standard primate feed containing fresh fruit. All animal care and handling were conducted in accordance with guidelines established by WuXi Biologics' IACUC.

6.2 Induction of diabetes in monkeys

To induce hyperglycemia, male cynomolgus monkeys (3 to 6 years old) were fasted overnight and administered STZ intravenously twice (with a 2-week interval). Prior to injection, STZ was reconstituted in sodium citrate buffer (pH 4.5) to a final concentration of 25 mg/ml. Tail tip blood glucose was measured four times daily, before and 2 hours after morning and afternoon feedings. The animal exogenous insulin dosage was determined based on pre-meal blood glucose levels.

6.3 Immunosuppression strategies

Immunosuppressive therapy was initiated 2 days prior to transplantation (Day 2). From Day 2 onwards, sirolimus (Pfizer) (0.5 mg, once daily (q.d.), orally) and mofretinal dispersible tablets (Roche) (62.5 mg, twice daily (b.i.d.), p.o.) were administered daily. Diclofenac sodium suppositories (Hubei Qianjiang) (50 mg, rectally, p.r.) were administered 30 minutes prior to transplantation. ATG (Genzyme) (12.5 mg, intravenously, i.v.) was injected 1 hour before and 48 hours after transplantation. Etanercept (Pfizer) (25 mg, subcutaneous injection) was administered 1 hour before transplantation and on days 3 and 7 after transplantation.

6.4 transplant surgery

Animals were fasted for at least 4 hours and anesthetized by intramuscular injection of Zoltil® 50 (VIRBAC) at a dose of 3 to 5 mg/kg. Heart rate, temperature, blood oxygen, and blood pressure were monitored continuously during the surgical procedure. Regenerated islet tissue was transplanted via percutaneous portal vein injection (6000 or 30000 IEQ) mediated by ultrasound. Antibiotic treatment continued for 7 days post-transplant.

Two diabetic monkeys received either 6,000 (monkey 1) or 30,000 (monkey 2) regenerated islet tissue transplants. Monkey 1 was used to test the feasibility of intravenous injection of regenerated islet tissue into the portal vein without DSA, while monkey 2 was used to evaluate the short-term safety and efficacy of the regenerated islet tissue.

As shown in Figure 9, during the experiment, no tumor formation or cystic/ductal structures indicating cell proliferation were detected in immunocompromised STZ-induced diabetic monkeys transplanted with EnSC or regenerated islet tissue.

Implementation Examples 7. Regenerated pancreatic islet tissue Transplanted into diabetic humanized mice

This example illustrates the functionality of regenerated pancreatic islet tissue when transplanted into diabetic humanized mice.

7.1 By implanting in humans PBMC Generate humanized mice

PBMCs from the same patient or unrelated volunteers in Example 1 were isolated from whole blood using a Fico gradient. Four hours prior to PBMC infusion, NCG-hIL15-positive female mice (6 weeks) were irradiated with 250 cGy. Five million PBMCs were injected into the lateral caudal vein of each mouse. The effectiveness of PBMC engraftment was assessed weekly by flow cytometry based on the ratio of mouse CD45 to human CD45 cells in the mouse blood. The percentage of human CD45 cells increased to >40% within two weeks.

As shown in Figure 6, the proportions of live cells (through SSC and FSC), human blood cells (hCD45+), and mouse blood cells (mCD45+) were similar in the three patient humanized mice and the three volunteer humanized mice (sub-figures a and b), indicating that the humanized mouse model was successfully generated.

As shown in Figure 6, the regenerated islet tissue grafts collected from the renal capsule of humanized mice contained human β cells (C-peptide+ and NKX6-1+), α cells (glucagon+), and δ cells (sitosterin+), as shown 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.

7.2 Induction, transplantation, and evaluation of diabetes

After identification of human PBMC implantation, humanized mice were treated with intraperitoneal injection of 170 mg/kg STZ (Sigma-Aldrich, S0130) after a 4-hour hunger period. One thousand regenerated islet tissues generated from the patient's EnSC using the method described in Example 3 were transplanted into either 1) NCG-hIL15 mice humanized with the patient's PBMCs or 2) NCG-hIL15 mice humanized with PBMCs from unrelated volunteers under the left renal capsule. Fasting blood glucose was measured every two days. Glucose-stimulated human C-peptide secretion was performed as described above. Animals were sacrificed 28 days post-transplantation, and transplant survival was assessed in tissue sections by immunofluorescence detection of islet markers (C-peptide, glucagon, PDX1, NKX6-1).

