WO2023221418A1 - 一种促进胰岛α细胞转化为β细胞的诱导方法及其应用 - Google Patents

一种促进胰岛α细胞转化为β细胞的诱导方法及其应用 Download PDF

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WO2023221418A1
WO2023221418A1 PCT/CN2022/131004 CN2022131004W WO2023221418A1 WO 2023221418 A1 WO2023221418 A1 WO 2023221418A1 CN 2022131004 W CN2022131004 W CN 2022131004W WO 2023221418 A1 WO2023221418 A1 WO 2023221418A1
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differentiation
stage
cells
agent
inducing
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French (fr)
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顾雨春
吴理达
王贯乔
安翠平
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呈诺再生医学科技(北京)有限公司
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Definitions

  • the invention belongs to the technical field of biomedicine. Specifically, the invention relates to an induction method for promoting the conversion of pancreatic islet ⁇ cells into ⁇ cells and its application.
  • Diabetes mellitus is a group of chronic metabolic diseases characterized by hyperglycemia caused by insufficient insulin secretion or insulin resistance. It is mainly divided into type 1 diabetes (Type 1 diabetes mellitus (T1DM)), type 2 diabetes (Type 2 diabetes mellitus (T2DM), gestational diabetes (Gestational diabetes mellitus (GDM)) and monogenic diabetes. According to statistics, as of 2019, the number of diabetic patients worldwide exceeded 463 million, and it is expected that the number will increase to 700 million in 2045. Among them, type 1 diabetes is an autoimmune disease mediated by T lymphocytes. Due to insufficient insulin secretion due to damage to pancreatic islet cells, patients with type 1 diabetes need to completely rely on exogenous insulin treatment.
  • the stem cells used for the treatment of type 1 diabetes at the laboratory level include embryonic stem cells, induced pluripotent stem cells (iPSCs), mesenchymal stem cells (Mesenchymal stem cells, MSCs), and bone marrow-derived hematopoietic stem cells.
  • iPSCs induced pluripotent stem cells
  • MSCs mesenchymal stem cells
  • HSCs bone marrow-derived hematopoietic stem cells.
  • HSCs induced pluripotent stem cells
  • multipotent precursor cells in the pancreas and ⁇ -cell precursor cells found outside the pancreas (spleen, liver, endometrium).
  • Stem cell therapy for type 1 diabetes is based on the self-renewal function and potential directional differentiation function of stem cells.
  • iPSCs Compared with complete allogeneic pancreatic tissue transplantation, strict quality control can be carried out at the molecular level before transplantation, and the quality of transplanted cells and immune rejection can be controlled. With effective evaluation, stem cell therapy can partially or completely avoid transplant failure and post-transplantation complications caused by immune rejection.
  • the treatment of type 1 diabetes with iPSCs belongs to the category of regenerative medicine and has huge potential in the treatment of type 1 diabetes.
  • the basic principle is to reverse the patient's autologous cells into early undifferentiated pluripotent stem cells, which are then treated with various cytokines to imitate The growth and development process of embryonic cells induces them to become pancreatic islet beta cells with the function of secreting insulin. Unlike embryonic stem cells, iPSCs can only form new tissues and organs, but not new individuals, so they can avoid potential medical ethical issues. And because the cells are derived from the patient himself, immune rejection can be reduced or completely avoided.
  • the formation of islets is basically through the endoderm, gastrut tube, posterior foregut, pancreatic progenitor cells, pancreatic endocrine precursors, immature This process of ⁇ cells and mature ⁇ cells is realized.
  • most of the islet cells obtained by transdifferentiation are islet multi-hormone cells, that is, they express islet cells at the same time. ⁇ cells, islet ⁇ cells, islet ⁇ cells, islet PP cells, etc.
  • islet ⁇ cells express limited and unstable hormones, and the expression level of islet ⁇ cells is significantly higher than that of islet ⁇ cells, and most of the islet ⁇ cells are immature. status, the expression level of glucagon is significantly higher than that of insulin, which seriously limits its application in the treatment of type 1 diabetes. Therefore, there is still an urgent need in this field for a standard induction method that can obtain functional islet ⁇ -like cells in large quantities and stably.
  • the present invention provides an induction method and its application to promote the transformation of pancreatic islet ⁇ cells into ⁇ cells.
  • the induction method includes artemether and GABAA activator NS11394. Currently, there is no method to activate artemether and GABAA. There are reports on the combined use of NS11394 and NS11394 to promote the conversion of pancreatic islet ⁇ cells into ⁇ cells.
  • the object of the present invention is to provide an induction method and application for promoting the transformation of pancreatic islet ⁇ cells into ⁇ cells.
  • the induction method is replaced with islet ⁇ cells on the 35th day of induction differentiation. Cells were transformed into ⁇ -cell medium, and artemether and GABAA activator NS11394 were added for induction until day 40. It has been verified that the induction method of the present invention can significantly increase the expression of pancreatic islet ⁇ cells, and has broad application prospects in the treatment of type 1 diabetes.
  • a first aspect of the present invention provides a differentiation-inducing agent that induces the differentiation of pluripotent stem cells into functional pancreatic islet ⁇ cells.
  • the inducing differentiation agent includes a first stage inducing differentiation agent, a second stage inducing differentiation agent, a third stage inducing differentiation agent, a fourth stage inducing differentiation agent, a fifth stage inducing differentiation agent, a sixth stage inducing differentiation agent, The seventh stage differentiation agent and the eighth stage differentiation agent;
  • the first-stage differentiation inducing agent includes the first-stage differentiation inducing agent A and the first-stage differentiation inducing agent B;
  • the first-stage differentiation inducing agent A includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, GDF8, and CHIR-99021;
  • the first-stage differentiation inducing agent B contains sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, and GDF8;
  • the second stage differentiation inducing agent includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, and FGF-7;
  • the third stage differentiation agent includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, FGF-7, SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine Additives, TPPB;
  • the fourth stage differentiation inducing agent includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, ascorbic acid, FGF-7, SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine Additives, TPPB;
  • the fifth stage differentiation agent includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive, and triiodothyronogen Amino acid, ALK5i II, zinc sulfate, heparin;
  • the sixth stage differentiation inducing agent includes the sixth stage differentiation inducing agent A and the sixth stage differentiation inducing agent B;
  • the sixth stage differentiation agent A includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, sulfuric acid Zinc, GSi XX, heparin;
  • the sixth stage differentiation agent B contains sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, sulfuric acid zinc, heparin;
  • the seventh stage differentiation agent includes sodium bicarbonate, glutamine, glucose, fetal bovine serum albumin, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5i II, zinc sulfate, and heparin , acetyl-l-cysteine, water-soluble vitamin E, R428;
  • the eighth stage differentiation agent includes artemether and NS11394;
  • the eighth stage differentiation agent also includes 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.
  • concentrations of each component in the first-stage differentiation agent are: (10-500) ng/mL GDF8, (0.5-20) ⁇ 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.
  • concentrations of each component in the first-stage differentiation agent are: 100ng/mL GDF8, 3 ⁇ M CHIR-99021, 0.5% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, and 1.5g/L hydrogen carbonate. sodium.
  • concentrations of each component in the second stage differentiation agent are: (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.
  • concentrations of each component in the second-stage differentiation agent are: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.5% fetal bovine serum albumin, 10mM glucose, 2mM glutamine, and 1.5g/L carbonic acid. Sodium hydrogen.
  • concentrations of each component in the third stage differentiation agent are: (0.01-5)mM ascorbic acid, (10-100)ng/mL FGF-7, (0.01-5) ⁇ M SANT-1, (0.01 -10) ⁇ 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.
  • concentrations of each component in the third stage differentiation agent are: 0.25mM ascorbic acid, 50ng/mL FGF-7, 0.25 ⁇ M SANT-1, 1 ⁇ 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.
  • concentrations of each component in the fourth stage differentiation agent are: (0.01-5)mM ascorbic acid, (0.01-10)ng/mL FGF-7, (0.01-5) ⁇ M SANT-1, (0.01 -10) ⁇ 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.
  • concentrations of each component in the fourth stage differentiation agent are: 0.25mM ascorbic acid, 2ng/mL FGF-7, 0.25 ⁇ M SANT-1, 1 ⁇ 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.
  • concentrations of each component in the fifth stage differentiation agent are: (0.01-5) ⁇ M SANT-1, (0.01-10) ⁇ M retinoic acid, (10-500) nM LDN193189, (0.01-5 )% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) ⁇ M triiodothyronine, (0.01-50) ⁇ M ALK5i II, (1-50) ⁇ M zinc sulfate, (1-50) ⁇ 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.
  • concentrations of each component in the fifth-stage differentiation agent are: 0.25 ⁇ M SANT-1, 0.1 ⁇ M retinoic acid, 100 nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, and 1 ⁇ M triiodothyronine Orogenic acid, 10 ⁇ M ALK5i II, 10 ⁇ M zinc sulfate, 10 ⁇ g/mL heparin, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin.
  • concentrations of each component in the sixth stage inducing differentiation agent are: (10-500) nM LDN193189, (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) ⁇ M Tris Iodothyronine, (0.01-50) ⁇ M ALK5i II, (1-50) ⁇ M zinc sulfate, (10-500)nM GSi XX, (1-50) ⁇ 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.
  • concentrations of each component in the sixth stage inducing differentiation agent are: 100nM LDN193189, 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 ⁇ M triiodothyronine, 10 ⁇ M ALK5i II, 10 ⁇ M zinc sulfate, 100nM GSi XX, 10 ⁇ g/mL heparin, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin.
  • concentrations of each component in the seventh stage differentiation agent are: (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (0.01-10) ⁇ M triiodothyronine, (0.01 -50) ⁇ M ALK5i II, (1-50) ⁇ M zinc sulfate, (1-50) ⁇ g/mL heparin, (0.01-10)mM acetyl-l-cysteine, (1-50) ⁇ M water-soluble vitamins E. (1-10) ⁇ 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 protein.
  • concentrations of each component in the seventh-stage differentiation agent are: 0.5% insulin-transferrin-selenium-ethanolamine additive, 1 ⁇ M triiodothyronine, 10 ⁇ M ALK5i II, 10 ⁇ M zinc sulfate, 10 ⁇ g/mL Heparin, 1mM acetyl-l-cysteine, 10 ⁇ M water-soluble vitamin E, 2 ⁇ M R428, 1.5g/L sodium bicarbonate, 2mM glutamine, 20mM glucose, 2% fetal bovine serum albumin.
  • concentrations of each component in the eighth stage differentiation agent are: (0.01-5)% insulin-transferrin-selenium-ethanolamine additive, (1-50) ⁇ M water-soluble vitamin E, (1-50) )mM nicotinamide, (1-50) ⁇ g/mL heparin, (0.01-10)U/mL deoxyribonuclease I, (10-500) ⁇ M Necrostatin-1, (0.01-5) ⁇ 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) ⁇ M artemether, (1-50) ⁇ M NS11394.
  • the concentrations of each component in the eighth stage induced differentiation culture medium are: 0.5% insulin-transferrin-selenium-ethanolamine additive, 10 ⁇ M water-soluble vitamin E, 10 mM nicotinamide, 10 ⁇ g/mL heparin, 1 U/mL Deoxyribonuclease I, 100 ⁇ M Necrostatin-1, 0.1 ⁇ M Pefabloc, 2mM glutamine, 2.5mM calcium chloride dihydrate, 10mM N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid, 2% fetus Bovine serum albumin, 10 ⁇ M artemether, 10 ⁇ M NS11394.
  • a second aspect of the present invention provides an induction differentiation medium for inducing the differentiation of pluripotent stem cells into functional pancreatic islet ⁇ cells.
  • the induced differentiation medium includes a first stage induced differentiation medium, a second stage induced differentiation medium, a third stage induced differentiation medium, a fourth stage induced differentiation medium, a fifth stage induced differentiation medium, and a third stage induced differentiation medium.
