Method for preparing pancreatic β cells by differentiation

Abstract

Provided is a method for preparing cells by differentiation. Specifically, provided is a method for preparing pancreatic β cells by differentiation, comprising the following step: adding specific small-molecule substances to differentiation media at different stages. The method can shorten the differentiation time and improve the differentiation purity and differentiation efficiency of pancreatic β cells.

Description

A method for differentiating and preparing pancreatic β cells

[0001] This application claims priority to Chinese application No. 202411954405.9, entitled "A method for differentiating and preparing pancreatic β cells", filed on December 26, 2024. Technical Field

[0002] This invention belongs to the field of cell differentiation, specifically relating to a method for preparing pancreatic β cells through differentiation. Background Technology

[0003] Diabetes mellitus is a metabolic disease characterized by hyperglycemia, which is caused by a deficiency in insulin secretion or impaired biological action, or both. Long-term hyperglycemia leads to chronic damage and functional impairment of various tissues, particularly the eyes, kidneys, heart, blood vessels, and nerves. Diabetes has become a serious global chronic disease burden with enormous harm. The 2021 Global Diabetes Atlas released by the International Diabetes Federation (IDF) shows that there are approximately 537 million (10.5%) people aged 20-79 with diabetes worldwide (one in ten people have diabetes), and this number is projected to increase to 643 million (11.3%) by 2030 and 783 million (12.2%) by 2045. In 2021, my country had 140 million people with diabetes, and this number is projected to reach 174 million by 2045, ranking first in the world. Diabetes is mainly divided into type 1 diabetes and type 2 diabetes, with type 1 diabetes accounting for approximately 5-10% of all diabetes cases.

[0004] The pathogenesis of type 1 diabetes is mainly due to the destruction of pancreatic beta cells by the autoimmune system, leading to their inability to produce or secrete insulin and thus causing elevated blood sugar. Currently, the clinical treatment principle for type 1 diabetes is lifelong insulin injection. However, adjusting the insulin dosage to precisely control blood sugar is very difficult. Patients need to adjust their insulin dosage in a timely manner based on individual factors such as diet, activity, and even emotions to maintain blood sugar levels under normal physiological conditions as much as possible. Inaccurate insulin dosage can cause patients to have chronically high or low blood sugar levels, ultimately leading to serious and even life-threatening complications. Current clinical research shows that islet cell replacement therapy, which involves transplanting islet cells from deceased donors into patients, can precisely respond to changes in blood sugar and achieve a functional cure for type 1 diabetes. However, due to the extreme scarcity of deceased donor islet cells, it cannot meet the huge clinical demand; therefore, alternative solutions are urgently needed.

[0005] Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell with characteristics similar to human embryonic stem cells, induced by reprogramming human adult somatic cells (such as skin fibroblasts or blood cells) through the introduction of stem cell-specific transcription factors Oct3 / 4, Sox2, c-Myc, and Klf4. iPSCs possess multipotent differentiation and strong self-proliferative potential, and under certain conditions, they can differentiate into various tissue-specific functional cell types, such as immune cells (CAR-T cells, CAR-NK cells), cardiomyocytes, nerve cells, and pancreatic β cells. They hold immense promise for applications in disease research, drug screening, and cell therapy.

[0006] The technique of in vitro inducing iPSC differentiation into pancreatic β cells targets specific signaling pathways, precisely mimicking the in vivo developmental process of pancreatic β cells, and efficiently and directionally differentiating iPSCs into pancreatic β cells. The resulting pancreatic β cells possess functional activity highly similar to primary human β cells, enabling effective glucose response and regulation both in vitro and in vivo, ultimately reshaping the secretion of endogenous insulin in the human body. iPSC-derived cell products (e.g., pancreatic islet cells) have the capability for large-scale, robust in vitro production. Furthermore, leveraging CRISPR / CAS site-directed gene editing systems or islet encapsulation technology, low-immunogenicity, universal iPSC-derived cell products can be developed, minimizing the cost of cell-based drugs and ensuring accessibility for widespread clinical application. However, current in vitro induction techniques for iPSC differentiation into pancreatic β cells still suffer from drawbacks such as low differentiation purity and long differentiation cycles, and differentiation efficiency needs further improvement. Summary of the Invention

[0007] To address the problems of low purity and long differentiation cycles in existing pancreatic β-cell differentiation methods, this disclosure provides a highly efficient method for preparing pancreatic β-cells. This method shortens the differentiation time and improves the differentiation efficiency of pancreatic β-cells. In some embodiments, the method provided by this disclosure reduces the time required for pluripotent stem cells to differentiate into pancreatic β-cells from the conventional approximately 30 days to 14-18 days, and increases the differentiation efficiency of pancreatic β-cells (C-Peptide+ / NKX6.1+ double positivity ratio) from 30-50% to 70-85%. The method provided by this disclosure significantly reduces the cost of islet preparation, facilitates large-scale production, reduces patient transplantation costs, and expands the accessibility of clinical applications.

[0008] This disclosure first provides a method for preparing pancreatic β cells, comprising the following steps:

[0009] (1) Culture pluripotent stem cells to differentiate them into a fixed endoderm;

[0010] (2) Differentiate the defined endoderm cells into gastrulator cells;

[0011] (3) Differentiate the primitive intestinal cells into posterior foregut cells;

[0012] (4) Differentiate the posterior foregut cells into pancreatic progenitor cells;

[0013] (5) Differentiate the pancreatic progenitor cells into endocrine progenitor cells; and

[0014] (6) Differentiate the endocrine precursor cells into pancreatic β cells.

[0015] In one or more embodiments, the stem cells are PSCs, such as iPSCs and ESCs.

[0016] In one or more embodiments, the stem cells do not differentiate into non-directed forms.

[0017] In one or more embodiments, the confluence of the stem cells is 60%-99%, preferably 80%.

[0018] In one or more embodiments, steps (1) through (6) are performed in a culture medium. Each of steps (1) through (6) is independently a serum-free culture medium.

[0019] In one or more embodiments, the serum-free culture medium comprises one or more selected from the following: MCDB131 or DMEM or RPMI1640, glucose, NaHCO3, BSA, ITS-X, Glutamax, vitamin C, antibiotics (e.g., penicillin and / or streptomycin), heparin or ZnSO4.

[0020] In one or more embodiments, the serum-free culture medium is selected from: Essential 8 medium, PluriSTEM medium, and mTeSR medium.

[0021] In one or more embodiments, the method includes reducing the size of the endoderm clusters, wherein adjusting the size of the cell clusters includes separating the clusters and re-aggregating them before they mature into β cells.

[0022] In one or more embodiments, the method includes reducing the cluster size of the stem cells, wherein adjusting the size of the cell clusters includes separating the clusters and re-aggregating them before they mature into β cells.

[0023] In one or more embodiments, the method includes reducing the cluster size of the stem cells in step (1). In one or more embodiments, reducing the cell cluster size includes digesting the stem cells into single cells. In one or more embodiments, the digestion uses trypsin, proteolytic enzymes, collagenases, or combinations thereof, such as EZ-LiFT, Accutase, or dispersant enzyme reagents, preferably Accutase.

[0024] In one or more embodiments, the method includes reducing the cluster size of the posterior foregut cells in step (4); preferably, adjusting the size of the cell clusters includes separating the clusters and re-aggregating them before maturing into β cells. In one or more embodiments, reducing the cell cluster size includes digesting the posterior foregut cells into single cells. In one or more embodiments, the digestion uses trypsin, proteolytic enzymes, collagenases, or combinations thereof, such as EZ-LiFT, Accutase, or dispersant agents, preferably Accutase.

[0025] In one or more embodiments, the time sufficient to form morphological endoderm cells, gastrulation cells, posterior foregut cells, pancreatic progenitor cells, and endocrine progenitor cells is between about 1 day and about 8 days (e.g., between 2 and 4 days), or the time sufficient to form β cells is between about 1 day and about 9 days (e.g., 3 to 6 days) or more than 9 days.

[0026] In one or more embodiments, the method does not include the use of one or more of Alk5i, thyroid hormones, serum, T3, N-acetylcysteine, Trolox, and R428.

[0027] In one or more embodiments, the pancreatic progenitor cells are not in contact with any one or more of serum, T3, N-acetylcysteine, Trolox, and R428.

[0028] In one or more embodiments, T3, N-acetylcysteine, Trolox, or R428 are not contained in the endodermal cells as they mature into β cells.

[0029] In one or more embodiments, the method enhances the function of pancreatic β cells.

[0030] In one or more embodiments, the PSC inoculation density in step (1) is 1×10 5 Up to 3×10 5 / cm 2 Preferably 2×10 5 / cm 2 .

[0031] In one or more embodiments, the environmental conditions for each step of the method are 5% CO2 and 37°C.

[0032] In one or more embodiments, steps (1)-(3) are 2D culture and steps (4)-(6) are 3D culture.

[0033] In one or more embodiments, in step (4), the posterior foregut cells are arranged at 5 × 10 6 / hole up to 10×10 6 / well inoculation.

[0034] In one or more embodiments, the culture medium in step (4) further comprises one or more of the following: (a) a p300 histone acetyltransferase inhibitor, (b) a selective inhibitor of EZH2 and EZH1, (c) a BET bromodomain inhibitor, (d) a p38 MAPK inhibitor, (e) a JNK inhibitor, (f) an HDAC inhibitor, (g) a G9a / GLP histone methyltransferase inhibitor, and (h) an Akt inhibitor. In one or more embodiments, the culture medium in step (4) further comprises (a) a selective inhibitor of EZH2 and EZH1, and / or (b) an HDAC inhibitor.

[0035] In one or more embodiments, the culture medium in step (6) further comprises one or more selected from: (a) an Axl inhibitor, (b) a MEK inhibitor, (c) a JAK / Aurora kinase inhibitor, (d) an ARK inhibitor, and (e) a histone demethylase inhibitor. In one or more embodiments, the culture medium in step (6) further comprises (a) a selective inhibitor of EZH2 and EZH1, and / or (b) a JAK2 / 3 inhibitor.

[0036] In one or more embodiments, the culture medium in step (4) further comprises one or more of the following: HAIII, L002, GSK126, EPZ-6438, DZNeP, UNC1999, I-BET151, SB203580, SP600125, VPA, SAHA, UNC0642, AT7867, AZD5363, TMP269, A-366. In one or more embodiments, the concentration of HAIII is 0.1 μM to 1 μM. In one or more embodiments, the concentration of L002 is 0.1 μM to 1 μM. In one or more embodiments, the concentration of GSK126 is 0.25 μM to 2.5 μM. In one or more embodiments, the concentration of EPZ-6438 is 0.1 μM to 1 μM. In one or more embodiments, the concentration of DZNeP is 0.1 μM to 1 μM. In one or more embodiments, the concentration of UNC1999 is 0.1 μM to 1 μM. In one or more embodiments, the concentration of I-BET151 is 0.1 μM to 1 μM. In one or more embodiments, the concentration of SB203580 is 0.1 μM to 1 μM. In one or more embodiments, the concentration of SP600125 is 0.1 μM to 1 μM. In one or more embodiments, the concentration of VPA is 50 μM to 500 μM. In one or more embodiments, the concentration of SAHA is 0.1 μM to 1 μM. In one or more embodiments, the concentration of UNC0642 is 0.1 μM to 1 μM. In one or more embodiments, the concentration of AT7867 is 0.1 μM to 1 μM. In one or more embodiments, the concentration of AZD5363 is 0.1 μM to 1 μM. In one or more embodiments, the concentration of TMP269 is 0.1 μM to 1 μM. In one or more embodiments, the concentration of A-366 is 0.1 μM to 1 μM.

[0037] In one or more embodiments, the culture medium in step (4) further comprises UNC1999 and / or SAHA. In one or more embodiments, the concentration of UNC1999 is 0.01 μM-0.25 μM, preferably 0.01 μM-0.05 μM, more preferably 0.025 μM-0.05 μM. In one or more embodiments, the concentration of SAHA is 0.01 μM-0.25 μM, preferably 0.01 μM-0.1 μM, more preferably 0.01 μM-0.05 μM.

[0038] In one or more embodiments, the culture medium in step (6) further comprises one or more of the following: AT9283, Danusertib, UNC1999, and CPI-455HCl. In one or more embodiments, the concentration of AT9283 is 0.05 μM to 2.5 μM. In one or more embodiments, the concentration of Danusertib is 0.05 μM to 2.5 μM. In one or more embodiments, the concentration of UNC1999 is 0.05 μM to 2.5 μM. In one or more embodiments, the concentration of CPI-455HCl is 0.05 μM to 2.5 μM.

[0039] In one or more embodiments, the culture medium in step (6) further comprises any one, two, or all three selected from: AT9283, Danusertib, and UNC1999. In one or more embodiments, the culture medium in step (6) further comprises any group selected from: 1) AT9283, 2) Danusertib, 3) AT9283 and Danusertib, 4) AT9283 and UNC1999; 5) UNC1999 and Danusertib; 6) UNC1999, AT9283, and Danusertib. In one or more embodiments, the concentration of UNC1999 is 0.25 μM to 5 μM, preferably 0.05 μM to 2.5 μM, more preferably 2.5 μM. In one or more embodiments, the concentration of AT9283 is 0.05 μM to 2.5 μM, preferably 0.1 μM to 0.5 μM, more preferably 0.25 μM. In one or more embodiments, the concentration of Danusertib is 0.05 μM to 2.5 μM, preferably 0.5 μM to 1.5 μM, and more preferably 1 μM.

[0040] In one or more embodiments, the culture medium in step (2) further comprises (a) an EZH2 and EZH1 selective inhibitor, and / or (b) an HDAC inhibitor.

[0041] In one or more embodiments, the culture medium in step (2) further comprises UNC1999 and / or SAHA. In one or more embodiments, the concentration of UNC1999 is 0.01 μM-0.25 μM, preferably 0.01 μM-0.05 μM, more preferably 0.025 μM-0.05 μM. In one or more embodiments, the concentration of SAHA is 0.01 μM-0.25 μM, preferably 0.01 μM-0.1 μM, more preferably 0.01 μM-0.05 μM.