As shown in Figure 4, patient-specific regenerated islet tissue survived and functioned in the renal capsules of diabetic immunodeficient mice humanized with the patient's own PBMCs, but was rejected by mice humanized with PBMCs from unrelated volunteers (subplot e-g), which suggests that autologous regenerated islet tissue may be tolerated by the patient's immune system.

As shown in Figure 4, the fasting blood glucose levels of STZ-induced diabetic humanized mice transplanted with patient-derived regenerated islet tissue were significantly lower than those of volunteer PBMC-derived diabetic mice transplanted with patient-derived regenerated islet tissue (subplot f), indicating that autologous regenerated islet tissue can significantly reduce fasting blood glucose levels in diabetic individuals.

As shown in Figure 4, on days 7 and 14 post-transplantation of regenerated islet tissue, 30 minutes after fasting and intraperitoneal glucose bolus injection, the secretion of human C-peptide in STZ-induced diabetic humanized mice transplanted with patient-derived regenerated islet tissue was significantly higher than that in volunteer PBMC-derived diabetic mice transplanted with patient-derived regenerated islet tissue (subplot g). This indicates that autologous regenerated islet tissue can function as islets to secrete insulin.

Implementation Examples 8. Clinical research

This example is a clinical study of regenerated islet tissue differentiated in vivo from autologous EnSCs in patients with impaired insulin secretion and type 2 diabetes mellitus (T2D).

8.1 Patient Information

The patient was a 59-year-old male with a 25-year history of type 2 diabetes (T2D). He developed end-stage diabetic nephropathy and underwent a kidney transplant in June 2017. His estimated glomerular filtration rate (eGFR) and serum creatinine (SCr) levels were maintained at 90–105 ml/(min·1.73 ^cm² ) and 45–72 μmol/L, respectively, indicating good donor organ viability and function. He received anti-rejection drugs (tacrolimus (Astellas) 1 mg bid and mofretinal (Roche) 0.5 mg bid), subcutaneous insulin injections of 20 U once daily at bedtime, and oral antidiabetic drugs (acarbose (Bayer) 50 mg three times daily (tid) and metformin (Merck) 0.75 g bid) (Figure 7 (subplot a)). However, the report indicated poor glycemic control since November 2019, characterized by an average blood glucose level of 7.8 ± 2 mmol/L (measured by a continuous glucose monitoring system/CGMS, ranging from 3.66 to 14.60 mmol/L, with an average glycemic deviation/MAGE of 5.54 mmol/L), a time within the range (TIR, 3.9–10.0 mM) of 87.7%, a time within the strict target range (TITR, 3.9–7.8 mM) of 56.7%, and 0.7 daily hyperglycemic events (> 10.0 mmol/L) and 0.3 daily hypoglycemic events (< 3.9 mmol/L) (Figure 15). Due to the significant issues surrounding insulin administration-induced hypoglycemia, the adverse effects of anti-rejection drugs on glycemic control, and the detrimental effects of poor glycemic control on the long-term survival of the donor kidney, the patient and the research team agreed to undergo autologous regenerated islet tissue transplantation.

8.2 Regenerated islet tissue transplantation

Figure 4 (subplot a) shows the process from when the patient obtains PBMCs until the final transplantation of regenerated islet tissue into the patient.

According to the regulatory guidelines of the Clinical Islet Transplantation Registry (CITR), patients underwent image-guided percutaneous transhepatic islet infusion into the main portal vein circulation under local anesthesia and with heparin administration. Portal vein patency was assessed by monitoring portal vein pressure during infusion and by post-infusion Doppler ultrasonography. A total of 1.2 million IEQ of regenerated islet tissue, produced as a single batch and meeting eligibility criteria, was directly delivered without prior cryopreservation. Portal venous angiography and portal vein pressure were monitored throughout the procedure to ensure there was no portal venous embolism or portal hypertension. Glucocorticoids were not used at any time. Post-operatively, patients were monitored overnight and allowed to get out of bed the following day. Patient adherence to scheduled appointments was 100% (some appointments were canceled or moved to local hospitals due to the COVID-19 pandemic).

Dosage design

The underlying principle behind the 1.2 million IEQ dose of regenerated islet tissue transplantation used in this T2D patient is based on the following facts: 1) Healthy individuals have approximately 4 to 6 million IEQ islets, and it is estimated that only 1/3 of the islets are functional under physiological conditions when stimulated by glucose, which means that approximately 1.5 million IEQ islets are sufficient for glycemic control. This is confirmed through clinical observation that transplanting 800,000 IEQ cadaveric islets usually eliminates the need for exogenous insulin in most patients with type 1 diabetes (see references below); 2) patients have significantly reduced endogenous β-cell quality (estimated to be at least 50%) based on pre- and post-meal C-peptide levels; and 3) a large amount of regenerated islet tissue can be lost during angiogenesis because regenerated islet tissue contains only pancreatic endodermal cells and not vascular endothelial cells, which are important for angiogenesis after cadaveric islet transplantation. The dosage of 1.2 million IEQ was chosen because it is estimated that 50% of the regenerated islet tissue may be lost during implantation, and the remaining 0.6 million IEQ is sufficient to supplement endogenous islet function.

