WO2023221565A1 - Method for differentiating induced pluripotent stem cell into pancreatic islet and use thereof in treating type i diabetes mellitus - Google Patents

Abstract

Provided are a method for differentiating an induced pluripotent stem cell into a pancreatic islet and use thereof in treating type I diabetes mellitus. By adopting the method, the induced differentiation efficiency of differentiating the induced pluripotent stem cell into the pancreatic islet can be remarkably improved, the maturation of cells for secreting insulin in vitro is promoted, and the GCG-/INS+ cell positive rate is significantly increased, providing a powerful support for cell therapy of type I diabetes mellitus, so that the method has a good clinical application prospect.

Description

A method of differentiating induced pluripotent stem cells into pancreatic islets and its application in the treatment of type I diabetes Technical field

The present invention belongs to the field of biomedicine technology. Specifically, the present invention relates to a method for differentiation of induced pluripotent stem cells into pancreatic islets and its application in the treatment of type I diabetes.

Background technique

Diabetes is a disease characterized by hyperglycemia, lack of insulin secretion or insulin resistance caused by various pathogenic factors, which in turn leads to glucose metabolism disorders. Currently, diabetes is divided into type 1 diabetes, type 2 diabetes, special types of diabetes and gestational diabetes. Among them, type 1 diabetes accounts for about 10% of diabetes patients. Type 1 diabetes is an organ-specific autoimmune disease mediated by T lymphocytes and characterized by the selective destruction of pancreatic beta cells. Type 1 diabetes The patient's own insulin secretion is absolutely lacking and requires lifelong dependence on insulin treatment. As the disease progresses, the patient eventually enters the "brittle diabetes stage." In addition, patients with type I diabetes have relatively insufficient insulin secretion due to the destruction of pancreatic beta cells, which exposes multiple organs of the body to a hyperglycemic environment for a long time, leading to severe cardiovascular, eye, renal and nervous system diseases. Improper or untimely treatment will cause serious complications, which may affect the patient's quality of life or lead to death.

At present, the standard treatment plan for type 1 diabetes is drug treatment, which uses oral hypoglycemic drugs, insulin injections, insulin pumps and other drug treatments. However, patients need to ensure uninterrupted high-quality insulin injection treatment throughout their lives. Type 1 diabetes patients are still incurable. Although drug treatment can effectively improve glucose metabolism and improve the patient's quality of life, lifelong drug treatment increases the risk of acute complications such as infection, ketoacidosis, and hypoglycemia, as well as retinal and neurological diseases. , the risk of chronic complications such as kidney, cardiovascular and cerebrovascular diseases, and large blood vessel plaques. Therefore, long-term exogenous injection of insulin can only alleviate the hyperglycemia symptoms of diabetes, but cannot fundamentally treat diabetes and achieve good intervention and treatment effects. Therefore, there is an urgent need in this field for a new and effective treatment strategy that can reduce or even eliminate acute complications and chronic complications for the treatment of type 1 diabetes.

In recent years, with the continuous development of islet transplantation and stem cell transplantation technology, new methods have been provided for the treatment of type I diabetes. Although islet transplantation is an ideal method to treat type I diabetes, most patients are unable to undergo islet transplantation due to the shortage of donor sources and immune rejection after transplantation. Therefore, the clinical application of this method is limited. Stem cell transplantation has gradually been used in the treatment of diabetes in recent years. However, the problem faced by this treatment method is the insufficient source of pancreatic beta cells that can secrete insulin (the only hormone that lowers blood sugar in the body) and the inability to maintain blood sugar regulation function in the body for a long time. Relevant studies have shown that functional insulin-secreting cells obtained through induction of differentiation, reprogramming and transdifferentiation of stem cells have broad prospects. Embryonic stem cells have the characteristics of unlimited proliferation and multi-lineage differentiation potential. However, the current clinical application of embryonic stem cells is restricted by ethics and morals, which makes induced pluripotent stem cells (iPSCs) an unlimited supply of insulin-secreting cells. source of seed cells and avoid potential medical ethical issues.

The latest research shows that induced pluripotent stem cells can not only generate specific cell types for patients, but also open up new avenues for innovation in disease treatment, drug screening and regenerative medicine. Induced pluripotent stem cells, as a type of stem cells with multi-directional differentiation potential, can obtain relatively mature and functional tissues or organs through the induction of relevant chemical small molecules and cell growth factors in vitro. The method of using stem cells to differentiate into tissues and organs can also be used in the treatment of diabetes, providing a new solution to the shortage of donors. However, at present, the differentiation of pluripotent stem cells into pancreatic endocrine cells is mainly achieved through the combined use of signaling molecules and their related inhibitors/agonists. They are generally differentiated according to 6-7 consecutive differentiation stages, namely: definitive endoderm, primitive Embryonic gut tube, posterior foregut, pancreatic endoderm, endocrine precursor cells and β-like early cells, and further differentiate into mature pancreatic islet β-like cells. However, the above-mentioned differentiation methods still produce immature β cells, and the single-positive rate of GCG-/INS+ cells is generally low. The hormones they express are limited and unstable, with various types and limited insulin content. They cannot be used for transplantation to treat diabetic patients.

It can be seen that insulin injection can cause various complications, the source of islet transplantation is limited, and the currently developed islet β-cell differentiation protocol is immature and small in number. The single-positive rate of GCG-/INS+ cells is generally low or does not have the ability to stimulate insulin secretion with glucose. (GSIS) function and other reasons, cell therapy for diabetes has been greatly restricted. Therefore, how to obtain a large number of functional, mature and stable pancreatic islet β cells from induced pluripotent stem cells is the field of cell therapy for type I diabetes. One of the main problems faced.

Contents of the invention

In order to solve the above-mentioned problems currently faced in this field, the purpose of the present invention is to provide a method for differentiation of induced pluripotent stem cells into pancreatic islets and its application in the treatment of type I diabetes.

The above objects of the present invention are achieved through the following technical solutions:

A first aspect of the present invention provides a differentiation-inducing agent that induces differentiation of iPSCs into functionally mature pancreatic islet β cells.

Further, the inducing differentiation agent includes a first stage inducing differentiation agent, a second stage inducing differentiation agent, a third stage inducing differentiation agent, a fourth stage inducing differentiation agent, a fifth stage inducing differentiation agent, a sixth stage inducing differentiation agent, Stage 7 differentiation agent;

Preferably, the first-stage differentiation inducing agent includes the first-stage differentiation inducing agent A and the first-stage differentiation inducing agent B;

More preferably, the first-stage differentiation inducing agent A includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, Activin A, and CHIR-99021;

More preferably, the first-stage differentiation inducing agent B includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, and Activin A;

Preferably, the second stage differentiation inducing agent includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, and FGF-7;

Preferably, the third stage differentiation inducing agent includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, FGF-7, insulin-transferrin-selenium, ethanolamine, SANT-1, retinoids Alcohol, LDN193189, TPPB;

Preferably, the fourth stage differentiation agent includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, FGF-7, insulin-transferrin-selenium, ethanolamine, SANT-1, retin Alcohol, LDN193189, TPPB;

Preferably, the fifth stage differentiation agent includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189, triiododine Thyroidine (T3), ALK5i II, zinc sulfate;

Preferably, the sixth stage differentiation inducing agent includes the sixth stage differentiation inducing agent A, the sixth stage differentiation inducing agent B, the sixth stage differentiation inducing agent C, and the sixth stage differentiation inducing agent D;

More preferably, the sixth stage differentiation agent A includes β-cellulin and erythrospongin A;

Most preferably, the sixth stage differentiation agent A also contains sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine ( T3), ALK5i II, zinc sulfate, GSi XX;

More preferably, the sixth stage differentiation agent B contains β-cellulin;

Most preferably, the sixth stage differentiation agent B also contains sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine ( T3), ALK5i II, zinc sulfate, GSi XX;

More preferably, the sixth stage differentiation inducing agent C includes β-cellulin, forskolin, and exenatide 4;

Most preferably, the sixth stage differentiation agent C also contains sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine ( T3), ALK5i II, zinc sulfate, GSi XX;

More preferably, the sixth stage differentiation inducing agent D includes β-cellulin, forskolin, exenatide 4, hepatocyte growth factor, and serotonin;

Most preferably, the sixth stage differentiation agent D also contains sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate , GSi XX, insulin-transferrin-selenium;

Preferably, the seventh stage differentiation inducing agent includes seventh stage differentiation inducing agent A and seventh stage differentiation inducing agent B;

More preferably, the seventh stage differentiation inducing agent A includes β-cellulin, forskolin, exenatide 4, hepatocyte growth factor, and serotonin;

Most preferably, the seventh stage differentiation agent A also contains sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, and triiodothyronine (T3) , ALK5i II, zinc sulfate, N-acetyl-L-cysteine, porcine intestinal mucosal heparin sodium, water-soluble vitamin E, R428;

More preferably, the seventh stage differentiation agent B includes glutamine, calcium chloride dihydrate, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, fetal bovine serum albumin, insulin- Transferrin-selenium, ethanolamine, water-soluble vitamin E, nicotinamide, heparin sodium, deoxyribonuclease I, Necrostatin-1, Pefabloc;

Most preferably, the concentrations of each component in the first-stage differentiation agent are: (10-500) ng/mL Activin A, (0.01-10) μM CHIR-99021, (0.01-10)% fetal bovine serum Albumin, (0.01-10)g/L sodium bicarbonate, (1-50)mM glucose, (0.01-5)mM glutamine;

Most preferably, the concentrations of each component in the first-stage differentiation agent are: 100ng/mL Activin A, 3μM CHIR-99021, 0.5% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 10mM glucose, 1mM glutamine;

More preferably, the concentrations of each component in the second stage differentiation agent are: (0.01-10)mM ascorbic acid, (10-200)ng/mL FGF-7, (0.01-10)% fetal bovine serum albumin Protein, (0.01-10)g/L sodium bicarbonate, (1-50)mM glucose, (0.01-5)mM glutamine;

Most preferably, the concentrations of each component in the second-stage differentiation agent are: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.5% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 10mM glucose, 1mM glutamine;

More preferably, the concentrations of each component in the third stage differentiation agent are: (0.01-5)mM ascorbic acid, (10-200)ng/mL FGF-7, (0.01-5)μM SANT-1, (0.01-5)μM retinol, (10-500)nM LDN193189, (50-500)nM TPPB, (0.01-10)% fetal bovine serum albumin, (0.01-10)g/L sodium bicarbonate, (1-100)mM glucose, (1-100)mM glutamine, (1-100)mg/L ethanolamine, (0.01-5)% insulin-transferrin-selenium;

Most preferably, the concentrations of each component in the third-stage differentiation agent are: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.25μM SANT-1, 1μM retinol, 100nM LDN193189, 200nM TPPB, 2% Fetal bovine serum albumin, 2.5g/L sodium bicarbonate, 10mM glucose, 1mM glutamine, 1mg/L ethanolamine, 0.5% insulin-transferrin-selenium;

More preferably, the concentrations of each component in the fourth stage differentiation agent are: (0.01-5)mM ascorbic acid, (0.01-10)ng/mL FGF-7, (0.01-5)μM SANT-1, (0.01-5)μM retinol, (50-500)nM LDN193189, (10-300)nM TPPB, (0.01-5)% fetal bovine serum albumin, (0.05-10)g/L sodium bicarbonate, (0.05-50)mM glucose, (0.01-5)mM glutamine, (0.01-5)mg/L ethanolamine, (0.01-5)% insulin-transferrin-selenium;

Most preferably, the concentrations of each component in the fourth stage inducing differentiation agent are: 0.25mM ascorbic acid, 2ng/mL FGF-7, 0.25μM SANT-1, 0.1μM retinol, 200nM LDN193189, 100nM TPPB, 2 % fetal bovine serum albumin, 2.5g/L sodium bicarbonate, 10mM glucose, 1mM glutamine, 1mg/L ethanolamine, 0.5% insulin-transferrin-selenium;

More preferably, the concentrations of each component in the fifth stage differentiation agent are: (0.01-5) μM SANT-1, (0.01-10) μM retinol, (10-500) nM LDN193189, (0.01 -5)μM triiodothyronine (T3), (1-50)μM ALK5i II, (1-50)μM zinc sulfate, (0.05-10)% fetal bovine serum albumin, (0.05-10)g /L sodium bicarbonate, (1-50)mM glucose, (0.01-10)mM glutamine, (0.01-5)% insulin-transferrin-selenium, (0.01-10) mg/L ethanolamine;

Most preferably, the concentrations of each component in the fifth stage differentiation agent are: 0.25 μM SANT-1, 0.05 μM retinol, 100 nM LDN193189, 1 μM triiodothyronine (T3), 10 μM ALK5i II, 10μM zinc sulfate, 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20mM glucose, 1mM glutamine, 0.5% insulin-transferrin-selenium, 1mg/L ethanolamine;

Most preferably, the concentrations of each component in the sixth stage inducing differentiation agent are: (10-500) nM LDN193189, (0.01-10) μM triiodothyronine (T3), (1-50) μM ALK5i II, (1-50)μM zinc sulfate, (0.05-10)% fetal bovine serum albumin, (0.01-5)g/L sodium bicarbonate, (1-100)mM glucose, (0.01-5)mM Glutamine, (0.01-5)mg/L ethanolamine, (50-300)nM GSi XX, (0.01-5)μM erythrospongin A, (10-500)ng/mL hepatocyte growth factor, (1- 50)μM serotonin, (1-50)ng/mL β-cellulin, (1-50)μM forskolin, (10-500)ng/mL exenatide 4;