[0042] In one or more embodiments, the culture medium in step (3) further comprises (a) an EZH2 and EZH1 selective inhibitor, and / or (b) an HDAC inhibitor.

[0043] In one or more embodiments, the culture medium in step (3) further comprises UNC1999 and / or SAHA. In one or more embodiments, the concentration of UNC1999 is 0.01 μM-0.25 μM, preferably 0.01 μM-0.05 μM, more preferably 0.025 μM-0.05 μM. In one or more embodiments, the concentration of SAHA is 0.01 μM-0.25 μM, preferably 0.01 μM-0.1 μM, more preferably 0.01 μM-0.05 μM.

[0044] In one or more embodiments, the culture medium in steps (2)-(4) further comprises (a) an EZH2 and EZH1 selective inhibitor and / or (b) an HDAC inhibitor, and the culture medium in step (6) further comprises (a) an EZH2 and EZH1 selective inhibitor and / or (b) a JAK2 / 3 inhibitor.

[0045] In one or more embodiments, the culture medium in steps (2)-(4) further comprises UNC1999 and SAHA. In one or more embodiments, the culture medium in steps (2)-(4) further comprises UNC1999 and SAHA, and the culture medium in step (6) further comprises 1) AT9283, 2) AT9283 and UNC1999, or 3) UNC1999, AT9283 and Danusertib. In one or more embodiments, the concentration of UNC1999 in steps (2)-(4) is 0.01-0.25 μM, preferably 0.01 μM-0.05 μM, more preferably 0.025 μM-0.05 μM. In one or more embodiments, the concentration of SAHA is 0.01 μM-0.25 μM, preferably 0.01 μM-0.1 μM, more preferably 0.01 μM-0.05 μM. In one or more embodiments, the concentration of UNC1999 in step (6) is 0.25 μM-5 μM, preferably 0.05 μM-2.5 μM, more preferably 2.5 μM. In one or more embodiments, the concentration of AT9283 is 0.05 μM-2.5 μM, preferably 0.1 μM-0.5 μM, more preferably 0.25 μM.

[0046] In one or more embodiments, the culture medium in steps (2) to (4) further comprises 0.01 μM to 0.25 μM (preferably 0.01 μM, 0.025 μM, 0.05 μM or 0.25 μM) of UNC1999 and 0.1 μM of SAHA, and the culture medium in step (6) further comprises 1 μM of AT9283 and 1 μM of UNC1999.

[0047] In one or more embodiments, the culture medium in steps (2) to (4) further comprises 0.1 μM of UNC1999 and 0.01 μM-0.25 μM (preferably 0.01 μM, 0.025 μM, 0.05 μM or 0.25 μM) of SAHA, and the culture medium in step (6) further comprises 1 μM of AT9283 and 1 μM of UNC1999.

[0048] In one or more embodiments, the culture medium in steps (2) to (4) further comprises 0.1 μM of UNC1999 and 0.1 μM of SAHA, and the culture medium in step (6) further comprises 0.05 μM to 2.5 μM (preferably 0.05 μM, 0.1 μM, 0.25 μM, 0.5 μM or 2.5 μM) of AT9283 and 1 μM of UNC1999.

[0049] In one or more embodiments, the culture medium in steps (2) to (4) further comprises 0.1 μM of UNC1999 and 0.1 μM of SAHA, and the culture medium in step (6) further comprises 1 μM of AT9283 and 0.25 μM to 5 μM (preferably 0.25 μM, 0.5 μM, 2.5 μM or 5 μM) of UNC1999.

[0050] In one or more embodiments, the culture medium in steps (2) to (4) further comprises 0.1 μM of UNC1999 and 0.1 μM of SAHA, and the culture medium in step (6) further comprises 1 μM of AT9283 and 1 μM of UNC1999.

[0051] In one or more embodiments, the culture medium in step (4) further comprises 0.1 μM of UNC1999 and 0.1 μM of SAHA, and the culture medium in step (6) further comprises 1 μM of AT9283 and 1 μM of UNC1999.

[0052] In one or more embodiments, the method takes a total of 14-21 days, preferably 14-18 days, and more preferably 18 days.

[0053] In one or more embodiments, the method achieves a pancreatic β-cell differentiation efficiency of 70%-85%.

[0054] In one or more embodiments, the proportion of pancreatic β cells expressing both C-Peptide and NKX6.1 positive by the method is 70%-85%.

[0055] In another aspect, this disclosure provides pancreatic β cells obtained by the methods provided herein.

[0056] In another aspect, this disclosure also provides pharmaceutical compositions comprising pancreatic β cells obtained by the methods described in any embodiment herein and pharmaceutically acceptable excipients.

[0057] This disclosure also provides, in another aspect, the use of pancreatic β-cells obtained by the methods described in any embodiment herein in the preparation of medicaments for treating diseases. In one or more embodiments, the diseases include pancreatic β-cell dysfunction disorders. Preferably, the pancreatic β-cell dysfunction disorders include one or more of the following benign lesions: type 1 diabetes, type 2 diabetes, insulinoma, hereditary pancreatic β-cell dysfunction, pancreatitis following total pancreatectomy, and benign pancreatic tumors.

[0058] This disclosure also provides a treatment method comprising: administering a therapeutically effective amount of pancreatic β cells obtained by the method described herein or a pharmaceutical composition described in any embodiment herein to a subject in need. Attached Figure Description

[0059] Figure 1 shows the C-Peptide+ / NKX6.1+ dual-positive flow cytometry results for verifying islet differentiation using the optimal small molecule combination.

[0060] Figure 2 shows the immunofluorescence detection results after islet differentiation using the optimal combination of small molecules.

[0061] Figure 3 is a schematic diagram of the process for further shortening the islet differentiation time. Detailed Implementation

[0062] This invention can be implemented in many different forms and is not limited to the embodiments described herein. It should be understood that these embodiments are provided so that a thorough and complete understanding of the disclosure of this invention will be achieved.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Although the numerical ranges and parameter approximations shown in the broad scope of the invention are provided, the values ​​shown in the specific embodiments are described as accurately as possible. However, any value inherently contains a certain degree of error due to the standard deviation present in their respective measurements. Furthermore, all scopes disclosed herein should be understood to encompass any and all subscopes contained herein.

[0064] This invention relates to a highly efficient method for preparing pancreatic β-cells through differentiation. Specifically, it involves adding a small molecule compound as an adjuvant to the conventional pancreatic islet cell differentiation process to shorten differentiation time and improve differentiation efficiency. This reduces the cost of islet preparation, facilitates large-scale production, lowers patient transplant costs, and expands accessibility for clinical applications.

[0065] definition

[0066] Stem cells and their differentiation

[0067] Stem cells are undifferentiated cells defined by their ability to both self-renew and differentiate at the single-cell level. Stem cells can produce daughter cells, including self-renewing progenitor cells, non-renewing progenitor cells, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells from multiple cell lineages derived from multiple germ layers (endoderm, mesoderm, and ectoderm). Stem cells also generate tissues from multiple germ layers after transplantation and, upon injection into the blastocyst, contribute to substantially to most (if not all) of the tissues.

[0068] Stem cells can be classified according to their differentiation potential. Differentiation is the process by which undifferentiated (“undirected”) or underdifferentiated cells acquire the characteristics of specialized cells (such as nerve cells or muscle cells). Differentiated cells are cells that have already occupied a more specialized (“directed”) position in a cell lineage. When applied to the process of differentiation, the term “directed” refers to a cell that has progressed to a certain point in the differentiation pathway, where, under normal circumstances, the cell will continue to differentiate into a specific cell type or a subpopulation of cell types, and under normal circumstances, it cannot differentiate into different cell types or revert to a less differentiated cell type. “Dedifferentiation” refers to the process by which a cell reverts to a less specialized (or directed) position in its cell lineage. As used herein, a “cell lineage” defines a cell’s heritability, i.e., which cells it comes from and what cells it can produce. Cell lineage positions a cell within the genetic program of development and differentiation.

[0069] Lineage-specific markers are characteristics that are definitively associated with the phenotype of cells in a lineage of interest and can be used to assess the differentiation of undifferentiated cells into that lineage. As used herein, a “marker” is a nucleic acid or polypeptide molecule that is differentially expressed in the cells of interest. In this context, differential expression means an elevated level for a positive marker and a decreased level for a negative marker compared to undifferentiated cells. Because the detectable levels of the marker nucleic acid or polypeptide in the cells of interest are sufficiently higher or lower than those in other cells, any of a variety of methods known in the art can be used to identify and distinguish the cells of interest from other cells. As used herein, a cell is “positive for that specific marker” or “positive” when a specific marker is adequately detected in the cell. Similarly, a cell is “negative” or “negative” for that specific marker when a specific marker is not adequately detected in the cell.

[0070] The term "pluripotent stem cells (PSCs)" refers to stem cells that can differentiate into various cell types in vitro but cannot develop into a complete individual. They occupy a higher level in the stem cell lineage, below totipotent stem cells but above multipotent stem cells. PSCs can differentiate into various tissue cells of the body, such as skin, muscle, and nerve cells, but their differentiation potential is limited. PSCs are of significant value in regenerative medicine and disease research, as they can be induced to differentiate into specific cell types for therapeutic purposes. These cells are generally divided into two categories: embryonic stem cells (ESCs) derived from blastocyst embryos and induced pluripotent stem cells (iPSCs), the latter being induced from adult somatic cells to become pluripotent. Typically, the production and culture of these two types of PSCs are for disease research, drug discovery, and studies of normal human development. PSCs can be derived from various species, including humans, rats, mice, pigs, or other non-mammals.

[0071] In this article, "induced pluripotent stem cells" (iPSCs) refer to pluripotent stem cells that are transformed from somatic cells (e.g., skin cells) into embryonic stem cells through gene reprogramming technology. These cells possess the ability to self-renew and differentiate into various cell types, and have great potential in fields such as disease modeling, drug screening, and regenerative medicine. Induced pluripotent stem cells (iPSCs) can be obtained by reprogramming somatic cells through ectopic expression of four transcription factors (OCT3 / 4, KLF4, SOX2, and c-MYC). In some embodiments, the iPSCs are derived from humans, i.e., human induced pluripotent stem cells (hiPSCs).

[0072] Pancreatic islet cells and their differentiation lineage

[0073] The term "islet cells" refers to a cluster of cells composed of endocrine gland cells. Islet cells mainly include pancreatic A cells (α cells), B cells (β cells), D cells (δ cells), and pancreatic PP cells. Dysfunction of islet cells can lead to metabolic diseases such as diabetes; therefore, studying the biological characteristics and regulatory mechanisms of islet cells is crucial for understanding and treating these diseases.

[0074] The term "pancreatic β-cells" refers to a cell type within the pancreatic islet cells. They are a crucial component of pancreatic endocrine cells, comprising approximately 60-70% of all islet cells. Primarily located in the islet region of the pancreas, they coexist with other types of endocrine cells, such as α-cells and δ-cells. Pancreatic β-cells are mainly responsible for synthesizing and secreting insulin, a hormone that plays a critical role in regulating blood glucose levels. For example, when blood glucose levels rise, β-cells secrete insulin, which promotes glucose uptake by cells (such as muscle and fat cells), thereby lowering blood glucose levels. Furthermore, insulin promotes the conversion of glucose into glycogen for storage in the liver and inhibits the liver's glucose production, helping to maintain blood glucose homeostasis. Pancreatic β-cells communicate with several other different types of islet cells through extracellular spaces and gap junctions, forming a complex cellular network. This arrangement allows the cellular products secreted by one type of cell to influence the function of downstream cells, synergistically regulating blood glucose. For instance, insulin secreted by β-cells can inhibit glucagon secretion by α-cells. The secretory function of pancreatic β cells is regulated by various factors, including blood glucose levels, hormones, and nerves. Impaired β cell function can lead to diabetes (especially type 1 and type 2 diabetes). Therefore, protecting and restoring pancreatic β cell function is an important research direction in diabetes treatment. Basic research and clinical applications of pancreatic β cells are of great significance.

[0075] When attempting to replicate the differentiation process of pluripotent stem cells in static in vitro cell cultures to obtain functional pancreatic endocrine cells, this differentiation process is typically viewed as proceeding sequentially in multiple consecutive stages. Specifically, this differentiation process is often considered to proceed in six to seven stages. In this stepwise process, "stage one" refers to the first step of the differentiation process, in which pluripotent stem cells differentiate into definitive endoderm cells (hereinafter also referred to as "stage one cells"). "Stage two" refers to the second step of the differentiation process, in which definitive endoderm cells differentiate into primitive gut tube cells (hereinafter also referred to as "stage two cells"). "Stage three" refers to the third step of the differentiation process, in which primitive gut tube cells differentiate into posterior foregut cells (hereinafter also referred to as "stage three cells"). "Stage four" refers to the fourth step of the differentiation process, in which posterior foregut cells differentiate into pancreatic progenitors (hereinafter also referred to as "stage four cells"). "Stage 5" refers to the fifth step in the differentiation process, in which pancreatic progenitor cells differentiate into endocrine progenitor cells (hereinafter also referred to as "stage 5 cells"). "Stage 6" refers to the sixth step in the differentiation process, in which endocrine progenitor cells differentiate into pancreatic islet-β cells (hereinafter also referred to as "stage 6 cells").

[0076] However, it should be noted that not all cells in a given cell population undergo all these differentiation stages at the same rate. Therefore, it is not uncommon to detect cells in in vitro cell cultures that are progressing earlier or later in the differentiation pathway than most cells in the cell population, especially in cultures at later stages of differentiation. For example, pancreatic β-cells are occasionally observed during the fifth stage of differentiation in cell cultures. For the purpose of illustrating the invention, characteristics of various cell types associated with the stages defined above are described herein.