8.3 Mixed diet tolerance test (MMTT)

After an overnight fast (≥10 hours), at 7:30 AM, patients were instructed to consume a standard mixed meal consisting of a 200 g steamed bun and 50 mL of water at a constant pace over 5 minutes. Blood samples were collected before intake (0 minutes) and at 15, 30, 60, 120, 180, and 240 minutes after intake. No degludec insulin was administered 24 hours prior to the MMTT, and no oral antidiabetic medications were administered 20 hours prior to the MMTT.

8.4 Evaluation of clinical outcomes

At designated consultations, patients were weighed and their Clark hypoglycemia awareness score was reported, along with routine and disease-specific assessments. Endocrine function and diabetes-specific parameters were assessed using the Mixed Diet Tolerance Test (MMTT) at baseline and at weeks 4, 8, 12, 16, 20, and 24, and every 12 weeks thereafter (Figure 7 (subplot a)). Blood glucose control was measured using a 24-hour real-time glucose monitoring system (Medtronic Guardian™ connected to a subcutaneous continuous glucose monitoring system/CGMS). All information from the CGMS device was centrally evaluated. Baseline and follow-up CGM glucose levels were measured during the first 52 weeks, with an average CGM device wearing duration of at least 3 days.

Security monitoring

Safety endpoints include treatment-induced adverse events (TEAEs), early discontinuation of treatment due to adverse events, and adjudication of adverse events. Tumor formation is monitored every three months by enhanced magnetic resonance imaging of the upper abdomen and measurement of serum cancer-related antigens.

Three major clinical outcomes were monitored over the first 116 weeks: glycemic targets, reduction in exogenous insulin, and circulating C-peptide/insulin levels under fasting and meal stimulation (follow-up assessment results are presented in Figures 13 and 14). Significant changes in glycemic control were observed as early as week 2 post-transplant, with MAGE decreasing from 5.50 mmol/L to 3.60 mmol/L, and notably, TITR rapidly increasing from 56.7% to 77.8% (Figure 4 (subplot h) and Figure 16). During the same time period, the time above the range (TAR) decreased by 55% relative to baseline, and events of severe hyperglycemia (>13.9 mM) and hypoglycemia (<3.9 mM) completely disappeared (Figure 8 (subplots a-b) and Figure 15).

Between weeks 4 and 12, a significant reduction in dynamic mean glucose variability (from 5.50 (baseline) to 2.6 mmol/L) (Figure 15) and a steady increase in TITR (from 81% to 90%) were observed (Figure 4 (subplot h), Figure 8 (subplots c-e), and Figure 15). After week 32, the patients' TITR reached 99% and remained thereafter (Figure 4 (subplots h and I) and Figure 15), while the meal glucose shift/MAGE (gold standard for glycemic variability) decreased from 5.50 mM (baseline) to 1.60 mM (Figure 7 (subplot d), Figure 8 (subplots h-l), and Figure 15). Importantly, no episodes of hypoglycemia or severe hyperglycemia were observed during the entire 116-week follow-up period post-surgery (Figures 8 and 15).

Furthermore, the MMTT revealed a trend towards stabilization of postoperative glycemic variability, as evidenced by stable fasting glucose concentrations and significant reductions in postprandial glucose concentrations (maximum of 21.3 mM at baseline, compared to a maximum of 9.1 mM at week 105) (Figure 4 (subplot j) and Figure 14). Consistently, the area under the curve (AUC) derived from 5-point intravenous glucose values decreased to 40% of baseline (Figure 7 (subplot b)), further confirmed by the AUC values obtained from a continuous glucose monitoring system (CGM) (Figure 7 (subplot c)). Heme A1c levels decreased from 6.6% (baseline) to 5.5% (week 85) and 4.6% (week 113) (Figure 4 (subplot h) and Figure 16).

Notably, insulin requirements gradually decreased until complete withdrawal was achieved at the end of week 11 (Figure 4 (subplot h)), and oral antidiabetic medications were gradually reduced starting from week 44 and discontinued at week 48 (acarbose) and week 56 (metformin) (Figure 7 (subplot a)).