  • the first-stage differentiation medium includes a basal medium MCDB131 and the first-stage differentiation agent described in the first aspect of the present invention
  • the second stage inducing differentiation medium includes the basal medium MCDB131 and the second stage inducing differentiation agent described in the first aspect of the present invention
  • the third stage induction differentiation medium includes basal medium MCDB131 and the third stage induction differentiation agent described in the first aspect of the present invention
  • the fourth stage inducing differentiation medium includes basal medium MCDB131 and the fourth stage inducing differentiation agent described in the first aspect of the present invention
  • the fifth stage inducing differentiation medium includes basal medium MCDB131 and the fifth stage inducing differentiation agent described in the first aspect of the present invention
  • the sixth stage inducing differentiation medium includes basal medium MCDB131 and the sixth stage inducing differentiation agent described in the first aspect of the present invention
  • the seventh stage inducing differentiation medium includes a basal medium MCDB131 and the seventh stage inducing differentiation agent described in the first aspect of the present invention
  • the eighth stage inducing differentiation medium includes 50% Ham’s F-12 medium, 50% medium 199, and the eighth stage inducing differentiation agent described in the first aspect of the present invention.
  • the third aspect of the present invention provides a method for inducing differentiation of pluripotent stem cells into functional pancreatic islet ⁇ cells.
  • the method includes the following steps:
  • Second stage induced differentiation using the second stage induced differentiation medium described in the second aspect of the present invention to induce the differentiation of definitive endoderm cells into primitive intestinal tube cells;
  • the complete culture medium described in step (1) is E8 complete culture medium.
  • E8 complete culture medium contains ROCK inhibitor.
  • ROCK inhibitors include Y-27632, GSK429286A, RKI-1447, Y-33075dihydrochloride, Thiazovivin, K-115, SLx-2119, Chroman1, SAR407899 and/or SR-3677.
  • ROCK inhibitor is Y-27632.
  • concentration of Y-27632 is 0.001-100 ⁇ M.
  • concentration of Y-27632 is 10 ⁇ M.
  • the cell density of the induced pluripotent stem cells described in step (1) is 0.1-10.0 ⁇ 10 5 cells/cm 2 .
  • the cell density of the induced pluripotent stem cells described in step (1) is 1.8-2.2 ⁇ 10 5 cells/cm 2 .
  • induced pluripotent stem cells described in step (1) are derived from mammals.
  • the induced pluripotent stem cells described in step (1) are derived from humans, mice, rats, goats, sheep, pigs, cats, rabbits, dogs, wolves, horses, or cattle.
  • induced pluripotent stem cells described in step (1) are derived from humans.
  • the induced pluripotent stem cells described in step (1) are human-derived induced pluripotent stem cells, and the induced pluripotent stem cells are derived from Beijing Chengnuo Medical Technology Co., Ltd., according to the company It is prepared by the method described in the previously applied patent (Application No.: 201910110768.7).
  • the time for inducing differentiation in the first stage is 3 days.
  • the first stage induction differentiation medium A was used to induce differentiation.
  • the first stage induction differentiation medium B was used to induce differentiation.
  • the second stage of differentiation induction time is 2 days.
  • the second stage induction differentiation medium was used to induce differentiation.
  • the time for inducing differentiation in the third stage is 2 days.
  • the third stage induction differentiation medium was used to induce differentiation.
  • the fourth stage of differentiation induction time is 3 days.
  • the fourth stage induction differentiation medium was used to induce differentiation.
  • the time for inducing differentiation in the fifth stage is 3 days.
  • the cells were seeded into a low-affinity culture plate, and on the 11th to 13th day of induction, the fifth-stage induction differentiation medium was used to induce differentiation.
  • the time for inducing differentiation in the sixth stage is 14 days.
  • the sixth stage induction differentiation medium A was used to induce differentiation.
  • the sixth stage induction differentiation medium B was used to induce differentiation.
  • the seventh stage of differentiation induction time is 13 days.
  • the seventh stage induction differentiation medium was used to induce differentiation.
  • the time for inducing differentiation in the eighth stage is 6 days.
  • the eighth stage induction differentiation medium was used to induce differentiation.
  • a fourth aspect of the present invention provides a functional pancreatic islet ⁇ cell or cell population derived from induced pluripotent stem cells.
  • the functional pancreatic islet ⁇ cells or cell population are obtained by inducing differentiation using the method described in the third aspect of the present invention.
  • pancreatic islet ⁇ cells or cell population are functional and stable pancreatic islet ⁇ cells or cell populations.
  • a fifth aspect of the invention provides a pharmaceutical composition for treating and/or preventing diabetes.
  • the pharmaceutical composition includes the cells or cell populations according to the fourth invention of the present invention.
  • composition further includes pharmaceutically acceptable carriers and/or excipients.
  • the diabetes includes type 1 diabetes, type 2 diabetes, gestational diabetes, and monogenic diabetes.
  • the diabetes is type 1 diabetes.
  • the pharmaceutically acceptable carriers and/or excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used to help the stability of the drug or help improve the effectiveness as needed.
  • the activity of the component (the cell or cell population according to the fourth invention of the present invention), the preparation that can be used in this pharmaceutical composition can be in the form of its original compound itself, or optionally its pharmaceutically acceptable salt
  • the pharmaceutical composition thus formulated can be administered by any appropriate administration method known to those skilled in the art as needed.
  • the pharmaceutically acceptable carrier and/or excipient may additionally contain liquids such as water, physiological saline, glycerin, and ethanol.
  • auxiliary substances such as wetting or emulsifying agents or pH buffering substances may be present in the pharmaceutical compositions.
  • suitable forms of administration include those suitable for parenteral administration, such as by injection or infusion, such as by bolus injection or continuous infusion, intravenous, inhalable or subcutaneous forms.
  • parenteral administration such as by injection or infusion, such as by bolus injection or continuous infusion, intravenous, inhalable or subcutaneous forms.
  • the product may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents such as suspending agents, preservatives, stabilizers and/or Dispersant.
  • the present invention also provides a biological preparation for treating and/or preventing diabetes.
  • the biological preparation includes the cells or cell populations described in the fourth aspect of the present invention and/or the pharmaceutical composition described in the fifth aspect of the present invention.
  • the biological preparation may also include any other reagents that can be used for the treatment and/or prevention, or auxiliary treatment and/or prevention of diabetes, including but not limited to small molecule compounds, antibodies, cytokines, etc.
  • the reagents in the above-mentioned biological preparations and the cells or cell populations or pharmaceutical compositions of the present invention can be used in combination or separately.
  • the small molecule compounds include but are not limited to sulfonylurea hypoglycemic drugs, biguanide hypoglycemic drugs, insulin sensitizers, and sulfonylurea drugs.
  • the sulfonylurea hypoglycemic drugs include, but are not limited to: Youjiangtang, Mepyrida (Udalin), Ketangli, Dameikang (gliclazide), and Tangsupin (gliquidone);
  • the biguanide hypoglycemic drugs include, but are not limited to: Jiangtangling (phenformin, DB1), metformin (Metformin tablets, Medican, Dihuatang tablets, Glucophage);
  • the insulin sensitizers include, but are not limited to : Thiazolidinediones (glitazones, such as rosiglitazone, pioglitazone), biguanides.
  • a sixth aspect of the invention provides a method for treating and/or preventing diabetes.
  • the method includes administering to a subject in need an effective amount of the cell or cell population described in the fourth aspect of the present invention and/or the pharmaceutical composition described in the fifth aspect of the present invention.
  • the diabetes includes type 1 diabetes, type 2 diabetes, gestational diabetes, and monogenic diabetes.
  • the diabetes is type 1 diabetes.
  • the subject suffers from or is at risk of developing diabetes, or suffers from or is at risk of suffering from a metabolic disorder.
  • pancreatic islet ⁇ cells or cell populations generated by the methods provided by the invention can be administered to a subject for the treatment of type 1 diabetes, type 2 diabetes, gestational diabetes, and monogenic diabetes.
  • the subject includes various types of mammals, preferably humans.
  • the functional islet beta cells or cell populations can be implanted as dispersed cells or formed into cell clusters, which can be infused into the hepatic portal vein.
  • cells may be provided in a biocompatible degradable polymeric support, in a porous non-degradable device, or encapsulated to protect them from the host immune response.
  • the cells can be implanted into a suitable site in the recipient. Implantation sites include, for example, the liver, native pancreas, renal subcapsular space, omentum, peritoneum, subserosal space, intestine, stomach or subcutaneous pocket.
  • the implanted functional islet beta cells or cell populations in vivo, other factors such as growth factors, antioxidants can be administered before, simultaneously with or after the administration of the functional islet beta cells or cell populations. or anti-inflammatory agents. These factors can be secreted by endogenous cells and exposed to the administered cells in situ, and the function of the implanted cells can be enhanced by any combination of endogenous and exogenously administered growth factors known in the art.
  • the amount of functional islet beta cells or cell populations used in the implantation and the manner in which they are administered depend on a number of different factors, including the patient's condition and degree of response to therapy, and can be determined by one of ordinary skill in the art.
  • the treatment method further includes incorporating the cells into a three-dimensional support prior to implantation.
  • Cells can be maintained on the support in vitro before implantation into a subject.
  • the cell-containing support can be implanted directly into the patient without additional in vitro culture.
  • the support may optionally incorporate at least one agent that promotes survival and function of the transplanted cells.
  • the functional islet beta cells or cell population, the pharmaceutical composition, or the biologic may be administered by any suitable means, such as by bolus infusion, delivered by injection, such as subcutaneous injection, It can also be delivered via islet cell transplantation.
  • the functional islet beta cells or cell population can be transplanted via portal vein puncture, and the functional islet beta cells or cell population are delivered to the liver, where the islets are capable of growing and secreting insulin.
  • the seventh aspect of the present invention provides the use of artemether and NS11394 in combination to promote the conversion of pancreatic islet ⁇ cells into pancreatic islet ⁇ cells.
  • the eighth aspect of the present invention provides the use of artemether and NS11394 in combination to induce the differentiation of pluripotent stem cells into functional pancreatic islet ⁇ cells.
  • a ninth aspect of the present invention provides the use of the differentiation agent described in the first aspect of the present invention in preparing an induction differentiation medium for inducing pluripotent stem cells to differentiate into functional pancreatic islet ⁇ cells.
  • a tenth aspect of the present invention provides the use of the differentiation-inducing agent described in the first aspect of the present invention in inducing the differentiation of pluripotent stem cells into functional pancreatic islet ⁇ cells.
  • An eleventh aspect of the present invention provides the use of the differentiation medium described in the second aspect of the present invention in inducing the differentiation of pluripotent stem cells into functional pancreatic islet ⁇ cells.
  • a twelfth aspect of the present invention provides the use of the cells or cell populations described in the fourth aspect of the present invention in the preparation of drugs for treating and/or preventing diabetes.
  • a thirteenth aspect of the present invention provides the use of the cells or cell populations described in the fourth aspect of the present invention in the treatment and/or prevention of diabetes.
  • the fourteenth aspect of the present invention provides the use of the pharmaceutical composition according to the fifth aspect of the present invention in the treatment and/or prevention of diabetes.
  • the diabetes includes type 1 diabetes, type 2 diabetes, gestational diabetes, and monogenic diabetes;
  • the diabetes is type 1 diabetes.
  • the present invention has the following advantages and beneficial effects:
  • the invention provides a novel induction method for promoting the transformation of pancreatic islet ⁇ cells into ⁇ cells.
  • the induction method is based on the traditional induction method and on the 35th day of induction differentiation, is replaced with the culture of pancreatic islet ⁇ cells into ⁇ cells. base, while adding artemether and GABAA activator NS11394 for induction until the 40th day. It has been verified that the expression level of the iconic molecules of pancreatic islet ⁇ cells prepared through the induction method provided by the invention is significantly increased, indicating that the induction method provided by the invention can significantly increase the expression level of pancreatic islet ⁇ cells, which is clinically 1 Provides scientific basis for the treatment of type 2 diabetes.
  • Figure 1 is a statistical diagram of the results of qPCR detection of the expression of islet maturation marker gene mRNA.
  • Group A control group: used the seventh stage induction differentiation medium on the 35th to 40th day of induction;
  • Group B used on the 35th day of induction -40 days using ⁇ cells to ⁇ cell culture medium;
  • Group C using ⁇ cells to ⁇ cell culture medium on days 35-40 of induction, and adding artemether and GABAA activator NS11394 at the same time.
  • diabetes refers to the state of chronic hyperglycemia, an excess of sugar in the blood caused by a relative or absolute lack of insulin action, a form of a large group of disorders in humans characterized by glycemic control. It is characterized by impaired or impaired control of glucose levels in the blood. Diabetes itself is a chronic hormonal disorder characterized by impaired metabolism of glucose and other energy-producing fuels, and the late development of severe vascular and neurological complications. Diabetes increases the risk of heart, brain, and peripheral vascular disease by 2-7 times and is Leading cause of neonatal morbidity and mortality. Currently, there are three basic types of diabetes, type 1 or insulin-dependent diabetes mellitus (IDDM), type 2 or non-insulin-dependent diabetes mellitus (NIDDM), gestational diabetes, and monogenic diabetes.
