CN114807015A - Induction method for promoting islet alpha cells to be converted into beta cells and application thereof - Google Patents

Induction method for promoting islet alpha cells to be converted into beta cells and application thereof Download PDF

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CN114807015A
CN114807015A CN202210557363.XA CN202210557363A CN114807015A CN 114807015 A CN114807015 A CN 114807015A CN 202210557363 A CN202210557363 A CN 202210557363A CN 114807015 A CN114807015 A CN 114807015A
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王贯乔
顾雨春
安翠平
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Chengnuo Regenerative Medical Technology Beijing Co ltd
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Chengnuo Regenerative Medical Technology Beijing Co ltd
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Abstract

The invention discloses an induction method for promoting islet alpha cells to be converted into beta cells and application thereof, wherein on the basis of a traditional induction method, a culture medium for converting the islet alpha cells into the beta cells is replaced by the traditional induction method on the 35 th day of induced differentiation, and artemether and a GABAA activator NS11394 are added for induction till the 40 th day. Experiments show that the induction method can obviously improve the expression quantity of the islet beta cells and is suitable for the production and clinical application of large-scale cell preparations.

Description

Induction method for promoting islet alpha cells to be converted into beta cells and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an induction method for promoting islet alpha cells to be converted into beta cells and application thereof.
Background
Diabetes Mellitus (DM) is a group of chronic metabolic diseases characterized by hyperglycemia due to insufficient insulin secretion or insulin resistance, and is mainly classified into Type 1Diabetes mellitus (T1 DM), Type 2Diabetes mellitus (T2 DM), Gestational Diabetes Mellitus (GDM), and monogenic Diabetes mellitus. Statistically, by 2019, the number of diabetics in the world is 4.63 hundred million, and is predicted to increase to 7 hundred million in 2045. Among them, type 1diabetes is an autoimmune disease mediated by T lymphocytes. Patients with type 1diabetes need to be completely dependent on exogenous insulin therapy due to insufficient insulin secretion caused by damaged islet cells. Although insulin therapy has been developed from "one injection before a meal every day" to "an artificial intelligent control insulin pump", it is not a radical cure nor prevention of a series of complications including retinopathy, neuropathy, and the like. In recent years, islet transplantation combined with immunosuppression has been successfully applied to the treatment of type 1diabetes, but clinical application thereof has been limited due to problems of severe donor organ deficiency, requirement of lifetime immunosuppression, and difficulty in long-term survival after islet transplantation. The pluripotent stem cells have great potential in solving the problem of donor islet shortage, induce and differentiate islet organoids, and differentiate into islet beta cells capable of secreting insulin in the islet organoids, so that an unlimited cell source can be provided for the diabetes replacement therapy. However, islet beta cells produced in vitro are not transcriptionally and functionally mature compared to native adult islet beta cells. Thus, mature islet beta cell functionality is crucial.
Regeneration and alternative treatment of islet beta cells is considered to be a promising approach for curing type 1 diabetes. Currently, the stem cells used for the treatment of type 1diabetes at the laboratory level are embryonic stem cells, Induced pluripotent stem cells (iPSCs), Mesenchymal Stem Cells (MSCs), bone marrow-derived Hematopoietic Stem Cells (HSCs), pluripotent precursor cells within the pancreas, and β -cell precursor cells present outside the pancreas (spleen, liver, endometrium). Stem cell therapy for type 1diabetes is based on the self-renewal function and potential directed differentiation function of stem cells, and compared with complete allogenic pancreatic tissue transplantation, strict quality control can be performed at a molecular level before transplantation, and the quality of transplanted cells and immune rejection can be effectively evaluated, so that stem cell therapy can partially or completely avoid graft failure and postoperative complications caused by immune rejection. The basic principle of iPSCs for treating type 1diabetes belongs to the field of regenerative medicine, and has great potential in treating type 1 diabetes. Unlike embryonic stem cells, iPSCs can only form new tissues and organs, but not new individuals, so potential medical ethical issues can be avoided, and immune rejection can be reduced or completely avoided as the cells originate from the patient himself.
At the present stage, there are various methods for successfully inducing the transdifferentiation of pluripotent stem cells into pancreatic islets, wherein the formation of pancreatic islets is basically realized by the process of endoderm, primitive gut tube, posterior foregut, pancreatic progenitor cells, pancreatic endocrine precursor, immature beta cells, mature beta cells, however, the majority of pancreatic islet cells induced by the transdifferentiation induced by the numerous techniques for inducing pluripotent stem cells to differentiate pancreatic islet cells, which have been developed so far, are islet multihormone cells, that is, islet alpha cells, islet beta cells, islet gamma cells, islet PP cells and the like are simultaneously expressed, wherein, the islet beta cells express limited and unstable hormone, the expression level of the islet alpha cells is obviously higher than that of the islet beta cells, and most of islet beta cells are in an immature state, the expression level of glucagon is obviously higher than that of insulin, and the application of the islet beta cells in the treatment of type 1diabetes is severely limited. Therefore, there is still a need in the art for a standard induction method capable of obtaining functional islet β -like cells in a large amount and stably. Based on the above, the invention provides an induction method for promoting the transformation of islet alpha cells into beta cells and an application thereof, wherein the induction method comprises artemether and a GABAA activator NS11394, and at present, no report about the combined application of artemether and the GABAA activator NS11394 to the transformation of the islet alpha cells into the beta cells exists.
Disclosure of Invention
In view of the above, the present invention aims to provide an induction method for promoting the transformation of islet alpha cells into beta cells and an application thereof, wherein the induction method is based on the traditional induction method, and is replaced by a culture medium for transforming islet alpha cells into beta cells at the 35 th day of induced differentiation, and simultaneously artemether and GABAA activator NS11394 are added for induction to the 40 th day. The verification shows that the induction method can obviously improve the expression quantity of the islet beta cells, and has wide application prospect in the treatment of type 1 diabetes.
The above object of the present invention is achieved by the following technical solutions:
in a first aspect of the present invention, there is provided an induction differentiation agent for inducing differentiation of pluripotent stem cells into functional islet beta cells.
Further, the induced differentiation agent comprises a first stage induced differentiation agent, a second stage induced differentiation agent, a third stage induced differentiation agent, a fourth stage induced differentiation agent, a fifth stage induced differentiation agent, a sixth stage induced differentiation agent, a seventh stage induced differentiation agent and an eighth stage induced differentiation agent;
preferably, the first-stage induction differentiation agent comprises a first-stage induction differentiation agent A and a first-stage induction differentiation agent B;
more preferably, the first stage induction differentiation agent a comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, GDF8, CHIR-99021;
more preferably, the first stage induction differentiation agent B comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, GDF 8;
preferably, the second stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, FGF-7;
preferably, the third stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, bovine serum albumin, ascorbic acid, FGF-7, SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive, TPPB;
preferably, said fourth stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, FGF-7, SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive, TPPB;
preferably, the fifth stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, zinc sulfate, heparin;
preferably, the sixth stage induction differentiation agent comprises a sixth stage induction differentiation agent a and a sixth stage induction differentiation agent B;
more preferably, the sixth stage induction differentiation agent a comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, zinc sulfate, GSi XX, heparin;
more preferably, the sixth stage induction differentiation agent B comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, zinc sulfate, heparin;
preferably, the seventh stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, zinc sulfate, heparin, acetyl-l-cysteine, water soluble vitamin E, R428;
preferably, the eighth stage induction differentiation agent comprises artemether and NS 11394;
more preferably, the eighth stage induction differentiation agent further comprises glutamine, calcium chloride dihydrate, N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid, fetal bovine serum albumin, insulin-transferrin-selenium-ethanolamine additive, water-soluble vitamin E, nicotinamide, heparin, deoxyribonuclease i, Necrostatin-1, Pefabloc;
most preferably, the concentrations of the ingredients in the first-stage induced differentiation agent are respectively: (10-500) ng/mL GDF8, (0.5-20) μ M CHIR-99021, (0.01-5)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the ingredients in the first-stage induced differentiation agent are respectively: 100ng/mL GDF8, 3. mu.M CHIR-99021, 0.5% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 1.5g/L sodium bicarbonate;
more preferably, the concentrations of the ingredients in the second-stage induction differentiation agent are respectively: (0.01-5) mM ascorbic acid, (10-100) ng/mL FGF-7, (0.01-5)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the ingredients in the second stage induction differentiation agent are respectively: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.5% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 1.5g/L sodium bicarbonate;
more preferably, the concentrations of the ingredients in the third stage induction differentiation agent are respectively: (0.01-5) mM ascorbic acid, (10-100) ng/mL FGF-7, (0.01-5) μ M SANT-1, (0.01-10) μ M retinoic acid, (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (50-500) nM TPPB, (0.05-10)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the ingredients in the third stage induction differentiation agent are respectively: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.25. mu.M SANT-1, 1. mu.M retinoic acid, 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 200nM TPPB, 2% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 2.5g/L sodium bicarbonate;
more preferably, the concentrations of the components in the fourth stage induction differentiation agent are respectively as follows: (0.01-5) mM ascorbic acid, (0.01-10) ng/mL FGF-7, (0.01-5) μ M SANT-1, (0.01-10) μ M retinoic acid, (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (50-500) nM TPPB, (0.05-10)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the components in the fourth stage induction differentiation agent are respectively as follows: 0.25mM ascorbic acid, 2ng/mL FGF-7, 0.25. mu.M SANT-1, 1. mu.M retinoic acid, 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 200nM TPPB, 2% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 2.5g/L sodium bicarbonate;
more preferably, the concentrations of the ingredients in the fifth stage induction differentiation agent are respectively as follows: (0.01-5) μ M SANT-1, (0.01-10) μ M retinoic acid, (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) μ M triiodothyronine, (0.01-50) μ M ALK5iII, (1-50) μ M zinc sulfate, (1-50) μ g/mL heparin, (0.05-10) g/L sodium bicarbonate, (0.05-10) mM glutamine, (1-50) mM glucose, (0.05-10)% fetal bovine serum albumin;
most preferably, the concentrations of the ingredients in the fifth stage induction differentiation agent are respectively: 0.25 μ M SANT-1, 0.1 μ M retinoic acid, 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 μ M triiodothyronine, 10 μ M ALK5i II, 10 μ M zinc sulfate, 10 μ g/mL heparin, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin;
most preferably, the concentrations of the ingredients in the sixth stage induction differentiation agent are respectively: (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) μ M triiodothyronine, (0.01-50) μ M ALK5i II, (1-50) μ M zinc sulfate, (10-500) nM GSi XX, (1-50) μ g/mL heparin, (0.05-10) g/L sodium bicarbonate, (0.05-10) mM glutamine, (1-50) mM glucose, (0.05-10)% fetal bovine serum albumin;
most preferably, the concentrations of the ingredients in the sixth stage induction differentiation agent are respectively: 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 μ M triiodothyronine, 10 μ M ALK5i II, 10 μ M zinc sulfate, 100nM GSi XX, 10 μ g/mL heparin, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin;
more preferably, the concentrations of the ingredients in the seventh stage induction differentiation agent are respectively: (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) μ M triiodothyronine, (0.01-50) μ M ALK5i II, (1-50) μ M zinc sulfate, (1-50) μ g/mL heparin, (0.01-10) mM acetyl-L-cysteine, (1-50) μ M water-soluble vitamin E, (1-10) μ M R428, (0.05-10) g/L sodium bicarbonate, (0.05-10) mM glutamine, (1-50) mM glucose, (0.05-10)% fetal bovine serum albumin;
most preferably, the concentrations of the ingredients in the seventh stage induction differentiation agent are respectively: 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 μ M triiodothyronine, 10 μ M ALK5i II, 10 μ M zinc sulfate, 10 μ g/mL heparin, 1mM acetyl-L-cysteine, 10 μ M water soluble vitamin E, 2 μ M MR428, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin;
more preferably, the concentrations of the ingredients in the eighth stage induction differentiation agent are respectively: (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (1-50) μ M water-soluble vitamin E, (1-50) mM nicotinamide, (1-50) μ g/mL heparin, (0.01-10) U/mL deoxyribonuclease I, (10-500) μ M Neostatin-1, (0.01-5) μ M Pefabloc, (0.01-10) mM glutamine, (0.01-10) mM calcium chloride dihydrate, (0.1-50) mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, (0.05-10)% fetal bovine serum albumin, (1-50) μ M artemether, (1-50) μ M NS 11394;
most preferably, the concentrations of the components in the eighth stage differentiation-inducing culture agent are respectively: 0.5% insulin-transferrin-selenium-ethanolamine additive, 10 μ M water soluble vitamin E, 10mM nicotinamide, 10 μ g/mL heparin, 1U/mL deoxyribonuclease I, 100 μ M Neostatin-1, 0.1 μ M Pefabloc, 2mM glutamine, 2.5mM calcium chloride dihydrate, 10mM N-2 hydroxyethyl piperazine-N-2-ethane sulfonic acid, 2% bovine serum albumin, 10 μ M artemether, 10 μ M NS 11394.