Compared with preoperative levels, the mean postoperative fasting C-peptide level (0.68 nmol/L) increased threefold (Figure 4 (subplot k) and Figure 15). Notably, the secretion of C-peptide (Figure 4 (subplot k)) and insulin (Figure 4 (subplot l)) measured by MMTT showed a significant increase compared with preoperative tests, which was confirmed by AUC (Figure 7 (subplot b)).

During the 116-week follow-up period, no tumor formation was detected by MRI of the upper abdomen or by measuring serum tumor-associated antigen markers. Treatment-induced adverse events (TEAEs) included: 1) temporary abdominal distension and loss of appetite over 4 to 8 weeks, relieved by methimazole; 2) recoverable weight loss of <5% (80 kg to 76 kg).

Data from the first 116 weeks revealed significant improvements in glycemic control and pancreatic function. The graft was well tolerated, with no tumor formation or serious transplant-related adverse events. These data indicate that stem cell-derived islet tissue can salvage pancreatic function in patients with advanced type 2 diabetes mellitus (T2D).

Although this disclosure has been specifically shown and described with reference to a particular embodiment, those skilled in the art will understand that various changes in form and detail may be made without departing from the spirit and scope of this disclosure as disclosed herein.

Figure 1 illustrates the quality control of EnSC. (a) Morphology of EnSC. (b) FACS results, revealing the ratio of SOX17+/FOXA1+ cells in EnSC. (c) Karyotype analysis results of WB20 EnSC cell line. (d) Growth curve of EnSC. (e) Acceptance criteria and pathogen test results of EnSC. (f) Circle diagram of parental PBMC (left) and EnSC (breeding line WB20) (right) regarding genotypic variation from WGS analysis. From the outside in, each of the nine circles represents a morphology of genotypic variation. Circle 1: Chromosome; Circle 2: Density map of single nucleotide variants (SNVs); Circle 3: Density map of indel insertions; Circle 4: Density map of indel deletions; Circle 5: Density map of mutation sites in coding regions; Circle 6: Density map of mutation sites in non-coding regions; Circle 7: Location map of copy number variants (CNVs), with red and blue bars indicating copy number increases and decreases, respectively; Circle 8: Location map of structural variants (SVs), with orange and green bars 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.

Figure 2 illustrates quality control of the intermediate pancreatic differentiation stage in EnSC. (a) and (b) Morphology and cellular composition of the differentiation culture at the pancreatic precursor cell (PP) stage. (a) is a representative phase-contrast image of EnSC-derived PP cells. (b) shows FACS data revealing the NKX6-1+ / PDX1+ PP cell ratio at this stage. (c) and (d) Morphology and cellular composition of the differentiation culture at the endocrine precursor cell (EP) stage. (c) is a representative phase-contrast image of EnSC-derived EP cells. (d) Presents FACS data revealing the proportion of NKX6-1+ / PDX1+ and CHGA- / NKX6-1+ PP cells differentiating into CHGA+ endocrine precursor cells in the early endocrine precursor phase (day 3 of the EP phase), and the proportion of CHGA+ endocrine precursor cells to emerging endocrine (C-peptide+ or glucagon+) cells in the late endocrine precursor phase (day 8 of the EP phase). Scale bar, 100 μm.

Figure 3 illustrates the quality control of regenerated islet tissue. (a) Morphology of regenerated islet tissue (scale bar, 100 μm). (b) Results of FACS analysis of pancreatic endocrine cells in regenerated islet tissue, where chromogranin A+ populations represent pan-endocrine compartments, C-peptide+glucagon- populations represent β cells, glucagon+ populations represent α cells, and somatostatin+ populations represent δ cells. (c) Immunofluorescence staining of regenerated islet tissue, showing C-peptide (CPEP) positive β cells, glucagon (GCG) positive α cells, and somatostatin (SST) positive δ cells (scale bar, 50 μm). (d) Expression levels of specified genes in regenerated islet tissue and adult islets, measured by quantitative RT-PCR (qRT-PCR). PDX1 , NKX6.1 , and insulin ( INS ) were expressed in adult β cells, while glucagon ( GCG ) and somatostatin ( SST ) were typically expressed in adult α and δ cells, respectively. (e) and (f) Clusters and gene expression in subpopulations of regenerated islet tissue, revealed by single-cell transcriptomic analysis (scRNA seq, 10× Genomics). (e) tSNE cluster data from scRNA seq, showing various subpopulations of regenerated islet tissue (2721 cells with low UMI excluded from 15244 sequenced cells). (f) Presentation of representative gene expression for cell types (represented by boxes with blue dashed lines) in regenerated islet tissue. (g) C-peptide secretion from human (primary) islets and 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 undetectable in regenerated islet tissue. (i) Pathogen testing criteria and results for regenerated islet tissue.