  • IDDM insulin-dependent diabetes mellitus
  • NIDDM non-insulin-dependent diabetes mellitus
  • Type 1 diabetes a disorder with little or no insulin secretory capacity, rely on exogenous insulin to prevent metabolic decompensation (such as ketoacidosis) and death.
  • metabolic decompensation such as ketoacidosis
  • diabetes develops suddenly (i.e., within days and weeks) in a previously healthy, nonobese child or young adult; in older age groups, it may develop gradually.
  • the typical patient is often ill, has significant symptoms (eg, polyuria, polydipsia, bulimia, and weight loss) and may demonstrate ketoacidosis.
  • Type 1 diabetes has a long asymptomatic preclinical phase, usually lasting several years, during which pancreatic beta cells are gradually destroyed by autoimmune attack by HLA and other genetic factors as well as environmental influences.
  • type 2 diabetes is by far the most common form of the disease. These patients retain significant levels of endogenous insulin secretory capacity. However, insulin levels were low relative to insulin resistance and peripheral glucose levels. People with type 2 diabetes do not rely on insulin for immediate survival and rarely develop ketosis except in situations of intense physical stress. However, these patients may require insulin therapy to control hyperglycemia. Typically, type 2 diabetes appears after age 40, has a high proportion of genetic penetrance unrelated to HLA genes, and is associated with obesity. Clinical features of type 2 diabetes may be mild (fatigue, weakness, dizziness, blurred vision, or other nonspecific complaints may predominate) or be tolerated for many years before the patient seeks medical attention. Additionally, if the levels of high blood sugar are not high enough to produce symptoms, the disease may only become apparent after complications develop.
  • gestational diabetes refers to female patients with impaired glucose tolerance that appears or is first detected during pregnancy.
  • Gestational diabetes usually occurs in the second and third trimesters, the time of pregnancy-related peak of insulin-antagonizing hormones. After delivery, glucose tolerance usually (but not always) returns to normal.
  • the diagnosis of diabetes is usually clear when the classic symptoms of polyuria, polydipsia, and weight loss are present. All that is required is that a random plasma glucose measurement of venous blood be 200 mg/dl or higher. If diabetes is suspected and cannot be confirmed by random glucose determination, then the screening test of choice is the overnight fasting plasma glucose level. The diagnosis is established if fasting glucose is equal to or greater than 126 mg/dl on at least two separate occasions.
  • the term “comprises” or “includes” refers to the compositions, methods, and their corresponding components present in a given embodiment, but is also open-ended to include unspecified elements.
  • treatment and/or prevention refers to the medical management of a patient with the purpose of curing, ameliorating, stabilizing or preventing a disease, pathological condition, disorder or related complications thereof.
  • the term includes active therapy, i.e., treatment specifically aimed at ameliorating the disease, pathological condition, condition, or its associated complications, and also includes etiological treatment, i.e., treatment aimed at removing the cause of the relevant disease, pathological condition, condition, or its associated complications. Purpose of treatment.
  • the term includes palliative care, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition, condition, or its associated complications; the term also includes preventive treatment, i.e., treatment designed to minimize or partially or completely suppress Treatment aimed at the development of a related disease, pathological condition, disorder or its related complications; and supportive care, i.e., used to complement another specific therapy aimed at improving the related disease, pathological state, disorder or its related complications Treatment.
  • palliative care i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition, condition, or its associated complications
  • preventive treatment i.e., treatment designed to minimize or partially or completely suppress Treatment aimed at the development of a related disease, pathological condition, disorder or its related complications
  • supportive care i.e., used to complement another specific therapy aimed at improving the related disease, pathological state, disorder or its related complications Treatment.
  • the diabetes-related diseases, pathological conditions, disorders, or complications thereof include hyperglycemia, suboptimal glycemic control, ketoacidosis, insulin resistance, elevated growth hormone levels, elevated glycosylated hemoglobin and hyperglycation end product (AGE) levels, dawn phenomenon, suboptimal lipid profile, vascular disease (e.g., atherosclerosis), microangiopathy, retinopathy (e.g., proliferative diabetic retinopathy), kidney disease, neuropathy, pregnancy complications (eg, early termination (pregnancy) and birth defects), etc.
  • hyperglycemia e.g., atherosclerosis
  • microangiopathy e.g., proliferative diabetic retinopathy
  • kidney disease e.g., neuropathy
  • pregnancy complications eg, early termination (pregnancy) and birth defects
  • End points included in the treatment definition are, for example, increased insulin sensitivity, decreased insulin administration while maintaining glycemic control, decreased HbA1c, improved glycemic control, decreased vascular, renal, neurological, retinal, and other diabetic complications. , prevention or reduction of the "dawn phenomenon," improved lipid profile, reduced pregnancy complications, and reduced ketoacidosis.
  • the term "effective amount” refers to an amount that produces a therapeutic effect on and is acceptable to humans and/or animals.
  • a therapeutically or pharmaceutically effective amount of a drug is the amount of drug required to produce the desired therapeutic effect, which may be reflected by the results of clinical trials, model animal studies, and/or in vitro studies.
  • the pharmaceutically effective dose depends on several factors, including but not limited to: characteristics of the treatment subject (such as the height, weight, gender, age and medication history of the treatment subject), and the severity of the disease.
  • the pharmaceutical active ingredients described in the present invention can form pharmaceutical compositions or pharmaceutical preparations.
  • the "effective amount” refers to an amount sufficient to inhibit, slow down or prevent the occurrence and development of diabetes and/or its related complications in a subject.
  • the term "effective amount” refers to a "therapeutically and/or prophylactically effective amount” and further refers to an amount sufficient to cause delay or prevention or elimination or alleviation of diabetes mellitus (type 1 or diabetes, type 2 diabetes, gestational diabetes, mono- The amount of functional pancreatic islet beta cells, pharmaceutical compositions, and biological preparations of the present invention that can cause the occurrence, recurrence, or onset of one or more symptoms of genetic diabetes).
  • the therapeutically and/or preventively effective amount preferably refers to the following amount of functional pancreatic islet beta cells, pharmaceutical compositions, and biological preparations according to the present invention, which delays the onset of diabetes or diabetes-related symptoms in the subject. At least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%.
  • the term "subject” refers to an animal, human or non-human to which functional pancreatic beta cells, pharmaceutical compositions, biologics of the present invention are provided for the treatment of diabetes.
  • the present disclosure contemplates both human and veterinary applications.
  • the term includes, but is not limited to: birds, reptiles, amphibians and mammals such as humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, horses, cattle, Cats, dogs, sheep, chickens and goats.
  • the subject is human, chicken, or mouse.
  • the subject is human. Both pediatric and adult subjects were included.
  • subjects described herein can be pediatric and adult subjects at least 6 months old (e.g., 6 months or older, 12 months or older, 18 months or older, 2 years old or older, 4 years or older, 6 years or older, 10 years or older, 13 years or older, 16 years or older, 18 years or older, 21 years or older, 25 years or older Older, 30 years or older, 35 years or older, 40 years or older, 45 years or older, 50 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, 80 years or older, 85 years or older, 90 years or older or 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 18, 20, 21, 24, 25, 27, 28, 30, 33, 35, 37, 39, 40, 42, 44, 45, 48, 50, 52, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 or more years old).
  • 6 months old e.g., 6 months or older, 12 months or older
  • the term "mammal” refers to any animal classified as a mammal, including, but not limited to: humans, rodents, sporting animals, zoo animals, pet animals and domestic or farm animals such as dogs, cats, cattle, Sheep, pigs, horses, and non-human primates such as monkeys.
  • the rodent is a mouse or rat.
  • the mammal is a human, also referred to herein as a patient or subject, and in specific embodiments of the invention, the mammal is preferably a human.
  • compositions and biological preparations provided by the present invention in some embodiments, it is expected to locally apply the functional islet ⁇ cells, pharmaceutical compositions and biological preparations provided by the present invention in the area that needs treatment.
  • Biological agents which may be achieved by, for example, but not limited to, local infusion, by injection or by means of an implant which is a porous, non-porous or gel-like material, including a membrane, for example an elastic membrane (sialastic membrane);
  • the functional islet beta cells, pharmaceutical compositions, and biological preparations provided by the invention can be delivered in vesicles (especially liposomes) (see Langer, Science 249:1527-1533 (1990); Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);
  • the present invention The functional pancreatic beta cells, pharmaceutical compositions, and biologics provided can be delivered in controlled release or sustained release systems.
  • a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRCCrit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574).
  • polymeric materials can be used to achieve controlled or sustained release of functional islet beta cells of the present invention (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem.23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg.
  • polymers used in sustained release formulations include, but are not limited to: poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate) ), poly(methacrylic acid), polyglycolide (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide ( PLA), poly(lactide-co-glycolide) (PLGA) and polyorthoesters.
  • the polymer used in the sustained release formulation is inert, free of leachable impurities, stable on storage, sterile and biodegradable.
  • a controlled or sustained release system can be placed near the target of treatment (i.e., the lungs), thus requiring only a portion of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, supra, vol. 2, pp.115-138(1984)).
  • Sustained release formulations containing one or more functional islet beta cells of the invention may be produced using any technique known to those skilled in the art. See, e.g., U.S. Patent No.
  • the functional islet beta cells, pharmaceutical compositions, and biologics of the present invention may be formulated to be compatible with their intended route of administration.
  • routes of administration include, but are not limited to, parenteral, eg, subcutaneous, intravenous (eg, hepatic portal vein), intradermal, oral, intranasal (eg, inhalation), transdermal (topical), transmucosal, and rectal administration.
  • the pharmaceutical composition is formulated according to conventional procedures as a pharmaceutical composition suitable for subcutaneous, intravenous (eg, hepatic portal vein), intramuscular, or oral administration to humans.
  • the pharmaceutical composition is formulated for subcutaneous administration to humans according to conventional procedures.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • solubilizers and local anesthetics such as lidocaine (lignocamne) may also be included in the pharmaceutical composition to reduce pain at the injection site.
  • the pharmaceutical composition may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, for example, in ampoules or multi-dose containers with an added preservative.
  • Pharmaceutical compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • beta cell function can be assessed before, during, and after treatment by the methods described herein or by any method known to one of ordinary skill in the art.
  • DCCT Diabetes Control and Complications Trial
  • HA1 and HA1c glycosylated hemoglobin percent monitoring as a standard for evaluating glycemic control
  • characterization of daily insulin requirements, C-peptide levels/responses, hypoglycemic episodes, and/or FPIR can be used as markers of ⁇ -cell function or used to establish therapeutic indicators (see respectively Keymeulen et al., 2005, N.Engl.J.Med.352:2598-2608; Herold et al., 2005, Diabetes 54:1763-1769; U.S. Patent Application Publication No. 2004/0038867A1; and Greenbaum et al., 2001, Diabetes 50:470 -476).
  • FPIR is calculated as the sum of insulin values at 1 minute and 3 minutes after IGTT, which was performed according to the Islet Cell Antibody Register User's Study protocol (see, e.g., Bingley et al., 1996, Diabetes 45: 1720-1728 and McCulloch et al., 1993, Diabetes Care 16:911-915).
  • the invention includes embodiments in which the stated endpoint is included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other endpoint is excluded. Unless otherwise specified, both endpoints should be assumed to be included. Furthermore, it should be understood that values expressed as ranges may assume any specific value within the stated range in various embodiments of the invention unless otherwise indicated or otherwise apparent from the context and understanding of one skilled in the art. A value or subrange up to one-tenth of the unit of the lower limit of the stated range, unless the context clearly dictates otherwise.
  • Example 1 Method for inducing differentiation of iPSCs into pancreatic islets
  • Human-derived iPSCs (from Beijing Chengnuo Medical Technology Co., Ltd., prepared according to the method described in the company's previously applied patent (201910110768.7)) are washed with DPBS phosphate buffer after the degree of polymerization reaches 70%-80% 2 times, and then digested with TrypLE Express digestion enzyme for 3-5 minutes at 37°C. Neutralize with DMEM/F12 culture medium at a ratio of 5:1 and centrifuge at 200g for 5 minutes. Use E8 complete medium + 10 ⁇ M Y-27632 (ROCK inhibitor) to resuspend and plate the seeds at a density of approximately 1.8-2.2 ⁇ 10 5 cells/cm 2 . Use 0.13-0.2 mg/mL for the 12-well cell plate 24-48 hours in advance. Matrigel coated.