[0077] As used herein, “defined endoderm cells” refers to cells that possess the characteristics of cells derived from the epiblast during gastrulation and form the gastrointestinal tract and its derivatives. Defined endoderm cells express at least one of the following markers: FOXA2 (also known as hepatocyte nuclear factor 3-β (“HNF3β”), GATA4, SOX17, CXCR4, Brachyury, Cerberus, OTX2, goosecoid, C-Kit, CD99, and MIXL1. Characteristic markers of defined endoderm cells include CXCR4, FOXA2, and SOX17. Therefore, defined endoderm cells are characterized by the expression of CXCR4, FOXA2, and SOX17.

[0078] As used in this article, "gastrulocytes" refers to cells derived from the fixed endoderm that produce all endoderm organs, such as the lungs, liver, pancreas, stomach, and intestine. Gastrulocytes are characterized by a significantly increased expression of HNF4α compared to that in fixed endoderm cells. For example, during the second phase, an increase in HNF4α expression in mRNA may be observed to be ten to forty times higher than before.

[0079] As used herein, “posterior foregut endoderm cells” refers to the endoderm cells that give rise to the esophagus, lungs, stomach, liver, pancreas, gallbladder, and part of the duodenum. Posterior foregut endoderm cells express at least one of the following markers: PDX1, FOXA2, CDX2, SOX2, and HNF4α. A characteristic of posterior foregut endoderm cells is increased PDX1 expression compared to archenterocytes. For example, more than 50 percent of cells in a stage III culture typically express PDX1.

[0080] As used herein, “pancreatic progenitor cells” refers to cells that express at least one of the following markers: PDX1, NKX6.1, HNF6, NGN3, SOX9, PAX4, PAX6, ISL1, gastrin, FOXA2, PTF1a, PROX1, and HNF4α. Pancreatic progenitor cells are characterized by positive expression of PDX1, NKX6.1, and SOX9.

[0081] As used herein, "endocrine precursor cells" refers to pancreatic endoderm cells capable of becoming pancreatic hormone-expressing cells. Endocrine precursor cells express at least one of the following markers: NGN3, NKX2.2, NeuroD1, ISL1, PAX4, PAX6, or ARX. Endocrine precursor cells may be characterized by the expression of NKX2.2 and NeuroD1.

[0082] As used in this article, “pancreatic β cells” refers to pancreatic endocrine cells that are capable of secreting insulin and at least one of the following transcription factors: PDX1, NKX2.2, NKX6.1, NeuroD1, ISL1, HNF3β, MAFA, and PAX6.

[0083] culture medium

[0084] The term "primary culture medium" refers to the culture medium used to incubate pluripotent stem cells (PSCs) before they enter the differentiation stage. The commonly used medium is DMEM / F12. DMEM / F12 contains various amino acids, vitamins, and inorganic salts, providing essential nutritional support for PSCs and maintaining cell survival and basic metabolism. In addition to the basal medium, serum or serum substitutes such as fetal bovine serum (FBS) or chemical serum substitutes (KSR) are usually added. Growth factors are also typically added to maintain the undifferentiated state of PSCs or improve cell viability.

[0085] In addition, there are existing serum-free culture media with clearly defined chemical compositions specifically designed for culturing PSCs, such as Essential8 medium, PluriSTEM medium, and mTeSR medium. These media contain various growth factors, nutrients, and other additives. For example, they contain components of recombinant human basic fibroblast growth factor (bFGF) and transforming growth factor-β (TGF-β) signaling pathways, which can effectively maintain the undifferentiated state and self-renewal capacity of PSCs. Compared to DMEM medium, these media with specific components can effectively maintain the undifferentiated state of cells, preventing premature differentiation, and serum-free media can avoid potential problems such as pathogen contamination risks from serum.

[0086] The term "differentiation medium" refers to a culture medium that mimics the signaling pathways of cell development in vivo after the cells enter the differentiation stage, enabling them to differentiate in a targeted manner. The direction of cell differentiation typically varies, and the composition of the medium also differs at different differentiation stages, with modifications or additions of specific substances depending on the needs. For example, during the targeted differentiation of PSCs into nerve cells, the differentiation medium typically contains neurotrophic factors such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF); during the targeted differentiation into cardiomyocytes, the medium typically contains bone morphogenetic protein 4 (BMP4) and fibroblast growth factor (FGF); during the targeted differentiation into pancreatic islet cells, the medium typically contains Activin A and glycogen synthesis kinase-3 (GSK-3) inhibitors; and during the targeted differentiation into hepatocytes, the medium typically contains hepatocyte growth factor (HGF) and oncogene M (OSM).

[0087] Pancreatic islet cell differentiation media typically use DMEM (Dulbecco's Modified Eagle Medium) or MCDB131 as a base, providing cells with essential energy and material support to create a favorable environment for islet cell differentiation. During differentiation, growth factors (such as Activin A and FGF) and small molecule compounds (such as CHIR99021, LDN193189, and Sant1) are added. Activin A plays a crucial role in the early stages of islet cell differentiation, inducing endoderm cells to differentiate into pancreatic cells and promoting pancreatic progenitor cell specialization. FGF-2 promotes cell proliferation and migration, contributing to the aggregation and morphogenesis of pancreatic progenitor cells in the early stages of islet cell differentiation. FGF-10 primarily functions in branching morphogenesis during pancreatic development, regulating pancreatic duct formation and cell differentiation. CHIR99021 is a glycogen synthesis kinase-3 (GSK-3) inhibitor. During islet cell differentiation, inhibiting GSK-3 activation of the Wnt / β-catenin signaling pathway promotes the production of endocrine cells such as pancreatic β cells. LDN193189 is an inhibitor of the bone morphogenetic protein (BMP) signaling pathway, reducing its adverse effects on islet cell differentiation by inhibiting the BMP signaling pathway. Sant1, by inhibiting the Shh signaling pathway, relieves its inhibitory effect on pancreatic development, thereby promoting the normal differentiation of pancreatic progenitor cells and facilitating islet cell formation. Through the synergistic effect of various growth factors and small molecule compounds, the signaling pathways of islet cell development in vivo are mimicked, prompting cells to gradually express islet cell-specific markers, such as insulin, glucagon, and somatostatin-related genes, ultimately forming islet cells with endocrine function.

[0088] In some embodiments, the original culture medium is StemFlex medium, StemScale medium, Essential 8 medium, PluriSTEM medium, or TeSR. TM -AOF or mTesR1 medium, preferably TeSR TM -AOF medium or mTesR1 medium, more preferably mTesR1 medium.

[0089] In some embodiments, the differentiation medium is based on MCDB131 medium, supplemented with growth factors and small molecule compounds, including: basic culture components, trace elements, putrescine, adenine, thymine, amino acids and vitamins, glycogen synthesis kinase-3 (GSK-3) inhibitor (CHIR99021), transforming growth factor-β (Activin A), keratinocyte growth factor (KGF), bone morphogenetic protein (BMP) signaling inhibitor (LDN193189), PKC protein activator (TPB), methyltransferase (UNC1999), protein deacetylase (SAHA), and dual inhibitors of JAK and Aurora kinases (AT9283), etc.

[0090] Characteristics, origins and culture of pluripotent stem cells

[0091] The characteristics of pluripotent stem cells are well known to those skilled in the art. Markers expressed by pluripotent stem cells include, for example, one or more of the following markers: ABCG2, cripto, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

[0092] Another ideal phenotype for proliferating pluripotent stem cells is the potential to differentiate into cells from all three germ layers: endoderm, mesoderm, and ectoderm. Stem cell pluripotency can be confirmed, for example, by injecting cells into severely combined immunodeficiency (“SCID”) mice, fixing the resulting teratomas with 4% paraformaldehyde, and then histologically examining them to identify evidence of cell types from these three germ layers. Alternatively, pluripotency can be determined by generating embryoids and assessing the presence of markers associated with the three germ layers within the embryoids.

[0093] Proliferating pluripotent stem cell lines can be karyotyped using standard G-banding techniques and compared with publicly available karyotypes of corresponding primate species. The aim is to obtain cells with a "normal karyotype" (meaning the cells are euploid), in which all human chromosomes are present and without significant alterations.

[0094] Exemplary types of pluripotent stem cells that can be used include established pluripotent cell lines, including pre-embryonic tissue (e.g., blastocyst), embryonic tissue, or fetal tissue obtained at any time during pregnancy, typically but not necessarily before approximately 10 to 12 weeks of gestation. Non-limiting examples are established human embryonic stem cell lines or human embryonic germ cell lines, such as human embryonic stem cell lines H1, H7, and H9 (WiCell Research Institute, Madison, WI, USA). Cells derived from pluripotent stem cell populations cultured without feeder cells are also suitable options. Inducible pluripotent cells (iPS) or reprogrammed pluripotent cells derived from adult human cells may also be used, utilizing the forced expression of a variety of pluripotency-associated transcription factors such as OCT4, NANOG, SOX2, KLF4, and ZFP42 (Annu RevGenomics Hum Genet 2011, 12: 165-185; see also IPS, Cell, 126(4): 663-676). The human embryonic stem cells used in the methods provided in this disclosure may also be prepared as described by Thomson et al. (US Patent 5,843,780; Science, 1998, 282:1145-1147; Curr Top Dev Biol 1998, 38:133-165; Proc Natl Acad SciU.SA 1995, 92:7844-7848). Mutated human embryonic stem cell lines, such as BG01v (BresaGen, Athens, Ga.), or those derived from adult somatic cells, such as those disclosed by Takahashi et al. in Cell 131:1-12 (2007), may also be used. In some embodiments, the pluripotent stem cells suitable for use in this invention may be obtained according to the methods described in the following literature: Li et al. (Cell Stem Cell 4: 16-19, 2009); Maherali et al. (Cell Stem Cell 1: 55-70, 2007); Stadtfeld et al. (Cell Stem Cell 2: 230-240); Nakagawa et al. (Nature Biotechnol 26: 101-106, 2008); Takahashi et al. (Cell 131: 861-872, 2007); and U.S. Patent Application Publication 2011 / 0104805. In some embodiments, the pluripotent stem cells may be of non-embryonic origin. All of these references, patents, and patent applications are incorporated herein by reference in their entirety, particularly those relating to the isolation, culture, expansion, and differentiation of pluripotent cells.

[0095] In one embodiment, pluripotent stem cells are typically cultured on a layer of feeder cells that can support the life activities of the pluripotent stem cells in a variety of ways. Alternatively, pluripotent stem cells are cultured in a culture system that is substantially devoid of feeder cells but still supports the proliferation of pluripotent stem cells without significant differentiation. Pluripotent stem cells can be supported to grow without differentiation in feeder cell-free cultures using a culture medium that has been conditioning by previously culturing another cell type. Alternatively, pluripotent stem cells can be supported to grow without differentiation in feeder cell-free cultures using a chemically defined culture medium.

[0096] Pluripotent cells can be easily expanded in cultures using multiple feeder layers or by using containers coated with matrix proteins. Alternatively, culture media with defined components, such as... 1. The chemical composition of the culture medium (StemCell Technologies, Vancouver, Canada) defines the surface for routine cell expansion. Pluripotent cells can be easily removed from the culture plate using enzymatic digestion, mechanical separation, or the use of various calcium chelating agents such as EDTA (ethylenediaminetetraacetic acid). Alternatively, pluripotent cells can be expanded in suspension in the absence of any matrix proteins or feeder layer.

[0097] Various methods for expanding and culturing pluripotent stem cells can be used in the present invention protected by the claims. For example, the methods of the present invention can be those of Reubinoff et al., Thompson et al., Richard et al., and U.S. Patent Application Publication 2002 / 0072117. Reubinoff et al. (Nature Biotechnology 18:399-404 (2000)) and Thompson et al. (Science 282:1145-1147 (1998)) disclosed the culture of pluripotent stem cell lines derived from human blastocysts using mouse embryonic fibroblast feeder cell layers. Richards et al. (Stem Cells 21:546-556, 2003) evaluated the ability of a group of eleven different adult, fetal, and neonatal feeder cell layers to support the culture of human pluripotent stem cells, noting that human embryonic stem cell lines cultured on adult skin fibroblast feeder cells maintained the morphology of human embryonic stem cells and preserved pluripotency. U.S. Patent Application Publication 2002 / 0072117 discloses cell lines for producing culture media that support the growth of primate pluripotent stem cells in feeder-free cultures. The cell lines used are mesenchymal cell lines and fibroblast-like cell lines derived from embryonic tissue or differentiated from embryonic stem cells. U.S. Patent Application Publication 2002 / 072117 also discloses the use of these cell lines as primary feeder cell layers.

[0098] Other suitable methods for expanding and culturing pluripotent stem cells have been disclosed in the literature, for example, by Wang et al., Stojkovic et al., Miyamoto et al., and Amit et al. Wang et al. (Stem Cells 23: 1221-1227, 2005) disclosed a method for long-term culture of human pluripotent stem cells on a feeder cell layer derived from human embryonic stem cells. Stojkovic et al. (Stem Cells 2005 23: 306-314, 2005) disclosed a feeder cell system derived from spontaneous differentiation of human embryonic stem cells. Miyamoto et al. (Stem Cells 22: 433-440, 2004) disclosed a feeder cell source obtained from human placenta. Amit et al. (Biol. Reprod 68: 2150-2156, 2003) disclosed a feeder cell layer derived from human foreskin.