Figure 4 illustrates the preclinical study and clinical results of autologous regenerated islet tissue transplantation in patients with type 2 diabetes mellitus (T2D). (a) A simplified procedure, illustrating the main procedures involved in the production and quality control of autologous regenerated islet tissue and the postoperative evaluation of the safety and efficacy of regenerated islet tissue transplantation. (b)-(d) Reversal of hyperglycemia in immunocompromised (SCID Beige) mice with STZ-induced diabetes. (b) Schematic illustration of regenerated islet tissue renal capsule transplantation into diabetic mice. (c) Fasting glucocorticoids in diabetic mice (blue line represents the sham treatment group, showing persistent hyperglycemia and death within 2 months; red line represents the regenerated islet tissue transplantation group, showing reversal of hyperglycemia within one month and loss of therapeutic effect after nephrectomy of the transplanted organ). (d) On days 90 and 180 after regenerated islet tissue transplantation, after fasting and 30 minutes after intraperitoneal glucose injection, STZ-induced secretion of human C-peptide in diabetic mice. (e)-(g) Immunogenicity of regenerated islet tissue in humanized mice. (e) Schematic illustration of transplantation of patient-specific regenerated islet tissue syngeneic and allogeneic renal capsules into patient- and volunteer-derived PBMC-humanized diabetic mice (NCG hIL 15, non-obese diabetic mice with severe complex immunodeficiency and interleukin-2 receptor γ deficiency). (f) Fasting glucocorticism in STZ-induced diabetic humanized mice (blue line represents the control group of three diabetic mice humanized with patient-derived PBMCs, and red line represents the group of three diabetic mice humanized with patient-derived PBMCs). (g) On days 7 and 14 post-transplantation of regenerated islet tissue, after fasting and 30 minutes after intraperitoneal glucose bolus, secretion of human C-peptide in humanized diabetic mice ([U.D.]: undetectable). (h) Clinical measurements of time within the target range (TITR), time within the range (TIR), and heme A1C (HbA1c) and insulin (degludec) doses over 116 weeks. Changes in degludec dosage (yellow line) are shown on the left y-axis. The ratios of time within the strict target range (TITR, 3.9 to 7.8 mM) (green line) and time within the range (TIR, 3.9 to 10.0 mM) (blue line) and serum heme A1c levels (red line) are shown on the right y-axis. (i) Continuous interstitial glucose fluctuations from CGM measurements at weeks 52 and 105 compared to pre-operative levels. The green lines at 3.9 mM and 7.8 mM delineate the target glucose range for health. The brown (preoperative)/green (week 52)/dark blue (week 105) lines, the medium brown (preoperative)/medium green (week 52)/medium blue (week 105) areas, and the light brown (preoperative)/light green (week 52)/light blue (week 105) areas represent the median (50%), 25-75% range, and 5-95% range, respectively. The results of the Mixed Diet Tolerance Test (MMTT) (j)-(l) were used to assess pancreatic function by monitoring serum levels of circulating glucose (j), C-peptide (k), and insulin (l) under fasting and dietary stimulation.

Figure 5 illustrates the improvement of STZ-induced diabetes in monkeys by regenerated islet tissue. (a) Schematic illustration of regenerated islet tissue implanted into the portal vein of STZ-induced diabetic monkeys. Two diabetic monkeys received either 6,000 (monkey 1) or 30,000 (monkey 2) regenerated islet tissues. Monkey 1 was used to test the feasibility of intravenous injection of regenerated islet tissue without digital subtraction angiography (DSA), while monkey 2 was used to evaluate the short-term safety and efficacy of regenerated islet tissue. Tail apex blood glucose was measured before morning and afternoon feedings. Animal exogenous insulin dosage was determined based on pre-meal blood glucose levels. (b) Daily pre-meal blood glucose levels (left) and insulin dosage (right) during the 28 days prior to and following surgery (before: before surgery; after: after 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 with transplanted regenerated islet tissue. The patterns of blood glucose (c) and human C-peptide (d) were monitored by IVGTT before diabetes model establishment/STZ treatment (before diabetes model establishment), 2 weeks before transplantation (2 weeks before Tx), and 2, 3, and 4 weeks after transplantation (2, 3, and 4 weeks after Tx).