  • Stage 1 Inducing iPSCs to differentiate into definitive endoderm cells
  • the first stage induction differentiation medium is composed of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin) (BSA), GDF8 and CHIR-99021.
  • the concentration of GDF8 is 100ng/mL
  • the concentration of CHIR-99021 is 3 ⁇ M
  • the concentration of fetal bovine serum albumin (BSA) is 0.5%
  • the concentration of glucose is 10mM
  • the concentration of glutamine is 2mM.
  • the concentration of sodium bicarbonate is 1.5g/L.
  • the first stage of induction lasted for 3 days. Among them, on the first day of induction (D1), the concentration of GDF8 in the culture medium was 100ng/mL; the concentration of CHIR-99021 was 3 ⁇ mol/L. On induction days 2-3 (D2-D3), the concentration of GDF8 in the culture medium was 100ng/mL. That is: the first stage medium A is used on the first day of induction.
  • the first stage medium A consists of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, and defatted BSA (fetal bovine serum albumin).
  • first-stage medium B which consists of basal medium MCDB131, sodium bicarbonate, and glutamine (GlutaMax) , glucose, defatted BSA (fetal bovine serum albumin) (BSA) and GDF8. Change the medium daily with fresh medium.
  • the second stage induction differentiation medium consists of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin) (BSA), ascorbic acid and fibroblast growth factor 7 (FGF-7) composition.
  • the concentration of ascorbic acid is 0.25mM
  • the concentration of fibroblast growth factor 7 (FGF-7) is 50ng/mL
  • the concentration of fetal bovine serum albumin (BSA) is 0.5%
  • the concentration of glucose is 10mM
  • the concentration of glutamine is 2mM
  • the concentration of sodium bicarbonate is 1.5g/L.
  • the duration of the second stage of induction is 2 days.
  • the concentration of ascorbic acid in the culture medium is 0.25mM
  • the concentration of fibroblast growth factor 7 (FGF-7) is 50ng/mL
  • that is, on the 4th to 5th day of induction Use the second stage of induction differentiation medium on the next day. Change the medium daily with fresh medium.
  • the third stage induction differentiation medium consists of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin) (BSA), ascorbic acid, fibroblast growth factor 7 (FGF-7), SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive and protein kinase C activator TPPB.
  • basal medium MCDB131 sodium bicarbonate
  • glucose defatted BSA (fetal bovine serum albumin) (BSA)
  • BSA fetal bovine serum albumin
  • FGF-7 fibroblast growth factor 7
  • SANT-1 retinoic acid
  • LDN193189 insulin-transferrin-selenium-ethanolamine additive
  • protein kinase C activator TPPB protein kinase C activator
  • the concentration of ascorbic acid is 0.25mM
  • the concentration of fibroblast growth factor 7 (FGF-7) is 50ng/mL
  • the concentration of SANT-1 is 0.25 ⁇ M
  • the concentration of retinoic acid is 1 ⁇ M
  • the concentration of LDN193189 Concentrations are 100nM
  • insulin-transferrin-selenium-ethanolamine additive is 0.5%
  • protein kinase C activator TPPB is 200nM
  • fetal bovine serum albumin is 2%
  • glucose is 10mM
  • glutamine The concentration of amide is 2mM and the concentration of sodium bicarbonate is 2.5g/L.
  • the third stage of induction lasts for 2 days.
  • D6-D7 the concentration of ascorbic acid in the culture medium is 0.25mM; the concentration of fibroblast growth factor 7 (FGF-7) is 50ng. /mL; the concentration of SANT-1 is 0.25 ⁇ M; the concentration of retinoic acid is 1 ⁇ M; the concentration of LDN193189 is 100 nM; the concentration of insulin-transferrin-selenium-ethanolamine additive is 0.5%, and the concentration of protein kinase C activator TPPB It is 200nM, that is, the third stage induction differentiation medium is used on the 6th to 7th day of induction, and fresh medium is replaced every day.
  • the induction differentiation medium of the fourth stage consists of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin) (BSA), ascorbic acid, fibroblast growth factor 7 (FGF-7), SANT-1, retinoic acid, LDN193189, insulin-transferrin-selenium-ethanolamine additive and protein kinase C activator TPPB.
  • basal medium MCDB131 sodium bicarbonate
  • glucose defatted BSA (fetal bovine serum albumin) (BSA)
  • BSA fetal bovine serum albumin
  • FGF-7 fibroblast growth factor 7
  • SANT-1 retinoic acid
  • LDN193189 insulin-transferrin-selenium-ethanolamine additive
  • protein kinase C activator TPPB protein kinase C activator
  • the concentration of ascorbic acid is 0.25mM
  • the concentration of fibroblast growth factor 7 (FGF-7) is 2ng/mL
  • the concentration of SANT-1 is 0.25 ⁇ M
  • the concentration of retinoic acid is 1 ⁇ M
  • the concentration of LDN193189 Concentrations are 100nM
  • insulin-transferrin-selenium-ethanolamine additive is 0.5%
  • protein kinase C activator TPPB is 200nM
  • fetal bovine serum albumin is 2%
  • glucose is 10mM
  • glutamine The concentration of amide is 2mM and the concentration of sodium bicarbonate is 2.5g/L.
  • the induction time of the fourth stage is 3 days.
  • D8-D10 the concentration of ascorbic acid in the culture medium is 0.25mM; the concentration of fibroblast growth factor 7 (FGF-7) is 2ng. /mL; the concentration of SANT-1 is 0.25 ⁇ M; the concentration of retinoic acid is 1 ⁇ M; the concentration of LDN193189 is 100 nM; the concentration of insulin-transferrin-selenium-ethanolamine additive is 0.5%; the concentration of protein kinase C activator TPPB It is 200 nM, that is, on the 8th to 10th day of induction, use the induction differentiation medium of the fourth stage, and replace the medium with fresh medium every day.
  • the fifth stage Inducing pancreatic progenitor cells to differentiate into endocrine progenitor cells
  • the induction differentiation medium of the fifth stage consists of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin) (BSA), SANT-1, retinoic acid, Composed of LDN193189, insulin-transferrin-selenium-ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate and heparin.
  • the concentration of SANT-1 is 0.25 ⁇ M
  • the concentration of retinoic acid is 0.1 ⁇ M
  • the concentration of LDN193189 is 100 nM
  • the concentration of insulin-transferrin-selenium-ethanolamine additive is 0.5%
  • the concentration of triiodothyronogen The concentration of amino acid is 1 ⁇ M
  • the concentration of ALK5 inhibitor ALK5i II is 10 ⁇ M
  • the concentration of zinc sulfate is 10 ⁇ M
  • the concentration of heparin is 10 ⁇ g/mL
  • the concentration of sodium bicarbonate is 1.5g/L
  • the concentration of glutamine is 2mM
  • the concentration of glucose was 20mM
  • the concentration of fetal bovine serum albumin was 2%.
  • the fifth stage of induction lasts for 3 days.
  • D11 On the 11th day of induction (D11), cells are seeded into a low-affinity culture plate using cell scrapers.
  • D11-D13 The concentration of SANT-1 in the culture medium is 0.25 ⁇ M; the concentration of retinoic acid is 0.1 ⁇ M; the concentration of LDN193189 is 100 nM; the concentration of insulin-transferrin-selenium-ethanolamine additive is 0.5%; the concentration of triiodothyronine is The concentration of ALK5 inhibitor ALK5i II is 10 ⁇ M; the concentration of zinc sulfate is 10 ⁇ M; the concentration of heparin is 10 ⁇ g/mL, that is, on days 11-13 of induction, use the induction differentiation medium of the fifth stage, every day Replace with fresh culture medium.
  • Stage six Inducing endocrine progenitor cells to differentiate into immature pancreatic islet ⁇ cells
  • the sixth stage induction differentiation medium consists 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.
  • basal medium MCDB131 sodium bicarbonate
  • glucose defatted BSA (fetal bovine serum albumin) (BSA)
  • BSA fetal bovine serum albumin
  • LDN193189 insulin-transferrin- Selenium-ethanolamine additive
  • triiodothyronine triiodothyronine
  • ALK5 inhibitor ALK5i II zinc sulfate
  • the concentration of LDN193189 is 100 nM
  • the concentration of insulin-transferrin-selenium-ethanolamine additive is 0.5%
  • the concentration of triiodothyronine is 1 ⁇ M
  • the concentration of ALK5 inhibitor ALK5i II is 10 ⁇ M
  • the concentration of sulfate The concentration of zinc is 10 ⁇ M
  • the concentration of ⁇ -secretase inhibitor GSi XX is 100 nM
  • the concentration of heparin is 10 ⁇ g/mL
  • the concentration of sodium bicarbonate is 1.5 g/L
  • the concentration of glutamine is 2mM
  • the concentration of glucose is 20mM
  • fetal bovine serum albumin concentration is 2%.
  • the duration of the sixth stage of induction is 14 days.
  • the concentration of LDN193189 in the culture medium is 100 nM; the concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%;
  • the concentration of triiodothyronine is 1 ⁇ M;
  • the concentration of ALK5 inhibitor ALK5i II is 10 ⁇ M;
  • the concentration of zinc sulfate is 10 ⁇ M;
  • the concentration of ⁇ -secretase inhibitor GSi XX is 100 nM, and the concentration of heparin is 10 ⁇ g/mL.
  • the concentration of LDN193189 in the culture medium was 100 nM; the concentration of insulin-transferrin-selenium-ethanolamine additive was 0.5%; the concentration of triiodothyronine was 1 ⁇ M; ALK5
  • the concentration of the inhibitor ALK5i II is 10 ⁇ M; the concentration of zinc sulfate is 10 ⁇ M; the concentration of heparin is 10 ⁇ g/mL, that is, on the 14th to 20th day of induction, the sixth stage medium A is used, and the sixth stage medium A is composed 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, ⁇ -secretase inhibitor
  • Stage 7 Inducing the differentiation of immature islet ⁇ cells into mature islet ⁇ cells.
  • the seventh stage induction differentiation medium consists of basal medium MCDB131, sodium bicarbonate, glutamine (GlutaMax), glucose, defatted BSA (fetal bovine serum albumin) (BSA), insulin-transferrin-selenium- Composed of ethanolamine additive, triiodothyronine, ALK5 inhibitor ALK5i II, zinc sulfate, heparin, acetyl-l-cysteine (N-Cys), water-soluble vitamin E and Axl inhibitor R428.
  • basal medium MCDB131 sodium bicarbonate
  • glutamine GlutaMax
  • glucose defatted BSA (fetal bovine serum albumin) (BSA)
  • BSA fetal bovine serum albumin
  • the concentration of the insulin-transferrin-selenium-ethanolamine additive is 0.5%
  • the concentration of triiodothyronine is 1 ⁇ M
  • the concentration of the ALK5 inhibitor ALK5i II is 10 ⁇ M
  • the concentration of zinc sulfate is 10 ⁇ M.
  • the concentration of heparin was 10 ⁇ g/mL
  • the concentration of acetyl-l-cysteine (N-Cys) was 1 mM
  • the concentration of water-soluble vitamin E was 10 ⁇ M
  • the concentration of Axl inhibitor R428 was 2 ⁇ M
  • the concentration of sodium bicarbonate was 1.5g/L
  • the concentration of glutamine is 2mM
  • the concentration of glucose is 20mM
  • the concentration of fetal bovine serum albumin is 2%.
  • the seventh stage induction time is 13 days.
  • the concentration of insulin-transferrin-selenium-ethanolamine additive in the culture medium is 0.5%; triiodothyronine
  • the concentration of ALK5 inhibitor ALK5i II is 10 ⁇ M; the concentration of zinc sulfate is 10 ⁇ M; the concentration of heparin is 10 ⁇ g/mL; the concentration of acetyl-l-cysteine (N-Cys) is 1mM; water-soluble
  • the concentration of vitamin E is 10 ⁇ M; the concentration of Axl inhibitor R428 is 2 ⁇ M, that is, the seventh stage induction differentiation medium is used on days 28-34 of induction. Change the medium daily with fresh medium.