[0099] An alternative culture system employs a serum-free medium supplemented with growth factors that promote the proliferation of embryonic stem cells. Examples of such culture systems include, but are not limited to, those disclosed in Cheon et al., Levenstein et al., and U.S. Patent Application Publication 2005 / 0148070. Cheon et al. (BioReprod DOI: 10.1095 / biolreprod.105.046870, October 19, 2005) disclosed a feeder-free serum-free culture system in which embryonic stem cells are maintained in an unconditioned serum replacement (SR) medium supplemented with different growth factors that induce the self-renewal of embryonic stem cells. Levenstein et al. (Stem Cells 24:568-574, 2006) disclosed a method for long-term culture of human embryonic stem cells in the absence of fibroblasts or conditioned media using a medium supplemented with bFGF. U.S. Patent Application Publication 2005 / 0148070 discloses a method for culturing human embryonic stem cells in a serum-free and fibroblast-free feeder culture medium. The method comprises culturing stem cells in a medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or a transferrin alternative, and at least one insulin or an insulin alternative. This medium is substantially free of mammalian fetal serum and contains at least about 100 ng / ml of fibroblast growth factor that activates the fibroblast growth factor signal transduction receptor, wherein the source of the growth factor is not solely a fibroblast feeder layer. This medium supports the proliferation of stem cells in an undifferentiated state in the absence of feeder cells or in a conditioned culture medium. Other suitable methods for culturing and expanding pluripotent stem cells are disclosed in U.S. Patent Application Publication 2005 / 0233446, U.S. Patent 6,800,480, U.S. Patent Application Publication 2005 / 0244962, and WO 2005 / 065354.

[0100] Pluripotent stem cells can be seeded onto a suitable culture medium. In one embodiment, a suitable culture medium is an extracellular matrix component, such as those derived from the basement membrane or those that can form part of an adhesion molecule receptor-ligand conjugate. In one embodiment, a suitable culture medium is MATRIGEL™ (Becton Dickenson). MATRIGEL™ is a soluble formulation derived from Engelbreth-Holm Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane. Other extracellular matrix components and mixtures of components are suitable as alternatives. Depending on the cell type being expanded, these alternatives may include individual laminins, fibronectin, proteoglycans, nestin, heparan sulfate, etc., or various combinations of these substances.

[0101] Differentiation of pluripotent stem cells

[0102] When pluripotent cells differentiate into β cells, they undergo multiple stages, each characterized by the presence or absence of specific markers. Cell differentiation to these stages is achieved under specific culture conditions, including the addition or absence of certain growth factors to the culture medium. Generally, this differentiation may involve pluripotent stem cells differentiating into morphological endoderm cells. These morphological endoderm cells can then further differentiate into gastrulation cells, which in turn differentiate into posterior foregut cells. Posterior foregut cells can differentiate into pancreatic progenitor cells, which can then differentiate into endocrine progenitor cells. These endocrine progenitor cells can subsequently differentiate into β-producing cells.

[0103] In some embodiments of the present invention, a scheme using pluripotent stem cells as a starting material is employed to obtain β cells. This scheme includes the following stages:

[0104] Phase 1: Treat cell culture lines obtained from pluripotent stem cells with appropriate growth factors and / or small molecules to induce them to differentiate into well-defined endoderm cells.

[0105] Second stage: Treat cells from the first stage with appropriate growth factors and / or small molecules to induce them to further differentiate into gastrulatory cells.

[0106] Phase 3: Treat cells from Phase 2 with appropriate growth factors and / or small molecules to induce them to further differentiate into foregut cells expressing the posterior end.

[0107] Phase 4: Treat cells from Phase 3 with appropriate growth factors and / or small molecules to induce them to further differentiate into pancreatic progenitor cells.

[0108] Phase 5: Treat cells from Phase 4 with appropriate growth factors and / or small molecules to induce them to further differentiate into endocrine precursor cells.

[0109] Stage 6: Treat cells from Stage 5 with appropriate factors and / or small molecules to induce them to further differentiate into β cells.

[0110] While in some embodiments the invention covers methods for differentiating pluripotent stem cells into β cells, the invention also covers methods for differentiating cells from other intermediate stages (e.g., endodermal cells, gastrula cells, posterior foregut cells, pancreatic progenitor cells, endocrine progenitor cells) into β cells. Furthermore, although the differentiation process is described above as separate stages, the process of handling and undergoing differentiation of cells may be sequential and uninterrupted.

[0111] Methods for assessing the expression levels of protein and nucleic acid markers in cultured or isolated cells are standard practices in the art. These methods include quantitative reverse transcription polymerase chain reaction (RT-PCR), Northern blotting, in situ hybridization (see, for example, Current Protocols in Molecular Biology (edited by Ausubel et al., Supplement 2001)), immunoassays (such as immunohistochemical analysis of tissue sections), Western blotting, and FACS methods for measuring readily available markers in intact cells (see, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)). Alternatively, differentiation efficiency can be determined by exposing treated cell populations to reagents (such as antibodies) that specifically recognize protein markers expressed by cells expressing characteristic markers of a target cell type.

[0112] Differentiated cells can also be further purified. For example, after treating pluripotent stem cells using the method of the present invention, the treated cell population can be exposed to a reagent (such as an antibody) that specifically recognizes protein markers characteristically expressed by the differentiated cells being purified, thereby purifying the differentiated cells.

[0113] Phase 1: Pluripotent stem cells differentiate into well-defined endoderm cells

[0114] Pluripotent stem cells can be differentiated into well-defined endoderm cells using any suitable method known in the art or any method proposed in this invention. Suitable methods for differentiating pluripotent stem cells into well-defined endoderm cells are disclosed in the following patents: U.S. Patent Application Publication 2007 / 0254359, U.S. Patent Application Publication 2009 / 0170198, U.S. Patent Application Publication 2009 / 0170198, U.S. Patent Application Publication 2011 / 0091971, U.S. Patent Application Publication 2010 / 0015711, U.S. Patent Application Publication 2010 / 0015711, U.S. Patent Application Publication 2012 / 0190111, U.S. Patent Application Publication 2012 / 0190112, and U.S. Patent Application Publication 2012 / 0190112. U.S. Patent Application Publication 2012 / 0196365, U.S. Patent Application Publication 2010 / 0015711, U.S. Patent Application Publication 2012 / 0190111, U.S. Patent Application Publication 2012 / 0190112, U.S. Patent Application Publication 2012 / 0196365, U.S. Patent Application Publication 2010 / 0015711, U.S. Patent Application Publication 2012 / 0190111, U.S. Patent Application Publication 2012 / 0190112, U.S. Patent Application Publication 2012 / 0196365, the full text of the above patents is incorporated herein by reference.

[0115] In one embodiment of the invention, pluripotent stem cells are treated with a culture medium supplemented with activin A to obtain well-defined endoderm cells. For example, such a culture medium may contain about 50 ng / ml to about 150 ng / ml, or about 75 ng / ml to about 125 ng / ml, or about 100 ng / ml of activin A. The pluripotent cells can be cultured for about two to five days, preferably about two to three days, to allow them to differentiate into well-defined endoderm cells.

[0116] In another embodiment of the invention, pluripotent stem cells are treated with a culture medium supplemented with activin A and CHIR99021 to obtain well-defined endoderm cells. For example, such a culture medium may contain about 50 ng / ml to about 150 ng / ml, or about 75 ng / ml to about 125 ng / ml, or about 100 ng / ml of activin A, or about 1 μM to about 5 μM, or about 2 μM to about 4 μM, or about 3 μM of CHIR99021.

[0117] The presence or absence of characteristic markers of morphologically defined endoderm cells can be used to determine whether cells expressing these markers have been generated. Pluripotent stem cells typically do not express these markers. Therefore, pluripotent cell differentiation can be detected once cells begin to express characteristic markers of morphologically defined endoderm cells.

[0118] Second stage: Differentiating the formed endoderm cells into gastrulator cells.

[0119] Defined endoderm cells can further differentiate into gastrulatory cells. In one embodiment, the formation of gastrulatory cells includes culturing cells expressing characteristic markers of defined endoderm cells in a medium containing fibroblast growth factor 7 (“FGF7” or “KGF”) or FGF10, preferably FGF7, to induce differentiation. For example, such a medium may contain about 25 ng / ml to about 75 ng / ml, or about 30 ng / ml to about 60 ng / ml, or about 50 ng / ml of FGF7. Cells expressing characteristic markers of defined endoderm cells may be cultured under these conditions for about two to three days, preferably about two days.

[0120] In another embodiment, differentiation of gastrulation cells includes culturing shaped endoderm cells in a medium containing FGF7 or FGF10 and ascorbic acid (vitamin C) to induce differentiation. This medium may contain about 0.1 mM to about 0.5 mM of ascorbic acid, or about 0.2 mM to about 0.4 mM, or about 0.25 mM of ascorbic acid. This medium may also contain about 25 ng / ml to about 75 ng / ml, or about 30 ng / ml to about 60 ng / ml, or about 50 ng / ml of FGF7 or FGF10, preferably FGF7. For example, this medium may contain about 0.25 mM of ascorbic acid and about 50 ng / ml of FGF7. In one embodiment, the shaped endoderm cells are treated with FGF7 and ascorbic acid for two days.

[0121] In some embodiments, the culture medium for culturing shaped endoderm cells is further supplemented with an EZH2 histone methyltransferase inhibitor and / or a histone deacetylase inhibitor. In some embodiments, the EZH2 histone methyltransferase inhibitor is selected from UNC1999, GSK126, EPZ-6438, and DZNeP; preferably UNC1999. For example, such a medium may contain UNC1999 at concentrations of about 0.01 to about 0.25 μM, or about 0.1 μM. In some embodiments, the histone deacetylase inhibitor is selected from SAHA, VPA, TSA, and TMP269; preferably SAHA. For example, such a medium may contain SAHA at concentrations of about 0.01 to about 0.25 μM, or about 0.1 μM.

[0122] Stage 3: Archenteric cells differentiate into posterior foregut cells.

[0123] The gastrulator cells can further differentiate into posterior foregut cells. In one embodiment, the second-stage cells are further differentiated into third-stage cells by placing the second-stage cells in a medium supplemented with a Sonic Hedgehog (“SHH”) signaling pathway antagonist (such as SMO antagonist 1 (“SANT-1”) ((E)-4-benzyl-N-((3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methylene-piperazin-1-amine)), retinoic acid, a protein kinase C (“PKC”) activator (such as (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzolactam (“TPB”)), FGF7 or FGF10, and ascorbic acid. The second-stage cells can be cultured for approximately two to four days, preferably approximately two days. In one embodiment, the medium is supplemented with approximately 0.1 μM to approximately 0.3 μM SANT-1, about 0.5 μM to about 3 μM retinoic acid, about 100 nM to about 300 nM TPB, about 15 ng / ml to about 75 ng / ml FGF7, and about 0.15 mM to about 0.35 mM ascorbic acid. In another embodiment, the culture medium is supplemented with about 0.25 μM SANT-1, about 1 μM retinoic acid, about 200 nM TPB, about 50 ng / ml FGF7, and about 0.25 mM ascorbic acid.

[0124] In some embodiments, the culture medium for culturing gastrulation cells is further supplemented with an EZH2 histone methyltransferase inhibitor and / or a histone deacetylase inhibitor. In some embodiments, the EZH2 histone methyltransferase inhibitor is selected from UNC1999, GSK126, EPZ-6438, and DZNeP; preferably UNC1999. For example, such a medium may contain UNC1999 at concentrations of about 0.01 to about 0.25 μM, or about 0.1 μM. In some embodiments, the histone deacetylase inhibitor is selected from SAHA, VPA, TSA, and TMP269; preferably SAHA. For example, such a medium may contain SAHA at concentrations of about 0.01 to about 0.25 μM, or about 0.1 μM.

[0125] Stage 4: The posterior foregut cells differentiate into pancreatic progenitor cells.

[0126] The posterior foregut cells can further differentiate into pancreatic progenitor cells. In one embodiment, the third-stage cells are further differentiated into fourth-stage cells by culturing them in a medium supplemented with an SHH signaling pathway antagonist (such as SANT-1), retinoic acid, a bone morphogenetic protein (“BMP”) inhibitor (such as LDN-193189, head protein, or notochordin), Nicotinamide, EGF7, and ascorbic acid. The third-stage cells can be cultured for approximately two to four days, preferably approximately three days. In one embodiment, the medium is supplemented with approximately 0.1 μM to approximately 0.3 μM SANT-1, approximately 0.5 μM to approximately 3 μM retinoic acid, approximately 100 nM to approximately 300 nM LDN-193189, approximately 5 mM to approximately 15 mM Nicotinamide, approximately 50 ng / ml to approximately 150 ng / ml EGF, and approximately 0.15 mM to approximately 0.35 mM ascorbic acid. In another embodiment, the culture medium is supplemented with approximately 0.25 μM SANT-1, approximately 1 μM retinoic acid, approximately 200 nM LDN-193189, approximately 10 mM Nicotinamide, approximately 100 ng / ml EGF, and approximately 0.25 mM ascorbic acid.

[0127] In some embodiments, the culture medium for culturing posterior foregut cells is further supplemented with an EZH2 histone methyltransferase inhibitor and / or a histone deacetylase inhibitor. In some embodiments, the EZH2 histone methyltransferase inhibitor is selected from UNC1999, GSK126, EPZ-6438, and DZNeP; preferably UNC1999. For example, such a medium may contain UNC1999 at a concentration of about 0.01 to about 0.25 μM, or about 0.1 μM. In some embodiments, the histone deacetylase inhibitor is selected from SAHA, VPA, TSA, and TMP269; preferably SAHA. For example, such a medium may contain SAHA at a concentration of about 0.01 μM to about 0.25 μM, or about 0.1 μM.

[0128] Stage 5: Pancreatic progenitor cells differentiate into endocrine progenitor cells

[0129] Pancreatic progenitor cells can further differentiate into endocrine progenitor cells. In one embodiment, stage 4 cells are further differentiated into stage 5 cells by culturing them in a medium supplemented with an SHH signaling pathway antagonist (such as SANT-1), a γ-secretase inhibitor (e.g., GSiXX), thyroid hormone T3 / T4 or a thyroid hormone receptor agonist (e.g., GC1 (Sobetirome)), a TGF-βRI (ALK5) inhibitor (e.g., Alk5i II), and ascorbic acid. Stage 4 cells can be cultured for approximately two to four days, preferably approximately two days. In one embodiment, the medium is supplemented with approximately 0.1 μM to approximately 0.3 μM SANT-1, approximately 50 nM to approximately 150 nM GSiXX, approximately 5 μM to approximately 20 μM Alk5i II, approximately 0.5 μM to approximately 2 μM GC1, and approximately 0.15 mM to approximately 0.35 mM ascorbic acid. In another embodiment, the culture medium is supplemented with approximately 0.25 μM SANT-1, approximately 100 nM GSiXX, approximately 10 μM Alk5i II, approximately 1 μM GCI, and approximately 0.25 mM ascorbic acid.