Most preferably, the concentrations of each component in the sixth-stage differentiation agent are: 100 nM LDN193189, 1 μM triiodothyronine (T3), 10 μM ALK5i II, 10 μM zinc sulfate, 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20mM glucose, 1mM glutamine, 1mg/L ethanolamine, 100nM GSi XX, 1μM erythrospongin A, 50ng/mL hepatocyte growth factor, 10μM serotonin, 20ng/mL β-cellulin, 10μM hair Throatin, 50ng/mL exenatide 4;

Most preferably, the concentrations of each component in the seventh stage differentiation agent A are: (0.05-10)% fetal bovine serum albumin, (0.05-10) g/L sodium bicarbonate, (5-50) mM glucose, (0.01-5)mM glutamine, (0.01-5)mg/L ethanolamine, (0.01-2.5)% insulin-transferrin-selenium, (0.01-5)μM triiodothyronine ( T3), (0.01-50)μM ALK5i II, (1-50)μM zinc sulfate, (0.01-5)mM N-acetyl-L-cysteine, (1-50)μg/mL porcine intestinal mucosal heparin Sodium, (1-50)μM water-soluble vitamin E, (1-50)μM R428, (10-100)ng/mL hepatocyte growth factor, (1-50)μM serotonin, (1-50)ng/ mL β-cellulin, (1-50) μM forskolin, (10-100) ng/mL exenatide 4;

Most preferably, the concentrations of each component in the seventh stage differentiation agent A are: 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20mM glucose, 1mM glutamine, 1mg/L ethanolamine, 0.5% insulin-transferrin-selenium, 1μM triiodothyronine (T3), 10μM ALK5i II, 10μM zinc sulfate, 1mM N-acetyl-L-cysteine, 10μg/mL porcine intestinal mucosal heparin sodium, 10μM water-soluble vitamin E, 2μM R428, 50ng/mL hepatocyte growth factor, 10μM serotonin, 20ng/mL β-cellulin, 10μM forskolin, 50ng/mL exenatide 4;

Most preferably, the concentrations of each component in the seventh stage differentiation agent B are: (0.01-10)mM glutamine, (0.01-10)mM calcium chloride dihydrate, (1-50)mM N -2hydroxyethylpiperazine-N-2-ethanesulfonic acid, (0.01-5)% fetal bovine serum albumin, (0.01-2)mL/L insulin-transferrin-selenium, (0.01-5) mg/L ethanolamine, (1-50)μM water-soluble vitamin E, (1-50)mM nicotinamide, (1-50)μg/mL heparin sodium, (0.01-2.5)U/mL deoxyribonuclease I, (50-500)μM Necrostatin-1, (0.001-2)μM Pefabloc;

Most preferably, the concentrations of each component in the seventh stage differentiation agent B are: 2mM glutamine, 2.5mM calcium chloride dihydrate, 10mM N-2 hydroxyethylpiperazine-N-2-ethane. Sulfonic acid, 2% fetal bovine serum albumin, 0.6mL/L insulin-transferrin-selenium, 1mg/L ethanolamine, 10μM water-soluble vitamin E, 10mM nicotinamide, 10μg/mL heparin sodium, 1U/mL DNA Enzyme I, 100μM Necrostatin-1, 0.1μM Pefabloc.

A second aspect of the present invention provides an induction differentiation medium for inducing iPSCs to differentiate into functionally mature pancreatic islet β cells.

Further, the induced differentiation medium includes a first stage induced differentiation medium, a second stage induced differentiation medium, a third stage induced differentiation medium, a fourth stage induced differentiation medium, a fifth stage induced differentiation medium, and a third stage induced differentiation medium. Six-stage induction differentiation medium, seventh-stage induction differentiation medium;

Preferably, the first-stage differentiation medium includes basal medium MCDB131 and the first-stage differentiation agent described in the first aspect of the present invention;

Preferably, the second-stage differentiation medium includes basal medium MCDB131 and the second-stage differentiation agent described in the first aspect of the present invention;

Preferably, the third stage inducing differentiation medium includes basal medium MCDB131 and the third stage inducing differentiation agent described in the first aspect of the present invention;

Preferably, the fourth stage inducing differentiation medium includes basal medium MCDB131 and the fourth stage inducing differentiation agent described in the first aspect of the present invention;

Preferably, the fifth stage inducing differentiation medium includes basal medium MCDB131 and the fifth stage inducing differentiation agent described in the first aspect of the present invention;

Preferably, the sixth stage inducing differentiation medium includes basal medium MCDB131 and the sixth stage inducing differentiation agent described in the first aspect of the present invention;

Preferably, the seventh stage induction differentiation medium includes basal medium MCDB131, 50% Ham’s F-12 medium, 50% medium 199, and the seventh stage induction differentiation agent described in the first aspect of the present invention.

The third aspect of the present invention provides a method for inducing iPSCs to differentiate into functionally mature pancreatic islet β cells.

Further, the method includes the following steps:

(1) Provide iPSCs and perform monolayer differentiation culture in complete culture medium;

(2) In the first stage of induction differentiation, the iPSCs obtained in step (1) are induced to differentiate into definitive endoderm cells using the first stage induction differentiation medium;

(3) In the second stage of induction differentiation, the second stage induction differentiation medium is used to induce the differentiation of definitive endoderm cells into primitive intestinal tube cells;

(4) In the third stage of induction differentiation, the third stage induction differentiation medium is used to induce the differentiation of the protogut tube cells into posterior foregut cells;

(5) The fourth stage induces differentiation, using the fourth stage induction differentiation medium to induce the posterior foregut cells to differentiate into pancreatic progenitor cells;

(6) Fifth stage induction differentiation, using the fifth stage induction differentiation medium to induce the differentiation of pancreatic progenitor cells into pancreatic endocrine progenitor cells;

(7) Sixth stage induction differentiation, using the sixth stage induction differentiation medium to induce the differentiation of pancreatic endocrine progenitor cells into pancreatic endocrine cells;

(8) Seventh-stage induction differentiation: Use the seventh-stage induction differentiation medium to induce the differentiation of pancreatic endocrine cells into mature islet cells to obtain functionally mature islet β cells.

Further, the complete culture medium described in step (1) is E8 complete culture medium;

Preferably, the E8 complete culture medium contains a ROCK inhibitor;

More preferably, the ROCK inhibitor includes Y27632, GSK429286A, RKI-1447, Y-33075dihydrochloride, Thiazovivin, K-115, SLx-2119, Chroman1, SAR407899 and/or SR-3677;

Most preferably, the ROCK inhibitor is Y27632;

Most preferably, the concentration of Y27632 is 0.001-100 μM;

Most preferably, the concentration of Y27632 is 10 μM;

Preferably, the iPSCs described in step (1) are derived from mammals;

More preferably, the iPSCs described in step (1) are derived from humans, mice, rats, goats, sheep, pigs, cats, rabbits, dogs, wolves, horses or cattle;

Most preferably, the iPSCs described in step (1) are derived from humans;

Most preferably, the culture time in step (1) is 3 days;

Most preferably, from Day-3 to Day-1, fresh medium is replaced every day;

Preferably, the first stage induction differentiation medium described in step (2) contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, activin, and GSK-3 inhibitor;

More preferably, the activin includes Activin A, Activin B, Activin AB, Activin AC;

Most preferably, the activin is Activin A;

More preferably, the GSK-3 inhibitor includes CHIR-99021, CHIR-98014, AZD-2858, SB-216763, AT-7519, TW-S119, KY-19382 (A3051), NP-031112, SB-415286 , AZD-1080, AR-A014418, TDZD-8, LY-2090314;

Most preferably, the GSK-3 inhibitor is CHIR-99021;

More preferably, the first stage of inducing differentiation medium includes the first stage of inducing differentiation medium A and the first stage of inducing differentiation medium B;

Most preferably, the first-stage induced differentiation medium A includes basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, Activin A, and CHIR-99021;

Most preferably, the first-stage induced differentiation medium B contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, and Activin A;

Most preferably, the concentrations of each component in the first stage induction differentiation medium are: (10-500) ng/mL Activin A, (0.01-10) μM CHIR-99021, (0.01-10)% fetal bovine Serum albumin, (0.01-10)g/L sodium bicarbonate, (1-50)mM glucose, (0.01-5)mM glutamine;

Most preferably, the concentrations of each component in the first stage induction differentiation medium are: 100ng/mL Activin A, 3μM CHIR-99021, 0.5% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 10mM glucose. , 1mM glutamine;

Most preferably, the time for inducing differentiation in the first stage is 3 days;

Most preferably, on the first day of induction, the first stage induction differentiation medium A is used to induce differentiation;

Most preferably, on the 2nd to 3rd day of induction, the first stage induction differentiation medium B is used to induce differentiation;

Most preferably, on Day0-Day2, fresh medium is replaced every day.

Further, the second stage induction differentiation medium described in step (3) includes basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, and fibroblast growth factor;

Preferably, the fibroblast growth factors include FGF-7, FGF-2, FGF-6, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16 , FGF-17, FGF-18, FGF-21, FGF-5, FGF-1, FGF-3, FGF-4, FGF-8, FGF-9, FGF-19, FGF-20;

More preferably, the fibroblast growth factor is FGF-7;

Most preferably, the concentrations of each component in the second stage induction differentiation medium are: (0.01-10)mM ascorbic acid, (10-200)ng/mL FGF-7, (0.01-10)% fetal bovine serum. Albumin, (0.01-10)g/L sodium bicarbonate, (1-50)mM glucose, (0.01-5)mM glutamine;

Most preferably, the concentrations of each component in the second stage induction differentiation medium are: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.5% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, and 10mM glucose. , 1mM glutamine;

Most preferably, the time for inducing differentiation in the second stage is 2 days;

Most preferably, on the 1st to 2nd day of induction, the second stage induction differentiation medium is used to induce differentiation;

Most preferably, on Day3-Day4, fresh culture medium is replaced every day;

Preferably, the third stage induction differentiation medium described in step (4) includes basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, fibroblast growth factor, insulin- Transferrin-Selenium, Ethanolamine, SANT-1, Retin Alcohol, BMP inhibitor, protein kinase C activator;

More preferably, the fibroblast growth factors include FGF-7, FGF-2, FGF-6, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF- 16. FGF-17, FGF-18, FGF-21, FGF-5, FGF-1, FGF-3, FGF-4, FGF-8, FGF-9, FGF-19, FGF-20;

Most preferably, the fibroblast growth factor is FGF-7;

More preferably, the BMP inhibitors include LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD161570;

Most preferably, the BMP inhibitor is LDN193189;

More preferably, the protein kinase C activator includes TPPB and PMA;

Most preferably, the protein kinase C activator is TPPB;

Most preferably, the concentrations of each component in the third stage induction differentiation medium are: (0.01-5)mM ascorbic acid, (10-200)ng/mL FGF-7, (0.01-5)μM SANT-1 , (0.01-5)μM retinol, (10-500)nM LDN193189, (50-500)nM TPPB, (0.01-10)% fetal bovine serum albumin, (0.01-10)g/L sodium bicarbonate , (1-100)mM glucose, (1-100)mM glutamine, (1-100)mg/L ethanolamine, (0.01-5)% insulin-transferrin-selenium;

Most preferably, the concentrations of each component in the third stage induction differentiation medium are: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.25μM SANT-1, 1μM retinol, 100nM LDN193189, 200nM TPPB, 2 % fetal bovine serum albumin, 2.5g/L sodium bicarbonate, 10mM glucose, 1mM glutamine, 1mg/L ethanolamine, 0.5% insulin-transferrin-selenium;

Most preferably, the time for inducing differentiation in the third stage is 2 days;

Most preferably, on the 1st to 2nd day of induction, the third stage induction differentiation medium is used to induce differentiation;

Most preferably, on Day 5-Day 6, fresh medium should be replaced every day.