In a second aspect of the present invention, there is provided an induced differentiation medium for inducing differentiation of pluripotent stem cells into functional islet beta cells.
Further, the induced differentiation culture medium comprises a first-stage induced differentiation culture medium, a second-stage induced differentiation culture medium, a third-stage induced differentiation culture medium, a fourth-stage induced differentiation culture medium, a fifth-stage induced differentiation culture medium, a sixth-stage induced differentiation culture medium, a seventh-stage induced differentiation culture medium and an eighth-stage induced differentiation culture medium;
preferably, the first stage induced differentiation medium comprises the basal medium MCDB131, the first stage induced differentiation agent as described in the first aspect of the invention;
preferably, the second stage induced differentiation medium comprises the basal medium MCDB131, the second stage induced differentiation agent described in the first aspect of the present invention;
preferably, the third stage induced differentiation medium comprises the basal medium MCDB131, the third stage induced differentiation agent as described in the first aspect of the present invention;
preferably, the fourth stage differentiation-inducing medium comprises the basal medium MCDB131, the fourth stage differentiation-inducing agent as described in the first aspect of the present invention;
preferably, the fifth stage differentiation-inducing medium comprises the basal medium MCDB131, the fifth stage differentiation-inducing agent described in the first aspect of the present invention;
preferably, the sixth stage differentiation-inducing medium comprises the basal medium MCDB131, the sixth stage differentiation-inducing agent as described in the first aspect of the present invention;
preferably, the seventh stage differentiation-inducing medium comprises the basal medium MCDB131, the seventh stage differentiation-inducing agent described in the first aspect of the present invention;
preferably, the eighth stage induction differentiation medium comprises 50% Ham's F-12medium, 50% medium 199, the eighth stage induction differentiation agent described in the first aspect of the invention.
In a third aspect of the present invention, there is provided an induced differentiation method for inducing differentiation of pluripotent stem cells into functional islet beta cells;
further, the method comprises the steps of:
(1) providing induced pluripotent stem cells and culturing in complete medium;
(2) inducing differentiation at a first stage, namely inducing the induced pluripotent stem cells obtained in the step (1) to differentiate towards definitive endoderm cells by adopting a first-stage induced differentiation culture medium;
(3) inducing differentiation in the second stage, inducing differentiation medium in the second stage to induce differentiation of definitive endoderm cell to original intestinal canal cell;
(4) inducing differentiation in the third stage, namely inducing the differentiation of primitive intestinal canal cells to backward foregut cells by adopting a third-stage inducing differentiation culture medium;
(5) inducing differentiation at a fourth stage, and inducing the foregut cells to differentiate into pancreatic progenitor cells by adopting a fourth-stage inducing differentiation culture medium;
(6) inducing differentiation in the fifth stage, namely inducing pancreatic progenitor cells to differentiate into endocrine progenitor cells by adopting a fifth-stage induced differentiation culture medium;
(7) inducing differentiation in a sixth stage, namely inducing the endocrine progenitor cells to differentiate into immature islet beta cells by adopting a sixth-stage induced differentiation culture medium;
(8) inducing differentiation at a seventh stage, namely inducing the immature islet beta cells to differentiate into mature islet beta cells by adopting a seventh-stage induced differentiation culture medium;
(9) and (3) inducing differentiation in an eighth stage, and further inducing the maturation of the islet beta cells by adopting an eighth stage induced differentiation culture medium to obtain the functional islet beta cells.
Further, the complete culture medium in the step (1) is E8 complete culture medium;
preferably, the E8 complete medium contains a ROCK inhibitor;
more preferably, the ROCK inhibitor comprises Y-27632, GSK429286A, RKI-1447, Y-33075dihydrochloride, Thiazovivin, K-115, SLx-2119, Chroman1, SAR407899, and/or SR-3677;
most preferably, the ROCK inhibitor is Y-27632;
most preferably, the concentration of Y-27632 is 0.001-100. mu.M;
most preferably, the concentration of Y-27632 is 10. mu.M;
preferably, the induced pluripotent stem cells in step (1) have a cell density of 0.1 to 10.0X 10 5 cells/cm 2 ；
More preferably, the induced pluripotent stem cells in step (1) have a cell density of 1.8 to 2.2X 10 5 cells/cm 2 ；
Preferably, the induced pluripotent stem cells in step (1) are derived from a mammal;
more preferably, the induced pluripotent stem cells described in step (1) are derived from a human, mouse, rat, goat, sheep, pig, cat, rabbit, dog, wolf, horse, or cow;
most preferably, the induced pluripotent stem cells in step (1) are derived from a human;
preferably, the first stage differentiation induction medium described in step (2) comprises a basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, growth differentiation factor, GSK-3 inhibitor;
more preferably, the growth differentiation factor comprises GDF8, GDF15, GDF9, GDF11, GDF8, GDF5, GDF1, GDF9B, GDF 6;
most preferably, the growth differentiation factor is GDF 8;
more preferably, the GSK-3 inhibitor comprises 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 differentiation-inducing medium described in step (2) comprises a first stage differentiation-inducing medium a, a first stage differentiation-inducing medium B;
most preferably, the first stage differentiation induction medium a comprises the basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, GDF8, CHIR-99021;
most preferably, the first stage differentiation induction medium B comprises the basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, GDF 8;
more preferably, the first stage induces differentiation for a period of 3 days;
most preferably, on the 1 st day of induction, the first stage induction differentiation medium A is used for induction differentiation;
most preferably, the first stage induction differentiation culture medium B is adopted for induction differentiation at the induction days 2-3;
most preferably, fresh medium is changed daily on days 2-3 of induction;
most preferably, the concentrations of the components in the first stage differentiation induction medium are respectively: (10-500) ng/mL GDF8, (0.5-20) μ M CHIR-99021, (0.01-5)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the components in the first stage differentiation induction medium are respectively: 100ng/mL GDF8, 3. mu.M CHIR-99021, 0.5% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 1.5g/L sodium bicarbonate.
Further, the second stage differentiation induction medium in step (3) comprises a basic medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid and fibroblast growth factor;
preferably, the fibroblast growth factor comprises 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 the components in the second-stage differentiation-inducing medium are respectively: (0.01-5) mM ascorbic acid, (10-100) ng/mL FGF-7, (0.01-5)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the components in the second-stage differentiation-inducing medium are respectively: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.5% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 1.5g/L sodium bicarbonate;
most preferably, the second stage induces differentiation for a period of 2 days;
most preferably, on days 4-5 of induction, inducing differentiation is performed using the second-stage differentiation-inducing medium;
most preferably, fresh medium is changed daily on days 4-5 of induction;
preferably, the third stage differentiation induction medium described in step (4) comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, fibroblast growth factor, SANT-1, retinoic acid, BMP inhibitor, insulin-transferrin-selenium-ethanolamine additive, protein kinase C activator;
more preferably, the fibroblast growth factor comprises 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, PD 161570;
most preferably, the BMP inhibitor is LDN 193189;
more preferably, the protein kinase C activator comprises TPPB, PMA;
most preferably, the protein kinase C activator is TPPB;
most preferably, the concentrations of the components in the third stage differentiation-inducing medium are: (0.01-5) mM ascorbic acid, (10-100) ng/mL FGF-7, (0.01-5) μ M SANT-1, (0.01-10) μ M retinoic acid, (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (50-500) nM TPPB, (0.05-10)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the components in the third stage differentiation-inducing medium are: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.25. mu.M SANT-1, 1. mu.M retinoic acid, 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 200nM TPPB, 2% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 2.5g/L sodium bicarbonate;
most preferably, the period of time for the third stage to induce differentiation is 2 days;
most preferably, on days 6-7 of induction, the third stage differentiation induction medium is used for induction differentiation;
most preferably, fresh medium is replaced daily between days 6 and 7 of induction.