[0130] Stage 6: Endocrine precursor cells differentiate into pancreatic β cells

[0131] Endocrine progenitor cells can further differentiate into pancreatic β cells. In one embodiment, stage 5 cells are further differentiated into pancreatic β cells by culturing them in a medium supplemented with a γ-secretase inhibitor (e.g., GSiXX), a TGF-βRI (ALK5) inhibitor (e.g., Alk5i II), and ascorbic acid. Stage 5 cells can be cultured for approximately two to five days, preferably approximately four days. In one embodiment, the medium is supplemented with approximately 50 nM to approximately 150 nM GSiXX, approximately 5 μM to approximately 20 μM Alk5i II, and approximately 0.15 mM to approximately 0.35 mM ascorbic acid. In another embodiment, the medium is supplemented with approximately 100 nM GSiXX, approximately 10 μM Alk5i II, and approximately 0.25 mM ascorbic acid.

[0132] In some embodiments, the culture medium for culturing endocrine precursor cells is further supplemented with AT9283 or Danusertib. In some embodiments, the concentration of AT9283 or Danusertib in the culture medium is about 0.05 to about 2.5 μM; preferably about 0.25 μM. In some embodiments, the culture medium for culturing endocrine precursor cells is further supplemented with UNC1999. In some embodiments, the concentration of UNC1999 in the culture medium is about 0.25 to about 5 μM; preferably about 2.5 μM.

[0133] In one or more embodiments, the pancreatic β-cell differentiation method comprises six stages, S1-S6, wherein...

[0134] The differentiation medium for the S1 stage contains (A) basic components: MCDB131, D-(+)-Glucose (5mM), NaHCO3 (1.5g / L), FAF-BSA (5g / L), Glutamax (2mM), Vitamin C (0.25mM), P / S (1%), CHIR9902 (3μM), and Activin A (100ng / ml), or (B) basic components: MCDB131, D-(+)-Glucose (5mM), NaHCO3 (1.5g / L), FAF-BSA (5g / L), Glutamax (2mM), Vitamin C (0.25mM), P / S (1%), and Activin A (100ng / ml);

[0135] The differentiation medium for the S2 stage contains (1) basic components: MCDB131, D-(+)-Glucose (5mM), NaHCO3 (1.5g / L), FAF-BSA (5g / L), Glutamax (2mM), Vitamin C (0.25mM), P / S (1%), KGF (50ng / ml), and (2) supplementary components: UNC1999 (0.01-0.25μM) and SAHA (0.01-0.25μM), wherein the concentration of UNC1999 is preferably 0.025μM and the concentration of SAHA is preferably 0.01μM;

[0136] The differentiation medium for the S3 stage contains: (1) basic components: MCDB131, D-(+)-Glucose (5mM), NaHCO3 (2.5g / L), FAF-BSA (20g / L), Glutamax (2mM), Vitamin C (0.25mM), ITS-X (0.5%), P / S (1%), Sant1 (0.25μM), RA (1μM), TPB (200nM), KGF (50ng / ml), and (2) supplementary components: UNC1999 (0.01-0.25μM) and SAHA (0.01-0.25μM), wherein the concentration of UNC1999 is preferably 0.025μM and the concentration of SAHA is preferably 0.01μM;

[0137] The differentiation medium for the S4 stage contains: (1) basic components: MCDB131, D-(+)-Glucose (5mM), NaHCO3 (2.5g / L), FAF-BSA (20g / L), Glutamax (2mM), Vitamin C (0.25mM), ITS-X (0.5%), P / S (1%), Sant1 (0.25μM), RA (100nM), LDN193189 (200nM), Nicotinamide (10mM), EGF (100ng / ml); (2) supplementary components: UNC1999 (0.01-0.25μM), SAHA (0.01-0.25μM), wherein the concentration of UNC1999 is preferably 0.025μM; and the concentration of SAHA is preferably 0.01μM.

[0138] The differentiation medium for stage S5 contains the following basic components: MCDB131, D-(+)-Glucose (15mM), NaHCO3 (1.5g / L), FAF-BSA (20g / L), Glutamax (2mM), Heparin (10μg / ml), ITS-X (0.5%), ZnSO4 (10μM), Vitamin C (0.25mM), P / S (1%), Sant1 (0.25μM), GSiXX (100nM), Alk5i II (10μM), and GCI (1μM).

[0139] The differentiation medium for stage S6 contained: (1) basic components: MCDB131, D-(+)-Glucose (15mM), NaHCO3 (1.5g / L), FAF-BSA (20g / L), Glutamax (2mM), Heparin (10g / ml), ITS-X (0.5%), ZnSO4 (10μM), Vitamin C (0.25mM), P / S (1%), Alk5i II (10 μM), GSiXX (100 nM), (2) an additive selected from any of the following groups: a. AT9283 (0.05-2.5 μM), UNC1999 (0.25-5 μM), wherein the concentration of AT9283 is preferably 0.25 μM and the concentration of UNC1999 is preferably 2.5 μM; or b. AT9283 (0.05-0.5 μM), wherein the concentration is preferably 0.25 μM; or c. no additive.

[0140] In some implementations, the "culture environment and method" for cell differentiation is static culture in a 5% CO2, 37°C incubator or shaker culture, wherein static culture is required for stages S1-S3 and culture is required on a horizontal shaker at 90-95 rpm for stages S4-S6.

[0141] In some implementations, the method takes 14-21 days, preferably 14-18 days, and more preferably 18 days.

[0142] In some implementations, the method achieves a pancreatic β-cell differentiation efficiency of 70%-85%.

[0143] Treatment methods and pharmaceutical compositions

[0144] This disclosure also provides, in one aspect, cells or cell populations that can be obtained through the methods provided herein. These cells or cell populations can be used to treat diseases related to pancreatic islet cells, including diabetes and islet cell tumors. Diabetes is classified into type 1 and type 2. Type 1 diabetes is an autoimmune disease in which the immune system attacks and destroys pancreatic β cells, leading to insulin deficiency and elevated blood sugar levels. Long-term hyperglycemia can easily trigger various complications. Type 2 diabetes is mainly caused by impaired pancreatic β cell function and insulin resistance, and is related to genetics, obesity, and unhealthy lifestyle. In the early stages, it can be controlled through diet, exercise, and oral hypoglycemic agents. Islet cell tumors are classified as functional or non-functional. Functional insulinomas can autonomously secrete large amounts of insulin, causing hypoglycemia symptoms, which can be life-threatening in severe cases. Non-functional islet cell tumors do not secrete or secrete small amounts of inactive hormones; tumor growth can compress surrounding tissues, leading to symptoms such as abdominal pain and abdominal masses.

[0145] This disclosure also provides a treatment method in one aspect. Specifically, the invention provides a method for treating patients with diabetes or at risk of developing diabetes. In one embodiment, the treatment method includes implanting cells obtainable by the method provided in this disclosure into the patient. In one embodiment, the treatment method includes differentiating pluripotent stem cells in vitro into first-stage, second-stage, third-stage, fourth-stage, fifth-stage, or sixth-stage cells, for example as described herein, and then implanting the differentiated cells into the patient. In one embodiment, the method further includes a step of culturing pluripotent stem cells, for example as described herein, prior to the step of differentiating the pluripotent stem cells.

[0146] In one embodiment, the method further includes a step of cell differentiation in vivo after the implantation step.

[0147] In one implementation, the patient is a mammal, preferably a human.

[0148] In one embodiment, differentiated cells can be implanted in a dispersed manner, or the cells can be first formed into cell clusters and then injected into the portal vein. Alternatively, differentiated cells can be introduced into a biodegradable biocompatible polymeric carrier, a non-degradable porous device, or the differentiated cells can be encapsulated to protect them from destruction by the host immune response. The cells can be implanted into appropriate sites within the recipient body. Implantation sites include, for example, the liver, the pre-existing pancreas, the subcapsular space of the kidney, the omentum, the peritoneum, the rectus abdominis muscle, the subserosal space, the intestine, the stomach, the subcutaneous tissue, and muscles.

[0149] To promote further differentiation of implanted cells in vivo and ensure their survival or activity remains at a certain level, additional factors, such as growth factors, antioxidants, or anti-inflammatory agents, can be administered to the recipient before, during, or after cell implantation. These factors can be secreted by endogenous cells and contact the implanted cells in situ. Differentiation of implanted cells can be induced by any combination of endogenous and exogenous growth factors known in the art.

[0150] The amount of cells transplanted depends on a variety of factors, including the patient’s physical condition and response to the therapy, and can be determined by those skilled in the art.

[0151] In one embodiment, the treatment method further includes binding cells to a three-dimensional carrier prior to transplantation. These cells can remain on the carrier in vitro before being transplanted into the patient. Alternatively, the carrier containing these cells can be directly implanted into the patient without additional in vitro culture. Optionally, at least one agent that promotes the survival and function of the transplanted cells may be incorporated into the carrier.

[0152] This disclosure also provides, in one aspect, a pharmaceutical composition comprising pancreatic β-cells obtained by the methods described herein, and pharmaceutically acceptable excipients, including but not limited to diluents, carriers, solubilizers, emulsifiers, preservatives, and / or adjuvants. The excipients are preferably non-toxic to the recipient at the doses and concentrations used. Such excipients include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. In some embodiments, the pharmaceutical composition may contain substances for improving, maintaining, or retaining, for example, the composition's pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeation. These substances are known in the art. The optimal pharmaceutical composition can be determined based on the intended route of administration, delivery method, and required dosage.

[0153] Pharmaceutical compositions intended for internal administration are typically provided in sterile formulations. Sterilization is achieved through filtration via a sterile filter membrane. When the composition is lyophilized, sterilization can be performed before or after lyophilization and rehydration. In the treatment methods described herein, the pharmaceutical compositions disclosed can be used for parenteral delivery, with specific dosage forms and delivery methods to be determined by those skilled in the art as needed. Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals, or as dehydrated or lyophilized powders. The formulations can be stored in ready-to-use form or rehydrated before administration (e.g., lyophilized). The present invention also provides kits for generating single-dose administration units.

[0154] Partial Specific Implementation Plan

[0155] This invention first provides a method for preparing pancreatic β cells, comprising the following steps: (1) contacting stem cells with (1) a TGFβ / activin agonist and / or (2) a glycogen synthase kinase 3 (GSK) inhibitor for a duration sufficient to form a definitive endoderm (DE); (2) contacting the definitive endoderm cells with an FGFR2 IIIb agonist for a duration sufficient to form a primitive gut tube (PGT) cell; and (3) contacting the primitive gut tube cells with a Hedgehog-Smoothened (HH / SMO) inhibitor, a retinoic acid receptor (RAR) agonist, a protein kinase C activator, and / or an FGFR2 IIIb agonist for a duration sufficient to form a posterior foregut (PGT). (4) The duration of time for which the posterior foregut (PF) cells (also known as early pancreatic progenitors) are exposed to Hedgehog-Smoothened (HH / SMO) inhibitors, retinoic acid receptor (RAR) agonists, BMP inhibitors, nicotinamide, and ErbB activators for a duration sufficient to form pancreatic progenitors (PP); (5) The duration of time for which the pancreatic progenitors are exposed to Hedgehog-Smoothened (HH / SMO) inhibitors, γ-secretase inhibitors, Alk5 inhibitors, and / or thyroid hormone receptor agonists for a duration sufficient to form endocrine progenitors (EP); and (6) The duration of time for which the endocrine progenitors are exposed to Alk5 inhibitors and γ-secretase inhibitors for a duration sufficient to form islet-β cells.

[0156] In one or more embodiments, the stem cells are PSCs, such as iPSCs and ESCs. In one or more embodiments, the stem cells are not non-directed differentiated. In one or more embodiments, the confluence of the stem cells is 60%-99%, preferably 80%. In one or more embodiments, the TGFβ / activin agonist is activin A. In one or more embodiments, the glycogen synthase kinase 3 (GSK) inhibitor is CHIR99021. In one or more embodiments, the HH / SMO inhibitor is Sant1. In one or more embodiments, the retinoic acid receptor (RAR) agonist is retinoic acid (RA). In one or more embodiments, the BMP inhibitor is LDN193189. In one or more embodiments, the protein kinase C activator is TBP (a precursor amyloid protein regulator). In one or more embodiments, the FGFR2 IIIb agonist is KGF. In one or more embodiments, the ErbB activator is EGF. In one or more embodiments, the γ-secretase inhibitor is GSiXX. In one or more embodiments, the Alk5 inhibitor is ALK5inhII. In one or more embodiments, the thyroid hormone receptor agonist is an alpha receptor agonist and a beta receptor agonist. In one or more embodiments, the thyroid hormone receptor agonist is GC1 (Sobetirome).