Further, the fourth stage induction differentiation medium described in step (5) includes basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, fibroblast growth factor, and insulin-transferase. Ferritin - selenium, ethanolamine, SANT-1, retinol, BMP inhibitor, protein kinase C activator;

Preferably, the fibroblast growth factors include FGF-7, FGF-2, FGF-6, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16 , FGF-17, FGF-18, FGF-21, FGF-5, FGF-1, FGF-3, FGF-4, FGF-8, FGF-9, FGF-19, FGF-20;

More preferably, the fibroblast growth factor is FGF-7;

Preferably, the BMP inhibitors include LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD161570;

More preferably, the BMP inhibitor is LDN193189;

Preferably, the protein kinase C activator includes TPPB and PMA;

More preferably, the protein kinase C activator is TPPB;

Most preferably, the concentrations of each component in the fourth stage induction differentiation medium are: (0.01-5) mM ascorbic acid, (0.01-10) ng/mL FGF-7, (0.01-5) μM SANT-1 , (0.01-5)μM retinol, (50-500)nM LDN193189, (10-300)nM TPPB, (0.01-5)% fetal bovine serum albumin, (0.05-10)g/L sodium bicarbonate , (0.05-50)mM glucose, (0.01-5)mM glutamine, (0.01-5)mg/L ethanolamine, (0.01-5)% insulin-transferrin-selenium;

Most preferably, the concentrations of each component in the fourth stage induction differentiation medium are: 0.25mM ascorbic acid, 2ng/mL FGF-7, 0.25μM SANT-1, 0.1μM retinol, 200nM LDN193189, 100nM TPPB, 2% fetal bovine serum albumin, 2.5g/L sodium bicarbonate, 10mM glucose, 1mM glutamine, 1mg/L ethanolamine, 0.5% insulin-transferrin-selenium;

Most preferably, the time for inducing differentiation in the fourth stage is 3 days;

Most preferably, on days 1-3 of induction, the fourth stage induction differentiation medium is used to induce differentiation;

Most preferably, on Day7-Day9, fresh medium is replaced every day;

Preferably, the fifth stage induction differentiation medium described in step (6) includes basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, BMP inhibitor, thyroid hormone, ALK5 inhibitor, zinc sulfate;

More preferably, the BMP inhibitors include LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD161570;

Most preferably, the BMP inhibitor is LDN193189;

More preferably, the thyroid hormones include triiodothyronine (T3) and tetraiodothyronine (T4);

Most preferably, the thyroid hormone is triiodothyronine (T3);

More preferably, the ALK5 inhibitor includes ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-β receptor inhibitor V, TGF-β receptor inhibitor TGF-β receptor inhibitor I, TGF-β receptor inhibitor IV, TGF-β receptor inhibitor VII, TGF-β receptor inhibitor VIII, TGF-β receptor inhibitor II, TGF-β receptor inhibitor VI and TGF-β receptor inhibitor III;

Most preferably, the ALK5 inhibitor is ALK5i II;

Most preferably, the concentrations of each component in the fifth stage induction differentiation medium are: (0.01-5) μM SANT-1, (0.01-10) μM retinol, (10-500) nM LDN193189, ( 0.01-5)μM triiodothyronine (T3), (1-50)μM ALK5i II, (1-50)μM zinc sulfate, (0.05-10)% fetal bovine serum albumin, (0.05-10) g/L sodium bicarbonate, (1-50)mM glucose, (0.01-10)mM glutamine, (0.01-5)% insulin-transferrin-selenium, (0.01-10) mg/L ethanolamine;

Most preferably, the concentrations of each component in the fifth stage induction differentiation medium are: 0.25 μM SANT-1, 0.05 μM retinol, 100 nM LDN193189, 1 μM triiodothyronine (T3), 10 μM ALK5i II , 10μM zinc sulfate, 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20mM glucose, 1mM glutamine, 0.5% insulin-transferrin-selenium, 1mg/L ethanolamine;

Most preferably, the time for inducing differentiation in the fifth stage is 3 days;

Most preferably, on days 1-3 of induction, the fifth stage induction differentiation medium is used to induce differentiation;

Most preferably, from Day 10 to Day 12, fresh medium should be replaced every day.

Further, the sixth stage induction differentiation medium described in step (7) includes basal medium MCDB131, BMP inhibitor, thyroid hormone, ALK5 inhibitor, zinc sulfate, fetal bovine serum albumin, sodium bicarbonate, glucose, glutathione. Aminoamide, ethanolamine, gamma-secretinase inhibitor, erythrospongin A, hepatocyte growth factor, serotonin, β-cellulin, forskolin, exenatide 4;

Preferably, the BMP inhibitors include LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD161570;

More preferably, the BMP inhibitor is LDN193189;

Preferably, the thyroid hormones include triiodothyronine (T3) and tetraiodothyronine (T4);

More preferably, the thyroid hormone is triiodothyronine (T3);

Preferably, the ALK5 inhibitor includes ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-β receptor inhibitor V, TGF-β receptor Inhibitor I, TGF-β receptor inhibitor IV, TGF-β receptor inhibitor VII, TGF-β receptor inhibitor VIII, TGF-β receptor inhibitor II, TGF-β receptor inhibitor VI and TGF -β-receptor inhibitor III;

More preferably, the ALK5 inhibitor is ALK5i II;

Preferably, the γ-secretase inhibitors include GSi XX, GSi IX, GSi XI, GSi XII, GSi XIII, GSi XIV, GSi XVI, GSi BMS-708163, LY411575, LY3039478;

More preferably, the γ-secretase inhibitor is GSiXX;

Preferably, the sixth stage inducing differentiation medium includes the sixth stage inducing differentiation medium A, the sixth stage inducing differentiation medium B, the sixth stage inducing differentiation medium C, and the sixth stage inducing differentiation medium D;

More preferably, the sixth stage induction differentiation medium A contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodine Thyroidine (T3), ALK5i II, zinc sulfate, GSi XX, β-cellulin, erythrospongin A;

More preferably, the sixth stage induction differentiation medium B contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodine Thyroidine (T3), ALK5i II, zinc sulfate, GSi XX, β-cellulin;

More preferably, the sixth stage induction differentiation medium C contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodine Thyroidine (T3), ALK5i II, zinc sulfate, GSi XX, β-cellulin, forskolin, exenatide 4;

More preferably, the sixth stage induction differentiation medium D contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX, insulin-transferrin-selenium, β-cellulin, forskolin, exenatide 4, hepatocyte growth factor, serotonin;

Most preferably, the concentrations of each component in the sixth stage induction differentiation medium are: (10-500) nM LDN193189, (0.01-10) μM triiodothyronine (T3), (1-50) μM ALK5i II, (1-50)μM zinc sulfate, (0.05-10)% fetal bovine serum albumin, (0.01-5)g/L sodium bicarbonate, (1-100)mM glucose, (0.01-5) mM glutamine, (0.01-5)mg/L ethanolamine, (50-300)nM GSi XX, (0.01-5)μM erythrospongin A, (10-500)ng/mL hepatocyte growth factor, (1 -50)μM serotonin, (1-50)ng/mL β-cellulin, (1-50)μM forskolin, (10-500)ng/mL exenatide 4;

Most preferably, the concentrations of each component in the sixth stage induction differentiation medium are: 100 nM LDN193189, 1 μM triiodothyronine (T3), 10 μM ALK5i II, 10 μM zinc sulfate, and 2% fetal bovine serum albumin. , 1.5g/L sodium bicarbonate, 20mM glucose, 1mM glutamine, 1mg/L ethanolamine, 100nM GSi XX, 1μM erythrospongin A, 50ng/mL hepatocyte growth factor, 10μM serotonin, 20ng/mL β-cellulin, 10μM forskolin, 50ng/mL exenatide 4;

Most preferably, the time for inducing differentiation in the sixth stage is 14 days;

Most preferably, on the first day of induction, the sixth stage induction differentiation medium A is used to induce differentiation;

Most preferably, on days 2-7 of induction, the sixth stage induction differentiation medium B is used to induce differentiation;

Most preferably, on the 8th to 9th day of induction, the sixth stage induction differentiation medium C is used to induce differentiation;

Most preferably, on the 10th to 14th day of induction, the sixth stage induction differentiation medium D is used to induce differentiation;

Most preferably, from Day 13 to Day 27, change the medium half every day;

Preferably, the seventh stage induction differentiation medium described in step (8) includes the seventh stage induction differentiation medium A and the seventh stage induction differentiation medium B;

More preferably, the seventh stage induction differentiation medium A contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, thyroid hormone, ALK5 Inhibitors, zinc sulfate, N-acetyl-L-cysteine, porcine intestinal mucosal heparin sodium, water-soluble vitamin E, AXL inhibitor, β-cellulin, forskolin, exenatide 4, hepatocyte growth factor , 5-hydroxytryptamine;

Most preferably, the thyroid hormones include triiodothyronine (T3) and tetraiodothyronine (T4);

Most preferably, the thyroid hormone is triiodothyronine (T3);

Most preferably, the ALK5 inhibitor includes ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-β receptor inhibitor V, TGF-β receptor inhibitor TGF-β receptor inhibitor I, TGF-β receptor inhibitor IV, TGF-β receptor inhibitor VII, TGF-β receptor inhibitor VIII, TGF-β receptor inhibitor II, TGF-β receptor inhibitor VI and TGF-β receptor inhibitor III;

Most preferably, the ALK5 inhibitor is ALK5i II;

Most preferably, the AXL inhibitor includes R428, BMS-907351, BMS-777607, XL184, TP-0903, XL092, LDC1267, LY2801653, CEP-40783, RU-301, S49076, ONO-7475, Ningetinib;

Most preferably, the AXL inhibitor is R428;

Most preferably, the concentrations of each component in the seventh stage induction differentiation medium A are: (0.05-10)% fetal bovine serum albumin, (0.05-10) g/L sodium bicarbonate, (5-50) )mM glucose, (0.01-5)mM glutamine, (0.01-5)mg/L ethanolamine, (0.01-2.5)% insulin-transferrin-selenium, (0.01-5)μM triiodothyronine (T3), (0.01-50)μM ALK5i II, (1-50)μM zinc sulfate, (0.01-5)mM N-acetyl-L-cysteine, (1-50)μg/mL porcine intestinal mucosa Heparin sodium, (1-50)μM water-soluble vitamin E, (1-50)μM R428, (10-100)ng/mL hepatocyte growth factor, (1-50)μM serotonin, (1-50)ng /mL β-cellulin, (1-50) μM forskolin, (10-100) ng/mL exenatide 4;

Most preferably, the concentrations of each component in the seventh stage induction differentiation medium A are: 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20mM glucose, 1mM glutamine, and 1mg/L ethanolamine. , 0.5% insulin-transferrin-selenium, 1μM triiodothyronine (T3), 10μM ALK5i II, 10μM zinc sulfate, 1mM N-acetyl-L-cysteine, 10μg/mL porcine intestinal mucosal heparin sodium , 10μM water-soluble vitamin E, 2μM R428, 50ng/mL hepatocyte growth factor, 10μM serotonin, 20ng/mL β-cellulin, 10μM forskolin, 50ng/mL exenatide 4;

More preferably, the seventh stage induction differentiation medium B contains Ham's F-12 medium, medium 199, glutamine, calcium chloride dihydrate, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid , fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, water-soluble vitamin E, nicotinamide, heparin sodium, deoxyribonuclease I, necroptosis inhibitor, serine protease inhibitor;

Most preferably, the necroptosis inhibitor includes Necrostatin-1 and Necrostatin-2;

Most preferably, the necroptosis inhibitor is Necrostatin-1;

Most preferably, the serine protease inhibitor includes Pefabloc, Benzamidine, MBTI, PMSF, LBTI;

Most preferably, the serine protease inhibitor is Pefabloc;

Most preferably, the concentrations of each component in the seventh stage induction differentiation medium B are: 50% Ham's F-12 medium, 50% medium 199, (0.01-10)mM glutamine, (0.01-10)mM Calcium chloride dihydrate, (1-50)mM N-2hydroxyethylpiperazine-N-2-ethanesulfonate Acid, (0.01-5)% fetal bovine serum albumin, (0.01-2) mL/L insulin-transferrin-selenium, (0.01-5) mg/L ethanolamine, (1-50) μM water-soluble vitamin E , (1-50)mM nicotinamide, (1-50)μg/mL heparin sodium, (0.01-2.5)U/mL deoxyribonuclease I, (50-500)μM Necrostatin-1, (0.001-2) μM Pefabloc;

Most preferably, the concentrations of each component in the seventh stage induction differentiation medium B are: 50% Ham's F-12 medium, 50% medium 199, 2mM glutamine, 2.5mM calcium chloride dihydrate, 10mM N- 2hydroxyethylpiperazine-N-2-ethanesulfonic acid, 2% fetal bovine serum albumin, 0.6mL/L insulin-transferrin-selenium, 1mg/L ethanolamine, 10μM water-soluble vitamin E, 10mM nicotinamide , 10μg/mL heparin sodium, 1U/mL deoxyribonuclease I, 100μM Necrostatin-1, 0.1μM Pefabloc;

Most preferably, the seventh stage differentiation induction time is 14 days;

Most preferably, on days 1-8 of induction, the seventh stage induction differentiation medium A is used to induce differentiation;

Most preferably, on the 9th to 11th day of induction, the seventh stage induction differentiation medium B is used to induce differentiation;

Most preferably, from Day 28 to Day 40, change the medium half every day.

The fourth aspect of the present invention provides a functionally mature pancreatic islet β cell or cell population derived from iPSCs.

Further, the cells or cell populations are obtained by inducing differentiation using the method described in the third aspect of the present invention;

Preferably, the islet beta cells or cell population are functional, stable, mature islet beta cells or cell populations;

Preferably, the single positive rate of GCG-/INS+ cells in the islet β cells or cell population is 95%.

A fifth aspect of the invention provides a pharmaceutical composition for treating and/or preventing diabetes.

Further, the pharmaceutical composition includes the cells or cell populations described in the fourth aspect of the present invention;

Preferably, the pharmaceutical composition also contains pharmaceutically acceptable carriers and/or excipients;

Preferably, the diabetes includes type 1 diabetes, type 2 diabetes, special types of diabetes, and gestational diabetes;

More preferably, the diabetes is type 1 diabetes.

The sixth aspect of the present invention provides applications in any of the following aspects:

(1) Application of β-cellulin, forskolin, exenatide 4, hepatocyte growth factor and serotonin in combination to induce differentiation of iPSCs into functional pancreatic islet β cells;

(2) The application of the differentiation-inducing agent described in the first aspect of the present invention in the preparation of differentiation-inducing medium for inducing iPSCs to differentiate into functional pancreatic islet β cells;

(3) The use of the differentiation-inducing agent described in the first aspect of the present invention in inducing the differentiation of iPSCs into functional pancreatic islet β cells;

(4) The application of the differentiation medium described in the second aspect of the present invention in inducing the differentiation of iPSCs into functional islet β cells;

(5) The use of the cells or cell populations described in the fourth aspect of the present invention in the preparation of drugs for treating and/or preventing diabetes;

(6) Application of the pharmaceutical composition according to the fifth aspect of the present invention in the treatment and/or prevention of diabetes;

Preferably, the diabetes includes type 1 diabetes, type 2 diabetes, special types of diabetes, and gestational diabetes;

More preferably, the diabetes is type 1 diabetes.