Further, the fourth stage differentiation induction medium described in the step (5) comprises a basal medium MCDB131, sodium bicarbonate, glutamine, glucose, bovine serum albumin, ascorbic acid, fibroblast growth factor, SANT-1, retinoic acid, BMP inhibitor, insulin-transferrin-selenium-ethanolamine additive, protein kinase C activator;
preferably, the fibroblast growth factor comprises 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, PD 161570;
more preferably, the BMP inhibitor is LDN 193189;
preferably, the protein kinase C activator comprises TPPB, PMA;
more preferably, the protein kinase C activator is TPPB;
most preferably, the concentrations of the components in the fourth stage differentiation induction medium are respectively: (0.01-5) mM ascorbic acid, (0.01-10) ng/mL FGF-7, (0.01-5) μ M SANT-1, (0.01-10) μ M retinoic acid, (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (50-500) nM TPPB, (0.05-10)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the components in the fourth stage differentiation induction medium are respectively: 0.25mM ascorbic acid, 2ng/mL FGF-7, 0.25. mu.M SANT-1, 1. mu.M retinoic acid, 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 200nM TPPB, 2% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 2.5g/L sodium bicarbonate;
most preferably, the fourth stage induces differentiation for a period of 3 days;
most preferably, on days 8-10 of induction, the fourth stage induction differentiation culture medium is used for induction differentiation;
most preferably, on days 8-10 of induction, fresh medium is changed daily;
preferably, the fifth stage differentiation-inducing medium described in step (6) comprises a basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, SANT-1, retinoic acid, BMP inhibitor, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor, zinc sulfate, heparin;
more preferably, the BMP inhibitors include LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD 161570;
most preferably, the BMP inhibitor is LDN 193189;
more preferably, the ALK5 inhibitors include ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-beta receptor inhibitor V, TGF-beta receptor inhibitor I, TGF-beta receptor inhibitor IV, TGF-beta receptor inhibitor VII, TGF-beta receptor inhibitor VIII, TGF-beta receptor inhibitor II, TGF-beta receptor inhibitor VI, and TGF-beta receptor inhibitor III;
most preferably, the ALK5 inhibitor is ALK5i II;
most preferably, the concentrations of the components in the fifth stage differentiation induction medium are respectively: (0.01-5) μ M SANT-1, (0.01-10) μ M retinoic acid, (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) μ M triiodothyronine, (0.01-50) μ MALK5i II, (1-50) μ M zinc sulfate, (1-50) μ g/mL heparin, (0.05-10) g/L sodium bicarbonate, (0.05-10) mM glutamine, (1-50) mM glucose, (0.05-10)% fetal bovine serum albumin;
most preferably, the concentrations of the components in the fifth stage differentiation induction medium are: 0.25 μ M SANT-1, 0.1 μ M retinoic acid, 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 μ M triiodothyronine, 10 μ M ALK5i II, 10 μ M zinc sulfate, 10 μ g/mL heparin, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin;
most preferably, the fifth stage induces differentiation for a period of 3 days;
most preferably, on day 11 of induction, cells are plated into low-affinity culture plates, and on days 11-13 of induction, differentiation is induced using the fifth-stage differentiation-inducing medium;
most preferably, fresh medium is replaced daily on days 11-13 of induction.
Further, the sixth stage differentiation induction medium described in step (7) comprises a basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, a BMP inhibitor, an insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, an ALK5 inhibitor, zinc sulfate, a gamma-secretase inhibitor, heparin;
preferably, the BMP inhibitors include LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD 161570;
more preferably, the BMP inhibitor is LDN 193189;
preferably, the ALK5 inhibitors include ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-beta receptor inhibitor V, TGF-beta receptor inhibitor I, TGF-beta receptor inhibitor IV, TGF-beta receptor inhibitor VII, TGF-beta receptor inhibitor VIII, TGF-beta receptor inhibitor II, TGF-beta receptor inhibitor VI, and TGF-beta receptor inhibitor III;
more preferably, the ALK5 inhibitor is ALK5i II;
preferably, the gamma-secretase inhibitor comprises GSi XX, GSi IX, GSi XI, GSi XII, GSi XIII, GSi XIV, GSi XVI, GSi XIX, GSi XVII, GSi XXI, RO4929097, LY450139, MK-0752, BMS-708163, LY411575, LY 3039478;
more preferably, the gamma-secretase inhibitor is GSi XX;
preferably, the sixth stage differentiation induction medium comprises a sixth stage differentiation induction medium a and a sixth stage differentiation induction medium B;
more preferably, said sixth stage differentiation induction medium a comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, zinc sulfate, GSi XX, heparin;
more preferably, said sixth stage differentiation induction medium B comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, zinc sulfate, heparin;
most preferably, the sixth stage induces differentiation for a period of 14 days;
most preferably, on the 14 th to 20 th days of induction, the sixth stage induction differentiation culture medium A is adopted for induction differentiation;
most preferably, on days 21-27 of induction, differentiation is induced using stage six differentiation induction medium B;
most preferably, fresh medium is changed daily between days 14-27 of induction;
most preferably, the concentrations of the components in the sixth stage differentiation induction medium are respectively: (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) μ M triiodothyronine, (0.01-50) μ M ALK5i II, (1-50) μ M zinc sulfate, (10-500) nM GSi XX, (1-50) μ g/mL heparin, (0.05-10) g/L sodium bicarbonate, (0.05-10) mM glutamine, (1-50) mM glucose, (0.05-10)% fetal bovine serum albumin;
most preferably, the concentrations of the components in the sixth stage differentiation induction medium are respectively: 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 μ M triiodothyronine, 10 μ M ALK5i II, 10 μ M zinc sulfate, 100nM GSi XX, 10 μ g/mL heparin, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin;
preferably, the seventh stage differentiation induction medium described in step (8) comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium-ethanolamine supplement, triiodothyronine, ALK5 inhibitor, zinc sulfate, heparin, acetyl-l-cysteine, water-soluble vitamin E, Axl inhibitor;
more preferably, the ALK5 inhibitors include ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-beta receptor inhibitor V, TGF-beta receptor inhibitor I, TGF-beta receptor inhibitor IV, TGF-beta receptor inhibitor VII, TGF-beta receptor inhibitor VIII, TGF-beta receptor inhibitor II, TGF-beta receptor inhibitor VI, and TGF-beta receptor inhibitor III;
most preferably, the ALK5 inhibitor is ALK5i II;
more preferably, the Axl inhibitors include 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 concentration of each component in the seventh stage differentiation induction medium is: (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) μ M triiodothyronine, (0.01-50) μ M ALK5i II, (1-50) μ M zinc sulfate, (1-50) μ g/mL heparin, (0.01-10) mM acetyl-L-cysteine, (1-50) μ M water-soluble vitamin E, (1-10) μ M R428, (0.05-10) g/L sodium bicarbonate, (0.05-10) mM glutamine, (1-50) mM glucose, (0.05-10)% fetal bovine serum albumin;
most preferably, the concentration of each component in the seventh stage differentiation induction medium is: 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 μ M triiodothyronine, 10 μ M ALK5i II, 10 μ M zinc sulfate, 10 μ g/mL heparin, 1mM acetyl-L-cysteine, 10 μ M water soluble vitamin E, 2 μ M R428, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin;
most preferably, the seventh stage induces differentiation for a period of 13 days;
most preferably, on days 28-34 of induction, the seventh stage differentiation induction medium is used for induction differentiation;
most preferably, fresh medium is changed daily between induction days 28-34;
preferably, the eighth stage differentiation induction medium described in step (9) comprises islet alpha cell beta cell transformation medium, artemether, GABAA activator;
more preferably, the islet alpha cell transbeta cell culture medium comprises Ham's F-12medium, medium 199, glutamine, calcium chloride dihydrate, N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid, fetal bovine serum albumin, insulin-transferrin-selenium-ethanolamine additive, water-soluble vitamin E, nicotinamide, heparin, deoxyribonuclease i, a necrotic apoptosis inhibitor, a serine protease inhibitor;
most preferably, the necrotic apoptosis inhibitor comprises Necrostatin-1, Necrostatin-2;
most preferably, the necrotic apoptosis inhibitor is Necrostatin-1;
most preferably, the serpin comprises Pefabloc, Benzamidine, MBTI, PMSF, LBTI;
most preferably, the serine protease inhibitor is Pefabloc;
more preferably, the GABAA activator comprises NS11394, R-16659, Muscimol, Arbaclofen, Propofol, Baclofen, Isoguvacine hydrochloride;
most preferably, the GABAA activator is NS 11394;
most preferably, the concentrations of the components in the eighth stage differentiation-inducing medium are respectively: 50% Ham's F-12medium, 50% medium 199, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (1-50) μ M water-soluble vitamin E, (1-50) mM nicotinamide, (1-50) μ g/mL heparin, (0.01-10) U/mL deoxyribonuclease I, (10-500) μ M Neostatin-1, (0.01-5) μ M Pefabloc, (0.01-10) mM glutamine, (0.01-10) mM calcium chloride dihydrate, (0.1-50) mM N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid, (0.05-10)% fetal bovine serum albumin, (1-50) μ M artemether, (1-50) μ M NS 11394;
most preferably, the concentrations of the components in the eighth stage differentiation-inducing medium are respectively: 50% Ham's F-12medium, 50% medium 199, 0.5% insulin-transferrin-selenium-ethanolamine additive, 10 μ M water-soluble vitamin E, 10mM nicotinamide, 10 μ g/mL heparin, 1U/mL deoxyribonuclease I, 100 μ M Neostatin-1, 0.1 μ M Pefabloc, 2mM glutamine, 2.5mM calcium chloride dihydrate, 10mM N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid, 2% fetal bovine serum albumin, 10 μ M artemether, 10 μ M NS 11394;
most preferably, the eighth stage induces differentiation for a period of 6 days;
most preferably, on the 35 th to 40 th days of induction, the eighth stage differentiation induction medium is used for induction differentiation;
most preferably, fresh medium is replaced daily on days 35-40 of induction.