Figure 6 illustrates the characteristics of humanized mice. (a) and (b) Characteristics of NCG-hIL15 mice humanized from patient (a) or volunteer (b) PBMCs by the presence of hCD45+ human cells in peripheral blood. (a) Ratio of live cells (via SSC and FSC), human blood cells (hCD45+), and mouse blood cells (mCD45+) in three patient humanized mice. (b) Ratio of live cells, human blood cells, and mouse blood cells in three volunteer humanized mice. (c) Immunofluorescence staining of grafts collected from the renal capsule of patient-humanized mice for the presence of human β cells (C-peptide+ and NKX6-1+), α cells (glucagon+) and δ cells (stostatin+); C-peptide (CPEP, red), glucagon (GCG, green), somatostatin (SST, purple) and NKX6-1 (cyan); the area delineated by the white dashed line indicates mouse kidney tissue; scale bar, 50 μm.

Figure 7 illustrates the clinical assessment and outcomes of glycemic control. (a) Follow-up time points for routine and disease-specific clinical assessments, and a schematic diagram of the treatments received by patients throughout the follow-up period. Endocrine function and diabetes-specific parameters were assessed using the Mixed Diet Tolerance Test (MMTT) at baseline and at weeks 4, 8, 12, 16, 20, 24, 36, and 48, and subsequently at designated time points. Glycemic control was measured using a 24-hour continuous glucose monitoring (CGM) system. Baseline and follow-up CGM glucose levels were measured during the first 52 weeks and subsequently at designated time points, with an average CGM device wearing duration of at least 3 days. The primary treatment regimen included antihyperglycemic agents and immunosuppressants. Antidiabetic treatment included metformin (0.75 g bid, tapered from week 44 and discontinued from week 56) and acarbose (50 mg tid, tapered from week 44 and discontinued from week 48). The insulin analog degludec was administered from 2021 (20 U once daily at bedtime), but was immediately discontinued after regenerated islet tissue transplantation, resumed and tapered from week 2, and discontinued at the end of week 11. Graft-versus-host disease was treated with mycophenolate mofetil (0.5 g bid after kidney transplantation) and tacrolimus (1 to 3 mg bid orally after kidney transplantation, depending on serum FK506 concentration). ☆ indicates the follow-up time point for clinical evaluation, and Δ indicates the CGMS monitoring stage. The grid pattern indicates the decreasing stage. (b) Area under the curve (AUC) of intravenous glucose (Fig. 4(j)), C-peptide (Fig. 4(k)), and insulin (Fig. 4(l)) values at 5 points (0, 30, 60, 120, 180 minutes) from the Mixed Diet Tolerance Test (MMTT). (c) Continuous glucose monitoring during the MMTT (0 to 240 minutes) measured every 5 minutes using a CGM device at each follow-up time point. The target glucose range is delineated by dashed horizontal lines at 3.9 and 10.0 mM. The AUC multiple (right figure area) is the under-curve product of the follow-up time points relative to the AUC standardized at week 2 (2W). (d) Meal glucose shift (mean glucose shift amplitude, MAGE), the gold standard for glycemic variability, is represented by the baseline and the mean glucose values (within the 95% range) at 1.5 hours before and 2 hours after each meal at weeks 52 and 105. The target glucose shift for healthy individuals is delineated by green horizontal lines at 3.9 and 7.8 mM.

Figure 8 illustrates continuous glucose monitoring at each follow-up time point. (a)-(l) Continuous glucose monitoring (CGM) traces before surgery (a) and at each follow-up time point (b)-(l). GCM data were collected for 72 consecutive hours at each follow-up time point. The median (dark blue line), 25-75% (medium blue area), and 5-95% (light blue area) ranges are shown, with the green horizontal line delineating the target glucose range (3.9 to 10.0 mM) for diabetic patients. Meal glucose offset (MAGE) calculated from the mean glucose value (within the 95% range) is shown in red on each graph area.

Figure 9 illustrates the analysis of teratoma formation.

Figure 10 shows the quality control standards for EnSC and regenerated islet tissue.

Figure 11 shows the inoculum list.

Figure 12 shows the antibody list.

Figure 13 shows key laboratory values before and after transplantation.

Figure 14 shows the main targets of the follow-up visits.

Figure 15 shows the exploratory objectives.

Claims (59)