[0157] In one or more embodiments, steps (1) through (6) are performed in a culture medium. Each of steps (1) through (6) is independently a serum-free culture medium. In one or more embodiments, the serum-free culture medium comprises one or more selected from: MCDB131 or DMEM or RPMI1640, glucose, NaHCO3, BSA, ITS-X, Glutamax, vitamin C, antibiotics (e.g., penicillin and / or streptomycin), heparin, or ZnSO4. In one or more embodiments, the serum-free culture medium is selected from: Essential8 medium, PluriSTEM medium, and mTeSR medium. In one or more embodiments, step (1) is performed in a culture medium containing: MCDB131, glucose, NaHCO3, BSA, Glutamax, and vitamin C. In one or more embodiments, step (2) is performed in a culture medium containing: MCDB131, glucose, NaHCO3, BSA, Glutamax, and vitamin C. In one or more embodiments, step (3) is performed in a culture medium containing MCDB131, glucose, NaHCO3, BSA, Glutamax, vitamin C, and ITS-X. In one or more embodiments, step (4) is performed in a culture medium containing MCDB131, glucose, NaHCO3, BSA, Glutamax, vitamin C, and ITS-X. In one or more embodiments, step (5) is performed in a culture medium containing MCDB131, glucose, NaHCO3, BSA, Glutamax, vitamin C, ITS-X, heparin, and ZnSO4. In one or more embodiments, step (6) is performed in a culture medium containing MCDB131, glucose, NaHCO3, BSA, Glutamax, vitamin C, ITS-X, heparin, and ZnSO4.

[0158] In one or more embodiments, the method includes reducing the cluster size of the endoderm, wherein adjusting the size of the cell clusters includes separating the clusters and re-aggregating them before they mature into β cells. In one or more embodiments, the method includes reducing the cluster size of the stem cells, wherein adjusting the size of the cell clusters includes separating the clusters and re-aggregating them before they mature into β cells.

[0159] In one or more embodiments, the method includes reducing the cluster size of the stem cells in step (1). In one or more embodiments, reducing the cell cluster size includes digesting the stem cells into single cells. In one or more embodiments, the digestion uses trypsin, proteolytic enzymes, collagenases, or combinations thereof, such as EZ-LiFT, Accutase, or dispersant enzyme reagents, preferably Accutase.

[0160] In one or more embodiments, the method includes reducing the cluster size of the posterior foregut cells in step (4); preferably, adjusting the size of the cell clusters includes separating the clusters and re-aggregating them before maturing into β cells. In one or more embodiments, reducing the cell cluster size includes digesting the posterior foregut cells into single cells. In one or more embodiments, the digestion uses trypsin, proteolytic enzymes, collagenases, or combinations thereof, such as EZ-LiFT, Accutase, or dispersant agents, preferably Accutase.

[0161] In one or more embodiments, the time sufficient to form morphological endoderm cells, gastrulation cells, posterior foregut cells, pancreatic progenitor cells, and endocrine progenitor cells is between about 1 day and about 8 days (e.g., between 2 and 4 days), or the time sufficient to form β cells is between about 1 day and about 9 days (e.g., 3 to 6 days) or more than 9 days.

[0162] In one or more embodiments, the method does not include the use of one or more of Alk5i, thyroid hormones, serum, T3, N-acetylcysteine, Trolox, and R428.

[0163] In one or more embodiments, the pancreatic progenitor cells are not in contact with any one or more of serum, T3, N-acetylcysteine, Trolox, and R428.

[0164] In one or more embodiments, T3, N-acetylcysteine, Trolox, or R428 are not contained in the endodermal cells as they mature into β cells.

[0165] In one or more embodiments, the method enhances the function of pancreatic β cells.

[0166] In one or more embodiments, the PSC inoculation density in step (1) is 1×10 5 Up to 3×10 5 / cm 2 Preferably 2×10 5 / cm 2 .

[0167] In one or more embodiments, the environmental conditions for each step of the method are 5% CO2 and 37°C.

[0168] In one or more embodiments, steps (1)-(3) are 2D culture and steps (4)-(6) are 3D culture.

[0169] In one or more embodiments, in step (4), the posterior foregut cells are arranged at 5 × 10 6 / hole up to 10×10 6 / well inoculation.

[0170] In one or more embodiments, the culture medium in step (4) further comprises one or more of the following: (a) a p300 histone acetyltransferase inhibitor, (b) a selective inhibitor of EZH2 and EZH1, (c) a BET bromodomain inhibitor, (d) a p38 MAPK inhibitor, (e) a JNK inhibitor, (f) an HDAC inhibitor, (g) a G9a / GLP histone methyltransferase inhibitor, and (h) an Akt inhibitor. In one or more embodiments, the culture medium in step (4) further comprises (a) a selective inhibitor of EZH2 and EZH1, and / or (b) an HDAC inhibitor.

[0171] In one or more embodiments, the culture medium in step (6) further comprises one or more selected from: (a) an Axl inhibitor, (b) a MEK inhibitor, (c) a JAK / Aurora kinase inhibitor, (d) an ARK inhibitor, and (e) a histone demethylase inhibitor. In one or more embodiments, the culture medium in step (6) further comprises (a) a selective inhibitor of EZH2 and EZH1, and / or (b) a JAK2 / 3 inhibitor.

[0172] In one or more embodiments, the culture medium in step (4) further comprises one or more of the following: HAIII, L002, GSK126, EPZ-6438, DZNeP, UNC1999, I-BET151, SB203580, SP600125, VPA, SAHA, UNC0642, AT7867, AZD5363, TMP269, A-366. In one or more embodiments, the concentration of HAIII is 0.1-1 μM. In one or more embodiments, the concentration of L002 is 0.1-1 μM. In one or more embodiments, the concentration of GSK126 is 0.25-2.5 μM. In one or more embodiments, the concentration of EPZ-6438 is 0.1-1 μM. In one or more embodiments, the concentration of DZNeP is 0.1-1 μM. In one or more embodiments, the concentration of UNC1999 is 0.1-1 μM. In one or more embodiments, the concentration of I-BET151 is 0.1-1 μM. In one or more embodiments, the concentration of SB203580 is 0.1-1 μM. In one or more embodiments, the concentration of SP600125 is 0.1-1 μM. In one or more embodiments, the concentration of VPA is 50-500 μM. In one or more embodiments, the concentration of SAHA is 0.1-1 μM. In one or more embodiments, the concentration of UNC0642 is 0.1-1 μM. In one or more embodiments, the concentration of AT7867 is 0.1-1 μM. In one or more embodiments, the concentration of AZD5363 is 0.1-1 μM. In one or more embodiments, the concentration of TMP269 is 0.1-1 μM. In one or more embodiments, the concentration of A-366 is 0.1-1 μM.

[0173] In one or more embodiments, the culture medium in step (4) further comprises UNC1999 and / or SAHA. In one or more embodiments, the concentration of UNC1999 is 0.01-0.25 μM, preferably 0.01-0.05 μM, more preferably 0.025 μM-0.05 μM. In one or more embodiments, the concentration of SAHA is 0.01-0.25 μM, preferably 0.01-0.1 μM, more preferably 0.01 μM-0.05 μM.

[0174] In one or more embodiments, the culture medium in step (6) further comprises one or more of the following: AT9283, Danusertib, UNC1999, and CPI-455HCl. In one or more embodiments, the concentration of AT9283 is 0.05-2.5 μM. In one or more embodiments, the concentration of Danusertib is 0.05-2.5 μM. In one or more embodiments, the concentration of UNC1999 is 0.05-2.5 μM. In one or more embodiments, the concentration of CPI-455HCl is 0.05-2.5 μM.

[0175] In one or more embodiments, the culture medium in step (6) further comprises any one, two, or all three selected from: AT9283, Danusertib, and UNC1999. In one or more embodiments, the culture medium in step (6) further comprises any group selected from: 1) AT9283, 2) Danusertib, 3) AT9283 and Danusertib, 4) AT9283 and UNC1999; 5) UNC1999 and Danusertib; 6) UNC1999, AT9283, and Danusertib. In one or more embodiments, the concentration of UNC1999 is 0.25-5 μM, preferably 0.05-2.5 μM, more preferably 2.5 μM. In one or more embodiments, the concentration of AT9283 is 0.05-2.5 μM, preferably 0.1-0.5 μM, more preferably 0.25 μM. In one or more embodiments, the concentration of Danusertib is 0.05-2.5 μM, preferably 0.5-1.5 μM, more preferably 1 μM.

[0176] In one or more embodiments, the culture medium in step (2) further comprises (a) an EZH2 and EZH1 selective inhibitor, and / or (b) an HDAC inhibitor.

[0177] In one or more embodiments, the culture medium in step (2) further comprises UNC1999 and / or SAHA. In one or more embodiments, the concentration of UNC1999 is 0.01-0.25 μM, preferably 0.01-0.05 μM, more preferably 0.025 μM-0.05 μM. In one or more embodiments, the concentration of SAHA is 0.01-0.25 μM, preferably 0.01-0.1 μM, more preferably 0.01 μM-0.05 μM.

[0178] In one or more embodiments, the culture medium in step (3) further comprises (a) an EZH2 and EZH1 selective inhibitor, and / or (b) an HDAC inhibitor.

[0179] In one or more embodiments, the culture medium in step (3) further comprises UNC1999 and / or SAHA. In one or more embodiments, the concentration of UNC1999 is 0.01-0.25 μM, preferably 0.01-0.05 μM, more preferably 0.025 μM-0.05 μM. In one or more embodiments, the concentration of SAHA is 0.01-0.25 μM, preferably 0.01-0.1 μM, more preferably 0.01 μM-0.05 μM.

[0180] In one or more embodiments, the culture medium in steps (2)-(4) further comprises (a) an EZH2 and EZH1 selective inhibitor and / or (b) an HDAC inhibitor, and the culture medium in step (6) further comprises (a) an EZH2 and EZH1 selective inhibitor and / or (b) a JAK2 / 3 inhibitor.

[0181] In one or more embodiments, the culture medium in steps (2)-(4) further comprises UNC1999 and SAHA. In one or more embodiments, the culture medium in steps (2)-(4) further comprises UNC1999 and SAHA, and the culture medium in step (6) further comprises 1) AT9283, 2) AT9283 and UNC1999, or 3) UNC1999, AT9283 and Danusertib. In one or more embodiments, the concentration of UNC1999 in steps (2)-(4) is 0.01-0.25 μM, preferably 0.01-0.05 μM, more preferably 0.025 μM-0.05 μM. In one or more embodiments, the concentration of SAHA is 0.01-0.25 μM, preferably 0.01-0.1 μM, more preferably 0.01 μM-0.05 μM. In one or more embodiments, the concentration of UNC1999 in step (6) is 0.25-5 μM, preferably 0.05-2.5 μM, more preferably 2.5 μM. In one or more embodiments, the concentration of AT9283 is 0.05-2.5 μM, preferably 0.1-0.5 μM, more preferably 0.25 μM.

[0182] In one or more embodiments, the culture medium in steps (2) to (4) further comprises 0.01 μM to 0.25 μM (preferably 0.01 μM, 0.025 μM, 0.05 μM or 0.25 μM) of UNC1999 and 0.1 μM of SAHA, and the culture medium in step (6) further comprises 1 μM of AT9283 and 1 μM of UNC1999.

[0183] In one or more embodiments, the culture medium in steps (2) to (4) further comprises 0.1 μM of UNC1999 and 0.01 μM-0.25 μM (preferably 0.01 μM, 0.025 μM, 0.05 μM or 0.25 μM) of SAHA, and the culture medium in step (6) further comprises 1 μM of AT9283 and 1 μM of UNC1999.

[0184] In one or more embodiments, the culture medium in steps (2) to (4) further comprises 0.1 μM of UNC1999 and 0.1 μM of SAHA, and the culture medium in step (6) further comprises 0.05 μM to 2.5 μM (preferably 0.05 μM, 0.1 μM, 0.25 μM, 0.5 μM or 2.5 μM) of AT9283 and 1 μM of UNC1999.

[0185] In one or more embodiments, the culture medium in steps (2) to (4) further comprises 0.1 μM of UNC1999 and 0.1 μM of SAHA, and the culture medium in step (6) further comprises 1 μM of AT9283 and 0.25 μM to 5 μM (preferably 0.25 μM, 0.5 μM, 2.5 μM or 5 μM) of UNC1999.

[0186] In one or more embodiments, the culture medium in steps (2) to (4) further comprises 0.1 μM of UNC1999 and 0.1 μM of SAHA, and the culture medium in step (6) further comprises 1 μM of AT9283 and 1 μM of UNC1999.

[0187] In one or more embodiments, the culture medium in step (4) further comprises 0.1 μM of UNC1999 and 0.1 μM of SAHA, and the culture medium in step (6) further comprises 1 μM of AT9283 and 1 μM of UNC1999.

[0188] In one or more embodiments, the method takes a total of 14-21 days, preferably 14-18 days, and more preferably 18 days.

[0189] In one or more embodiments, the method achieves a pancreatic β-cell differentiation efficiency of 70%-85%.

[0190] In one or more embodiments, the proportion of pancreatic β cells expressing both C-Peptide and NKX6.1 positive by the method is 70%-85%.

[0191] This invention provides pancreatic β cells obtained by the method described herein.

[0192] The present invention also provides pharmaceutical compositions comprising pancreatic β cells obtained by the methods described in any embodiment herein and pharmaceutically acceptable excipients.

[0193] This invention also provides the use of pancreatic β cells obtained by the method described in any embodiment herein in the preparation of medicaments for treating diseases. In one or more embodiments, the diseases include pancreatic β cell dysfunction disorders. Preferably, the pancreatic β cell dysfunction disorders include one or more of the following benign lesions: type 1 diabetes, type 2 diabetes, insulinoma, hereditary pancreatic β cell dysfunction, pancreatitis following total pancreatectomy, and benign pancreatic tumors.

[0194] This invention provides a treatment method comprising: administering a therapeutically effective amount of pancreatic β-cells obtained by the method of this invention or a pharmaceutical composition as described in any embodiment herein to a subject in need. In one or more embodiments, the disease includes pancreatic β-cell dysfunction disorders. Preferably, the pancreatic β-cell dysfunction disorders include one or more of the following: type 1 diabetes, type 2 diabetes, insulinoma, hereditary pancreatic β-cell dysfunction (such as adult-onset diabetes), pancreatitis following total pancreatectomy, and benign pancreatic tumors.