In a fourth aspect of the invention there is provided a functional islet beta cell or cell population derived from an induced pluripotent stem cell.
Further, the cell or cell population is obtained by inducing differentiation using the method of the third aspect of the present invention;
preferably, the islet beta cell or cell population is a functional, stable islet beta cell or cell population.
In a fifth aspect of the present invention, there is provided a pharmaceutical composition for the treatment and/or prevention of diabetes.
Further, the pharmaceutical composition comprises a cell or cell population according to the fourth invention of the present invention;
preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant;
preferably, the diabetes comprises type 1diabetes, type 2diabetes, gestational diabetes, monogenic diabetes;
more preferably, the diabetes is type 1 diabetes.
A sixth aspect of the invention provides the use of any one of the following:
(1) the application of the combination of artemether and NS11394 in promoting the transformation of islet alpha cells into islet beta cells;
(2) the application of the combination of artemether and NS11394 in inducing the differentiation of pluripotent stem cells into functional islet beta cells;
(3) the use of an induction differentiation agent according to the first aspect of the present invention in the preparation of an induction differentiation medium for inducing pluripotent stem cells to differentiate into functional islet beta cells;
(4) the use of an induction differentiation agent according to the first aspect of the present invention for inducing differentiation of pluripotent stem cells into functional islet beta cells;
(5) the use of an induced differentiation medium according to the second aspect of the present invention for inducing differentiation of pluripotent stem cells into functional islet beta cells;
(6) use of a cell or population of cells according to the fourth aspect of the invention in the manufacture of a medicament for the treatment and/or prophylaxis of diabetes;
(7) the use of a pharmaceutical composition according to the fifth aspect of the invention for the treatment and/or prevention of diabetes;
preferably, the diabetes comprises type 1diabetes, type 2diabetes, gestational diabetes, monogenic diabetes;
more preferably, the diabetes is type 1 diabetes.
Compared with the prior art, the invention has the advantages and beneficial effects that:
the invention provides a novel induction method for promoting islet alpha cells to be converted into beta cells, which is characterized in that on the basis of the traditional induction method, a culture medium for converting the islet alpha cells into the beta cells is replaced when the islet alpha cells are induced to differentiate on the 35 th day, and artemether and GABAA activator NS11394 are added for induction till the 40 th day. The verification shows that the expression level of the marker molecules of the islet beta cells prepared by the induction method provided by the invention is obviously increased, which indicates that the induction method provided by the invention can obviously improve the expression level of the islet beta cells and provides scientific basis for treating type 1diabetes clinically.
Drawings
FIG. 1 is a statistical chart of results of qPCR detection of the expression of pancreatic islet maturation marker gene mRNA, wherein group A (control group): using a seventh stage induction differentiation medium on the induction days 35-40; group B: using alpha cell to beta cell culture medium at 35-40 days of induction; group C: alpha cell transbeta cell medium was used on days 35-40 of induction with the addition of artemether and GABAA activator NS 11394.
Detailed Description
The invention is further illustrated below with reference to specific examples, which are intended to be purely exemplary of the invention and are not to be interpreted as limiting the same. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biomaterials, etc. used in the following examples are commercially available unless otherwise specified.
Example 1 method for inducing differentiation of iPSCs into pancreatic islets
1. Experimental Material
The experimental materials referred to in the examples of the present invention are shown in Table 1.
TABLE 1 Experimental materials
Figure BDA0003655439530000191
Figure BDA0003655439530000201
2. Method for inducing differentiation of iPSCs into islet beta cells
Human-derived iPSCs (from zeono medical science and technology ltd, beijing, prepared according to the method described in the earlier patent (201910110768.7) of the company) having a degree of polymerization of 70% to 80% were washed 2 times with DPBS phosphate buffer and then digested with TrypLE Express digestive enzyme at 37 ℃ for 3 to 5 min. The mixture was neutralized with DMEM/F12 medium at a ratio of 5:1 and centrifuged at 200g for 5 min. The seed was resuspended in E8 complete Medium + 10. mu. M Y-27632(ROCK inhibitor) at a density of about 1.8-2.2X 10 5 cells/cm 2 12-well cell plates were coated 24-48 hours in advance with 0.13-0.2mg/mL Matrigel.
(1) The first stage is as follows: inducing iPSCs to differentiate to definitive endoderm
The differentiation induction medium of the first stage consists of a basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (bovine serum albumin) (BSA), GDF8, and CHIR-99021.
In this medium, GDF8 was present at a concentration of 100ng/mL, CHIR-99021 was present at a concentration of 3. mu.M, Bovine Serum Albumin (BSA) was present at a concentration of 0.5%, glucose was present at a concentration of 10mM, glutamine was present at a concentration of 2mM, and sodium bicarbonate was present at a concentration of 1.5 g/L.
The time for the first stage induction was 3 days. Wherein, on induction day 1 (D1), the concentration of GDF8 in the medium is 100 ng/mL; the concentration of CHIR-99021 was 3. mu. mol/L. On induction days 2-3 (D2-D3), the concentration of GDF8 in the medium was 100 ng/mL. Namely: induction day 1, using first stage medium a consisting of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal Bovine Serum Albumin) (BSA), GDF8, and CHIR-99021; day 2-3 of induction, first stage medium B was used, consisting of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal Bovine Serum Albumin) (BSA) and GDF 8. Fresh medium was changed daily.
(2) And a second stage: inducing differentiation of definitive endoderm cells to primitive gut tube cells
The induced differentiation medium of the second stage consists of a basic medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (bovine serum albumin fetal (BSA)), ascorbic acid and fibroblast growth factor 7 (FGF-7).
In this medium, the concentration of ascorbic acid was 0.25mM, the concentration of fibroblast growth factor 7(FGF-7) was 50ng/mL, the concentration of fetal Bovine Serum Albumin (BSA) was 0.5%, the concentration of glucose was 10mM, the concentration of glutamine was 2mM, and the concentration of sodium bicarbonate was 1.5 g/L.
The time for the second phase induction was 2 days. Wherein, on days 4-5 of induction (D4-D5), the concentration of ascorbic acid in the medium is 0.25 mM; the concentration of fibroblast growth factor 7(FGF-7) is 50 ng/mL; namely: the second stage differentiation-inducing medium was used on induction days 4-5. Fresh medium was changed daily.
(3) And a third stage: inducing differentiation of primitive gut tube cells towards posterior foregut cells
The induced differentiation culture medium of the third stage consists of a basic culture medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (bovine serum albumin) (BSA), ascorbic acid, fibroblast growth factor 7(FGF-7), SANT-1, retinoic acid, LDN193189, an insulin-transferrin-selenium-ethanolamine additive and a protein kinase C activator TPPB.
In the medium, the concentration of ascorbic acid was 0.25mM, the concentration of fibroblast growth factor 7(FGF-7) was 50ng/mL, the concentration of SANT-1 was 0.25. mu.M, the concentration of retinoic acid was 1. mu.M, the concentration of LDN193189 was 100nM, the concentration of insulin-transferrin-selenium-ethanolamine additive was 0.5%, the concentration of protein kinase C activator TPPB was 200nM, the concentration of bovine serum albumin was 2%, the concentration of glucose was 10mM, the concentration of glutamine was 2mM, and the concentration of sodium bicarbonate was 2.5 g/L.
The induction time of the third stage is 2 days, and the ascorbic acid concentration in the culture medium is 0.25mM on the induction 6-7 days (D6-D7); the concentration of fibroblast growth factor 7(FGF-7) is 50 ng/mL; the concentration of SANT-1 is 0.25. mu.M; retinoic acid concentration is 1 μ M; the concentration of LDN193189 is 100 nM; the concentration of the insulin-transferrin-selenium-ethanolamine additive was 0.5% and the concentration of protein kinase C activator TPPB was 200nM, i.e.: day 6-7 of induction the differentiation induction medium of the third stage was used, with fresh medium being changed daily.
(4) A fourth stage: induction of differentiation of posterior foregut cells into pancreatic progenitor cells
The induced differentiation culture medium of the fourth stage consists of a basic culture medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (bovine serum albumin) (BSA), ascorbic acid, fibroblast growth factor 7(FGF-7), SANT-1, retinoic acid, LDN193189, an insulin-transferrin-selenium-ethanolamine additive and a protein kinase C activator TPPB.
In the medium, the concentration of ascorbic acid was 0.25mM, the concentration of fibroblast growth factor 7(FGF-7) was 2ng/mL, the concentration of SANT-1 was 0.25. mu.M, the concentration of retinoic acid was 1. mu.M, the concentration of LDN193189 was 100nM, the concentration of insulin-transferrin-selenium-ethanolamine additive was 0.5%, the concentration of protein kinase C activator TPPB was 200nM, the concentration of bovine serum albumin was 2%, the concentration of glucose was 10mM, the concentration of glutamine was 2mM, and the concentration of sodium bicarbonate was 2.5 g/L.
The fourth stage induction time is 3 days, and the ascorbic acid concentration in the culture medium is 0.25mM on induction days 8-10 (D8-D10); the concentration of fibroblast growth factor 7(FGF-7) is 2 ng/mL; the concentration of SANT-1 is 0.25. mu.M; retinoic acid concentration is 1 μ M; the concentration of LDN193189 is 100 nM; the concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5 percent; the concentration of protein kinase C activator TPPB was 200nM, i.e.: the induced differentiation medium of the fourth stage was used on the induction days 8 to 10, and the fresh medium was changed every day.
(5) The fifth stage: induction of pancreatic progenitor cell differentiation into endocrine progenitor cells
The differentiation induction culture medium of the fifth stage consists of a basic culture medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (bovine serum albumin) (BSA), SANT-1, retinoic acid, LDN193189, an insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, an ALK5 inhibitor ALK5i II, zinc sulfate and heparin.