Compared with the existing technology, the present invention has the following advantages and beneficial effects:

The invention provides a method for differentiation of induced pluripotent stem cells into pancreatic islets and its application in the treatment of type I diabetes. The method uses the small molecule erythrospongin A that inhibits skeleton proteins on the 13th day, and on the 20th day β-cellulin, forskolin, and exenatide 4 were used, in the 22nd On day 35, hepatocyte growth factor HGF and serotonin were used, and on day 35, 50% Ham's F-12 medium, 50% medium 199, glutamine, calcium chloride dihydrate, and N-2 hydroxyethylpiperazine- N-2-Ethanesulfonic acid, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, water-soluble vitamin E, nicotinamide, heparin, deoxyribonuclease I, necroptosis inhibitor Necrostatin-1 and the serine protease inhibitor Pefabloc. The method can significantly improve the induction differentiation efficiency of iPSCs, promote the maturation of insulin-secreting cells in vitro, and can significantly increase the single-positive rate of GCG-/INS+ cells. Through this method, a large number of functional cells can be obtained. mature, stable islet β cells.

Description of the drawings

Figure 1 shows the results of cell seeding area screening and Matrigel gel concentration screening. Figure A: the results obtained by seeding in 12-well plates, 6-well plates, and 10cm culture dishes respectively. Figure B: the Matrigel gel concentration screening results. , Picture C: Statistical chart of Matrigel concentration screening results;

Figure 2 shows the detection results of the first stage of definitive endoderm induction. Picture A: qPCR detection of negative control group (iPSC, iPS), Activin A group (Act-A) and GDF-8 group (GDF-8). Statistical chart showing the expression levels of endoderm markers SOX17, FOXA2 and CRCX4. Panel B: Results of immunofluorescence detection of the positive rate of endoderm marker FOXA2 in Activin A group (Act-A) and GDF-8 group (GDF-8). picture;

Figure 3 shows the detection results of the second stage of gastrointestinal tube stage induction. Picture A: the results of immunofluorescence detection of the positive rate of the primitive intestinal tube markers HNF1β and FOXA2. Picture B: the positive rate of the primitive intestinal tube markers HNF1β and FOXA2 detected by qPCR. The result statistics chart;

Figure 4 shows the detection results of the third stage of post-foregut stage induction. Figure A: Immunofluorescence detection of the positive rate of markers PDX-1 and SOX9 in the post-foregut stage. Figure B: qPCR detection of post-foregut stage. Statistical chart showing the positive rates of markers PDX-1 and SOX9;

Figure 5 is a graph showing the detection results of induction of pancreatic progenitor cells in the fourth stage. Figure A: Immunofluorescence detection of the positive rate of post-foregut stage markers PDX-1 and SOX9. Figure B: qPCR detection of post-foregut stage. Statistical chart showing the positive rates of markers PDX-1 and SOX9;

Figure 6 shows the detection results of the fifth stage pancreatic endocrine progenitor cell stage induction, in which the pancreatic endocrine progenitor cell markers NKX6.1 and NGN3 in the control group (iPSC, iPS) and the fifth stage induction group (Islet) were detected by qPCR. , detect the expression level of PDX-1mRNA;

Figure 7 shows the detection results of the sixth stage of pancreatic endocrine cell stage induction and the seventh stage of mature islet cell stage induction. Picture A: qPCR detection of islet cell markers INS, Statistical chart of the expression levels of GCG and NKX6.1 mRNA. Panel B: Results of immunofluorescence detection of the positive rates of islet cell markers INS, GCG, NKX6.1, and MAFA in Group A and Group B.

Detailed ways

The present invention will be further described below with reference to specific examples, which are only used to explain the present invention and cannot be understood as limiting the present invention. Those of ordinary skill in the art can understand that various changes, modifications, substitutions and modifications can be made to these embodiments without departing from the principles and purposes of the invention. The scope of the invention is defined by the claims and their equivalents. . The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Example 1 Method for inducing differentiation of iPSCs into pancreatic islets

1. Experimental reagents and experimental materials

The experimental reagents and experimental materials involved in the embodiments of the present invention are shown in Table 1 and Table 2 respectively.

Table 1 Experimental reagents

Table 2 Experimental materials

2. iPSCs recovery

The induced pluripotent stem cells (iPSCs) described in the present invention are derived from Beijing Yuchennuo Medical Technology Co., Ltd. and are prepared using the iPSCs preparation method described in the company's previously applied patent 201910110768.7.

(1) According to the amount of culture medium required for recovery, prepare E8 complete culture medium containing ROCKi, add 1 μL ROCKi storage solution to each ml of culture medium, and preheat.

(2) Prepare 10-15mL of E8 complete culture medium and preheat it at 37°C.

(3) Take out a cryopreservation tube from the liquid nitrogen tank, immediately put it into a 38-39°C hot water bucket, and shake it back and forth for 60-90 seconds to completely melt the frozen cell suspension.

(4) Once the cells in the cryopreservation tube are completely melted (liquid state), immediately take them out of the hot water bucket, thoroughly disinfect the surface of the cryopreservation tube with 75% alcohol, and place it in a clean workbench. When the frozen tube is about to thaw, sterilize the centrifuge tube containing E8 culture medium that has been preheated to 37°C with alcohol and place it on a clean workbench.

(5) Under strict aseptic operating conditions, take out the cell suspension from the cryopreservation tube, inject preheated E8 culture base into a 15mL centrifuge tube, gently pipet 2-3 times, centrifuge at 200g×5min, and discard the supernatant after centrifugation. .

(6) Add ROCKi-containing culture medium, gently pipet 2-3 times, and take out the Matrigel-coated well plate/culture bottle from the incubator. Aspirate away the coating solution and gently add the cell suspension along the uncoated side (do not add directly to the coating layer). Place the culture dish in the incubator for culture.

Note: During recovery, choose the container for inoculation according to the number of cells. Generally, 500,000-800,000 cells are inoculated in a 6-well plate, and 800,000-1 million cells are inoculated in T25 bottles. 1 million to 2 million cells are inoculated into T75 bottles.

3. Passaging of iPSCs

(1) Prepare 0.5mM EDTA working solution: Pipette 5mL of DPBS into a new 15mL centrifuge tube, then add 5μL of 0.5M EDTA stock solution, and mix to create a 0.5mM EDTA working solution. The digestive juice is ready for use.

(2) According to the amount of culture medium required for passaging, prepare E8 complete culture medium containing ROCKi, and add 1 μL ROCKi storage solution per ml of culture medium.

(3) Take out the well plate/culture bottle to be passaged from the incubator, discard the supernatant, and wash it twice with DPBS (the amount of DPBS used each time is not less than the amount of the original culture medium), 1 minute each time (when washing, the Leave DBPS in the well/bottle for 30-45 seconds before aspirating).

(4) After adding EDTA working solution, add about 6mL EDTA working solution to the T75 bottle, place it in the incubator and incubate for 5 minutes. During this period, you can observe it under the microscope. When all the cells in the colony begin to become round and no larger clumps fall off, gently remove the EDTA working solution. Aspirate off the EDTA without disturbing the cells.

(5) Add E8 complete culture medium to terminate the digestion. When it is terminated, it is advisable to add the culture medium quickly to make the iPSCs fall off in the form of clumps. Do not pipet the cells repeatedly to avoid blowing the cells into individual cells, and try to keep the cells in cell clumps.

(6) After balancing, centrifuge at 200g for 5 minutes. After centrifugation, discard the supernatant, gently shake the bottom of the centrifuge tube, add 5 mL of E8 complete culture medium containing ROCKi to resuspend, and gently pipette the cell pellet with a Pasteur dropper (do not pipette). More than 3 times), inoculate the cell suspension into a T25 bottle coated with Matrigel, and place the culture bottle in a 37°C, 5% _CO2 incubator for static culture.

(7) After 24 hours of culture, replace the entire medium with fresh E8 complete medium.

(8) Thereafter, complete medium replacement operations were performed every day, and passage was performed when the cell confluence was about 70%-80%.

(9) The number of iPSCs cells to be seeded is about 8000 cells/cm ² . According to different iPSCs lines, the passage density can be adjusted so that the iPSCs cell passage interval is 5-7 days.

4. Prepare 60x Matrigel-coated cell culture plate

(1) Pre-cool the 12-well plate and 25mL DMEM\F12 culture medium at -20°C for about 20 minutes in advance.

(2) In the ice box, use a hemostatic forceps to open the 1.5mL EP tube containing Matrigel. Pay attention to aseptic operation and do not touch it with your hands to prevent rewarming.

(3) Add 400 μL of pre-cooled coating solution into the EP tube containing Matrigel. After repeated pipetting, suck the supernatant back into the pre-cooled centrifuge tube. This process needs to be repeated several times to make the Matrigel dissolve quickly and do not rewarm.

(4) Take out the pre-cooled object to be coated and place it on the ice box. Use an electric pipette to mix the coating solution, and then use a suitable measuring device to coat in the following volume: 12-well plate: 1 mL per well.

(5) Place the coated culture bottle/plate in a 37°C, 5% CO ₂ incubator and incubate overnight.

5. Screening of cell seeding area

Figure 1 shows that the higher the rate of positive cells in the cell protrusion. By screening different culture areas, including: 12-well plates, 6-well plates, and 10cm culture dishes, and seeding them separately, the results show that the larger the culture area, the harder it is for cells to To form a ball, the 12-well plate has the best effect (see Figure 1A).

6. Matrigel concentration screening

12-well plate, Matrigel concentration 0.05-0.1mg/mL (Group A), 12-well plate, Matrigel concentration 0.1-0.2mg/mL (Group B). In the initial plating process, different gel concentrations were used in Group A and Group B respectively. The islet production was observed at the end of induction. The results showed that when the Matrigel concentration was 0.1-0.2 mg/mL, the induction production was the highest (see Figure 1B and Figure 1C). At this point, the present invention has formed an optimal initial induction method, that is, using Add 0.1-0.2mg/mL Matrigel to the 12-well plate for induction.

7. Establishment of iPSC monolayer differentiation (Day-3 to Day-1)

This stage consists of 2 mL E8 complete medium, 10 μM Y27632. Remove the plated cells from the 37°C, 5% _CO2 cell culture incubator Matrigel-coated 12-well plate, remove the liquid, and add 2mL E8Medium+10μM Y27632 to each well;

The amplification time is 3 days. On the 1st day of amplification, the concentration of Y27632 in the culture medium is 10 μM; on the 2nd to 3rd days of induction, the culture medium only replaces E8 complete culture medium; fresh culture medium is replaced every day.

8. The first stage: induction of definitive endoderm (Day0-2)

The first stage is the differentiation of iPSCs into definitive endoderm cells. The first stage induction differentiation medium consists of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), Activin A and CHIR-99021.

In this medium, the concentration of Activin A is 100ng/mL, the concentration of CHIR-99021 is 3μM, the concentration of fetal bovine serum albumin is 0.5%, the concentration of sodium bicarbonate is 1.5g/L, and the final concentration of glucose is 10mM, the concentration of glutamine is 1mM.

The first stage of induction lasts for 3 days. On the first day of induction, the concentration of Activin A in the culture medium is 100ng/mL; the concentration of CHIR-99021 is 3 μM; on the 2nd to 3rd day of induction, the concentration of Activin A in the culture medium is 100ng/mL; The concentration is 100ng/mL; that is, on the first day of induction, use the first-stage induction differentiation medium A, which consists of the basic medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), and glucose. , defatted BSA (fetal bovine serum albumin), Activin A and CHIR-99021; on the 2-3rd day of induction, use the first-stage induction differentiation medium B, which is composed of the basic medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin) and Activin A. Change the medium daily with fresh medium.

In this stage, the GDF-8 group was defined as group B, and Activin A (Act-A) was defined as group A. The positive rate of endodermal cell markers OX17 and FOXA2 obtained after induction was detected by qPCR.

9. The second stage: induction of gastrut tube stage (Day3-4)

The second stage is the differentiation of definitive endoderm cells into gastrula tube cells. The second stage induction differentiation medium consists of basic medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), and ascorbic acid. and FGF-7.

In this culture medium, the concentration of ascorbic acid is 0.25mM, the concentration of FGF-7 is 50ng/mL, the concentration of fetal bovine serum albumin is 0.5%, the concentration of sodium bicarbonate is 1.5g/L, and the final concentration of glucose is 10mM, the concentration of glutamine is 1mM.

The second stage of induction lasted for 2 days. On the 1st and 2nd days of induction, fresh medium was replaced every day.

10. The third stage: induction of posterior foregut stage (Day5-6)

The third stage is the differentiation of gastrointestinal tube cells into posterior foregut cells. The third stage induction differentiation medium consists of basic medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), Composed of ascorbic acid, FGF-7, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189 and TPPB.

In this medium, the concentration of ascorbic acid is 0.25mM, the concentration of FGF-7 is 50ng/mL, the concentration of SANT-1 is 0.25μM, the concentration of retinol is 1μM, the concentration of LDN193189 is 100nM, and the concentration of TPPB is 200nM, fetal bovine serum albumin concentration 2%, sodium bicarbonate concentration 2.5g/L, glucose final concentration 10mM, glutamine concentration 1mM, ethanolamine concentration 1mg/L, insulin-transduced The concentration of ferritin-selenium is 0.5%.

The third stage of induction lasted for 2 days. On the 1st and 2nd days of induction, fresh medium was replaced every day.

11. The fourth stage: induction of pancreatic progenitor cell stage (Day7-9)

The fourth stage is the differentiation of posterior foregut cells into pancreatic progenitor cells. The fourth stage induction differentiation medium consists of basic medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), Composed of ascorbic acid, FGF-7, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189, and TPPB.