  • the eighth stage of induction differentiation medium uses pancreatic islet A cell to ⁇ cell medium, which includes 50% Ham's F-12 medium, 50% medium 199, glutamine, calcium chloride dihydrate, N- 2hydroxyethylpiperazine-N-2-ethanesulfonic acid, defatted BSA (fetal bovine serum albumin) (BSA), insulin-transferrin-selenium-ethanolamine additive, water-soluble vitamin E, nicotinamide, heparin, It is composed of deoxyribonuclease I, necroptosis inhibitor Necrostatin-1, and serine protease inhibitor Pefabloc. Artemether and GABAA activator NS11394 are additional small molecules.
  • the concentration of insulin-transferrin-selenium-ethanolamine additive is 0.5%
  • the concentration of water-soluble vitamin E is 10 ⁇ M
  • the concentration of nicotinamide is 10mM
  • the concentration of heparin is 10 ⁇ g/mL
  • the concentration of deoxyribonuclease The concentration of I is 1U/mL
  • the concentration of necroptosis inhibitor Necrostatin-1 is 100 ⁇ M
  • the concentration of serine protease inhibitor Pefabloc is 0.1 ⁇ M
  • the concentration of glutamine is 2mM
  • the concentration of calcium chloride dihydrate is 2.5mM.
  • the concentration of N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid was 10mM
  • the concentration of fetal bovine serum albumin was 2%
  • the concentration of artemether was 10 ⁇ M
  • the concentration of GABAA activator NS11394 was 10 ⁇ M.
  • the eighth stage of induction lasts for 6 days.
  • the concentration of insulin-transferrin-selenium-ethanolamine additive in the culture medium is 0.5%; the concentration of water-soluble vitamin E is The concentration of nicotinamide is 10mM; the concentration of heparin is 10 ⁇ g/mL; the concentration of deoxyribonuclease I is 1U/mL; the concentration of necroptosis inhibitor Necrostatin-1 is 100 ⁇ M; the concentration of serine protease inhibitor Pefabloc The concentration of the additional small molecule artemether is 10 ⁇ M; the concentration of the GABAA activator NS11394 is 10 ⁇ M; that is, the eighth stage induction differentiation medium is used on days 35-40 of induction. Change the medium daily with fresh medium.
  • Insulin Insulin
  • GCG Glucagon
  • MAFA MAFA
  • Insulin refers to insulin, a polypeptide hormone composed of ⁇ and ⁇ double chains secreted by pancreatic ⁇ cells. It consists of 51 amino acids and has a molecular weight of approximately 5800 Da. Insulin is the only hormone in the body that lowers blood sugar. It is a marker gene for pancreatic beta cells;
  • Glucagon refers to glucagon, a linear polypeptide hormone composed of 29 amino acids secreted by pancreatic islet ⁇ cells, with a molecular weight of 3485 Da. It has the effect of raising blood sugar in the body;
  • MAFA refers to the v-maf musculoaponeurotic fibrosarcoma oncogene homologue A gene (v-maf musculoaponeurotic fibrosarcoma oncogene homologue A), which is a transcription factor with a leucine zipper structure.
  • MAFA is the only insulin gene-activating transcription factor discovered so far in pancreatic beta cells.
  • the maturation and maintenance of function of pancreatic ⁇ -cells depend on the normal expression of MAFA protein. Therefore, MAFA is a maturation marker gene of pancreatic ⁇ -cells.
  • RNA is present in the aqueous phase.
  • the capacity of the water phase layer is approximately 60% of the added RL volume. Transfer the water phase to a new tube and proceed to the next step;
  • the elution volume the higher the elution efficiency. If a higher RNA concentration is required, the elution volume can be appropriately reduced, but the minimum volume is preferably not less than 30 ⁇ L. If the volume is too small, the RNA elution efficiency will be reduced and the RNA will be reduced. Yield.
  • Total RNA integrity test Take 6 ⁇ L of RNA sample, conduct 1% agarose gel electrophoresis at 150V ⁇ 10min, observe and take pictures with a gel imaging system, the total RNA has 5s rRNA, 18s rRNA and 28s rRNA bands, three strips If it is complete, it can prove that the total RNA extraction is relatively complete.
  • the primer sequence information for qPCR detection genes INS, GCG, and MAFA is shown in Table 2 below;
  • the templates are as follows: Forward Primer (10 ⁇ M) 0.4 ⁇ L, Reverse Primer (10 ⁇ M) 0.4 ⁇ L, 2xTransStar Top/Tip Green qPCR SuperMix 10 ⁇ L, Nuclease-free Water 8.2 ⁇ L, cDNA 1 ⁇ L, Total volume 20 ⁇ L.
  • the template is as follows Temp Time 94°C30s 94°C5s 45cycles 55°C15s 72°C10s.
  • Control group A Use the seventh stage induction differentiation medium on the 35th to 40th day of induction;
  • Group B Use a-to- ⁇ medium on the 35th to 40th day of induction
  • Group C On the 35th to 40th day of induction, a-to- ⁇ medium was used, and artemether and GABAA activator NS11394 were added at the same time.
  • the concentration of artemether was 10 ⁇ M and the concentration of GABAA activator NS11394 was 10 ⁇ M.
  • the qPCR test results are shown in Figure 1.
  • the results showed that the mRNA expression levels of INS, GCG, and MAFA genes in group C were significantly higher than those in other groups (p ⁇ 0.05).
  • the mRNA expression level of INS gene in islet cells obtained after induction in group C increased 27.76 times compared with group A
  • the mRNA expression level of GCG gene increased 14.82 times compared with group A
  • the mRNA expression level of MAFA gene increased 4.34 times compared with group A, indicating that During the islet maturation stage, iPSCs are induced using ⁇ -cell to ⁇ -cell medium combined with artemether and GABAA activator NS11394, which can promote ⁇ -cell maturation and obtain a larger number of islet ⁇ -cells, which means the induction efficiency is higher.
  • pancreatic islet ⁇ cells The differentiation path of pancreatic islet ⁇ cells is iPSC-pancreatic progenitor cells-naive ⁇ cells- ⁇ cells.
  • the differentiation process into pancreatic ⁇ cells is carried out as a whole.
  • mature ⁇ cells When mature ⁇ cells are generated, mature ⁇ cells are also generated, and Mature ⁇ cells cannot transform into ⁇ cells.