In the medium, the concentration of SANT-1 was 0.25. mu.M, that of retinoic acid was 0.1. mu.M, that of LDN193189 was 100nM, that of insulin-transferrin-selenium-ethanolamine additive was 0.5%, that of triiodothyronine was 1. mu.M, that of ALK5 inhibitor ALK5i II was 10. mu.M, that of zinc sulfate was 10. mu.M, that of heparin was 10. mu.g/mL, that of sodium bicarbonate was 1.5g/L, that of glutamine was 2mM, that of glucose was 20mM, and that of fetal bovine serum albumin was 2%.
The induction time of the fifth stage is 3 days, on the induction day 11 (D11), the cells are plated into low-affinity culture plates by cell scraping, and on the induction days 11-13 (D11-D13), the concentration of SANT-1 in the culture medium is 0.25 μ M; retinoic acid concentration is 0.1 μ M; the concentration of LDN193189 is 100 nM; the concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5 percent; the concentration of triiodothyronine is 1 μ M; the concentration of ALK5 inhibitor ALK5i II was 10 μ M; the concentration of zinc sulfate is 10 MuM; the concentration of heparin was 10 μ g/mL, i.e.: the differentiation induction medium of the fifth stage was used on induction days 11 to 13, and fresh medium was changed daily.
(6) The sixth stage: induction of endocrine progenitor cell differentiation into immature islet beta cells
The differentiation induction medium of the sixth stage consists of a basic medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (bovine serum albumin) (BSA), LDN193189, an insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, an ALK5 inhibitor ALK5i II, zinc sulfate, a gamma-secretase inhibitor GSi XX and heparin.
In the culture medium, the concentration of LDN193189 is 100nM, the concentration of insulin-transferrin-selenium-ethanolamine additive is 0.5%, the concentration of triiodothyronine is 1 μ M, the concentration of ALK5 inhibitor ALK5iII is 10 μ M, the concentration of zinc sulfate is 10 μ M, the concentration of gamma-secretase inhibitor GSi XX is 100nM, the concentration of heparin is 10 μ g/mL, the concentration of sodium bicarbonate is 1.5g/L, the concentration of glutamine is 2mM, the concentration of glucose is 20mM, and the concentration of fetal bovine serum albumin is 2%.
The induction time of the sixth stage is 14 days, and the concentration of LDN193189 in the culture medium is 100nM on induction days 14-20 (D14-D20); the concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5 percent; the concentration of triiodothyronine is 1 μ M; the concentration of ALK5 inhibitor ALK5i II was 10 μ M; the concentration of zinc sulfate is 10 MuM; the gamma-secretase inhibitor GSi XX is 100nM, and the heparin concentration is 10. mu.g/mL. The concentration of LDN193189 in the medium was 100nM on induction days 21-27 (D21-D27); the concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5 percent; the concentration of triiodothyronine is 1 μ M; the concentration of ALK5 inhibitor ALK5i II was 10 μ M; the concentration of zinc sulfate is 10 MuM; the concentration of heparin was 10 μ g/mL, i.e.: induction days 14-20, using sixth stage medium a consisting of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal Bovine Serum Albumin) (BSA), LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, gamma-secretase inhibitor GSi XX, and heparin; induction days 21-27, a sixth stage medium B was used consisting of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal Bovine Serum Albumin) (BSA), LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, and heparin. Fresh medium was changed daily.
(7) A seventh stage: inducing differentiation of immature islet beta cells into mature islet beta cells
The differentiation induction medium of the seventh stage consists of a basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal Bovine Serum Albumin) (BSA), insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, heparin, acetyl-l-cysteine (N-Cys), water-soluble vitamin E, and Axl inhibitor R428.
In this medium, the concentration of insulin-transferrin-selenium-ethanolamine additive was 0.5%, the concentration of triiodothyronine was 1 μ M, the concentration of ALK5 inhibitor ALK5i II was 10 μ M, the concentration of zinc sulfate was 10 μ M, the concentration of heparin was 10 μ g/mL, the concentration of acetyl-L-cysteine (N-Cys) was 1mM, the concentration of water-soluble vitamin E was 10 μ M, the concentration of Axl inhibitor R428 was 2 μ M, the concentration of sodium bicarbonate was 1.5g/L, the concentration of glutamine was 2mM, the concentration of glucose was 20mM, and the concentration of fetal bovine serum albumin was 2%.
The induction time of the seventh stage is 13 days, and the concentration of the insulin-transferrin-selenium-ethanolamine additive in the culture medium is 0.5% on the induction days 28-34 (D28-D34); the concentration of triiodothyronine is 1 μ M; the concentration of ALK5 inhibitor ALK5i II was 10 μ M; the concentration of zinc sulfate is 10 MuM; the concentration of heparin is 10 mug/mL; the concentration of acetyl-l-cysteine (N-Cys) was 1 mM; the concentration of the water-soluble vitamin E is 10 mu M; the concentration of Axl inhibitor R428 was 2 μ M, i.e.: differentiation-inducing medium of the seventh stage was used on days 28-34 of induction. Fresh medium was changed daily.
(8) An eighth stage: further inducing maturation of islet beta cells
The culture medium for inducing differentiation of the eighth stage uses a culture medium for islet a cell to convert beta cells, the culture medium comprises 50% Ham's F-12medium, 50% medium 199, glutamine, calcium chloride dihydrate, N-2 hydroxyethyl piperazine-N-2-ethane sulfonic acid, defatted BSA (bovine serum albumin) (BSA), insulin-transferrin-selenium-ethanolamine additive, water-soluble vitamin E, nicotinamide, heparin, deoxyribonuclease I, necrotizing apoptosis inhibitor Necrostatin-1 and serine protease inhibitor Pefabloc, and artemether and GABAA activator NS11394 are small molecules additionally added.
In the culture medium, the concentration of insulin-transferrin-selenium-ethanolamine additive is 0.5%, the concentration of water-soluble vitamin E is 10 muM, the concentration of nicotinamide is 10mM, the concentration of heparin is 10 mug/mL, the concentration of deoxyribonuclease I is 1U/mL, the concentration of necrotizing apoptosis inhibitor Necrostatin-1 is 100 muM, the concentration of serine protease inhibitor Pefabloc is 0.1 muM, the concentration of glutamine is 2mM, calcium chloride dihydrate is 2.5mM, the concentration of N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid is 10mM, the concentration of fetal bovine serum albumin is 2%, the concentration of artemether is 10 muM, and the concentration of GABAA activator NS11394 is 10 muM.
The induction time of the eighth stage is 6 days, and the concentration of the insulin-transferrin-selenium-ethanolamine additive in the culture medium is 0.5% on the induction days 35-40 (D35-D40); the concentration of the water-soluble vitamin E is 10 mu M; the concentration of nicotinamide is 10 mM; the concentration of heparin is 10 mug/mL; the concentration of the DNase I is 1U/mL; the concentration of necroptosis inhibitor Necrostatin-1 is 100 mu M; the concentration of the serine protease inhibitor Pefabloc is 0.1 mu M, and the concentration of the additionally added small molecular artemether is 10 mu M; GABAA activator NS11394 at a concentration of 10 μ M; namely: differentiation-inducing medium of the eighth stage was used on days 35-40 of induction. Fresh medium was changed daily.
Example 2 qPCR detection of expression of mRNA of pancreatic islet maturation marker Gene
1. Experimental methods
After the iPSCs are induced, insulin (INSULIN) (INS), glucagon (GCG) and MAFA are used as islet markers, and the expression condition of the islet marker mRNA in the islet cells obtained through induced differentiation of the iPSCs is detected.
Wherein, Insulin (INS) refers to insulin, a polypeptide hormone consisting of alpha and beta double chains secreted by islet beta cells, consists of 51 amino acids, and has a molecular weight of about 5800 Da. Insulin is the only blood glucose lowering hormone in the body. A marker gene for islet beta cells;
glucagon (gcg) refers to glucagon, a linear polypeptide hormone consisting of 29 amino acids secreted by islet alpha cells and having a molecular weight of 3485 Da. Has the function of increasing blood sugar in vivo;
MAFA refers to the sarcoplasmic fibrosarcoma oncogene homolog A gene (v-maf mucouloaponeuronic fibrosarcoma oncogene homolog A), which is a transcription factor having a leucine zipper structure. MAFA is the only islet β cell insulin gene-activating transcription factor discovered to date. The maturation and functional maintenance of pancreatic β -cells are dependent on the normal expression of MAFA proteins. Thus, MAFA is a maturation marker gene for pancreatic islet β cells.
The specific experimental method is as follows:
1.1 Total RNA extraction and inversion
(1) After washing the cell sample in the cell culture dish twice with PBS, sucking up the PBS with a 1mL gun, adding 1mL Trizol (Invitrogen) solution, blowing, mixing uniformly, sucking into a 1.5mL RNase free EP tube to fully crack the cells, and standing for 5 minutes at room temperature;
(2) 0.2mL of chloroform was added per 1mL of Trizol. The sample tube cap was closed, shaken vigorously for 15 seconds and incubated for 3 minutes at room temperature;
(3) after centrifugation at 12,000rpm for 10 minutes at 4 ℃ the sample will separate 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 aqueous layer was about 60% of the volume of RL added, the aqueous phase was transferred to a new tube and the next operation was carried out;
(4) 1-fold volume of 70% ethanol was added (please first check if absolute ethanol had been added!), and the mixture was mixed by inversion (precipitation may occur). Transferring the obtained solution and possible precipitate into adsorption column RA, centrifuging at 10,000rpm for 45 s, discarding waste liquid, and sleeving the adsorption column back to the collecting tube;
(5) adding 500 mu L deproteinized liquid RE, centrifuging at 12,000rpm for 45 s, and discarding waste liquid;
(6) adding 700 μ L of rinsing solution RW (please check whether absolute ethanol is added), centrifuging at 12,000rpm for 60 s, and discarding the waste solution;
(7) adding 500 μ L of rinsing solution RW, centrifuging at 12,000rpm for 60 s, and discarding the waste liquid;
(8) putting the adsorption column RA back into an empty collection pipe, centrifuging at 12,000rpm for 2 minutes, and removing the rinsing liquid as much as possible so as to prevent residual ethanol in the rinsing liquid from inhibiting downstream reaction;
(9) taking out the adsorption column RA, placing into an RNase free centrifuge tube, adding 50-80 μ L of RNase free water in the middle part of the adsorption membrane according to the expected RNA yield, and heating in water bath at 65-70 deg.C in advance to obtain better heating effect;
(10) the mixture was left at room temperature for 2 minutes and centrifuged at 12,000rpm for 1 minute. If more RNA is needed, the obtained solution can be added into the centrifugal adsorption column again and centrifuged for 1 minute, or 30 μ L of RNase free water is added additionally and centrifuged for 1 minute, and the two eluates are combined;
(11) the larger the elution volume, the higher the elution efficiency, and if the RNA concentration is required to be high, the elution volume can be reduced appropriately, but the minimum volume is preferably not less than 30. mu.L, and the smaller the volume, the lower the RNA elution efficiency, and the RNA yield.