In this medium, the concentration of ascorbic acid is 0.25mM, the concentration of FGF-7 is 2ng/mL, the concentration of SANT-1 is 0.25μM, the concentration of retinol is 0.1μM, the concentration of LDN193189 is 200nM, and the concentration of TPPB is is 100nM, and the concentration of fetal bovine serum albumin is 2%, the concentration of sodium bicarbonate is 2.5g/L, the final concentration of glucose is 10mM, the concentration of glutamine is 1mM, the concentration of ethanolamine is 1mg/L, and the concentration of insulin-transferrin-selenium is 0.5%.

The fourth stage of induction lasted for 3 days. On days 1-3 of induction, fresh medium was replaced every day.

12. The fifth stage: induction of pancreatic endocrine progenitor cell stage (Day10-12)

The fifth stage is the differentiation of pancreatic progenitor cells into pancreatic endocrine progenitor cells. The fifth stage induction differentiation medium consists of basic medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), Insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189, thyroid hormone (T3), ALK5i II, zinc sulfate.

In this medium, the concentration of SANT-1 is 0.25 μM, the concentration of retinol is 0.05 μM, the concentration of LDN193189 is 100 nM, the concentration of thyroid hormone (T3) is 1 μM, the concentration of ALK5i II is 10 μM, and the concentration of zinc sulfate is The concentration is 10 μM, the concentration of fetal bovine serum albumin is 2%, the concentration of sodium bicarbonate is 1.5g/L, the final concentration of glucose is 20mM, the concentration of glutamine is 1mM, and the concentration of insulin-transferrin-selenium is 0.5%, the concentration of ethanolamine is 1mg/L.

The fifth stage of induction lasted for 3 days. On days 1-3 of induction, fresh medium was replaced every day.

13. Stage Six: Induction of Pancreatic Endocrine Cell Stage (Day13-27)

The sixth stage is the differentiation of pancreatic endocrine progenitor cells into pancreatic endocrine cells. The induction time at this stage is 14 days. The preparations for the first day of induction are as follows:

(1) Add 500 μL of anti-low adsorption liquid to the 24-well 400 μM microplate. Balance and centrifuge at 1300g for 5 minutes; remove air bubbles. If there are still air bubbles in the micropores, centrifuge again and discard the liquid;

(2) Use basal culture medium or DPBS to rinse each well, 2mL of the 24-well plate, and discard the liquid when using;

(3) Configure TD digestion solution Tryple: DPBS (TD) = 1:1;

(4) Wash the cells once with DPBS, digest them with TD digestion solution for 5 minutes, add 500 μL digestion solution to 1 well, digest 1 well first, use M3 main culture to terminate, then count, estimate the total number of cells, 400 μM micropores of 24-well plate There are about 1500-2000 cells in each microwell of the plate;

(5) After digesting all the cells in the 12-well plate, quickly use a suction pump to aspirate the digested liquid, use a pipette tip to absorb the main culture well one by one, and then collect it into a 50mL centrifuge tube, centrifuge for 5 minutes, and then use the complete culture The base was resuspended and seeded into an anti-adsorption-coated microplate, 1 mL per well, and placed in an incubator to settle for 20 minutes, 300g, and 5 minutes. After centrifugation, place it in an incubator and change the medium half a day every day for 1-14 days.

The sixth stage induction differentiation medium of group A at this stage from days 1 to 14 consists of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), insulin-transferrin Protein - selenium, ethanolamine, LDN193189, thyroid hormone (T3), ALK5i II, zinc sulfate, gamma-secretinase inhibitor;

The sixth stage induction differentiation medium on day 1 of Group B at this stage consists of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), insulin-transferrin- Composed of selenium, ethanolamine, LDN193189, thyroid hormone (T3), ALK5i II, zinc sulfate, γ-secretinase inhibitor, β-cellulin, and rhospongin A;

The sixth stage induction differentiation medium on days 2-7 of group B was the same as on day 1, but did not contain erythromongin A;

The sixth stage induction differentiation medium of group B on days 8-9 consisted of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), insulin-transferrin- Composed of 4 components: selenium, ethanolamine, LDN193189, thyroid hormone (T3), ALK5i II, zinc sulfate, γ-secretinase inhibitor, β-cellulin, forskolin, and exenatide;

The sixth stage induction differentiation medium of group B on days 10-14 consists of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), ethanolamine, LDN193189, thyroid hormone (T3), ALK5i II, zinc sulfate, gamma-secretinase inhibitor, insulin-transferrin-selenium, β-cellulin, forskolin, exenatide 4, hepatocyte growth factor, and serotonin.

In this medium, the concentration of LDN193189 is 100 nM, the concentration of thyroid hormone (T3) is 1 μM, the concentration of ALK5i II is 10 μM, the concentration of zinc sulfate is 10 μM, the concentration of fetal bovine serum albumin is 2%, and the concentration of sodium bicarbonate is The concentration of glucose is 1.5g/L, the final concentration of glucose is 20mM, the concentration of glutamine is 1mM, the concentration of ethanolamine is 1mg/L, the concentration of γ-secretinase inhibitor is 100nM, and the concentration of rhospongin A is 1μM, the concentration of hepatocyte growth factor is 50ng/mL, the concentration of serotonin is 10μM, the concentration of β-cellulin is 20ng/mL, the concentration of forskolin is 10μM, and the concentration of exenatide 4 is 50ng/mL.

14. Stage 7: Induction of mature islet cell stage (Day28-40)

The seventh stage is the differentiation of pancreatic endocrine cells into mature islet cells.

The seventh stage induction differentiation medium of group A at this stage from days 1 to 11 consists of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), insulin-transferrin Protein - composed of selenium, ethanolamine, thyroid hormone, ALK5i II, zinc sulfate, N-acetyl-L-cysteine, porcine intestinal mucosal heparin sodium, water-soluble vitamin E, AXL inhibitor R428, and the medium is changed half a day.

The seventh stage induction differentiation medium of group B on days 1-8 of this stage consists of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin), insulin-transferrin Protein - selenium, ethanolamine, thyroid hormone, ALK5i II, zinc sulfate, N-acetyl-L-cysteine, porcine intestinal mucosal heparin sodium, water-soluble vitamin E, AXL inhibitor R428, β-cellulin, forskolin, It is composed of exenatide 4, hepatocyte growth factor, and serotonin, and the medium is changed half a day.

In this medium, the concentration of fetal bovine serum albumin is 2%, the concentration of sodium bicarbonate is 1.5g/L, the final concentration of glucose is 20mM, the concentration of glutamine is 1mM, and the concentration of ethanolamine is 1mg/L , the concentration of insulin-transferrin-selenium is 0.5%, the concentration of thyroid hormone is 1μM, the concentration of ALK5i II is 10μM, the concentration of zinc sulfate is 10μM, the concentration of N-acetyl-L-cysteine is 1mM, The concentration of heparin sodium in porcine intestinal mucosa is 10μg/mL, the concentration of water-soluble vitamin E is 10μM, the concentration of AXL inhibitor R428 is 2μM, the concentration of hepatocyte growth factor is 50ng/mL, the concentration of 5-hydroxytryptamine is 10μM, β-cell The concentration of forskolin was 20 ng/mL, the concentration of forskolin was 10 μM, and the concentration of exenatide 4 was 50 ng/mL.

The seventh stage induction differentiation medium of group B on days 9-11 of this stage consists of 50% Ham's F-12 medium, 50% medium 199, glutamine, calcium chloride dihydrate, N-2 hydroxyethylpiperazine-N -2-Ethanesulfonic acid, defatted BSA (fetal bovine serum albumin) (BSA), insulin-transferrin-selenium, ethanolamine, water-soluble vitamin E, nicotinamide, heparin, deoxyribonuclease I, necrotizing apoptosis It is composed of apoptosis inhibitor Necrostatin-1 and serine protease inhibitor Pefabloc, and the medium is changed half a day.

In this culture medium, the concentration of glutamine is 2mM, the concentration of calcium chloride dihydrate is 2.5mM, the concentration of N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid is 10mM, and the concentration of defatted BSA ( The concentration of fetal bovine serum albumin (BSA) is 2%, the concentration of insulin-transferrin-selenium is 0.6mL/L, the concentration of ethanolamine is 1mg/L, the concentration of water-soluble vitamin E is 10μM, and the concentration of nicotinamide The concentration is 10mM, the concentration of heparin sodium is 10μg/mL, the concentration of deoxyribonuclease I is 1U/mL, the concentration of necroptosis inhibitor Necrostatin-1 is 100μM, and the concentration of serine protease inhibitor Pefabloc is 0.1μM.

Example 2 qPCR detection of the expression of marker gene mRNA in each induction differentiation stage, and immunofluorescence detection of the cell positivity rate in each induction differentiation stage

1. Experimental methods

1.1 RNA extraction

(1) Add 500 μL Trizol solution to the islet cell sample collected above, lyse it at room temperature for 5 minutes, add 200 μL chloroform, shake vigorously and mix for 15 seconds, and incubate at room temperature for 3 minutes;

(2) Centrifuge at 4°C and 12,000 rpm for 10 minutes. The sample will be divided into three layers: the lower organic phase, the middle layer and the upper colorless aqueous phase. RNA is present in the aqueous phase. The capacity of the water phase layer is approximately 60% of the added RL volume. Transfer the water phase to a new tube and proceed to the next step;

(3) Add 1 times the volume of 70% ethanol (please check whether absolute ethanol has been added first!), invert and mix (precipitation may occur at this time). The obtained solution and possible precipitates are transferred to the adsorption column RA and centrifuged at 10,000 rpm for 45 seconds. The waste liquid is discarded and the adsorption column is put back into the collection tube;

(4) Add 500 μL of protein-removing solution RE, centrifuge at 12,000 rpm for 45 seconds, and discard the waste liquid;

(5) Add 700 μL of rinse solution RW (please check whether absolute ethanol has been added first!), centrifuge at 12,000 rpm for 60 seconds, and discard the waste liquid;

(6) Add 500 μL of rinse solution RW, centrifuge at 12,000 rpm for 60 seconds, and discard the waste liquid;

(7) Put the adsorption column RA back into the empty collection tube, centrifuge at 12,000 rpm for 2 minutes, and try to remove the rinse liquid to prevent residual ethanol in the rinse liquid from inhibiting the downstream reaction;

(8) Take out the adsorption column RA and put it into an RNase free centrifuge tube. Add 50 μL RNase free wate to the middle of the adsorption membrane according to the expected RNA yield (incubate at 65-70°C in advance);

(9) Leave at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute. If more RNA is needed, add the obtained solution back to the centrifugal adsorption column, centrifuge for 1 minute, or add an additional 30 μL RNase free water, centrifuge for 1 minute, and combine the two eluates;

(10) The larger the elution volume, the higher the elution efficiency. If a higher RNA concentration is required, the elution volume can be appropriately reduced, but the minimum volume is preferably not less than 50 μL. If the volume is too small, the RNA elution efficiency will be reduced and the RNA will be reduced. Yield.

1.2 Reverse transcription

(1) Calculate based on total RNA concentration, and invert based on 1 μg concentration;

(2) Premix the reagents and total RNA according to the reaction system described in Table 3 below;

Table 3 Reaction system

(3) Transfer the premix to the PCR machine and use the cDNA template to perform the reaction (incubate at 42°C for 30 minutes and 85°C for 5 seconds);

(4) Quickly transfer the reversed cDNA and place it on ice for 1 minute to cool down;

(5) Store at -20°C and dilute to the required concentration before use. (Note: Before placing it in the PCR machine, check whether the centrifuge tube is tightly covered to prevent high temperature evaporation; after taking it out of the PCR machine, check whether the centrifuge tube cover has collapsed due to high temperature.

1.3 qPCR experimental methods

1.3.1 Primer test

(1) Primers designed based on DNA need to be tested by qPCR for specificity and amplification efficiency before the formal experiment. The specific reaction system and reaction conditions are as in the formal experiment, and each pair of primers needs to be compared with template water.

(2) After obtaining the results, first judge the primer specificity based on the melting curve. The selection criteria are: single peak and narrow peak shape, and no obvious primer-dimer melting curve peak in the water control. If the melting curves of multiple pairs of primers designed all show good specificity, the amplification curves of each primer should be compared, and primers with small Ct values and high amplification efficiency should be selected first for formal experiments. The detailed information of primer sequences for qPCR detection genes is shown in Table 4.

Table 4 Primer sequence information for qPCR detection genes

1.3.2 Sample discharge

(1) The addition of a sample should be arranged in the same row as much as possible;

(2) If the three repetitions of the formal experiment cannot be arranged on the same plate, the experiments in the three repetitions need to be separated, but the same repeated experiment cannot be separated into two plates.

1.3.3 Formal experiment

(1) Premix the reaction system shown in Table 5 below;

Table 5 Reaction system

(2) Add the premixed reagent into eight consecutive tubes, 18 μL per well;

(3) Quickly add 2μL of cDNA to the eight-serial tube, cover the lid, and place each row of eight-serial tubes on a handheld centrifuge for a few seconds;

(4) Put it into the Light cycler instrument and carry out the reaction according to the 3-step method, with the number of cycles being 40;

The template is shown in Table 6 below;

Table 6 Template

Note: cDNA is diluted 1:4 with sterilized pure water. If you encounter samples with low gene expression, cDNA can be added to the newly prepared reaction system after the cDNA is arranged in a certain order. After adding the sample, cover the eight-tube cap and mark the sequence 1-12 on the uppermost edge of the eight-tube cap (do not write the mark on the lid of the reaction tube. Avoid touching the middle of the eight-tube cap with bare hands. A transparent fluorescence collection area, and ensure that each well is tightly capped, otherwise reproducibility will be affected or melting curve peak drift may occur.