  • the medium of group C + artemether + GABAA activator promotes the transformation of naive ⁇ cells into ⁇ cells and also promotes the transformation of naive ⁇ cells into ⁇ cells.
  • ⁇ cells transform into mature ⁇ cells.
  • the results in the middle picture of Figure 1 shows that the medium + artemether + GABAA activation described in group C
  • the agent can significantly promote the transformation of islet ⁇ cells into islet ⁇ cells and also promote the transformation of immature ⁇ cells into mature ⁇ cells
  • the results in the left picture of Figure 1 showed that the medium + artemether + GABAA activator described in Group C can significantly promote the conversion of islet ⁇ cells into islet ⁇ cells, that is, the use of ⁇ cell to ⁇ cell medium combined with artemether and GABAA activator NS11394 on iPSCs
  • Induction can significantly promote ⁇ -cell maturation and obtain a larger number of functional islet ⁇ -cells, that is, the induction efficiency is significantly higher.

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Abstract

提供了一种促进胰岛α细胞转化为β细胞的诱导方法及其应用，该诱导方法在传统诱导方法的基础上，在诱导分化第35天时，更换为胰岛α细胞转化为β细胞的培养基，同时添加蒿甲醚和GABAA激活剂NS11394诱导至第40天。该诱导方法能够提高胰岛β细胞的表达量。

Description

一种促进胰岛α细胞转化为β细胞的诱导方法及其应用
相关申请的交叉引用
本申请要求享有以下申请文件的优先权：2022年5月20日提交的申请号为202210557363.X名称为“一种促进胰岛α细胞转化为β细胞的诱导方法及其应用”的专利申请，其内容以全文引用的方式并入。
序列表以引用的方式并入，本申请与电子格式的序列表一起提交。
技术领域
本发明属于生物医药技术领域，具体地，本发明涉及一种促进胰岛α细胞转化为β细胞的诱导方法及其应用。
背景技术
糖尿病(Diabetes mellitus，DM)是由于胰岛素分泌不足或胰岛素抵抗引起的以高血糖为特征的一组慢性代谢性疾病，主要分为1型糖尿病(Type 1 diabetes mellitus，T1DM)、2型糖尿病(Type 2 diabetes mellitus，T2DM)、妊娠糖尿病(Gestational diabetes mellitus，GDM)和单基因糖尿病。据统计，截止至2019年全球糖尿病患者超4.63亿，预计2045年将增至7亿。其中，1型糖尿病是一种由T淋巴细胞介导的自身免疫性疾病。由于胰岛细胞受损而导致胰岛素分泌不足，1型糖尿病患者需要完全依赖外源胰岛素治疗。虽然胰岛素治疗已从过去的“每日餐前一针”发展到“人工智能控制的胰岛泵”，但并不能根治，也不能防止包括视网膜病变、神经病变等一系列并发症的发生。近年来，胰岛移植结合免疫抑制已成功应用于1型糖尿病的治疗中，但由于供体器官严重缺乏、需要终身免疫抑制以及胰岛移植后难以长期存活等问题限制了其临床应用。多能干细胞在解决供体胰岛短缺方面有着巨大的潜力，多能干细胞诱导分化胰岛类器官，在胰岛类器官中分化为能够分泌胰岛素的胰岛β细胞可以为糖尿病替代治疗提供无限的细胞来源。然而与天然成人胰岛β细胞相比，体外生成的胰岛β细胞在转录和功能上并不成熟。因此，成熟胰岛β细胞功能性是至关重要的。
胰岛β细胞的再生和替代治疗被认为有望成为治愈1型糖尿病的有效途径。目前，在实验室水平上用于1型糖尿病治疗的干细胞有胚胎干细胞、诱导性多能干细胞(Induced pluripotent stem cell，iPSCs)、间充质干细胞(Mesenchymal stem cell，MSCs)、骨髓来源的造血干细胞(Hematopoietic stem cell，HSCs)、胰腺内的多能前体细胞以及存在于胰腺外(脾脏、肝脏、子宫内膜)的β细胞前体细胞。干细胞治疗1型糖尿病是基于干细胞的自我更新功能和潜在的定向分化功能，相较于完全异体胰腺组织移植，移植前能够在分子水平进行严格的质量控制，并能对移植细胞质量和免疫排斥反应进行有效评估，因而干细胞疗法可以部分或完全避免免疫排斥反应所导致的移植失败和移植术后并发症。其中，iPSCs治疗1型糖尿病属于再生医学范畴，在治疗1型糖尿病方面具有巨大的潜力，其基本原理是将患者自体细胞逆转为早期未分化的多能干细胞，再经各种细胞因子处理以模仿胚胎细胞的生长发育过程，诱导其成为具有分泌胰岛素功能的胰岛β细胞。与胚胎干细胞不同，iPSCs只能形成新的组织和器官，而不能形成新的个体，所以能够避免潜在的医学伦理问题，且因细胞来源于患者本身，所以还可减少或完全避免免疫排斥反应。
现阶段有多种方法能够使多能干细胞成功诱导转分化为胰岛，所述方法中胰岛的形成基本均是通过内胚层、原肠管、后前肠、胰腺祖细胞、胰腺内分泌前体、未成熟的β细胞、成熟的β细胞这一过程实现的，然而通过目前已经开发的众多多能干细胞诱导分化胰岛细胞的技术诱导转分化得到的胰岛细胞大多数为胰 岛多激素细胞，也即同时表达胰岛α细胞、胰岛β细胞、胰岛γ细胞、胰岛PP细胞等，其中，胰岛β细胞表达激素有限且不稳定，胰岛α细胞的表达量显著高于胰岛β细胞，且大部分胰岛β细胞处于未成熟状态，胰高血糖素的表达量明显高于胰岛素的表达量，严重限制了其在1型糖尿病治疗中的应用。因此，目前本领域仍亟需一种能够大量并稳定的获得功能性胰岛β样细胞的标准诱导方法。基于此，本发明提供了一种促进胰岛α细胞转化为β细胞的诱导方法及其应用，所述诱导方法中包含蒿甲醚和GABAA激活剂NS11394，目前，尚未有将蒿甲醚和GABAA激活剂NS11394两者联合应用于促进胰岛α细胞转化为β细胞的相关报道。
发明内容
有鉴于此，本发明的目的在于提供一种促进胰岛α细胞转化为β细胞的诱导方法及其应用，所述诱导方法在传统诱导方法的基础上，在诱导分化第35天时，更换为胰岛α细胞转化为β细胞的培养基，同时添加蒿甲醚和GABAA激活剂NS11394诱导至第40天。经验证发现，采用本发明所述的诱导方法能够显著提高胰岛β细胞的表达量，在1型糖尿病治疗中的应用前景广阔。
本发明的上述目的通过以下技术方案得以实现：
本发明的第一方面提供了一种诱导多能干细胞分化为功能性胰岛β细胞的诱导分化剂。
进一步，所述诱导分化剂包括第一阶段诱导分化剂、第二阶段诱导分化剂、第三阶段诱导分化剂、第四阶段诱导分化剂、第五阶段诱导分化剂、第六阶段诱导分化剂、第七阶段诱导分化剂、第八阶段诱导分化剂；
所述第一阶段诱导分化剂包括第一阶段诱导分化剂A、第一阶段诱导分化剂B；
所述第一阶段诱导分化剂A包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、GDF8、CHIR-99021；
所述第一阶段诱导分化剂B包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、GDF8；
所述第二阶段诱导分化剂包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、抗坏血酸、FGF-7；
所述第三阶段诱导分化剂包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、抗坏血酸、FGF-7、SANT-1、视黄酸、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、TPPB；
所述第四阶段诱导分化剂包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、抗坏血酸、FGF-7、SANT-1、视黄酸、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、TPPB；
所述第五阶段诱导分化剂包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、SANT-1、视黄酸、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、三碘甲状腺原氨酸、ALK5i II、硫酸锌、肝素；
所述第六阶段诱导分化剂包括第六阶段诱导分化剂A、第六阶段诱导分化剂B；
所述第六阶段诱导分化剂A包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、三碘甲状腺原氨酸、ALK5i II、硫酸锌、GSi XX、肝素；
所述第六阶段诱导分化剂B包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、三碘甲状腺原氨酸、ALK5i II、硫酸锌、肝素；
所述第七阶段诱导分化剂包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、胰岛素-转铁蛋白-硒-乙醇胺添加剂、三碘甲状腺原氨酸、ALK5i II、硫酸锌、肝素、乙酰-l-半胱氨酸、水溶性维生素E、R428；
所述第八阶段诱导分化剂包含蒿甲醚和NS11394；
所述第八阶段诱导分化剂还包含谷氨酰胺、二水氯化钙、N-2羟乙基哌嗪-N-2-乙烷磺酸、胎牛血清白蛋白、胰岛素-转铁蛋白-硒-乙醇胺添加剂、水溶性维生素E、烟酰胺、肝素、脱氧核糖核酸酶Ⅰ、Necrostatin-1、 Pefabloc。
进一步，所述第一阶段诱导分化剂中各成分的浓度分别为：(10-500)ng/mL GDF8、(0.5-20)μM CHIR-99021、(0.01-5)％胎牛血清白蛋白、(1-50)mM葡萄糖、(0.05-10)mM谷氨酰胺、(0.05-10)g/L碳酸氢钠。
进一步，所述第一阶段诱导分化剂中各成分的浓度分别为：100ng/mL GDF8、3μM CHIR-99021、0.5％胎牛血清白蛋白、10mM葡萄糖、2mM谷氨酰胺、1.5g/L碳酸氢钠。
进一步，所述第二阶段诱导分化剂中各成分的浓度分别为：(0.01-5)mM抗坏血酸、(10-100)ng/mL FGF-7、(0.01-5)％胎牛血清白蛋白、(1-50)mM葡萄糖、(0.05-10)mM谷氨酰胺、(0.05-10)g/L碳酸氢钠。
进一步，所述第二阶段诱导分化剂中各成分的浓度分别为：0.25mM抗坏血酸、50ng/mL FGF-7、0.5％胎牛血清白蛋白、10mM葡萄糖、2mM谷氨酰胺、1.5g/L碳酸氢钠。
进一步，所述第三阶段诱导分化剂中各成分的浓度分别为：(0.01-5)mM抗坏血酸、(10-100)ng/mL FGF-7、(0.01-5)μM SANT-1、(0.01-10)μM视黄酸、(10-500)nM LDN193189、(0.01-5)％胰岛素-转铁蛋白-硒-乙醇胺添加剂、(50-500)nM TPPB、(0.05-10)％胎牛血清白蛋白、(1-50)mM葡萄糖、(0.05-10)mM谷氨酰胺、(0.05-10)g/L碳酸氢钠。
进一步，所述第三阶段诱导分化剂中各成分的浓度分别为：0.25mM抗坏血酸、50ng/mL FGF-7、0.25μM SANT-1、1μM视黄酸、100nM LDN193189、0.5％胰岛素-转铁蛋白-硒-乙醇胺添加剂、200nM TPPB、2％胎牛血清白蛋白、10mM葡萄糖、2mM谷氨酰胺、2.5g/L碳酸氢钠。
进一步，所述第四阶段诱导分化剂中各成分的浓度分别为：(0.01-5)mM抗坏血酸、(0.01-10)ng/mL FGF-7、(0.01-5)μM SANT-1、(0.01-10)μM视黄酸、(10-500)nM LDN193189、(0.01-5)％胰岛素-转铁蛋白-硒-乙醇胺添加剂、(50-500)nM TPPB、(0.05-10)％胎牛血清白蛋白、(1-50)mM葡萄糖、(0.05-10)mM谷氨酰胺、(0.05-10)g/L碳酸氢钠。
进一步，所述第四阶段诱导分化剂中各成分的浓度分别为：0.25mM抗坏血酸、2ng/mL FGF-7、0.25μM SANT-1、1μM视黄酸、100nM LDN193189、0.5％胰岛素-转铁蛋白-硒-乙醇胺添加剂、200nM TPPB、2％胎牛血清白蛋白、10mM葡萄糖、2mM谷氨酰胺、2.5g/L碳酸氢钠。
进一步，所述第五阶段诱导分化剂中各成分的浓度分别为：(0.01-5)μM SANT-1、(0.01-10)μM视黄酸、(10-500)nM LDN193189、(0.01-5)％胰岛素-转铁蛋白-硒-乙醇胺添加剂、(0.01-10)μM三碘甲状腺原氨酸、(0.01-50)μM ALK5i II、(1-50)μM硫酸锌、(1-50)μg/mL肝素、(0.05-10)g/L碳酸氢钠、(0.05-10)mM谷氨酰胺、(1-50)mM葡萄糖、(0.05-10)％胎牛血清白蛋白。