1.2 Total RNA purity and integrity assays
(1) And (3) purity detection: taking 1 microliter of RNA sample, determining OD value on a nucleic acid protein detector, wherein the ratio of OD260/OD280 is 2, which indicates that the prepared RNA is relatively pure and has no protein pollution;
(2) total RNA integrity test: taking 6 mu L of RNA sample, carrying out electrophoresis on 1% agarose gel for 150V multiplied by 10min, observing by using a gel imaging system and taking pictures, and if three strips of 5s rRNA, 18s rRNA and 28s rRNA of the total RNA are complete, the extraction of the total RNA can be proved to be complete.
1.3RNA inversion
(1) Measuring and calculating the RNA concentration by using a spectrophotometer, and reversing according to the concentration of 500 ng;
(2) reagents were premixed with total RNA as follows: 500ng of Total RNA, 1. mu.L of Oligo dT, 10. mu.L of 2XTS Reaction Mix, 1. mu.L of RI/RT Enzyme Mix, 1. mu.L of gDNA Remover, and 20. mu.L of Total volume adjusted by RNase-free Water;
(3) transferring the premixed solution into a PCR instrument, and carrying out reaction by using a cDNA template (incubation at 42 ℃ for 30min and incubation at 85 ℃ for 5 s);
(4) rapidly transferring the reversed cDNA onto ice for 1min for cooling;
(5) storing at-20 deg.C, and diluting according to required concentration before use.
1.4 fluorescent quantitative PCR (qPCR) experimental method
The primer sequence information of the qPCR detection genes INS, GCG and MAFA is shown in the following table 2;
TABLE 2 primer sequence information for qPCR detection of genes INS, GCG, MAFA
Figure BDA0003655439530000281
The template is as follows: forward Primer (10. mu.M) 0.4. mu.L, Reverse Primer (10. mu.M) 0.4. mu.L, 2xTransStar Top/Tip Green qPCR SuperMix 10. mu.L, nucleic-free Water 8.2. mu.L, cDNA 1. mu.L, Total volume 20. mu.L.
(1) Firstly, premixing a reagent and a primer according to an upper template, and adding the reagent and the primer into an eight-connected tube, wherein each hole is 18 mu L;
(2) quickly adding 2 mu L of cDNA into the eight-connected tube, and covering a cover to perform instant separation;
(3) putting the mixture into a Light cycler instrument to react according to a 3-step method, wherein the cycle number is 40;
(4) the template is as follows Temp Time 94 ℃ 30s 94 ℃ 5s 45cycles 55 ℃ 15s 72 ℃ 10 s.
1.5 the specific grouping is as follows:
group a (control group): using induction differentiation culture medium of seventh stage at 35-40 days;
group B: using alpha-transbeta medium at 35-40 days of induction;
group C: on induction days 35-40, a-transbeta medium was used, with the addition of artemether and GABAA activator NS11394, wherein the concentration of artemether was 10 μ M and the concentration of GABAA activator NS11394 was 10 μ M.
2. Results of the experiment
The results of the qPCR assay are shown in FIG. 1, and the results show that the mRNA expression levels of INS, GCG and MAFA genes in group C are significantly higher than those in other groups (p < 0.05). The mRNA expression level of INS gene of islet cells obtained after induction of the group C is increased by 27.76 times compared with that of the group A, the mRNA expression level of GCG gene is increased by 14.82 times compared with that of the group A, and the mRNA expression level of MAFA gene is increased by 4.34 times compared with that of the group A, which shows that in the islet maturation stage, iPSCs are induced by adopting alpha cell to beta cell culture medium in combination with artemether and GABAA activator NS11394, the maturation of beta cells can be promoted, more islet beta cells can be obtained, and the induction efficiency is higher.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
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Claims (10)

1. An induction differentiation agent for inducing the differentiation of pluripotent stem cells into functional islet beta cells, which is characterized by comprising a first-stage induction differentiation agent, a second-stage induction differentiation agent, a third-stage induction differentiation agent, a fourth-stage induction differentiation agent, a fifth-stage induction differentiation agent, a sixth-stage induction differentiation agent, a seventh-stage induction differentiation agent and an eighth-stage induction differentiation agent;
preferably, the first-stage induction differentiation agent comprises a first-stage induction differentiation agent A and a first-stage induction differentiation agent B;
more preferably, the first stage induction differentiation agent a comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, GDF8, CHIR-99021;
more preferably, the first stage induction differentiation agent B comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, GDF 8;
preferably, the second stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, FGF-7;
preferably, the third stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, bovine serum albumin, ascorbic acid, FGF-7, SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive, TPPB;
preferably, the fourth stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, FGF-7, SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive, TPPB;
preferably, the fifth stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, zinc sulfate, heparin;
preferably, the sixth stage induction differentiation agent comprises a sixth stage induction differentiation agent a and a sixth stage induction differentiation agent B;
more preferably, the sixth stage induction differentiation agent a comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, zinc sulfate, GSi XX, heparin;
more preferably, the sixth stage induction differentiation agent B comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, zinc sulfate, heparin;
preferably, the seventh stage induction differentiation agent comprises sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium-ethanolamine supplement, triiodothyronine, ALK5iII, zinc sulfate, heparin, acetyl-l-cysteine, water soluble vitamin E, R428;
preferably, the eighth stage induction differentiation agent comprises artemether and NS 11394;
more preferably, the eighth stage induction differentiation agent further comprises glutamine, calcium chloride dihydrate, N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid, fetal bovine serum albumin, insulin-transferrin-selenium-ethanolamine additive, water-soluble vitamin E, nicotinamide, heparin, deoxyribonuclease i, Necrostatin-1, Pefabloc;
most preferably, the concentrations of the ingredients in the first-stage induction differentiation agent are: (10-500) ng/mL GDF8, (0.5-20) μ M CHIR-99021, (0.01-5)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the ingredients in the first-stage induced differentiation agent are respectively: 100ng/mL GDF8, 3. mu.M CHIR-99021, 0.5% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 1.5g/L sodium bicarbonate;
more preferably, the concentrations of the ingredients in the second-stage induction differentiation agent are respectively: (0.01-5) mM ascorbic acid, (10-100) ng/mL FGF-7, (0.01-5)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the ingredients in the second stage induction differentiation agent are respectively: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.5% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 1.5g/L sodium bicarbonate;
more preferably, the concentrations of the ingredients in the third stage induction differentiation agent are respectively: (0.01-5) mM ascorbic acid, (10-100) ng/mL FGF-7, (0.01-5) μ M SANT-1, (0.01-10) μ M retinoic acid, (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (50-500) nM TPPB, (0.05-10)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the ingredients in the third stage induction differentiation agent are respectively: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.25. mu.M SANT-1, 1. mu.M retinoic acid, 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 200nM TPPB, 2% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 2.5g/L sodium bicarbonate;
more preferably, the concentrations of the components in the fourth stage induction differentiation agent are respectively as follows: (0.01-5) mM ascorbic acid, (0.01-10) ng/mL FGF-7, (0.01-5) μ M SANT-1, (0.01-10) μ M retinoic acid, (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (50-500) nM TPPB, (0.05-10)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the components in the fourth stage induction differentiation agent are respectively as follows: 0.25mM ascorbic acid, 2ng/mL FGF-7, 0.25. mu.M SANT-1, 1. mu.M retinoic acid, 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 200nM TPPB, 2% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 2.5g/L sodium bicarbonate;
more preferably, the concentrations of the ingredients in the fifth stage induction differentiation agent are respectively as follows: (0.01-5) μ MSANT-1, (0.01-10) μ M retinoic acid, (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) μ M triiodothyronine, (0.01-50) μ M ALK5iII, (1-50) μ M zinc sulfate, (1-50) μ g/mL heparin, (0.05-10) g/L sodium bicarbonate, (0.05-10) mM glutamine, (1-50) mM glucose, (0.05-10)% fetal bovine serum albumin;
most preferably, the concentrations of the ingredients in the fifth stage induction differentiation agent are respectively: 0.25 μ M SANT-1, 0.1 μ M retinoic acid, 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 μ M triiodothyronine, 10 μ M ALK5iII, 10 μ M zinc sulfate, 10 μ g/mL heparin, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin;
most preferably, the concentrations of the ingredients in the sixth stage induction differentiation agent are respectively: (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) μ M triiodothyronine, (0.01-50) μ M ALK5iII, (1-50) μ M zinc sulfate, (10-500) nM GSiXX, (1-50) μ g/mL heparin, (0.05-10) g/L sodium bicarbonate, (0.05-10) mM glutamine, (1-50) mM glucose, (0.05-10)% fetal bovine serum albumin;
most preferably, the concentrations of the ingredients in the sixth stage induction differentiation agent are respectively: 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 μ M triiodothyronine, 10 μ M ALK5i II, 10 μ M zinc sulfate, 100nM GSi XX, 10 μ g/mL heparin, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin;
more preferably, the concentrations of the ingredients in the seventh stage induction differentiation agent are respectively: (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) μ M triiodothyronine, (0.01-50) μ MALK5i II, (1-50) μ M zinc sulfate, (1-50) μ g/mL heparin, (0.01-10) mM acetyl-L-cysteine, (1-50) μ M water-soluble vitamin E, (1-10) μ M R428, (0.05-10) g/L sodium bicarbonate, (0.05-10) mM glutamine, (1-50) mM glucose, (0.05-10)% fetal bovine serum albumin;
most preferably, the concentrations of the ingredients in the seventh stage induction differentiation agent are respectively: 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 μ M triiodothyronine, 10 μ M ALK5iII, 10 μ M zinc sulfate, 10 μ g/mL heparin, 1mM acetyl-L-cysteine, 10 μ M water-soluble vitamin E, 2 μ M MR428, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin;
more preferably, the concentrations of the ingredients in the eighth stage induction differentiation agent are respectively: (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (1-50) μ M water-soluble vitamin E, (1-50) mM nicotinamide, (1-50) μ g/mL heparin, (0.01-10) U/mL deoxyribonuclease I, (10-500) μ M Microstatin-1, (0.01-5) μ M Pefabloc, (0.01-10) mM glutamine, (0.01-10) mM calcium chloride dihydrate, (0.1-50) mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, (0.05-10)% fetal bovine serum albumin, (1-50) μ M artemether, (1-50) μ M NS 11394;
most preferably, the concentrations of the components in the eighth stage differentiation-inducing culture agent are respectively: 0.5% insulin-transferrin-selenium-ethanolamine additive, 10 μ M water soluble vitamin E, 10mM nicotinamide, 10 μ g/mL heparin, 1U/mL deoxyribonuclease I, 100 μ M Neostatin-1, 0.1 μ M Pefabloc, 2mM glutamine, 2.5mM calcium chloride dihydrate, 10mM N-2 hydroxyethyl piperazine-N-2-ethane sulfonic acid, 2% bovine serum albumin, 10 μ M artemether, 10 μ M NS 11394.