[0195] Example

[0196] Example 1, Materials and Methods

[0197] Islet cell differentiation

[0198] Reagents:

[0199] Table 1. Differentiation reagents

[0200] Table 2. Small molecules selected for optimization

[0201] Table 3. Components of the islet differentiation reagent

[0202] method:

[0203] S1 (Day 1-3): When pluripotent stem cells (PSCs) (see "Thermo Fisher Scientific, Epi 5")TM The Episomal iPSC Reprogramming Kit (catalog number A15960) outlines methods for obtaining iPSCs or promoting somatic cell reprogramming. Cells in good condition without undirected differentiation can begin pancreatic β-cell differentiation. Once PSC confluence reaches approximately 80%, they are digested into single cells using Accutase and seeded in Lamnin 521-coated culture dishes at a density of 2 × 10⁶ cells / mL. 5 / cm 2 The culture medium was mTesR1. After static incubation at 37°C with 5% CO2 for 24 hours, the medium was replaced with the S1 stage Day 1 differentiation medium. Thereafter, the S1 stage Day 2 and Day 3 differentiation media were replaced every 24 hours.

[0204] S2 (Day 4-6): Replace the S1 stage medium with the S2 stage differentiation medium and incubate statically in a 5% CO2, 37℃ environment.

[0205] S3 (Day 7-8): Replace the S2 stage medium with the S3 stage differentiation medium and incubate statically in a 5% CO2, 37℃ environment.

[0206] S4 (Days 9-12): Replace the S3 differentiation medium with the S4 differentiation medium. The S4 stage requires converting the 2D differentiation culture to a 3D differentiation culture state: Digest the cells in the S4 differentiation process into single cells using Accutase (37°C, 8 minutes), mix the single-cell suspension thoroughly, count the cells, and use 5 × 10⁶ viable cells per cell. 6 Up to 10×10 6 / well inoculated into AggreWell TM The cells were centrifuged at 300g for 3 min on a 400 6-well plate (STEMCELL Technologies, 34425) and then transferred to a 5% CO2 incubator at 37°C for static culture. After 24 hours of culture, the cells formed 3D cell spheroids resembling pancreatic islets.

[0207] S5 (Days 13-16): This section moves from the S4 stage to AggreWell. TM Cell spheres from the 400 plate were transferred to an Ultra-Low Attachment 6-well plate (Corning, 3471), and the S4 differentiation medium was replaced with the S5 differentiation medium. The plates were then placed on a horizontal shaker at 90-95 rpm and cultured in a 5% CO2 incubator at 37°C.

[0208] S6 (Days 17-21): The S5 differentiation medium was replaced with the S6 differentiation medium, and the cells were cultured on a horizontal shaker at 90-95 rpm in a 5% CO2 incubator at 37°C. After the S6 differentiation culture was completed, the pancreatic islet cells were digested into single cells for relevant detection and analysis.

[0209] Flow cytometry

[0210] Reagents:

[0211] Table 4. Flow Cytometry Detection Reagents

[0212] method:

[0213] 1. Digest pancreatic islet cells into single cells using Accutase (37°C, 15 minutes), count the cells, and transfer the required number of islet cells into 1.5 mL centrifuge tubes. The required total cell suspension volume (mL) = 2 × 10⁻⁶ 5 (cells) / Viable cell density (cells / mL)

[0214] 2. Add 1 mL of Staining Buffer to each tube to resuspend the cells, and centrifuge at 180 G for 5 minutes at room temperature.

[0215] 3. Add 0.5 mL of 4% PFA (paraformaldehyde) to each tube, pipette and aspirate 3-5 times to mix, incubate on wet ice for 10 minutes, then centrifuge to remove the supernatant.

[0216] 4. Add 0.5 mL of Foxp3 fixation / membrane breaking working solution to each tube, pipette and aspirate 3-5 times to mix, incubate on wet ice for 20 minutes, then centrifuge at 180 G for 5 minutes at room temperature.

[0217] 5. Add 1 mL of 1X membrane rupture buffer to each tube, pipette and aspirate 3-5 times to mix, then centrifuge.

[0218] 6. Add 50uL of blocking buffer to each tube, mix well, and incubate at room temperature for 40 minutes.

[0219] 7. Add 1 mL of 1X membrane rupture buffer to each tube, pipette and aspirate 3-5 times to mix, then centrifuge.

[0220] 8. Add Anti-C-Peptide, Anti-NKX6.1 antibody and corresponding isotype antibody to the corresponding centrifuge tubes and incubate at room temperature for 60 minutes.

[0221] 9. Wash 2-3 times with 1X membrane rupture buffer and then discard the supernatant.

[0222] 10. Resuspend the cells in 200 μL of Staining Buffer, aspirate 3-5 times with a pipette to mix, and store on ice in the dark.

[0223] 11. The Cytoflex S flow cytometer (Beckman, DL230324-0146) was used for the analysis.

[0224] Immunofluorescence detection

[0225] Reagents:

[0226] Table 5. Immunofluorescence detection reagents

[0227] method:

[0228] After the differentiated islet bodies are embedded, they are frozen sections to ensure that the islet bodies remain morphologically intact for subsequent fluorescent staining and identification experiments with relevant antibodies.

[0229] 1. Embedding the sample: Fix with 4% PFA for 1 hour, replace with 5-10 times the volume of 30% sucrose, dehydrate again at 4 degrees Celsius for 12-24 hours until the cell spheres settle to the bottom.

[0230] 2. Cryosectioning and Embedding: Start the cryostat and pre-cool. Set the chamber temperature to -21°C and the blade temperature to -20°C (adjust as needed based on the actual slicing situation). Remove the dehydrated islet bodies and allow them to stand on clean paper to remove excess moisture. Embed the OCTs in the mold, avoiding air bubbles. Quickly place the sample in the quick-freeze position of the cryostat and freeze until the OCTs are completely solidified. The embedded sample can be stored at -80°C.

[0231] 3. Frozen Sectioning: Remove the sample from the mold and fix it to the base using OCT according to the desired sectioning direction. Place it on a quick-freezing stage for solidification. Lock the crank handle and secure the blade, sample, and anti-roll plate. Adjust the distance and angle between the sample and the blade to ensure they are parallel to the blade and the cutting direction. Set the thickness of the final section to 15-30 micrometers. For RNA in situ hybridization, the thickness is typically 12-20 micrometers; for immunohistochemistry slides, it is typically 30-50 micrometers, and for slides, it is typically 20-30 micrometers. Use a small brush to spread the slide and attach it to a glass slide. Let the slide dry flat for 30 minutes.

[0232] 4. Immunofluorescence staining: Fix with 4% PFA for 1 hour, dehydrate with 30% PFA for 48 hours. Staining is performed using the immunohistochemical pen circle, with an action volume of 300 μl per slide. After OCT removal and blocking for 1 hour, C-Peptide, NKX6.1, PDX1, MAFA, and Glucagon antibodies are incubated with the samples overnight at 4°C. Wash three times with 500 μl PBST for 5 min each time; wash with secondary antibody at room temperature for 45 minutes, then wash three times with 500 μl PBST for 5 min each time; mount and photograph.

[0233] Functional detection of insulin secretion by pancreatic islet cells stimulated by glucose

[0234] reagents

[0235] Table 6. Reagents for detecting insulin secretion from pancreatic islet cells stimulated by glucose.

[0236] method:

[0237] 1. After the pancreatic islet cell spheroids have completed the S6 stage of islet differentiation, wash them three times with KrB buffer (containing 1 mM Na2HPO4, 128 mM NaCl, 5 mM NaHCO3, 5 mM KCl, 2.7 mM CaCl2·2H2O, 1.2 mM MgSO4, 1.2 mM KH2PO4, 10 mM HEPES, and 0.1% BSA).

[0238] 2. Transfer to KrB buffer containing 2.8 mM glucose and starve for 1 hour, then wash three times with KrB buffer;

[0239] 3. Transfer to KrB buffer containing 2.8 mM glucose, collect the supernatant after 1 hour, and wash three times with KrB buffer;

[0240] 4. Transfer to KrB buffer containing 16.7 mM glucose, collect the supernatant after 1 hour, and wash three times with KrB buffer;

[0241] 5. Transfer to a KrB buffer containing 30 mM KCL, and collect the supernatant after 1 hour.

[0242] All stimulation experiments were performed incubated at 37°C in a 5% CO2 incubator. The collected supernatant samples were stored at -80°C, and the amount of insulin secreted by islet cells under low / high glucose concentration (2.8 mM / 16.7 mM) stimulation was detected using a human insulin ELISA kit.

[0243] Example 2: Optimization of the S4 stage of pancreatic islet differentiation

[0244] Using the basic islet differentiation method described above, and adding the corresponding small molecules listed in Table 2 in the S4 stage for small molecule screening (concentrations used are shown in Table 7), the differentiation efficiency of pancreatic β cells was optimized. The expression ratio of the pancreatic β cell marker C-Peptide+ / NKX6.1+ was detected at the differentiation endpoint. The experimental results are shown in Table 7. UNC1999 showed better C-Peptide+ expression levels compared to other EZH2 histone methyltransferase inhibitors (GSK126, EPZ-6438, and DZNeP), and the concentration of UNC1999 (0.1 uM-1 uM) was lower than that of GSK126 (0.25 uM-2.5 uM); while SAHA showed the best C-Peptide+ / NKX6.1+ double positive expression levels compared to other histone deacetylase (HDAC) inhibitors (VPA, TSA, and TMP269). Compared with the control condition (no small molecules added in S4 stage, C-Peptide+: 65%; C-Peptide+ / NKX6.1+: 36.2%), considering the expression levels of C-Peptide+ and NKX6.1+ of each small molecule, the addition of small molecules UNC1999 (C-Peptide+: 87.9%; C-Peptide+ / NKX6.1+: 51.8%) and SAHA (C-Peptide+: 83.9%; C-Peptide+ / NKX6.1+: 54.6%) in S4 stage was more conducive to improving the purity of pancreatic β-cell differentiation.

[0245] Table 7. Results of small molecule screening affecting pancreatic islet differentiation in stage S4.

[0246] Example 3: Optimization of the S6 stage of pancreatic islet differentiation

[0247] Using the basic islet differentiation method described above, small molecules listed in Table 2 were added in the S6 stage for small molecule screening (all concentrations were 1 μM) to optimize the differentiation efficiency of pancreatic β cells. The expression ratio of the pancreatic β cell marker C-Peptide+ / NKX6.1+ was detected at the differentiation endpoint. The experimental results are shown in Table 8. The results showed that the small molecule AT9283 had the highest expression ratios of C-Peptide+ and NKX6.1+, with a C-Peptide+ single positivity rate of 79.7% and a C-Peptide+ / NKX6.1+ double positivity rate of 57.3%; followed by Danusertib (C-Peptide+ / NKX6.1+ double positivity rate of 47.1%). AT9283 showed better performance than the commonly used classic small molecule R428 (C-Peptide+ / NKX6.1+ double positivity rate of 43.5%) in islet differentiation. AT9283 is a dual inhibitor of JAK and Aurora kinases, and it shows superior efficacy compared to other JAK inhibitors in this optimization, such as Pyridone 6 (C-Peptide+ / NKX6.1+ dual positivity rate of 31.1%), Aurora kinase inhibitors in this optimization, such as Aurora kinase Inhibitor II (C-Peptide+ / NKX6.1+ dual positivity rate of 34.8%), Danusertib (C-Peptide+ / NKX6.1+ dual positivity rate of 47.1%), and ZM447439 (C-Peptide+ / NKX6.1+ dual positivity rate of 35.1%).

[0248] Table 8. Results of small molecule screening affecting pancreatic islet differentiation in stage S6.

[0249] Example 4: Further optimization and combination of small molecules screened in stages S2-4 and S6.

[0250] The preferred small molecules and their combinations screened in Example 2 were further extended to the S2-S4 stage for optimization of islet differentiation. Specifically, six conditions were added to the S2-4, S3-4 and S4 stages of the above basic islet differentiation method, respectively: (1) UNC1999 (0.1 uM), (2) SAHA (0.1 uM), (3) UNC0642 (0.1 uM), (4) UNC1999 (0.1 uM) + UNC0642 (0.1 uM), (5) SAHA (0.1 uM) + UNC0642 (0.1 uM) and (6) UNC1999 (0.1 uM) + SAHA (0.1 uM). After differentiation reached the S6 endpoint, the expression ratio of the pancreatic β cell marker C-Peptide+ / NKX6.1+ was detected by flow cytometry. The experimental results are shown in Table 9. The results show that the addition of the UNC1999+SAHA combination in the S2-4 stage can achieve the optimal pancreatic β-cell optimization effect. Compared with the control condition (without the addition of any small molecules) C-Peptide+ / NKX6.1+ expression ratio (33.9%), the expression ratio of C-Peptide+ / NKX6.1+ under the UNC1999+SAHA combination condition is 40.1%.

[0251] The superior small molecules and their combinations selected using the S6 stage were further optimized for pancreatic islet differentiation. Meanwhile, the UNC1999 small molecule was extended to the S6 stage for optimization, namely: (1) R428 (1uM), (2) AT9283 (1uM), (3) Danusertib (1uM), (4) UNC1999 (1uM), (5) CPI-455HCl (1uM) + UNC1999 (1uM), (6) AT9283 (1uM) + Danusertib (1uM). Nine conditions, including (7) AT9283(1uM)+UNC1999(1uM), (8) UNC1999(1uM)+Danusertib(1uM) and (9) AT9283(1uM)+UNC1999(1uM)+Danusertib(1uM), were added to the S6 stage of the differentiation method in Experiment 1 (Islet Differentiation Method, Table 3). After differentiation reached the endpoint, the expression ratio of the pancreatic β cell marker C-Peptide+ / NKX6.1+ was detected by flow cytometry. The experimental results are shown in Table 10. The results show that AT9283 is still superior to R428, while the optimization effect of adding UNC1999 alone is lower than that of the control condition. However, unexpectedly, the optimization of the combination of AT9283 and UNC1999 is not only better than the control condition, but also significantly better than the condition of adding AT9283 alone, achieving the best optimization effect in this study, that is, the expression ratio of C-Peptide+ / NKX6.1+ reaches 51.9%.