1.3.4 Getting on the computer

Turn on the power switch of the qPCR instrument; open the sample rack, put in eight consecutive tubes, close the sample rack, and set the template; click the Start button to start running the program; after the program is finished, take out the eight consecutive tubes on the sample rack; mark on the software Know the sample name of each reaction well and the name of the detected gene, and classify and save the result files; after the reaction is completed, the eight consecutive tubes should be placed in a sealed bag for storage.

1.4 Immunofluorescence

(1) Wash the cells at each stage for identification three times with DPBS, fix them with 4% PFA at room temperature for 40 minutes, and protect them from light (PFA returns to room temperature before use);

(2) Wash three times with DPBS, 0.5% TritonX-100 (DPBS dissolved), and punch for 20 minutes;

(3) 5% BSA blocking solution + 0.1% TritonX-100, blocking overnight at 4°C;

Prepare solution: PBST-1 (for cleaning): DPBS+0.1% TritonX-100; PBST-2 (for dissolving primary and secondary antibodies): DPBS+0.1% TritonX-100+1% BSA;

Incubate with primary antibody, primary antibody: PBST-2=1:200, overnight at 4°C;

Recover the primary antibody solution and wash it three times on PBST-1 shaker, 10 minutes each time;

Incubate with secondary antibody, secondary antibody: PBST-2=1:500, overnight at 4°C, protected from light. (If you are in a hurry, you can also use room temperature for 1 hour on a shaker.) Starting from adding the fluorescent secondary antibody, all subsequent steps should be protected from light;

The PBST-1 shaker is cleaned three times, 10 minutes each time;

DAPI is diluted 1000× and incubated for 2-3 minutes, protected from light. Wash with DPBS 1 to 2 times;

Take photos and observe directly.

2. Experimental results

(1) The first-stage detection results are shown in Figure 2A and Figure 2B. The results showed that the morphology of iPSC cells changed after induction of differentiation. The cell density continued to increase at this stage, which was also accompanied by cell death. Compared with undifferentiated stem cells, the nuclear-cytoplasmic ratio of endodermal cells was significantly reduced at this time, and the ratio of cells There are clear boundaries between contacts;

After iPSC induction, SOX17 and FOXA2 were used as endoderm markers, and the positive rate of endoderm was higher than 50%. In the embodiment of the present invention, the GDF-8 group was defined as group B, and Activin A was defined as group A. The endodermal cells obtained after induction were tested by qPCR and found that the SOX17 gene expressed in group A was 4.5 times that of group B and 500 times that of iPSCs in the negative control group; the FOXA2 gene expressed in group A was 7 times that of group B, which was a negative control. The expression of CRCX4 gene in group A was 110 times that of iPSCs in group B and 3 times that of iPSCs in the negative control group. After testing FOXA2/DAPI (immunofluorescence), the expression rate was >50%. It shows that the induction of iPSCs using Activin A and CHIR-99021 is more efficient, the number of endoderm cells obtained is larger, and the positive rate of SOX17 and FOXA2 is higher, which is significantly higher than the induction effect of GDF-8.

(2) The second stage detection results are shown in Figure 3A and Figure 3B. The results showed that the morphology of iPSC cells changed after induction of differentiation, and cord-shaped and spherical cells began to appear at the end of this stage. Positive cells in cord-shaped and globular cell clusters expressed the highest expression;

At this stage, HNF1β and FOXA2 are used as markers for the original intestinal tube. The positive rate of the original intestinal tube is higher than 60%. The endodermal cells obtained after induction were tested by qPCR and found that the positive rate of HNF1β gene was about 60% and the positive rate of FOXA2 gene was about 60%. is 80%.

(3) The third stage test results are shown in Figure 4A and Figure 4B. The results show that the morphology of iPSC cells after induction of differentiation changes, and cord-shaped and globular cell clusters continue to increase at this stage;

At this stage, PDX-1 and SOX9 are used as markers for the posterior foregut stage. The positive rate at the posterior foregut stage is higher than 70%. The posterior foregut cells obtained after induction were tested by qPCR and found that the positive rate of PDX-1 gene was high. The positive rate of SOX9 gene is higher than 60%, and the positive rate of SOX9 gene is higher than 25%.

(4) The test results of the fourth stage are shown in Figure 5A and Figure 5B. The results show that the morphology of cells after induction of differentiation changes. At this stage, globular structures continue to form in large numbers and gradually become denser;

At this stage, PDX-1 and SOX9 are used as markers for the posterior foregut stage. The positive rate at the posterior foregut stage is higher than 70%. The posterior foregut cells obtained after induction were tested by qPCR and found that the positive rate of PDX-1 gene was high. More than 90%, SOX9 gene positivity rate is higher than 50%.

(5) The fifth stage test results are shown in Figure 6. The results showed that the morphology of iPSC cells after induced differentiation changed, the cells continued to grow in stacks, and the globular structures were connected into sheets;

At this stage, NKX6.1, NGN3, and PDX-1 are used as endocrine progenitor cell phenotypes. It can be seen that the expression level of the marker gene NKX6.1 mRNA of the cells induced by the fifth stage is approximately 50 times that of iPSC, and the expression level of the marker gene NGN3 mRNA The expression level of PDX-1 is about 80 times that of iPSCs, and the expression level of marker gene PDX-1 mRNA is about 200 times that of iPSCs.

(6) The test results of the sixth and seventh stages are shown in Figure 7A and Figure 7B. The results show that at this stage, INS, GCG, NKX6.1, and MAFA are used as islet cell phenotypes, and the marker gene NKX6.1 mRNA of group B is The expression level was approximately 1.1 times that of group A, the expression level of the marker gene GCG mRNA of group B was approximately 2 times that of group A, and the expression level of the marker gene INS mRNA of group B was approximately 17 times that of group A. Immunofluorescence results showed that the single-positive rate of INS in group B was about 95%, while INS and GCG were co-expressed in group A, that is, the single-positive rate of GCG-/INS+ cells in group B was significantly increased.

The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be noted that those of ordinary skill in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims (10)