2. An induced differentiation medium for inducing pluripotent stem cells to differentiate into functional islet beta cells is characterized by comprising 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, a sixth-stage induced differentiation medium, a seventh-stage induced differentiation medium and an eighth-stage induced differentiation medium;
preferably, the first stage induced differentiation medium comprises a basal medium MCDB131, the first stage induced differentiation agent described in claim 1;
preferably, the second-stage induced differentiation medium comprises a basal medium MCDB131, the second-stage induced differentiation agent described in claim 1;
preferably, the third stage induced differentiation medium comprises a basal medium MCDB131, the third stage induced differentiation agent described in claim 1;
preferably, the fourth stage induced differentiation medium comprises a basal medium MCDB131, the fourth stage induced differentiation agent described in claim 1;
preferably, the fifth stage differentiation-inducing medium comprises a basal medium MCDB131, the fifth stage differentiation-inducing agent described in claim 1;
preferably, the sixth stage differentiation-inducing medium comprises a basal medium MCDB131, the sixth stage differentiation-inducing agent described in claim 1;
preferably, the seventh stage induced differentiation medium comprises basal medium MCDB131, the seventh stage induced differentiation agent described in claim 1;
preferably, the eighth stage induction differentiation medium comprises 50% Ham's F-12medium, 50% medium 199, the eighth stage induction differentiation agent described in claim 1.
3. An induced differentiation method for inducing differentiation of pluripotent stem cells into functional islet beta cells, comprising the steps of:
(1) providing induced pluripotent stem cells and culturing in complete medium;
(2) inducing differentiation at a first stage, namely inducing the induced pluripotent stem cells obtained in the step (1) to differentiate towards definitive endoderm cells by adopting a first-stage induced differentiation culture medium;
(3) inducing differentiation in the second stage, inducing differentiation medium in the second stage to induce differentiation of definitive endoderm cell to original intestinal canal cell;
(4) inducing differentiation in the third stage, namely inducing the differentiation of primitive intestinal canal cells to backward foregut cells by adopting a third-stage inducing differentiation culture medium;
(5) a fourth stage of induced differentiation, wherein the foregut cells are induced to differentiate into pancreatic progenitor cells by adopting a fourth stage of induced differentiation culture medium;
(6) inducing differentiation in the fifth stage, namely inducing pancreatic progenitor cells to differentiate into endocrine progenitor cells by adopting a fifth-stage induced differentiation culture medium;
(7) inducing differentiation in a sixth stage, namely inducing the endocrine progenitor cells to differentiate into immature islet beta cells by adopting a sixth-stage induced differentiation culture medium;
(8) inducing differentiation at a seventh stage, namely inducing the immature islet beta cells to differentiate into mature islet beta cells by adopting a seventh-stage induced differentiation culture medium;
(9) and (3) inducing differentiation in an eighth stage, and further inducing the maturation of the islet beta cells by adopting an eighth stage induced differentiation culture medium to obtain the functional islet beta cells.
4. The method according to claim 3, wherein the complete medium in step (1) is E8 complete medium;
preferably, the E8 complete medium contains a ROCK inhibitor;
more preferably, the ROCK inhibitor comprises Y-27632, GSK429286A, RKI-1447, Y-33075dihydrochloride, Thiazovivin, K-115, SLx-2119, Chroman1, SAR407899, and/or SR-3677;
most preferably, the ROCK inhibitor is Y-27632;
most preferably, the concentration of Y-27632 is 0.001-100. mu.M;
most preferably, the concentration of Y-27632 is 10. mu.M;
preferably, the induced pluripotent stem cells in step (1) have a cell density of 0.1 to 10.0X 10 5 cells/cm 2 ；
More preferably, the induced pluripotent stem cells in step (1) have a cell density of 1.8 to 2.2X 10 5 cells/cm 2 ；
Preferably, the induced pluripotent stem cells in step (1) are derived from a mammal;
more preferably, the induced pluripotent stem cells described in step (1) are derived from a human, mouse, rat, goat, sheep, pig, cat, rabbit, dog, wolf, horse, or cow;
most preferably, the induced pluripotent stem cells in step (1) are derived from a human;
preferably, the first stage differentiation induction medium described in step (2) comprises a basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, growth differentiation factor, GSK-3 inhibitor;
more preferably, the growth differentiation factor comprises GDF8, GDF15, GDF9, GDF11, GDF8, GDF5, GDF1, GDF9B, GDF 6;
most preferably, the growth differentiation factor is GDF 8;
more preferably, the GSK-3 inhibitor comprises 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 differentiation-inducing medium described in step (2) comprises a first stage differentiation-inducing medium a, a first stage differentiation-inducing medium B;
most preferably, the first stage differentiation induction medium a comprises the basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, GDF8, CHIR-99021;
most preferably, the first stage differentiation induction medium B comprises the basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, GDF 8;
most preferably, the first stage induces differentiation for a period of 3 days;
most preferably, on the 1 st day of induction, the first stage induction differentiation medium A is used for induction differentiation;
most preferably, the first stage induction differentiation culture medium B is adopted for induction differentiation at the induction days 2-3;
most preferably, the concentrations of the components in the first stage differentiation induction medium are respectively: (10-500) ng/mL GDF8, (0.5-20) μ M CHIR-99021, (0.01-5)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the components in the first stage differentiation induction medium are respectively: 100ng/mL GDF8, 3. mu.M CHIR-99021, 0.5% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 1.5g/L sodium bicarbonate.
5. The method according to claim 3, wherein the second stage differentiation induction medium in step (3) comprises a basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, fibroblast growth factor;
preferably, the fibroblast growth factor comprises 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 the components in the second-stage differentiation-inducing medium are respectively: (0.01-5) mM ascorbic acid, (10-100) ng/mL FGF-7, (0.01-5)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the components in the second stage differentiation-inducing medium are: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.5% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 1.5g/L sodium bicarbonate;
most preferably, the period of time for said second stage to induce differentiation is 2 days;
most preferably, on days 4-5 of induction, inducing differentiation is performed using the second-stage differentiation-inducing medium;
preferably, the third stage differentiation induction medium described in step (4) comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, fibroblast growth factor, SANT-1, retinoic acid, BMP inhibitor, insulin-transferrin-selenium-ethanolamine additive, protein kinase C activator;
more preferably, the fibroblast growth factor comprises 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, PD 161570;
most preferably, the BMP inhibitor is LDN 193189;
more preferably, the protein kinase C activator comprises TPPB, PMA;
most preferably, the protein kinase C activator is TPPB;
most preferably, the concentrations of the components in the third stage differentiation-inducing medium are: (0.01-5) mM ascorbic acid, (10-100) ng/mL FGF-7, (0.01-5) μ M SANT-1, (0.01-10) μ M retinoic acid, (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (50-500) nM TPPB, (0.05-10)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the components in the third stage differentiation-inducing medium are: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.25. mu.M SANT-1, 1. mu.M retinoic acid, 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 200nM TPPB, 2% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 2.5g/L sodium bicarbonate;
most preferably, the period of time for the third stage to induce differentiation is 2 days;
most preferably, the third-stage differentiation-inducing medium is used for inducing differentiation on days 6 to 7.