[0252] Table 9. Optimization results of islet differentiation in stages S2-S4

[0253] Table 10. Optimization results of islet differentiation in stage S6.

[0254] Example 5: Integration and optimization of superior small molecules screened in stages S2-4 and S6.

[0255] The small molecule combinations optimized in the S4 stage and the small molecules optimized in the S6 stage of Example 3 are further integrated and optimized, including the following four groups:

[0256] (1) The UNC1999 (0.1 uM) + SAHA (0.1 uM) small molecule combination was added to the S2-4 stages of the above-mentioned basic islet differentiation method.

[0257] (2) The UNC1999 (0.1 uM) + SAHA (0.1 uM) small molecule combination was added to stages S2-4 of the above basic islet differentiation method, and AT9283 (1 uM) was added to stage S6 of the differentiation method in experimental method 1 (islet differentiation method, Table 3).

[0258] (3) The UNC1999 (0.1 μM) + SAHA (0.1 μM) small molecule combination was added to stages S2-4 of the above-mentioned basic islet differentiation method, and AT9283 (1 μM) + UNC1999 (1 μM) was added to stage S6 of the differentiation method in experimental method 1 (islet differentiation method, Table 3), and

[0259] (4) The small molecule combination of UNC1999 (0.1 uM) + SAHA (0.1 uM) was added to the S2-4 stages of the above basic islet differentiation method, and AT9283 (1 uM) + UNC1999 (1 uM) + Danusertib (1 uM) was added to the S6 stage of the differentiation method in Experimental Method 1 (Islet Differentiation Method, Table 3).

[0260] After differentiating cells from each group to the endpoint, the expression ratio of the pancreatic β-cell marker C-Peptide+ / NKX6.1+ was detected by flow cytometry. The experimental results are shown in Table 11. The results show that adding the combination of small molecules UNC1999 (0.1 uM) + SAHA (0.1 uM) in stages S2-4, and adding the combination of small molecules AT9283 (1 uM) + UNC1999 (1 uM) in stage S6, can achieve the best pancreatic β-cell optimization effect, with the expression ratio of C-Peptide+ / NKX6.1+ reaching 79.5%.

[0261] Table 11. Results of small molecule combination optimization affecting islet differentiation

[0262] Example 6: Validation of optimal small molecule combination for pancreatic islet differentiation

[0263] The optimal differentiation combination from Example 4 was further validated for islet differentiation (n=2). The basic islet differentiation method described above was used, with the addition of a small molecule combination of UNC1999 (0.1 μM) + SAHA (0.1 μM) in stage S4, and another small molecule combination of AT9283 (1 μM) + UNC1999 (1 μM) in stage S6. After differentiation reached the endpoint, the expression ratio of the islet β-cell marker C-Peptide+ / NKX6.1+ was detected by flow cytometry. The experimental results are shown in Figure 1. The results show that under the currently selected optimal differentiation conditions, without any sorting or enrichment, the expression ratio of the islet β-cell marker C-Peptide+ / NKX6.1+ can reach nearly 85%, which is significantly better than the proportion of islet β-cells produced by current mainstream islet differentiation methods (30-50%).

[0264] Example 7: Immunofluorescence detection of pancreatic islet differentiation using the optimal combination of small molecules.

[0265] The optimal differentiation combination from Example 5 was further differentiated into islet cells. The basic islet differentiation method described above was employed, with the addition of a small molecule combination of UNC1999 (0.1 μM) + SAHA (0.1 μM) in stage S4, and an addition of a small molecule combination of AT9283 (1 μM) + UNC1999 (1 μM) in stage S6. After differentiation reached the endpoint, immunofluorescence was used to detect the expression of key biomarkers of islet β cells. The experimental results are shown in Figure 2. The results show that the islet cells expressed key islet biomarkers C-Peptide, NKX6.1, PDX1, and GCG (islet α cells), and also expressed the islet β cell maturation biomarker MAFA. The experimental results indicate that the islet cells produced by this innovative approach largely replicate the islet cells found in primary human pancreatic tissue.

[0266] Example 8: Functional testing of pancreatic islet differentiation using the optimal combination of small molecules.

[0267] The optimal differentiation combination method from Example 5 was used for islet differentiation, while the aforementioned basic islet differentiation method was used as a control. Specifically, the basic islet differentiation method was used, with or without the addition of the small molecule UNC1999 (0.1 μM) + SAHA (0.1 μM) combination in stage S4, and with or without the addition of the small molecule AT9283 (1 μM) + UNC1999 (1 μM) combination in stage S6. After differentiation reached the endpoint, the amount of insulin secreted by islet cells under low / high glucose concentration (2.8 mM / 16.7 mM) stimulation was detected using a human insulin ELISA kit to assess the islet cell function in response to high and low glucose concentration stimulation. The experimental results are shown in Table 12, where A represents the insulin detection results of the control method, and B represents the insulin detection results with the addition of the optimal small molecule combination. The results showed that under low / high glucose concentration (2.8 mM / 16.7 mM) stimulation, the glucose stimulation index (the ratio of insulin secretion by islet cells stimulated by high and low glucose concentrations, respectively) of the control condition was only 0.89 times (Table 12), indicating that the islet cells differentiated under the control condition did not have the function of secreting higher insulin in response to high glucose concentration (16.7 mM). However, the glucose stimulation index of the condition with the addition of the optimal small molecule combination was 2.03 times, indicating that the islet cells differentiated under this condition had the function of responding to both high and low glucose concentrations. That is, under the condition of switching from low to high glucose concentration, the islet cells could secrete higher concentrations of insulin, thereby mimicking the physiological function of islet cells in pancreatic tissue in response to glucose in the human body and achieving the purpose of controlling blood sugar.

[0268] Table 12. Functional detection results of the optimal small molecule combination after pancreatic islet differentiation.

[0269] Example 9: Optimizing the concentration of small molecule combinations

[0270] The optimal islet differentiation method described in Example 6 was used, with optimized concentrations of small molecules as shown in differentiation conditions 1-19 in Table 13. After differentiation reached the endpoint, the expression ratio of the pancreatic β-cell marker C-Peptide+ / NKX6.1+ was detected by flow cytometry. The experimental results are shown in Tables 14-15. The results showed that in stages S2-4 of pancreatic β-cell differentiation, the low concentration of the small molecule combination UNC1999 (0.025uM-0.05uM) + SAHA (0.01uM-0.05uM) exhibited better differentiation effects; in stage S6 of pancreatic β-cell differentiation, the small molecule combination UNC1999 (0.05uM-2.5uM) + AT9283 (0.1uM-0.5uM) exhibited better differentiation effects.

[0271] Table 13, Optimized usage concentration of small molecule combinations

[0272] Table 14. Optimized concentrations of small molecule combinations in stage S2-4

[0273] Table 15. Optimized concentration of small molecule combinations in stage S6

[0274] Example 10: Further shortening the islet differentiation time under optimal small molecule combination and concentration conditions.

[0275] The optimized small molecule concentrations from Example 9 were used, specifically the addition of the small molecule combination UNC1999 (0.025 μM) + SAHA (0.01 μM) during stages S2-4 of pancreatic β-cell differentiation, and the addition of the small molecule combination UNC1999 (2.5 μM) + AT9283 (0.25 μM) during stage S6. Further investigation was conducted to determine whether the optimal small molecule combination and concentration conditions of this protocol could shorten the islet differentiation time while maintaining a high differentiation efficiency of pancreatic β-cells (as shown in Figure 3). Using this experimental protocol, the expression ratio of the pancreatic β-cell marker C-Peptide+ / NKX6.1+ was detected by flow cytometry on days 14, 16, and 18 of islet differentiation. The experimental results are shown in Table 16 (n=3). The results indicate that using the optimal combination and concentration of small molecules in this scheme further shortens the islet differentiation time to days 14, 16, and 18, with the expression rates of the pancreatic β-cell marker C-Peptide+ / NKX6.1+ reaching 65.4%, 71.6%, and 73.9%, respectively. This optimization of islet β-cell differentiation demonstrates that, without sorting and enriching the final islet β-cell product, this innovative scheme can significantly improve the differentiation purity of islet β-cells (C-Peptide+ / NKX6.1+ double positivity rate reaching 75%-85%), while greatly shortening the differentiation time (14-18 days). This scheme suggests that it can minimize the labor and material costs for large-scale production of universal islet cells, meeting the broad accessibility requirements for clinical applications.

[0276] Table 16. Differentiation time of the optimal small molecule combination

Claims

1. A method, the method comprising: Culture pluripotent stem cells to differentiate them into posterior foregut cells; The posterior foregut cells were differentiated into pancreatic progenitor cells by treating them with a culture medium supplemented with EZH2 histone methyltransferase inhibitors and / or histone deacetylase inhibitors; and The pancreatic progenitor cells are differentiated into pancreatic β cells.

2. The method of claim 1, wherein the EZH2 histone methyltransferase inhibitor is selected from UNC1999, GSK126, EPZ-6438 and DZNeP; preferably UNC1999.

3. The method of claim 2, wherein the concentration of UNC1999 in the culture medium is about 0.01 μM to about 0.25 μM; preferably about 0.1 μM.

4. The method of claim 1, wherein the histone deacetylase inhibitor is selected from SAHA, VPA, TSA and TMP269; preferably SAHA.

5. The method of claim 4, wherein the concentration of SAHA in the culture medium is about 0.01 μM to about 0.25 μM; preferably about 0.1 μM.

6. A method, the method comprising: Culture pluripotent stem cells to differentiate them into endocrine precursor cells; and The endocrine precursor cells were differentiated into pancreatic β cells by treating them with a culture medium supplemented with AT9283 or Danusertib.

7. The method of claim 6, wherein the concentration of AT9283 or Danusertib in the culture medium is about 0.05 μM to about 2.5 μM; preferably about 0.25 μM.

8. The method of claim 7, wherein the culture medium is further supplemented with UNC1999.

9. The method of claim 8, wherein the concentration of UNC1999 in the culture medium is about 0.25 μM to about 5 μM; preferably about 2.5 μM.

10. A method, the method comprising: (1) Culture pluripotent stem cells to differentiate them into a fixed endoderm; (2) Differentiate the defined endoderm cells into gastrulator cells; (3) Differentiate the primitive intestinal cells into posterior foregut cells; (4) The posterior foregut cells are differentiated into pancreatic progenitor cells by treating them with a culture medium supplemented with one or more small molecules selected from the following: (a) p300 histone acetyltransferase inhibitor, (b) Selective inhibitors of EZH2 and EZH1 (c) BET bromodomain inhibitors, (d)p38 MAPK inhibitor, (e) JNK inhibitors, (f) HDAC inhibitors, (g) G9a / GLP histone methyltransferases, histone methyltransferase inhibitors, and (h)Akt inhibitors; (5) Differentiate the pancreatic progenitor cells into endocrine progenitor cells; and (6) The endocrine precursor cells were differentiated into pancreatic β cells by treating them with a culture medium supplemented with AT9283 or Danusertib.

11. The method of claim 10, wherein the culture medium used in step (4) comprises one or more small molecules selected from the group consisting of: HAIII, L002, GSK126, EPZ-6438, DZNeP, UNC1999, I-BET151, SB203580, SP600125, VPA, SAHA, UNC0642, AT7867, AZD5363, TMP269, and A-366.

12. The method of claim 11, wherein the concentrations of the small molecules in the culture medium are as follows: HAIII 0.1 μM-1 μM, L002 0.1 μM-1 μM, GSK126 0.25 μM-2.5 μM, EPZ-6438 0.1 μM-1 μM, DZNeP 0.1 μM-1 μM, UNC1999 0.1 μM-1 μM, and I-BET151 0.1 μM-1 μM. The concentrations of SB203580, SP600125, VPA, SAHA, UNC0642, AT7867, AZD5363, TMP269, and A-366 are 0.1 μM-1 μM.

13. The method according to any one of claims 10-13, wherein Step (2) includes treatment with a culture medium supplemented with EZH2 histone methyltransferase inhibitors and / or histone deacetylase inhibitors, and / or Step (3) involves treatment with a culture medium supplemented with EZH2 histone methyltransferase inhibitors and / or histone deacetylase inhibitors.

14. The method of any one of claims 10-13, wherein the culture medium used in step (6) is further supplemented with UNC1999.

15. The method according to any one of claims 1-14, wherein the proportion of pancreatic β cells expressing both C-Peptide and NKX6.1 is 70%-85%.

16. The method of any one of claims 1-14, wherein the culture medium is a serum-free culture medium. Preferably, the serum-free culture medium comprises one or more selected from the following: MCDB131, DMEM, RPMI1640, glucose, NaHCO3, BSA, ITS-X, Glutamax, vitamin C, antibiotics (e.g., penicillin and / or streptomycin), heparin, and ZnSO4.

17. The method of any one of claims 1-14, wherein the culture medium comprises one or more selected from: TGFβ / activin agonist, glycogen synthase kinase 3 inhibitor, Smoothened (HH / SMO) inhibitor, retinoic acid receptor (RAR) agonist, BMP inhibitor, protein kinase C activator, FGFR2 IIIb, EGFR / ErbB activator, γ-secretase inhibitor, Alk5 inhibitor, and thyroid hormone receptor agonist.

18. The method of any one of claims 1-14, wherein the time taken for pluripotent stem cells to differentiate into pancreatic β cells is 14 to 18 days.

19. The use of pancreatic β cells obtained by the method of any one of claims 1-18 in the preparation of medicaments for treating diseases. Preferably, the disease is suitable for pancreatic β-cell therapy. More preferably, the diseases include benign lesions such as type 1 diabetes, type 2 diabetes, insulinoma, hereditary pancreatic β-cell dysfunction, pancreatitis after total pancreatectomy, and benign pancreatic tumors.

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