An inducing differentiation agent that induces iPSCs to differentiate into functionally mature pancreatic islet beta cells, characterized in that the inducing differentiation agent includes a first-stage differentiation agent, a second-stage differentiation agent, a third-stage differentiation agent, and a third-stage differentiation agent. Four-stage differentiation inducing agent, fifth-stage differentiation inducing agent, sixth-stage differentiation inducing agent, and seventh-stage differentiation inducing agent; Preferably, the first-stage differentiation inducing agent includes the first-stage differentiation inducing agent A and the first-stage differentiation inducing agent B; More preferably, the first-stage differentiation inducing agent A includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, Activin A, and CHIR-99021; More preferably, the first-stage differentiation inducing agent B includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, and Activin A; Preferably, the second stage differentiation inducing agent includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, and FGF-7; Preferably, the third stage differentiation inducing agent includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, FGF-7, insulin-transferrin-selenium, ethanolamine, SANT-1, retinoids Alcohol, LDN193189, TPPB; Preferably, the fourth stage differentiation agent includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, FGF-7, insulin-transferrin-selenium, ethanolamine, SANT-1, retin Alcohol, LDN193189, TPPB; Preferably, the fifth stage differentiation agent includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, LDN193189, triiododine Thyroidine (T3), ALK5i II, zinc sulfate; Preferably, the sixth stage differentiation inducing agent includes the sixth stage differentiation inducing agent A, the sixth stage differentiation inducing agent B, the sixth stage differentiation inducing agent C, and the sixth stage differentiation inducing agent D; More preferably, the sixth stage differentiation agent A includes β-cellulin and erythrospongin A; Most preferably, the sixth stage differentiation agent A also contains sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine ( T3), ALK5i II, zinc sulfate, GSi XX; More preferably, the sixth stage differentiation agent B contains β-cellulin; Most preferably, the sixth stage differentiation agent B also contains sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine ( T3), ALK5i II, zinc sulfate, GSi XX; More preferably, the sixth stage differentiation inducing agent C includes β-cellulin, forskolin, and exenatide 4; Most preferably, the sixth stage differentiation agent C also contains sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodothyronine ( T3), ALK5i II, zinc sulfate, GSi XX; More preferably, the sixth stage differentiation inducing agent D includes β-cellulin, forskolin, exenatide 4, hepatocyte growth factor, and serotonin; Most preferably, the sixth stage differentiation agent D also contains sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate , GSi XX, insulin-transferrin-selenium; Preferably, the seventh stage differentiation inducing agent includes seventh stage differentiation inducing agent A and seventh stage differentiation inducing agent B; More preferably, the seventh stage differentiation inducing agent A includes β-cellulin, forskolin, exenatide 4, hepatocyte growth factor, and serotonin; Most preferably, the seventh stage differentiation inducing agent A also contains sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, pancreatic islets Vitamin-transferrin-selenium, ethanolamine, triiodothyronine (T3), ALK5i II, zinc sulfate, N-acetyl-L-cysteine, porcine intestinal mucosal heparin sodium, water-soluble vitamin E, R428; More preferably, the seventh stage differentiation agent B includes glutamine, calcium chloride dihydrate, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, fetal bovine serum albumin, insulin- Transferrin-selenium, ethanolamine, water-soluble vitamin E, nicotinamide, heparin sodium, deoxyribonuclease I, Necrostatin-1, Pefabloc; Most preferably, the concentrations of each component in the first-stage differentiation agent are: (10-500) ng/mL Activin A, (0.01-10) μM CHIR-99021, (0.01-10)% fetal bovine serum Albumin, (0.01-10)g/L sodium bicarbonate, (1-50)mM glucose, (0.01-5)mM glutamine; Most preferably, the concentrations of each component in the first-stage differentiation agent are: 100ng/mL Activin A, 3μM CHIR-99021, 0.5% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 10mM glucose, 1mM glutamine; More preferably, the concentrations of each component in the second stage differentiation agent are: (0.01-10)mM ascorbic acid, (10-200)ng/mL FGF-7, (0.01-10)% fetal bovine serum albumin Protein, (0.01-10)g/L sodium bicarbonate, (1-50)mM glucose, (0.01-5)mM glutamine; Most preferably, the concentrations of each component in the second-stage differentiation agent are: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.5% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 10mM glucose, 1mM glutamine; More preferably, the concentrations of each component in the third stage differentiation agent are: (0.01-5)mM ascorbic acid, (10-200)ng/mL FGF-7, (0.01-5)μM SANT-1, (0.01-5)μM retinol, (10-500)nM LDN193189, (50-500)nM TPPB, (0.01-10)% fetal bovine serum albumin, (0.01-10)g/L sodium bicarbonate, (1-100)mM glucose, (1-100)mM glutamine, (1-100)mg/L ethanolamine, (0.01-5)% insulin-transferrin-selenium; Most preferably, the concentrations of each component in the third-stage differentiation agent are: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.25μM SANT-1, 1μM retinol, 100nM LDN193189, 200nM TPPB, 2% Fetal bovine serum albumin, 2.5g/L sodium bicarbonate, 10mM glucose, 1mM glutamine, 1mg/L ethanolamine, 0.5% insulin-transferrin-selenium; More preferably, the concentrations of each component in the fourth stage differentiation agent are: (0.01-5)mM ascorbic acid, (0.01-10)ng/mL FGF-7, (0.01-5)μM SANT-1, (0.01-5)μM retinol, (50-500)nM LDN193189, (10-300)nM TPPB, (0.01-5)% fetal bovine serum albumin, (0.05-10)g/L sodium bicarbonate, (0.05-50)mM glucose, (0.01-5)mM glutamine, (0.01-5)mg/L ethanolamine, (0.01-5)% insulin-transferrin-selenium; Most preferably, the concentrations of each component in the fourth stage inducing differentiation agent are: 0.25mM ascorbic acid, 2ng/mL FGF-7, 0.25μM SANT-1, 0.1μM retinol, 200nM LDN193189, 100nM TPPB, 2 % fetal bovine serum albumin, 2.5g/L sodium bicarbonate, 10mM glucose, 1mM glutamine, 1mg/L ethanolamine, 0.5% insulin-transferrin-selenium; More preferably, the concentrations of each component in the fifth stage differentiation agent are: (0.01-5) μM SANT-1, (0.01-10) μM retinol, (10-500) nM LDN193189, (0.01 -5)μM triiodothyronine (T3), (1-50)μM ALK5i II, (1-50)μM zinc sulfate, (0.05-10)% fetal bovine serum albumin, (0.05-10)g /L sodium bicarbonate, (1-50)mM glucose, (0.01-10)mM glutamine, (0.01-5)% insulin-transferrin-selenium, (0.01-10) mg/L ethanolamine; Most preferably, the concentrations of each component in the fifth stage differentiation agent are: 0.25 μM SANT-1, 0.05 μM retinol, 100 nM LDN193189, 1 μM triiodothyronine (T3), 10 μM ALK5i II, 10μM zinc sulfate, 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20mM glucose, 1mM glutamine, 0.5% insulin-transferrin-selenium, 1mg/L ethanolamine; Most preferably, the concentrations of each component in the sixth stage differentiation agent are: (10-500) nM LDN193189, (0.01-10) μM triiodothyronine (T3), (1-50) μM ALK5i II, (1-50)μM zinc sulfate, (0.05-10)% fetal bovine serum albumin, (0.01-5)g/L sodium bicarbonate, (1-100)mM glucose, (0.01-5)mM Glutamine, (0.01-5)mg/L ethanolamine, (50-300)nM GSi XX, (0.01-5) μM erythrospongin A, (10-500) ng/mL hepatocyte growth factor, (1-50) μM serotonin, (1-50) ng/mL β-cellulin, (1-50) μM forskolin, ( 10-500)ng/mL exenatide 4; Most preferably, the concentrations of each component in the sixth-stage differentiation agent are: 100 nM LDN193189, 1 μM triiodothyronine (T3), 10 μM ALK5i II, 10 μM zinc sulfate, 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20mM glucose, 1mM glutamine, 1mg/L ethanolamine, 100nM GSi XX, 1μM erythrospongin A, 50ng/mL hepatocyte growth factor, 10μM serotonin, 20ng/mL β-cellulin, 10μM Forskolin, 50ng/mL exenatide 4; Most preferably, the concentrations of each component in the seventh stage differentiation agent A are: (0.05-10)% fetal bovine serum albumin, (0.05-10) g/L sodium bicarbonate, (5-50) mM glucose, (0.01-5)mM glutamine, (0.01-5)mg/L ethanolamine, (0.01-2.5)% insulin-transferrin-selenium, (0.01-5)μM triiodothyronine ( T3), (0.01-50)μM ALK5i II, (1-50)μM zinc sulfate, (0.01-5)mM N-acetyl-L-cysteine, (1-50)μg/mL porcine intestinal mucosal heparin Sodium, (1-50)μM water-soluble vitamin E, (1-50)μM R428, (10-100)ng/mL hepatocyte growth factor, (1-50)μM serotonin, (1-50)ng/ mL β-cellulin, (1-50) μM forskolin, (10-100) ng/mL exenatide 4; Most preferably, the concentrations of each component in the seventh stage differentiation agent A are: 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20mM glucose, 1mM glutamine, 1mg/L ethanolamine, 0.5% insulin-transferrin-selenium, 1μM triiodothyronine (T3), 10μM ALK5i II, 10μM zinc sulfate, 1mM N-acetyl-L-cysteine, 10μg/mL porcine intestinal mucosal heparin sodium, 10μM water-soluble vitamin E, 2μM R428, 50ng/mL hepatocyte growth factor, 10μM serotonin, 20ng/mL β-cellulin, 10μM forskolin, 50ng/mL exenatide 4; Most preferably, the concentrations of each component in the seventh stage differentiation agent B are: (0.01-10)mM glutamine, (0.01-10)mM calcium chloride dihydrate, (1-50)mM N -2hydroxyethylpiperazine-N-2-ethanesulfonic acid, (0.01-5)% fetal bovine serum albumin, (0.01-2)mL/L insulin-transferrin-selenium, (0.01-5) mg/L ethanolamine, (1-50)μM water-soluble vitamin E, (1-50)mM nicotinamide, (1-50)μg/mL heparin sodium, (0.01-2.5)U/mL deoxyribonuclease I, (50-500)μM Necrostatin-1, (0.001-2)μM Pefabloc; Most preferably, the concentrations of each component in the seventh stage differentiation agent B are: 2mM glutamine, 2.5mM calcium chloride dihydrate, 10mM N-2 hydroxyethylpiperazine-N-2-ethane. Sulfonic acid, 2% fetal bovine serum albumin, 0.6mL/L insulin-transferrin-selenium, 1mg/L ethanolamine, 10μM water-soluble vitamin E, 10mM nicotinamide, 10μg/mL heparin sodium, 1U/mL DNA Enzyme I, 100μM Necrostatin-1, 0.1μM Pefabloc. An induction differentiation medium that induces iPSCs to differentiate into functionally mature pancreatic islet beta cells, characterized in that the induction differentiation medium includes a first-stage induction differentiation medium, a second-stage induction differentiation medium, and a third-stage induction medium. Differentiation medium, fourth stage induction differentiation medium, fifth stage induction differentiation medium, sixth stage induction differentiation medium, seventh stage induction differentiation medium; Preferably, the first-stage differentiation medium includes basal medium MCDB131 and the first-stage differentiation agent described in claim 1; Preferably, the second-stage differentiation medium includes basal medium MCDB131 and the second-stage differentiation agent described in claim 1; Preferably, the third stage inducing differentiation medium includes basal medium MCDB131 and the third stage inducing differentiation agent described in claim 1; Preferably, the fourth stage inducing differentiation medium includes basal medium MCDB131 and the fourth stage inducing differentiation agent described in claim 1; Preferably, the fifth stage inducing differentiation medium includes basal medium MCDB131 and the fifth stage inducing differentiation agent described in claim 1; Preferably, the sixth stage inducing differentiation medium includes basal medium MCDB131 and the sixth stage inducing differentiation agent described in claim 1; Preferably, the seventh stage induction differentiation medium includes basal medium MCDB131, 50% Ham’s F-12 medium, 50% medium 199, and the seventh stage induction differentiation agent described in claim 1. A method for inducing iPSCs to differentiate into functionally mature pancreatic islet beta cells, characterized in that the method includes the following steps: (1) Provide iPSCs and perform monolayer differentiation culture in complete culture medium; (2) In the first stage of induction differentiation, the iPSCs obtained in step (1) are induced to differentiate into definitive endoderm cells using the first stage induction differentiation medium; (3) In the second stage of induction differentiation, the second stage induction differentiation medium is used to induce the differentiation of definitive endoderm cells into primitive intestinal tube cells; (4) In the third stage of induction differentiation, the third stage induction differentiation medium is used to induce the differentiation of the protogut tube cells into posterior foregut cells; (5) The fourth stage induces differentiation, using the fourth stage induction differentiation medium to induce the posterior foregut cells to differentiate into pancreatic progenitor cells; (6) Fifth stage induction differentiation, using the fifth stage induction differentiation medium to induce the differentiation of pancreatic progenitor cells into pancreatic endocrine progenitor cells; (7) Sixth stage induction differentiation, using the sixth stage induction differentiation medium to induce the differentiation of pancreatic endocrine progenitor cells into pancreatic endocrine cells; (8) Seventh-stage induction differentiation: Use the seventh-stage induction differentiation medium to induce the differentiation of pancreatic endocrine cells into mature islet cells to obtain functionally mature islet β cells. The method according to claim 3, characterized in that the complete culture medium described in step (1) is E8 complete culture medium; Preferably, the E8 complete culture medium contains a ROCK inhibitor; More preferably, the ROCK inhibitor includes Y27632, GSK429286A, RKI-1447, Y-33075 dihydrochloride, Thiazovivin, K-115, SLx-2119, Chroman1, SAR407899 and/or SR-3677; Most preferably, the ROCK inhibitor is Y27632; Most preferably, the concentration of Y27632 is 0.001-100 μM; Most preferably, the concentration of Y27632 is 10 μM; Preferably, the iPSCs described in step (1) are derived from mammals; More preferably, the iPSCs described in step (1) are derived from humans, mice, rats, goats, sheep, pigs, cats, rabbits, dogs, wolves, horses or cattle; Most preferably, the iPSCs described in step (1) are derived from humans; Most preferably, the culture time in step (1) is 3 days; Most preferably, from Day-3 to Day-1, fresh medium is replaced every day; Preferably, the first stage induction differentiation medium described in step (2) contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, activin, and GSK-3 inhibitor; More preferably, the activin includes Activin A, Activin B, Activin AB, Activin AC; Most preferably, the activin is Activin A; More preferably, the GSK-3 inhibitor includes CHIR-99021, CHIR-98014, AZD-2858, SB-216763, AT-7519, TW-S119, KY-19382 (A3051), NP-031112, SB-415286 , AZD-1080, AR-A014418, TDZD-8, LY-2090314; Most preferably, the GSK-3 inhibitor is CHIR-99021; More preferably, the first stage induction differentiation medium includes the first stage induction differentiation medium A and the first stage induction differentiation medium B; Most preferably, the first-stage induced differentiation medium A includes basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, Activin A, and CHIR-99021; Most preferably, the first-stage induced differentiation medium B contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, and Activin A; Most preferably, the concentrations of each component in the first stage induction differentiation medium are: (10-500) ng/mL Activin A, (0.01-10) μM CHIR-99021, (0.01-10)% fetal bovine Serum albumin, (0.01-10)g/L sodium bicarbonate, (1-50)mM glucose, (0.01-5)mM glutamine; Most preferably, the concentrations of each component in the first stage induction differentiation medium are: 100ng/mL Activin A, 3μM CHIR-99021, 0.5% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 10mM glucose. , 1mM glutamine; Most preferably, the time for inducing differentiation in the first stage is 3 days; Most preferably, on the first day of induction, the first stage induction differentiation medium A is used to induce differentiation; Most preferably, on the 2nd to 3rd day of induction, the first stage induction differentiation medium B is used to induce differentiation; Most preferably, on Day0-Day2, fresh medium is replaced every day. The method according to claim 3, wherein the second stage induction differentiation medium in step (3) contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, Ascorbic acid, fibroblast growth factor; Preferably, the fibroblast growth factors include FGF-7, FGF-2, FGF-6, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16 , FGF-17, FGF-18, FGF-21, FGF-5, FGF-1, FGF-3, FGF-4, FGF-8, FGF-9, FGF-19, FGF-20; More preferably, the fibroblast growth factor is FGF-7; Most preferably, the concentrations of each component in the second stage induction differentiation medium are: (0.01-10)mM ascorbic acid, (10-200)ng/mL FGF-7, (0.01-10)% fetal bovine serum. Albumin, (0.01-10)g/L sodium bicarbonate, (1-50)mM glucose, (0.01-5)mM