6. The method according to claim 3, wherein the fourth stage differentiation induction medium in step (5) comprises a basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, fibroblast growth factor, SANT-1, retinoic acid, BMP inhibitor, insulin-transferrin-selenium-ethanolamine additive, protein kinase C activator;
preferably, the fibroblast growth factor comprises 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 inhibitor comprises LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD 161570;
more preferably, the BMP inhibitor is LDN 193189;
preferably, the protein kinase C activator comprises TPPB, PMA;
more preferably, the protein kinase C activator is TPPB;
most preferably, the concentrations of the components in the fourth stage differentiation induction medium are respectively: (0.01-5) mM ascorbic acid, (0.01-10) ng/mL FGF-7, (0.01-5) μ M SANT-1, (0.01-10) μ M retinoic acid, (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (50-500) nM TPPB, (0.05-10)% fetal bovine serum albumin, (1-50) mM glucose, (0.05-10) mM glutamine, (0.05-10) g/L sodium bicarbonate;
most preferably, the concentrations of the components in the fourth stage differentiation induction medium are respectively: 0.25mM ascorbic acid, 2ng/mL FGF-7, 0.25. mu.M SANT-1, 1. mu.M retinoic acid, 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 200nM TPPB, 2% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, 2.5g/L sodium bicarbonate;
most preferably, the fourth stage induces differentiation for a period of 3 days;
most preferably, on days 8-10 of induction, the fourth stage induction differentiation culture medium is used for induction differentiation;
preferably, the fifth stage differentiation-inducing medium described in step (6) comprises a basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, SANT-1, retinoic acid, BMP inhibitor, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor, zinc sulfate, heparin;
more preferably, the BMP inhibitors include LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD 161570;
most preferably, the BMP inhibitor is LDN 193189;
more preferably, the ALK5 inhibitors include ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-beta receptor inhibitor V, TGF-beta receptor inhibitor I, TGF-beta receptor inhibitor IV, TGF-beta receptor inhibitor VII, TGF-beta receptor inhibitor VIII, TGF-beta receptor inhibitor II, TGF-beta receptor inhibitor VI, and TGF-beta receptor inhibitor III;
most preferably, the ALK5 inhibitor is ALK5i II;
most preferably, the concentrations of the components in the fifth stage differentiation induction medium are respectively: (0.01-5) μ M SANT-1, (0.01-10) μ M retinoic acid, (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) μ M triiodothyronine, (0.01-50) μ M ALK5iII, (1-50) μ M zinc sulfate, (1-50) μ g/mL heparin, (0.05-10) g/L sodium bicarbonate, (0.05-10) mM glutamine, (1-50) mM glucose, (0.05-10)% fetal bovine serum albumin;
most preferably, the concentrations of the components in the fifth stage differentiation induction medium are respectively: 0.25 μ M SANT-1, 0.1 μ M retinoic acid, 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 μ M triiodothyronine, 10 μ M ALK5i II, 10 μ M zinc sulfate, 10 μ g/mL heparin, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin;
most preferably, the fifth stage induces differentiation for a period of 3 days;
most preferably, cells are plated on day 11 of induction into low affinity culture plates and induced to differentiate using the fifth stage differentiation induction medium on days 11-13 of induction.
7. The method according to claim 3, wherein the sixth stage differentiation induction medium in step (7) comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, BMP inhibitor, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor, zinc sulfate, gamma-secretase inhibitor, heparin;
preferably, the BMP inhibitors include LDN193189, LDN212854, UK383367, LDN214117, GW788388, SM1-71, ER50891, DMH-1, LDN193189, K02288, PD 161570;
more preferably, the BMP inhibitor is LDN 193189;
preferably, the ALK5 inhibitors include ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-beta receptor inhibitor V, TGF-beta receptor inhibitor I, TGF-beta receptor inhibitor IV, TGF-beta receptor inhibitor VII, TGF-beta receptor inhibitor VIII, TGF-beta receptor inhibitor II, TGF-beta receptor inhibitor VI, and TGF-beta receptor inhibitor III;
more preferably, the ALK5 inhibitor is ALK5i II;
preferably, the gamma-secretase inhibitor comprises GSi XX, GSi IX, GSi XI, GSi XII, GSi XIII, GSi XIV, GSi XVI, GSi XIX, GSi XVII, GSi XXI, RO4929097, LY450139, MK-0752, BMS-708163, LY411575, LY 3039478;
more preferably, the gamma-secretase inhibitor is GSi XX;
preferably, the sixth stage differentiation induction medium comprises a sixth stage differentiation induction medium a and a sixth stage differentiation induction medium B;
more preferably, said sixth stage differentiation induction medium a comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, zinc sulfate, GSi XX, heparin;
more preferably, said sixth stage differentiation induction medium B comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, zinc sulfate, heparin;
most preferably, the sixth stage induces differentiation for a period of 14 days;
most preferably, on the 14 th to 20 th days of induction, the sixth stage induction differentiation culture medium A is adopted for induction differentiation;
most preferably, on days 21-27 of induction, differentiation is induced using stage six differentiation induction medium B;
most preferably, the concentrations of the components in the sixth stage differentiation induction medium are respectively: (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) μ M triiodothyronine, (0.01-50) μ M ALK5i II, (1-50) μ M zinc sulfate, (10-500) nM GSi XX, (1-50) μ g/mL heparin, (0.05-10) g/L sodium bicarbonate, (0.05-10) mM glutamine, (1-50) mM glucose, (0.05-10)% fetal bovine serum albumin;
most preferably, the concentrations of the components in the sixth stage differentiation induction medium are respectively: 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 μ M triiodothyronine, 10 μ M ALK5i II, 10 μ M zinc sulfate, 100nM GSi XX, 10 μ g/mL heparin, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin;
preferably, the seventh stage differentiation induction medium described in step (8) comprises basal medium MCDB131, sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium-ethanolamine supplement, triiodothyronine, ALK5 inhibitor, zinc sulfate, heparin, acetyl-l-cysteine, water-soluble vitamin E, Axl inhibitor;
more preferably, the ALK5 inhibitors include ALK5i II, R-268712, SB505124, GW788388, SD208, SB431542, ITD-1, LY2109761, A83-01, LY2157299, TGF-beta receptor inhibitor V, TGF-beta receptor inhibitor I, TGF-beta receptor inhibitor IV, TGF-beta receptor inhibitor VII, TGF-beta receptor inhibitor VIII, TGF-beta receptor inhibitor II, TGF-beta receptor inhibitor VI, and TGF-beta receptor inhibitor III;
most preferably, the ALK5 inhibitor is ALK5i II;
more preferably, the Axl inhibitors include 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 concentration of each component in the seventh stage differentiation induction medium is: (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) μ M triiodothyronine, (0.01-50) μ M ALK5i II, (1-50) μ M zinc sulfate, (1-50) μ g/mL heparin, (0.01-10) mM acetyl-L-cysteine, (1-50) μ M water-soluble vitamin E, (1-10) μ M R428, (0.05-10) g/L sodium bicarbonate, (0.05-10) mM glutamine, (1-50) mM glucose, (0.05-10)% fetal bovine serum albumin;
most preferably, the concentration of each component in the seventh stage differentiation induction medium is: 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 μ M triiodothyronine, 10 μ M ALK5i II, 10 μ M zinc sulfate, 10 μ g/mL heparin, 1mM acetyl-L-cysteine, 10 μ M water soluble vitamin E, 2 μ M R428, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin;
most preferably, the seventh stage induces differentiation for a period of 13 days;
most preferably, on days 28-34 of induction, the seventh stage differentiation induction medium is used for induction differentiation;
preferably, the eighth stage differentiation induction medium described in step (9) comprises islet alpha cell beta cell transformation medium, artemether, GABAA activator;
more preferably, the islet alpha cell transbeta cell culture medium comprises Ham's F-12medium, medium 199, glutamine, calcium chloride dihydrate, N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid, fetal bovine serum albumin, insulin-transferrin-selenium-ethanolamine additive, water-soluble vitamin E, nicotinamide, heparin, deoxyribonuclease i, a necrotic apoptosis inhibitor, a serine protease inhibitor;
most preferably, the necrotic apoptosis inhibitor comprises Necrostatin-1, Necrostatin-2;
most preferably, the necrotic apoptosis inhibitor is Necrostatin-1;
most preferably, the serpin comprises Pefabloc, Benzamidine, MBTI, PMSF, LBTI;
most preferably, the serine protease inhibitor is Pefabloc;
more preferably, the GABAA activator comprises NS11394, R-16659, Muscimol, Arbaclofen, Propofol, Baclofen, Isoguvacine hydrochloride;
most preferably, the GABAA activator is NS 11394;
most preferably, the concentrations of the components in the eighth stage differentiation-inducing medium are respectively: 50% Ham's F-12medium, 50% medium 199, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (1-50) μ M water-soluble vitamin E, (1-50) mM nicotinamide, (1-50) μ g/mL heparin, (0.01-10) U/mL deoxyribonuclease I, (10-500) μ M Neostatin-1, (0.01-5) μ M Pefabloc, (0.01-10) mM glutamine, (0.01-10) mM calcium chloride dihydrate, (0.1-50) mM N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid, (0.05-10)% fetal bovine serum albumin, (1-50) μ M artemether, (1-50) μ M NS 11394;
most preferably, the concentrations of the components in the eighth stage differentiation-inducing medium are respectively: 50% Ham's F-12medium, 50% medium 199, 0.5% insulin-transferrin-selenium-ethanolamine additive, 10 μ M water-soluble vitamin E, 10mM nicotinamide, 10 μ g/mL heparin, 1U/mL deoxyribonuclease I, 100 μ M Neostatin-1, 0.1 μ M Pefabloc, 2mM glutamine, 2.5mM calcium chloride dihydrate, 10mM N-2 hydroxyethylpiperazine-N-2-ethanesulfonic acid, 2% fetal bovine serum albumin, 10 μ M artemether, 10 μ M NS 11394;
most preferably, the eighth stage induces differentiation for a period of 6 days;
most preferably, the eighth stage differentiation-inducing medium is used for inducing differentiation at days 35 to 40.
8. An induced pluripotent stem cell-derived functional islet beta cell or cell population, wherein the cell or cell population is induced to differentiate by the method of any one of claims 3-7;
preferably, the islet beta cell or cell population is a functional, stable islet beta cell or cell population.
9. A pharmaceutical composition for the treatment and/or prevention of diabetes, characterized in that it comprises the cell or cell population according to claim 8;
preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant;
preferably, the diabetes comprises type 1diabetes, type 2diabetes, gestational diabetes, monogenic diabetes;
more preferably, the diabetes is type 1 diabetes.
10. The use of any one of the following aspects, wherein said use comprises:
(1) the application of the combination of artemether and NS11394 in promoting the transformation of islet alpha cells into islet beta cells;
(2) the application of the combination of artemether and NS11394 in inducing the differentiation of pluripotent stem cells into functional islet beta cells;
(3) use of the induction differentiation agent according to claim 1 for preparing an induction differentiation medium for inducing differentiation of pluripotent stem cells into functional islet beta cells;
(4) use of the induction differentiation agent as described in claim 1 for inducing differentiation of pluripotent stem cells into functional islet beta cells;
(5) use of the differentiation-inducing medium according to claim 2 for inducing differentiation of pluripotent stem cells into functional islet beta cells;
(6) use of the cell or cell population of claim 8 in the manufacture of a medicament for the treatment and/or prevention of diabetes;
(7) the use of a pharmaceutical composition according to claim 9 for the treatment and/or prevention of diabetes;
preferably, the diabetes comprises type 1diabetes, type 2diabetes, gestational diabetes, monogenic diabetes;
more preferably, the diabetes is type 1 diabetes.
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