进一步，所述第五阶段诱导分化剂中各成分的浓度分别为：0.25μM SANT-1、0.1μM视黄酸、100nM LDN193189、0.5％胰岛素-转铁蛋白-硒-乙醇胺添加剂、1μM三碘甲状腺原氨酸、10μM ALK5i II、10μM硫酸锌、10μg/mL肝素、1.5g/L碳酸氢钠、2mM谷氨酰胺、20mM葡萄糖、2％胎牛血清白蛋白。
进一步，所述第六阶段诱导分化剂中各成分的浓度分别为：(10-500)nM LDN193189、(0.01-5)％胰岛素-转铁蛋白-硒-乙醇胺添加剂、(0.01-10)μM三碘甲状腺原氨酸、(0.01-50)μM ALK5i II、(1-50)μM硫酸锌、(10-500)nM GSi XX、(1-50)μg/mL肝素、(0.05-10)g/L碳酸氢钠、(0.05-10)mM谷氨酰胺、(1-50)mM葡萄糖、(0.05-10)％胎牛血清白蛋白。
进一步，所述第六阶段诱导分化剂中各成分的浓度分别为：100nM LDN193189、0.5％胰岛素-转铁蛋白-硒-乙醇胺添加剂、1μM三碘甲状腺原氨酸、10μM ALK5i II、10μM硫酸锌、100nM GSi XX、10μg/mL肝素、1.5g/L碳酸氢钠、2mM谷氨酰胺、20mM葡萄糖、2％胎牛血清白蛋白。
进一步，所述第七阶段诱导分化剂中各成分的浓度分别为：(0.01-5)％胰岛素-转铁蛋白-硒-乙醇胺添加 剂、(0.01-10)μM三碘甲状腺原氨酸、(0.01-50)μM ALK5i II、(1-50)μM硫酸锌、(1-50)μg/mL肝素、(0.01-10)mM乙酰-l-半胱氨酸、(1-50)μM水溶性维生素E、(1-10)μM R428、(0.05-10)g/L碳酸氢钠、(0.05-10)mM谷氨酰胺、(1-50)mM葡萄糖、(0.05-10)％胎牛血清白蛋白。
进一步，所述第七阶段诱导分化剂中各成分的浓度分别为：0.5％胰岛素-转铁蛋白-硒-乙醇胺添加剂、1μM三碘甲状腺原氨酸、10μM ALK5i II、10μM硫酸锌、10μg/mL肝素、1mM乙酰-l-半胱氨酸、10μM水溶性维生素E、2μM R428、1.5g/L碳酸氢钠、2mM谷氨酰胺、20mM葡萄糖、2％胎牛血清白蛋白。
进一步，所述第八阶段诱导分化剂中各成分的浓度分别为：(0.01-5)％胰岛素-转铁蛋白-硒-乙醇胺添加剂、(1-50)μM水溶性维生素E、(1-50)mM烟酰胺、(1-50)μg/mL肝素、(0.01-10)U/mL脱氧核糖核酸酶Ⅰ、(10-500)μM Necrostatin-1、(0.01-5)μM Pefabloc、(0.01-10)mM谷氨酰胺、(0.01-10)mM二水氯化钙、(0.1-50)mM N-2羟乙基哌嗪-N-2-乙烷磺酸、(0.05-10)％胎牛血清白蛋白、(1-50)μM蒿甲醚、(1-50)μM NS11394。
进一步，所述第八阶段诱导分化培养剂中各成分的浓度分别为：0.5％胰岛素-转铁蛋白-硒-乙醇胺添加剂、10μM水溶性维生素E、10mM烟酰胺、10μg/mL肝素、1U/mL脱氧核糖核酸酶Ⅰ、100μM Necrostatin-1、0.1μM Pefabloc、2mM谷氨酰胺、2.5mM二水氯化钙、10mM N-2羟乙基哌嗪-N-2-乙烷磺酸、2％胎牛血清白蛋白、10μM蒿甲醚、10μM NS11394。
本发明的第二方面提供了一种诱导多能干细胞分化为功能性胰岛β细胞的诱导分化培养基。
进一步，所述诱导分化培养基包括第一阶段诱导分化培养基、第二阶段诱导分化培养基、第三阶段诱导分化培养基、第四阶段诱导分化培养基、第五阶段诱导分化培养基、第六阶段诱导分化培养基、第七阶段诱导分化培养基、第八阶段诱导分化培养基；
所述第一阶段诱导分化培养基包括基础培养基MCDB131、本发明第一方面中所述的第一阶段诱导分化剂；
所述第二阶段诱导分化培养基包括基础培养基MCDB131、本发明第一方面中所述的第二阶段诱导分化剂；
所述第三阶段诱导分化培养基包括基础培养基MCDB131、本发明第一方面中所述的第三阶段诱导分化剂；
所述第四阶段诱导分化培养基包括基础培养基MCDB131、本发明第一方面中所述的第四阶段诱导分化剂；
所述第五阶段诱导分化培养基包括基础培养基MCDB131、本发明第一方面中所述的第五阶段诱导分化剂；
所述第六阶段诱导分化培养基包括基础培养基MCDB131、本发明第一方面中所述的第六阶段诱导分化剂；
所述第七阶段诱导分化培养基包括基础培养基MCDB131、本发明第一方面中所述的第七阶段诱导分化剂；
所述第八阶段诱导分化培养基包括50％Ham’s F-12medium、50％medium 199、本发明第一方面中所述的第八阶段诱导分化剂。
本发明的第三方面提供了一种诱导多能干细胞分化为功能性胰岛β细胞的诱导分化方法。
进一步，所述方法包括如下步骤：
(1)提供诱导多能干细胞并在完全培养基中进行培养；
(2)第一阶段诱导分化，采用本发明第二方面所述的第一阶段诱导分化培养基诱导步骤(1)得到的诱导多能干细胞向定型内胚层细胞分化；
(3)第二阶段诱导分化，采用本发明第二方面所述的第二阶段诱导分化培养基诱导定型内胚层细胞向原始肠管细胞分化；
(4)第三阶段诱导分化，采用本发明第二方面所述的第三阶段诱导分化培养基诱导原始肠管细胞向后前肠细胞分化；
(5)第四阶段诱导分化，采用本发明第二方面所述的第四阶段诱导分化培养基诱导后前肠细胞向胰腺祖细胞分化；
(6)第五阶段诱导分化，采用本发明第二方面所述的第五阶段诱导分化培养基诱导胰腺祖细胞向内分泌祖细胞分化；
(7)第六阶段诱导分化，采用本发明第二方面所述的第六阶段诱导分化培养基诱导内分泌祖细胞向未成熟胰岛β细胞分化；
(8)第七阶段诱导分化，采用本发明第二方面所述的第七阶段诱导分化培养基诱导未成熟胰岛β细胞向成熟胰岛β细胞分化；
(9)第八阶段诱导分化，采用本发明第二方面所述的第八阶段诱导分化培养基进一步诱导胰岛β细胞的成熟，得到功能性胰岛β细胞。
进一步，步骤(1)中所述的完全培养基为E8完全培养基。
进一步，所述E8完全培养基中含有ROCK抑制剂。
进一步，所述ROCK抑制剂包括Y-27632、GSK429286A、RKI-1447、Y-33075dihydrochloride、Thiazovivin、K-115、SLx-2119、Chroman1、SAR407899和/或SR-3677。
进一步，所述ROCK抑制剂为Y-27632。
进一步，所述Y-27632的浓度为0.001-100μM。
进一步，所述Y-27632的浓度为10μM。
进一步，步骤(1)中所述的诱导多能干细胞的细胞密度为0.1-10.0×10 5cells/cm 2。
进一步，步骤(1)中所述的诱导多能干细胞的细胞密度为1.8-2.2×10 5cells/cm 2。
进一步，步骤(1)中所述的诱导多能干细胞来源于哺乳动物。
进一步，步骤(1)中所述的诱导多能干细胞来源于人类、小鼠、大鼠、山羊、绵羊、猪、猫、兔、狗、狼、马、或牛。
进一步，步骤(1)中所述的诱导多能干细胞来源于人类。
在本发明的具体实施方案中，步骤(1)中所述的诱导多能干细胞为来源于人类的诱导多能干细胞，所述诱导多能干细胞来源于北京呈诺医学科技有限公司，根据本公司在先申请的专利(申请号：201910110768.7)中所述的方法制备得到。
进一步，所述第一阶段诱导分化的时间为3天。
进一步，在诱导第1天，采用第一阶段诱导分化培养基A进行诱导分化。
进一步，在诱导第2-3天，采用第一阶段诱导分化培养基B进行诱导分化。
进一步，所述第二阶段诱导分化的时间为2天。
进一步，在诱导第4-5天，采用所述第二阶段诱导分化培养基进行诱导分化。
进一步，所述第三阶段诱导分化的时间为2天。
进一步，在诱导第6-7天，采用所述第三阶段诱导分化培养基进行诱导分化。
进一步，所述第四阶段诱导分化的时间为3天。
进一步，在诱导第8-10天，采用所述第四阶段诱导分化培养基进行诱导分化。
进一步，所述第五阶段诱导分化的时间为3天。
进一步，在诱导第11天，将细胞铺种到低亲附性培养板中，在诱导第11-13天，采用所述第五阶段诱导分化培养基进行诱导分化。
进一步，所述第六阶段诱导分化的时间为14天。
进一步，在诱导第14-20天，采用第六阶段诱导分化培养基A进行诱导分化。
进一步，在诱导第21-27天，采用第六阶段诱导分化培养基B进行诱导分化。
进一步，所述第七阶段诱导分化的时间为13天。
进一步，在诱导第28-34天，采用所述第七阶段诱导分化培养基进行诱导分化。
进一步，所述第八阶段诱导分化的时间为6天。
进一步，在诱导第35-40天，采用所述第八阶段诱导分化培养基进行诱导分化。
本发明的第四方面提供了一种诱导多能干细胞来源的功能性胰岛β细胞或细胞群体。
进一步，所述功能性胰岛β细胞或细胞群体为采用本发明第三方面所述的方法诱导分化得到的。
进一步，所述功能性胰岛β细胞或细胞群体为功能性的、稳定的胰岛β细胞或细胞群体。
本发明的第五方面提供了一种用于治疗和/或预防糖尿病的药物组合物。
进一步，所述药物组合物包含本发明第四发明所述的细胞或细胞群体。
进一步，所述药物组合物还包含药学上可接受的载体和/或辅料。
进一步，所述糖尿病包括1型糖尿病、2型糖尿病、妊娠糖尿病、单基因糖尿病。
进一步，所述糖尿病为1型糖尿病。
进一步，所述药学上可接受的载体和/或赋形剂在Remington's Pharmaceutical Sciences(19th ed.,1995)中有详细的记载，这些物质根据需要用于帮助药物的稳定性或有助于提高有效成分(本发明第四发明所述的细胞或细胞群体)的活性，在这种药物组合物中可以使用的制剂可以是其原始化合物本身的形式，或任选地使用其药物学可接受的盐的其他形式，如此配制得到的药物组合物根据需要可选择本领域技术人员已知的任何适当的给药方式将所述药物进行给药。
在一些实施方案中，所述药学上可接受的载体和/或辅料可另外含有液体，诸如水、生理盐水、甘油和乙醇。另外，诸如湿润剂或乳化剂或pH缓冲物质的辅助物质可存在于所述药物组合物中。这些载剂使得药物组合物能够配制成片剂、丸剂、糖衣丸、胶囊、液体、凝胶、糖浆、浆液和悬浮液，以便患者摄入。
在一些实施方案中，合适的施用形式包括适用于肠胃外施用的形式，例如通过注射或输注，例如通过快速注射或连续输液、静脉内、可吸入或皮下形式。在产品用于注射或输注的情况下，其可采用在油性或水性媒介物中的悬浮液、溶液或乳液的形式并且其可含有配制试剂，诸如悬浮剂、防腐剂、稳定剂和/或分散剂。
本发明还提供了一种用于治疗和/或预防糖尿病的生物制剂。
进一步，所述生物制剂包含本发明第四方面所述的细胞或细胞群体和/或本发明第五方面所述的药物组合物。
进一步，所述生物制剂还可包含其它能够用于治疗和/或预防、或辅助治疗和/或预防糖尿病的任何试剂，所述试剂包括但不限于小分子化合物、抗体、细胞因子等。其中，上述生物制剂中的试剂和本发明所述细胞或细胞群体或药物组合物可联合使用，也可分开使用。
进一步，所述小分子化合物包括但不限于磺脲类降糖药、双胍类降糖药、胰岛素增敏剂、磺酰脲类药物。所述磺脲类降糖药包括但不限于：优降糖、美吡达(优达灵)、克糖利、达美康(格列齐特)、糖适平(格列喹酮)；所述双胍类降糖药包括但不限于：降糖灵(苯乙双胍、DBl)、二甲双胍(二甲双胍片、美迪康、迪化糖锭、格华止)；所述胰岛素增敏剂包括但不限于：噻唑烷二酮类(格列酮类，如罗格列酮、吡格列酮)、双胍类。
本发明的第六方面提供了一种用于治疗和/或预防糖尿病的方法。
进一步，所述方法包括给有需要的受试者施用有效量的本发明第四方面所述的细胞或细胞群体和/或本发明第五方面所述的药物组合物。
进一步，所述糖尿病包括1型糖尿病、2型糖尿病、妊娠糖尿病、单基因糖尿病。
进一步，所述糖尿病为1型糖尿病。
进一步，所述受试者患有糖尿病或具有患有糖尿病的风险，或患有代谢障碍或具有患有代谢障碍的风险。
在一些实施方案中，通过本发明提供的方法产生的功能性胰岛β细胞或细胞群体可以给药到受试者，用于治疗1型糖尿病、2型糖尿病、妊娠糖尿病、单基因糖尿病。
在一些实施方案中，所述受试者包括各种类型的哺乳动物，优选为人。
在一些实施方案中，所述功能性胰岛β细胞或细胞群体可以作为分散的细胞或形成细胞簇植入，其可以输注到肝门静脉中。在某些情况下，细胞可以被提供在生物相容的可降解聚合支持物中、多孔的不可降解的装置中，或者被包封以保护其免于宿主免疫应答。细胞可以被植入到接受者中的适合位点内。植入位点包括例如肝、天然胰腺、肾被膜下空间、网膜、腹膜、浆膜下空间、肠、胃或皮下囊袋。
为了进一步增强植入的功能性胰岛β细胞或细胞群体在体内的功能、存活或活性，可以在功能性胰岛β细胞或细胞群体给药之前、同时或之后给药其他因子例如生长因子、抗氧化剂或抗炎剂。这些因子可以由内源性细胞分泌并在原位暴露于给药的细胞，可以通过本领域中已知的内源和外源给药的生长因子的任何组合来增强植入的细胞的功能。
在植入中使用的功能性胰岛β细胞或细胞群体的量以及其施用方式取决于多种不同因素，包括患者的状况和对疗法的响应程度，并且可以由本领域常规技术人员进行确定。
在一些实施方案中，所述治疗方法还包括在植入之前将所述细胞并入到三维支持物中。在植入到受试者体内之前，细胞可以在体外维持在该支持物上。或者，可以将含有细胞的支持物直接植入到患者体内而不需另外的体外培养。所述支持物可以任选地合并有至少一种促进移植的细胞的存活和功能的药剂。
在一些实施方案中，可以将所述功能性胰岛β细胞或细胞群体、所述药物组合物或所述生物制剂通过任何合适的方式施用，例如通过推注输注，通过注射例如皮下注射递送，还可以通过胰岛细胞移植的方式递送。在一些实施方案中，可以将所述功能性胰岛β细胞或细胞群体通过门静脉穿刺进行移植，将所述功能性胰岛β细胞或细胞群体输送至肝脏上，胰岛能够在肝脏上生长并分泌胰岛素。
本发明的第七方面提供了蒿甲醚和NS11394联合在促进胰岛α细胞转化为胰岛β细胞中的应用。
本发明的第八方面提供了蒿甲醚和NS11394联合在诱导多能干细胞分化为功能性胰岛β细胞中的应用。
本发明的第九方面提供了本发明第一方面所述的诱导分化剂在制备诱导多能干细胞分化为功能性胰岛 β细胞的诱导分化培养基中的应用。