glutamine; Most preferably, the concentrations of each component in the second stage induction differentiation medium are: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.5% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, and 10mM glucose. , 1mM glutamine; Most preferably, the time for inducing differentiation in the second stage is 2 days; Most preferably, on the 1st to 2nd day of induction, the second stage induction differentiation medium is used to induce differentiation; Most preferably, on Day3-Day4, fresh culture medium is replaced every day; Preferably, the third stage induction differentiation medium described in step (4) includes basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, fibroblast growth factor, insulin- Transferrin - selenium, ethanolamine, SANT-1, retinol, BMP inhibitor, protein kinase C activator; More preferably, the fibroblast growth factors include FGF-7, FGF-2, FGF-6, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF- 16. FGF-17, FGF-18, FGF-21, FGF-5, FGF-1, FGF-3, FGF-4, FGF-8, FGF-9, FGF-19, FGF-20; Most preferably, the fibroblast growth factor is FGF-7; More preferably, the BMP inhibitors include LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD161570; Most preferably, the BMP inhibitor is LDN193189; More preferably, the protein kinase C activator includes TPPB and PMA; Most preferably, the protein kinase C activator is TPPB; Most preferably, the concentrations of each component in the third stage induction differentiation medium are: (0.01-5)mM ascorbic acid, (10-200)ng/mL FGF-7, (0.01-5)μM SANT-1 , (0.01-5)μM retinol, (10-500)nM LDN193189, (50-500)nM TPPB, (0.01-10)% fetal bovine serum albumin, (0.01-10)g/L sodium bicarbonate , (1-100)mM glucose, (1-100)mM glutamine, (1-100)mg/L ethanolamine, (0.01-5)% insulin-transferrin-selenium; Most preferably, the concentrations of each component in the third stage induction differentiation medium are: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.25μM SANT-1, 1μM retinol, 100nM LDN193189, 200nM TPPB, 2 % fetal bovine serum albumin, 2.5g/L sodium bicarbonate, 10mM glucose, 1mM glutamine, 1mg/L ethanolamine, 0.5% insulin-transferrin-selenium; Most preferably, the time for inducing differentiation in the third stage is 2 days; Most preferably, on the 1st to 2nd day of induction, the third stage induction differentiation medium is used to induce differentiation; Most preferably, on Day 5-Day 6, fresh medium should be replaced every day. The method according to claim 3, characterized in that the fourth stage induction differentiation medium in step (5) contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, Ascorbic acid, fibroblast growth factor, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, BMP inhibitor, protein kinase C activator; Preferably, the fibroblast growth factors include FGF-7, FGF-2, FGF-6, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16 , FGF-17, FGF-18, FGF-21, FGF-5, FGF-1, FGF-3, FGF-4, FGF-8, FGF-9, FGF-19, FGF-20; More preferably, the fibroblast growth factor is FGF-7; Preferably, the BMP inhibitors include LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD161570; More preferably, the BMP inhibitor is LDN193189; Preferably, the protein kinase C activator includes TPPB and PMA; More preferably, the protein kinase C activator is TPPB; Most preferably, the concentrations of each component in the fourth stage induction differentiation medium are: (0.01-5)mM ascorbic acid, (0.01-10)ng/mL FGF-7, (0.01-5)μM SANT-1 , (0.01-5)μM retinol, (50-500)nM LDN193189, (10-300)nM TPPB, (0.01-5)% fetal bovine serum albumin, (0.05-10)g/L sodium bicarbonate , (0.05-50)mM glucose, (0.01-5)mM glutamine, (0.01-5)mg/L ethanolamine, (0.01-5)% insulin-transferrin-selenium; Most preferably, the concentrations of each component in the fourth stage induction differentiation medium are: 0.25mM ascorbic acid, 2ng/mL FGF-7, 0.25μM SANT-1, 0.1μM retinol, 200nM LDN193189, 100nM TPPB, 2% fetal bovine serum albumin, 2.5g/L sodium bicarbonate, 10mM glucose, 1mM glutamine, 1mg/L ethanolamine, 0.5% insulin-transferrin-selenium; Most preferably, the time for inducing differentiation in the fourth stage is 3 days; Most preferably, on days 1-3 of induction, the fourth stage induction differentiation medium is used to induce differentiation; Most preferably, on Day7-Day9, fresh medium is replaced every day; Preferably, the fifth stage induction differentiation medium described in step (6) includes basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, SANT-1, retinol, BMP inhibitor, thyroid hormone, ALK5 inhibitor, zinc sulfate; More preferably, the BMP inhibitors include LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD161570; Most preferably, the BMP inhibitor is LDN193189; More preferably, the thyroid hormones include triiodothyronine (T3) and tetraiodothyronine (T4); Most preferably, the thyroid hormone is triiodothyronine (T3); More preferably, the ALK5 inhibitor includes ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-β receptor inhibitor V, TGF-β receptor inhibitor TGF-β receptor inhibitor I, TGF-β receptor inhibitor IV, TGF-β receptor inhibitor VII, TGF-β receptor inhibitor VIII, TGF-β receptor inhibitor II, TGF-β receptor inhibitor VI and TGF-β receptor inhibitor III; Most preferably, the ALK5 inhibitor is ALK5i II; Most preferably, the concentrations of each component in the fifth stage induction differentiation medium are: (0.01-5) μM SANT-1, (0.01-10) μM retinol, (10-500) nM LDN193189, ( 0.01-5)μM triiodothyronine (T3), (1-50)μM ALK5i II, (1-50)μM zinc sulfate, (0.05-10)% fetal bovine serum albumin, (0.05-10) g/L sodium bicarbonate, (1-50)mM glucose, (0.01-10)mM glutamine, (0.01-5)% insulin-transferrin-selenium, (0.01-10) mg/L ethanolamine; Most preferably, the concentrations of each component in the fifth stage induction differentiation medium are: 0.25 μM SANT-1, 0.05 μM retinol, 100 nM LDN193189, 1 μM triiodothyronine (T3), 10 μM ALK5i II , 10μM zinc sulfate, 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20mM glucose, 1mM glutamine, 0.5% insulin-transferrin-selenium, 1mg/L ethanolamine; Most preferably, the time for inducing differentiation in the fifth stage is 3 days; Most preferably, on days 1-3 of induction, the fifth stage induction differentiation medium is used to induce differentiation; Most preferably, from Day 10 to Day 12, fresh medium should be replaced every day. The method according to claim 3, characterized in that the sixth stage induction differentiation medium in step (7) contains basal medium MCDB131, BMP inhibitor, thyroid hormone, ALK5 inhibitor, zinc sulfate, fetal bovine Serum albumin, sodium bicarbonate, glucose, glutamine, ethanolamine, gamma-secretinase inhibitor, erythrospongin A, hepatocyte growth factor, serotonin, β-cellulin, forskolin, exenatide 4 ; Preferably, the BMP inhibitors include LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD161570; More preferably, the BMP inhibitor is LDN193189; Preferably, the thyroid hormones include triiodothyronine (T3) and tetraiodothyronine (T4); More preferably, the thyroid hormone is triiodothyronine (T3); Preferably, the ALK5 inhibitor includes ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-β receptor inhibitor V, TGF-β receptor Inhibitor I, TGF-β receptor inhibitor IV, TGF-β receptor inhibitor VII, TGF-β receptor inhibitor VIII, TGF-β receptor inhibitor II, TGF-β receptor inhibitor VI and TGF -β receptor inhibition Agent III; More preferably, the ALK5 inhibitor is ALK5i II; Preferably, the γ-secretase inhibitors include GSi XX, GSi IX, GSi XI, GSi XII, GSi XIII, GSi XIV, GSi XVI, GSi BMS-708163, LY411575, LY3039478; More preferably, the γ-secretase inhibitor is GSiXX; Preferably, the sixth stage inducing differentiation medium includes the sixth stage inducing differentiation medium A, the sixth stage inducing differentiation medium B, the sixth stage inducing differentiation medium C, and the sixth stage inducing differentiation medium D; More preferably, the sixth stage induction differentiation medium A contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodine Thyroidine (T3), ALK5i II, zinc sulfate, GSi XX, β-cellulin, erythrospongin A; More preferably, the sixth stage induction differentiation medium B contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodine Thyroidine (T3), ALK5i II, zinc sulfate, GSi XX, β-cellulin; More preferably, the sixth stage induction differentiation medium C contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, LDN193189, triiodine Thyroidine (T3), ALK5i II, zinc sulfate, GSi XX, β-cellulin, forskolin, exenatide 4; More preferably, the sixth stage induction differentiation medium D contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ethanolamine, LDN193189, triiodothyronine (T3), ALK5i II, zinc sulfate, GSi XX, insulin-transferrin-selenium, β-cellulin, forskolin, exenatide 4, hepatocyte growth factor, serotonin; Most preferably, the concentrations of each component in the sixth stage induction differentiation medium are: (10-500) nM LDN193189, (0.01-10) μM triiodothyronine (T3), (1-50) μM ALK5i II, (1-50)μM zinc sulfate, (0.05-10)% fetal bovine serum albumin, (0.01-5)g/L sodium bicarbonate, (1-100)mM glucose, (0.01-5) mM glutamine, (0.01-5)mg/L ethanolamine, (50-300)nM GSi XX, (0.01-5)μM erythrospongin A, (10-500)ng/mL hepatocyte growth factor, (1 -50)μM serotonin, (1-50)ng/mL β-cellulin, (1-50)μM forskolin, (10-500)ng/mL exenatide 4; Most preferably, the concentrations of each component in the sixth stage induction differentiation medium are: 100 nM LDN193189, 1 μM triiodothyronine (T3), 10 μM ALK5i II, 10 μM zinc sulfate, and 2% fetal bovine serum albumin. , 1.5g/L sodium bicarbonate, 20mM glucose, 1mM glutamine, 1mg/L ethanolamine, 100nM GSi XX, 1μM erythrospongin A, 50ng/mL hepatocyte growth factor, 10μM serotonin, 20ng/mL β-cellulin, 10μM forskolin, 50ng/mL exenatide 4; Most preferably, the time for inducing differentiation in the sixth stage is 14 days; Most preferably, on the first day of induction, the sixth stage induction differentiation medium A is used to induce differentiation; Most preferably, on days 2-7 of induction, the sixth stage induction differentiation medium B is used to induce differentiation; Most preferably, on the 8th to 9th day of induction, the sixth stage induction differentiation medium C is used to induce differentiation; More preferably, on the 10th to 14th day of induction, the sixth stage induction differentiation medium D is used to induce differentiation; Most preferably, from Day 13 to Day 27, change the medium half every day; Preferably, the seventh stage induction differentiation medium described in step (8) includes the seventh stage induction differentiation medium A and the seventh stage induction differentiation medium B; More preferably, the seventh stage induction differentiation medium A contains basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, thyroid hormone, ALK5 Inhibitors, zinc sulfate, N-acetyl-L-cysteine, porcine intestinal mucosal heparin sodium, water-soluble vitamin E, AXL inhibitor, β-cellulin, forskolin, exenatide 4, hepatocyte growth factor , 5-hydroxytryptamine; Most preferably, the thyroid hormones include triiodothyronine (T3) and tetraiodothyronine (T4); Most preferably, the thyroid hormone is triiodothyronine (T3); Most preferably, the ALK5 inhibitor includes ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-β receptor inhibitor V, TGF-β receptor inhibitor TGF-β receptor inhibitor I, TGF-β receptor inhibitor IV, TGF-β receptor inhibitor VII, TGF-β receptor inhibitor VIII, TGF-β receptor inhibitor II, TGF-β receptor inhibitor VI and TGF-β receptor inhibitor III; Most preferably, the ALK5 inhibitor is ALK5i II; Most preferably, the AXL inhibitor includes R428, BMS-907351, BMS-777607, XL184, TP-0903, XL092, LDC1267, LY2801653, CEP-40783, RU-301, S49076, ONO-7475, Ningetinib; Most preferably, the AXL inhibitor is R428; Most preferably, the concentrations of each component in the seventh stage induction differentiation medium A are: (0.05-10)% fetal bovine serum albumin, (0.05-10) g/L sodium bicarbonate, (5-50) )mM glucose, (0.01-5)mM glutamine, (0.01-5)mg/L ethanolamine, (0.01-2.5)% insulin-transferrin-selenium, (0.01-5)μM triiodothyronine (T3), (0.01-50)μM ALK5i II, (1-50)μM zinc sulfate, (0.01-5)mM N-acetyl-L-cysteine, (1-50)μg/mL porcine intestinal mucosa Heparin sodium, (1-50)μM water-soluble vitamin E, (1-50)μM R428, (10-100)ng/mL hepatocyte growth factor, (1-50)μM serotonin, (1-50)ng /mL β-cellulin, (1-50) μM forskolin, (10-100) ng/mL exenatide 4; Most preferably, the concentrations of each component in the seventh stage induction differentiation medium A are: 2% fetal bovine serum albumin, 1.5g/L sodium bicarbonate, 20mM glucose, 1mM glutamine, and 1mg/L ethanolamine. , 0.5% insulin-transferrin-selenium, 1μM triiodothyronine (T3), 10μM ALK5i II, 10μM zinc sulfate, 1mM N-acetyl-L-cysteine, 10μg/mL porcine intestinal mucosal heparin sodium , 10μM water-soluble vitamin E, 2μM R428, 50ng/mL hepatocyte growth factor, 10μM serotonin, 20ng/mL β-cellulin, 10μM forskolin, 50ng/mL exenatide 4; More preferably, the seventh stage induction differentiation medium B contains Ham's F-12 medium, medium 199, glutamine, calcium chloride dihydrate, N-2-hydroxyethylpiperazine-N-2-ethanesulfonate Acid, fetal bovine serum albumin, insulin-transferrin-selenium, ethanolamine, water-soluble vitamin E, nicotinamide, heparin sodium, deoxyribonuclease I, necroptosis inhibitor, serine protease inhibitor; Most preferably, the necroptosis inhibitor includes Necrostatin-1 and Necrostatin-2; Most preferably, the necroptosis inhibitor is Necrostatin-1; Most preferably, the serine protease inhibitor includes Pefabloc, Benzamidine, MBTI, PMSF, LBTI; Most preferably, the serine protease inhibitor is Pefabloc; Most preferably, the concentrations of each component in the seventh stage induction differentiation medium B are: 50% Ham's F-12 medium, 50% medium 199, (0.01-10)mM glutamine, (0.01-10) mM calcium chloride dihydrate, (1-50)mM N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid, (0.01-5)% fetal bovine serum albumin, (0.01-2)mL/ L insulin-transferrin-selenium, (0.01-5)mg/L ethanolamine, (1-50)μM water-soluble vitamin E, (1-50)mM nicotinamide, (1-50)μg/mL heparin sodium, (0.01-2.5) U/mL deoxyribonuclease I, (50-500) μM Necrostatin-1, (0.001-2) μM Pefabloc; Most preferably, the concentrations of each component in the seventh stage induction differentiation medium B are: 50% Ham's F-12 medium, 50% medium 199, 2mM glutamine, 2.5mM calcium chloride dihydrate, 10mM N -2hydroxyethylpiperazine-N-2-ethanesulfonic acid, 2% fetal bovine serum albumin, 0.6mL/L insulin-transferrin-selenium, 1mg/L ethanolamine, 10μM water-soluble vitamin E, 10mM tobacco Amide, 10μg/mL heparin sodium, 1U/mL deoxyribonuclease I, 100μM Necrostatin-1, 0.1μM Pefabloc; Most preferably, the seventh stage differentiation induction time is 14 days; Most preferably, on days 1-8 of induction, the seventh stage induction differentiation medium A is used to induce differentiation; Most preferably, on the 9th to 11th day of induction, the seventh stage induction differentiation medium B is used to induce differentiation; Most preferably, from Day 28 to Day 40, change the medium half every day. A functionally mature pancreatic islet β cell or cell population derived from iPSCs, characterized in that the cell or cell population is obtained by inducing differentiation using the method described in any one of claims 3-7; Preferably, the pancreatic islet β cells or cell population are functional, stable, mature pancreatic islet β cells or cell populations; preferably, the pancreatic islet β cells or cell population have a single positive rate of GCG-/INS+ cells of 95%. . A pharmaceutical composition for treating and/or preventing diabetes, characterized in that the pharmaceutical composition contains the cells or cell populations described in claim 8; Preferably, the pharmaceutical composition also contains pharmaceutically acceptable carriers and/or excipients; Preferably, the diabetes includes type 1 diabetes, type 2 diabetes, special types of diabetes, and gestational diabetes; More preferably, the diabetes is type 1 diabetes. The application of any of the following aspects, characterized in that the application includes: (1) Application of β-cellulin, forskolin, exenatide 4, hepatocyte growth factor and serotonin in combination to induce differentiation of iPSCs into functional pancreatic islet β cells; (2) The use of the differentiation agent according to claim 1 in preparing an induction medium for inducing iPSCs to differentiate into functional pancreatic islet β cells; (3) The use of the differentiation inducing agent according to claim 1 in inducing differentiation of iPSCs into functional pancreatic islet β cells; (4) Application of the differentiation medium described in claim 2 in inducing differentiation of iPSCs into functional islet β cells; (5) Use of the cells or cell populations according to claim 8 in the preparation of drugs for treating and/or preventing diabetes; (6) Use of the pharmaceutical composition according to claim 9 in the treatment and/or prevention of diabetes; Preferably, the diabetes includes type 1 diabetes, type 2 diabetes, special types of diabetes, and gestational diabetes; More preferably, the diabetes is type 1 diabetes.