A method for generating an endoderm stem cell population, comprising: a) providing a pluripotent stem cell population; b) culturing the pluripotent stem cell population in a first culture medium containing Nodal signaling agonist and WNT signaling agonist; c) culturing the cells in a second culture medium containing Nodal signaling agonist and fibroblast growth factor (FGF); d) culturing the cells in a third culture medium containing a factor belonging to the TGF-β superfamily, FGF, hepatocyte growth factor (HGF), and VEGF; and e) The cells were cultured in a fourth medium containing a WNT signaling agonist, a TGF-β inhibitor, and epidermal growth factor (EGF) to generate an endoderm stem cell population. The method of claim 1, wherein the fourth culture medium does not contain FGF2 and/or Chir99021. The method of any of the aforementioned claims, wherein the Nodal signaling agonist is selected from the group consisting of: activator A, Nodal and GDF-8. The method of claim 3, wherein the Nodal signaling agonist is activator A. The method of any of the aforementioned claims, wherein the WNT signal transduction agonist is selected from the group consisting of: CHIR99021, Wnt3A, Wnt3a-AFM, R-Spondin-1 and BIO (6-bromoindorubin-3'-oxime), SKL2001, BML-284, CP21R7 and SB 216763. The method of claim 5, wherein the WNT signal transduction activator is CHIR99021 or Wnt3A. The method of any of the aforementioned 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 of request item 7, wherein the FGF is bFGF. The method of any of the aforementioned requests, wherein the factor belonging to the TGF-β superfamily is selected from the group consisting of BMP4, BMP2 and BMP7. As in the method of Request 9, the factor belonging to the TGF-β superfamily is BMP4. The method of any of the aforementioned claims, wherein the TGF-β inhibitor is selected from the group consisting of: A83-01, SB431542, ALK5 inhibitor, LDN-193189, Galunisertib (LY2157299), LY2109761, SB525334, SB505124, GW788388, LY364947, RepSox, LDN-193189, K02288, LDN-214117, SD-208, and Vactosertib. (TEW-7197), ML347, LDN-212854, DMH1, Pirfenidone, Alantolactone, SIS3, Hesperetin, and Dorsomorphin. The method of claim 11, wherein the TGF-β inhibitor is A83-01. The method of any of the preceding claims, wherein the pluripotent stem cell is selected from embryonic stem cells (ESCs) and induced pluripotent stem cells. The method of any of the preceding claims comprises: a) providing an induced pluripotent stem cell population; b) culturing the cells in a first medium containing activin A and CHIR99021; c) culturing the cells in a second medium containing activin A and bFGF; d) culturing the cells in a third medium containing BMP4, bFGF, HGF and VEGF; and e) culturing the cells in a fourth medium containing Wnt3A, A83-01 and EGF; thereby generating an endoderm stem cell population. The method of any of the preceding claims, wherein the second culture medium further comprises VEGF, ascorbic acid and/or glutamax. The method of any of the aforementioned claims, wherein the third culture medium further comprises TGF-α and/or dexamethasone. The method of any of the preceding claims, wherein the fourth culture medium further comprises Rspondin1, ascorbic acid and/or glutamic acid. As in any of the aforementioned requests, the culture period for step b is 1 to 2 days (preferably 1 day). As in any of the aforementioned requests, the culture period for step c is 2 to 6 days (preferably 4 days). The method described in any of the aforementioned requests, wherein the culture period for step d is 2 to 6 days (preferably 4 days). The method of any of the aforementioned requests, 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%). 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 expresses SOX17, CDX2, SOX9, GATA6, and/or FOXA1. The method of any 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 medium, MCDB131, DMEM/F12 medium, StemPro34-SFM, RPMI1640, IMDM, DMEM, Ham's F12 and CMRL1066. The method of any 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 substrate-free. An endoderm stem cell population produced according to any of the methods described in the preceding claims. A method for generating 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 niacinamide to generate a pancreatic precursor (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 precursor (EP) cell population; and d) The EP cell population was cultured in the presence of the factors involved in step c, as well as T3 and niacinamide, thereby generating a pancreatic endocrine cell population. The method of claim 26, wherein the endoderm stem cell population comprises the endoderm stem cell population of claim 25. The method of claim 26 or 27, wherein the BMP inhibitor is selected from the group consisting of: Noggin, doxorubicin and LDN-193189. The method of claim 28, wherein the BMP inhibitor includes Noggin. 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 inhibitor, LDN-193189, goncitabine (LY2157299), LY2109761, SB525334, SB505124, GW788388, LY364947, RepSox, LDN-193189, K02288, LDN-214117, SD-208, vatochrome (TEW-7197), ML347, LDN-212854, DMH1, pirfenidone, oleuropein, SIS3, and hesperidin and doxorubicin. The method of claim 30, wherein the TGF-β inhibitor comprises A83-01. 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 dibenzopyrene (DBZ). The method of any of claims 26 to 32, wherein the Nodal signaling agonist is selected from the group consisting of activator A, Nodal and GDF-8. The method of claim 33, wherein the Nodal signaling agonist is activator A. The method of any of claims 26 to 34 comprises: 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 niacinamide to generate a pancreatic precursor (PP) cell population; c) culturing the PP cell population in the presence of Noggin, A83-01, and GSI-XX to generate an endocrine precursor (EP) cell population; and d) culturing the EP cell population in the presence of T3 and niacinamide; thereby generating a pancreatic endocrine cell population. The method of any 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, niacinamide and at least one of Rspondin1, LDN-193189 and TPPB to generate a pancreatic precursor (PP) cell population. The method of any of claims 26 to 36, wherein step c includes culturing PP cell populations in the presence of Noggin, A83-01, GSI-XX and at least one of retinoic acid and SANT1 to generate endocrine precursor (EP) cell populations. The method of any of claims 26 to 37, wherein step d includes culturing the EP cell population in the presence of T3, nicotinamide and BMP4. If the method described in any of the requests 26 to 38 is followed, the culture period for step b is 2 to 6 days. If the method described in any of the requests 26 to 39 is followed, the culture period for step c is 3 to 8 days. If the method described in any of the requests 26 to 40 is followed, the culture period for step d is 7 to 21 days. The method of any of claims 26 to 41, wherein steps b through d are performed independently in a two-dimensional (2D) or three-dimensional (3D) environment. A population of pancreatic endocrine cells produced according to any one of claims 26 to 42. A pharmaceutical composition comprising a pancreatic endocrine cell population as described in any of claims 26 to 43 and a pharmaceutically acceptable culture medium. A method for treating a disease or condition associated with impaired pancreatic function in an individual, comprising: administering to the individual an effective amount of pancreatic endocrine cells derived from an endoderm stem cell population (i.e., regenerated islet tissue) to treat the individual's disease or condition associated with impaired pancreatic function. The method of claim 45, wherein the endoderm stem cell population is generated according to the method of any one of claims 1 to 25. The method of claim 46, wherein the pluripotent stem cell population is autologous or allogeneic. The method described in any of claims 45 to 47 includes hepatic hilar vein infusion, renal capsule transplantation, rectus abdominis muscle injection, subcutaneous implantation, mesenteric injection, retroperitoneal injection, hepatic artery injection, or iliac fossa injection. The method of any of the requests 45 to 48, wherein the individual is a human. The method of any of claims 45 to 48, wherein the effective quantity is about 0.5 to 3.0 million islet equivalents (IEQ) units. The method of any of the claims 45 to 50, wherein the disease or symptom associated with impaired pancreatic function is diabetes, such as type 1 diabetes (T1D), type 2 diabetes (T2D), type 3c diabetes, or mature-onset diabetes in young adults (MODY). The use of an endoderm stem cell population for the production of regenerated islet tissue to treat diseases or symptoms associated with impaired islet function in individuals in need. For the purposes of claim 52, the manufacture includes the steps defined in any of claims 26 to 42. A kit for generating endoderm stem cell populations from pluripotent stem cell populations, wherein the kit comprises a first group of factors, a second group of factors, a third group of factors, and a fourth group of factors, wherein the first group of factors comprises a Nodal signaling agonist and a WNT signaling agonist, the second group of factors comprises a Nodal signaling agonist and fibroblast growth factor (FGF), the third group of factors comprises factors belonging to the TGF-β superfamily, FGF, hepatocyte growth factor (HGF), and VEGF, and the fourth group of factors comprises a WNT signaling agonist, a TGF-β inhibitor, and epidermal growth factor (EGF). For example, in the set of request item 54, the fourth factor does not include FGF2 and/or Chir99021. A kit for generating pancreatic endocrine cell populations from endoderm stem cell populations, 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 comprises a BMP inhibitor, a Nodal signaling agonist, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and/or niacinamide; the sixth group of factors comprises a BMP inhibitor, a TGF-β inhibitor, and/or a γ-secretase inhibitor; and the seventh group of factors comprises T3 and/or niacinamide. A kit for generating pancreatic endocrine cell populations from pluripotent stem cell populations, comprising factors in groups one through seven. Group one comprises Nodal signaling agonists and WNT signaling agonists; group two comprises Nodal signaling agonists and fibroblast growth factor (FGF); and group three comprises factors belonging to the TGF-β superfamily, FGF, and hepatocyte growth factor (HGF). The fourth group of factors includes WNT signaling promoters, TGF-β inhibitors, and epidermal growth factor (EGF); the fifth group of factors includes BMP inhibitors, Nodal signaling promoters, FGF10, EGF, SANT1, retinoic acid, ascorbic acid, and niacinamide; the sixth group of factors includes BMP inhibitors, TGF-β inhibitors, and γ-secretase inhibitors; and the seventh group of factors includes T3 and niacinamide. For example, in the set of request item 57, the fourth factor does not include FGF2 and/or Chir99021. A kit for generating pancreatic endocrine cell populations from PP cell populations, wherein the kit contains a set of factors including a BMP inhibitor, a TGF-β inhibitor, and a γ-secretase inhibitor.