本发明的第十方面提供了本发明第一方面所述的诱导分化剂在诱导多能干细胞分化为功能性胰岛β细胞中的应用。
本发明的第十一方面提供了本发明第二方面所述的诱导分化培养基在诱导多能干细胞分化为功能性胰岛β细胞中的应用。
本发明的第十二方面提供了本发明第四方面所述的细胞或细胞群体在制备治疗和/或预防糖尿病的药物中的应用。
本发明的第十三方面提供了本发明第四方面所述的细胞或细胞群体在治疗和/或预防糖尿病中的应用。
本发明的第十四方面提供了本发明第五方面所述的药物组合物在治疗和/或预防糖尿病中的应用。
进一步，所述糖尿病包括1型糖尿病、2型糖尿病、妊娠糖尿病、单基因糖尿病；
进一步，所述糖尿病为1型糖尿病。
相对于现有技术，本发明具有的优点和有益效果：
本发明提供了一种新型的促进胰岛α细胞转化为β细胞的诱导方法，所述诱导方法在传统诱导方法的基础上，在诱导分化第35天时，更换为胰岛α细胞转化为β细胞的培养基，同时添加蒿甲醚和GABAA激活剂NS11394诱导至第40天。经验证发现，经过本发明提供的诱导方法制备得到的胰岛β细胞的标志性分子的表达量显著升高，表明了本发明提供的诱导方法能够显著提高胰岛β细胞的表达量，为临床上1型糖尿病的治疗提供了科学依据。
附图说明
图1为qPCR检测胰岛成熟标志基因mRNA的表达情况的结果统计图，其中，A组(对照组)：在诱导第35-40天使用第七阶段诱导分化培养基；B组：在诱导第35-40天使用α细胞转β细胞培养基；C组：在诱导第35-40天使用α细胞转β细胞培养基，并同时添加蒿甲醚和GABAA激活剂NS11394。
具体实施方式
下面结合具体实施例，进一步阐述本发明，仅用于解释本发明，而不能理解为对本发明的限制。本领域的普通技术人员可以理解为：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的试剂、生物材料等，如无特殊说明，均可从商业途径得到。
为了更好的阐述本发明，此处对本发明中涉及到的专业术语进行如下解释：
如本文中使用，术语“糖尿病”是指慢性高血糖症的状态，即因相对或绝对的胰岛素作用缺乏而引起的血中过量的糖，是人类一大组紊乱的一种形式，以血糖控制受损或血液中葡萄糖水平控制受损为特征。糖尿病本身是以葡萄糖和其它产能燃料的代谢受损，以及晚期发展出严重的血管和神经并发症为特征的慢性激素紊乱，糖尿病使心脏、脑和外周血管病变的风险增加2-7倍且是新生儿发病和死亡的主要原因。目前，有三种糖尿病的基本类型，1型或胰岛素依赖型糖尿病(IDDM)、2型或非胰岛素依赖型糖尿病(NIDDM)、妊娠糖尿病、单基因糖尿病。
其中，具有1型糖尿病这种紊乱的患者胰岛素分泌能力很低或无并且依赖于外来胰岛素预防代谢的代偿失调(如酮症酸中毒)和死亡。通常但不是总是，以前健康的无肥胖儿童或年轻成人突然出现糖尿病(即数日和数周内)；在较大年龄群中，它可能逐渐起病。在初次估计时，典型患者常常出现疾病，具有明显症状(如多尿、烦渴、贪食和体重减轻)，并且可能证明酮症酸中毒。1型糖尿病具有很长的无症状临床前期，通 常持续数年，在此期间胰腺β细胞逐渐由受到HLA和其它遗传因子以及环境影响的自身免疫攻击所破坏。
其中，研究发现糖尿病患者群体中90％以上是2型糖尿病，即，至今该疾病最普通的形式。这些患者保留了明显水平的内源胰岛素分泌能力。然而，胰岛素水平相对于抗胰岛素性和周围葡萄糖水平的值为低。2型糖尿病患者不依赖于胰岛素进行立即存活并很少发展为酮症，除了在强大身体压力的情况下。然而，这些患者可能需要胰岛素治疗来控制高血糖。典型地，2型糖尿病在40岁以后出现，与HLA基因不相关的遗传外显率的比例高，并且伴有肥胖。2型糖尿病的临床特征可能是轻微的(疲劳、虚弱、头晕、视力模糊或其它非特殊主诉可能占优势)或在患者求医前可以被忍受很多年。此外，如果高血糖的水平不足以产生症状，该疾病可能仅在发生并发症后才变得明显。
其中，妊娠糖尿病患者是指在妊娠期出现或首次检测到葡萄糖耐量受损的女性患者。妊娠糖尿病通常在第2和第3个三个月，即，妊娠相关的胰岛素拮抗激素峰的时间出现。分娩后，葡萄糖耐量通常(但不总是)恢复正常。当存在多尿、烦渴和体重减轻的典型症状时，糖尿病的诊断通常很明确。仅需要的是静脉血随机血浆葡萄糖测定是200mg/dl或更高。如果糖尿病是猜测的，由随机葡萄糖测定不能证实，那么所选择的筛选测试是过夜空腹血浆葡萄糖水平。如果在至少两个分开的时间下，空腹葡萄糖等于或高于126mg/dl，那么诊断确立。
如本文中使用，术语“包含”或“包括”是指在给定实施方案中存在的组合物、方法及其相应组分，但是也是开放式的，包含未指定的要素。
如本文中使用，术语“治疗和/或预防”是指以治愈、改善、稳定或预防疾病、病理状、病症或其相关并发症为目的对患者进行的医学管理。该术语包括积极疗法，即专门以改善疾病、病理状态、病症或其相关并发症为目的的治疗，并且还包括病因治疗，即以除去相关疾病、病理状态、病症或其相关并发症的病因为目的的治疗。此外，该术语还包括姑息治疗，即设计用于缓解症状而非治愈疾病、病理状态、病症或其相关并发症的治疗；该术语还包括预防性治疗，即以最大程度降低或者部分或完全抑制相关疾病、病理状态、病症或其相关并发症的发展为目的的治疗；以及支持性治疗，即用于补充另一种以改善相关疾病、病理状态、病症或其相关并发症为目的的特定疗法的治疗。
在一些实施方案中，所述糖尿病相关疾病、病理状态、病症或其相关并发症包括高血糖症、不理想的糖血(glycemic)控制、酮症酸中毒、胰岛素抗性、升高的生长激素水平、升高的糖基化血红蛋白和高度糖基化终产物(AGE)水平、黎明现象、不理想的脂类谱、血管病(例如，动脉粥样硬化)、微血管病、视网膜病(例如，增殖性糖尿病性视网膜病变)、肾脏疾病、神经病、怀孕并发症(例如，早期终止(妊娠)和出生缺陷)等。治疗定义中包括的结束点为，例如，胰岛素敏感性增加、维持糖血控制的同时胰岛素给药减少、HbA1c降低、改善的糖血控制、减少的血管、肾脏、神经、视网膜和其他糖尿病并发症、预防或减少“黎明现象”、改善的脂类谱、减少的怀孕并发症、和减少的酮症酸中毒。
如本文中使用，术语“有效量”是指可对人和/或动物产生治疗效果且可被人和/或动物所接受的量。例如，药物治疗上或药学上有效量是指产生需要的治疗效果所需要的药物的量，治疗效果可以通过临床试验结果、模型动物研究和/或体外研究的结果来反映。药学上有效量取决于几个因素，包括但不限于：治疗对象的特征因素(如治疗对象的身高、体重、性别、年龄和用药史等)、罹患疾病的严重程度。本发明中所述的药物活性成分(采用本发明所述的方法制备得到的功能性胰岛β细胞)与药学上可接受的载体和/或辅料(用于治疗给药的载体和/或载体，它们本身并不是必要的活性成分，且施用后没有过分的毒性)可以组成药物组合物或药物制剂。在本发明的具体实施方式中，所述“有效量”是指足以抑制、减缓或预防受试者糖尿病和/或其相关并发症发生和发展的量。
在一些实施方案中，术语“有效量”是指“治疗和/或预防有效量”，进一步是指足以导致延迟或预防或消除或减轻糖尿病(1型或糖尿病、2型糖尿病、妊娠糖尿病、单基因糖尿病)的一种或更多症状的发生、 复发或发作的本发明所述的功能性胰岛β细胞、药物组合物、生物制剂的量。
在一些实施方案中，治疗和/或预防有效量优选是指本发明所述的功能性胰岛β细胞、药物组合物、生物制剂的以下量，其将受试者的糖尿病发作延迟或糖尿病相关症状减轻至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％。
如本文中使用，术语“受试者”是指向其提供本发明所述的功能性胰岛β细胞、药物组合物、生物制剂以用于治疗糖尿病的动物、人类或非人类。本公开预期了人类和兽医学应用。该术语包括但不限于：鸟类、爬行动物、两栖动物和哺乳动物，例如人类、其他灵长类动物、猪、啮齿动物，例如小鼠和大鼠、兔、豚鼠、仓鼠、马、牛、猫、狗、绵羊、鸡和山羊。在一些实施方案中，所述受试者是人、鸡或小鼠。在优选的实施方案中，所述受试者是人。儿科和成人受试者均包括在内。例如，在本文中描述的受试者可以是至少6个月大的儿科和成人受试者(例如，6个月或更大、12个月或更大、18个月或更大、2岁或更大、4岁或更大、6岁或更大、10岁或更大、13岁或更大、16岁或更大、18岁或更大、21岁或更大、25岁或更大、30岁或更大、35岁或更大、40岁或更大、45岁或更大、50岁或更大、60岁或更大、65岁或更大、70岁或更大、75岁或更大、80岁或更大、85岁或更大、90岁或更大或2、3、4、5、6、7、8、9、10、12、13、14、15、16、18、20、21、24、25、27、28、30、33、35、37、39、40、42、44、45、48、50、52、55、60、65、70、75、80、85、90、95、96、97、98、99、100、101、102、103、104或更多岁)。
如本文中使用，术语“哺乳动物”是指任何分类为哺乳动物的动物，包括但不限于：人类、啮齿动物、运动动物、动物园动物、宠物动物和家畜或农场动物例如犬、猫、牛、绵羊、猪、马、和非人灵长类，例如猴类。优选地，啮齿动物为小鼠或大鼠。优选地，哺乳动物是人，本文也称之为患者或受试者，在本发明的具体实施方案中，所述哺乳动物优选为人。
关于本发明提供的功能性胰岛β细胞、药物组合物、生物制剂的施用方式，在一些实施方案中，可期望在需要治疗的区域局部施用本发明提供的功能性胰岛β细胞、药物组合物、生物制剂，这可以通过例如但不限于局部输注、通过注射或借助于植入物来实现，所述植入物是多孔的、无孔的或凝胶状的材料，包括膜，例如弹性膜(sialastic membrane)；在一些实施方案中，本发明提供的功能性胰岛β细胞、药物组合物、生物制剂可以在囊泡(特别是脂质体)中递送(参见Langer,Science 249:1527-1533(1990)；Treat et al.,Liposomes in the Therapy ofInfectious Disease and Cancer,Lopez-Berestein and Fidler(eds.),Liss,New York,pp.353-365(1989)；在一些实施方案中，本发明提供的功能性胰岛β细胞、药物组合物、生物制剂可以以受控释放或持续释放系统递送。
在一个实施方案中，可以使用泵来实现受控或持续释放(参见Langer,同上；Sefton,1987,CRCCrit.Ref.Biomed.Eng.14:20；Buchwald et al.,1980,Surgery 88:507；Saudek et al.,1989,N.Engl.J.Med.321:574)。
在另一个实施方案中，聚合物材料可用于实现本发明所述的功能性胰岛β细胞受控或持续释放(参见例如，Medical Applications of ControlledRelease,Langer and Wise(eds.),CRC Pres.,Boca Raton,Fla.(1974)；Controlled DrugBioavailability,Drug Product Design and Performance,Smolen and Ball(eds.),Wiley,New York(1984)；Ranger and Peppas,1983,J.,Macromol.Sci.Rev.Macromol.Chem.23:61；还参见Levy et al.,1985,Science 228:190；During et al.,1989,Ann.Neurol.25:351；Howard et al.,1989,J.Neurosurg.71:105)；美国专利No.5,679,377；美国专利No.5,916,597；美国专利No.5,912,015；美国专利No.5,989,463；美国专利No.5,128,326；PCT公开No.WO 99/15154；以及PCT公开No.WO 99/20253。
用于持续释放制剂的聚合物的实例包括但不限于：聚(甲基丙烯酸2-羟基乙酯)、聚(甲基丙烯酸甲酯)、聚(丙烯酸)、聚(乙烯-共-乙酸乙烯酯)、聚(甲基丙烯酸)、聚乙交酯(PLG)、聚酸酐、聚(N-乙烯基吡咯烷酮)、 聚(乙烯醇)、聚丙烯酰胺、聚(乙二醇)、聚丙交酯(PLA)、聚(丙交酯-共-乙交酯)(PLGA)和聚原酸酯。
在一个优选实施方案中，用于持续释放制剂的聚合物是惰性的，不含可浸出的杂质，在储存时稳定，无菌且可生物降解的。在又一个实施方案中，可将受控或持续释放系统放置在治疗靶标(即肺)附近，因此仅需要全身剂量的一部分(参见例如，Goodson,Medical Applications of Controlled Release,同上,vol.2,pp.115-138(1984))。
受控释放系统在Langer的综述(1990,Science 249:1527-1533)中有详细的介绍。可以使用本领域技术人员已知的任何技术来产生包含一种或更多种本发明所述的功能性胰岛β细胞的持续释放制剂。参见例如，美国专利No.4,526,938；PCT公开WO 91/05548；PCT公开WO 96/20698；Ning et al.,1996,Radiotherapy&Oncology 39:179-189；Song et al.,1995,PDAJournal of Pharmaceutical Science&Technology 50:372-397；Cleek et al.,1997,Pro.Int'l.Symp.Control.Rel.Bioact.Mater.24:853-854；以及Lam et al.,1997,Proc.Int'l.Symp.Control Rel.Bioact.Mater.24:759-760，其每一个通过引用整体并入本文。
可以将本发明所述的功能性胰岛β细胞、药物组合物、生物制剂配制成与其预期的施用途径相容。施用途径的实例包括但不限于肠胃外，例如皮下、静脉内(如肝门静脉)、皮内、经口、鼻内(例如吸入)、透皮(表面)、透粘膜和直肠施用。在一个具体实施方案中，根据常规程序将所述药物组合物配制为适于向人皮下、静脉内(如肝门静脉)、肌内、经口施用的药物组合物。
在一个优选实施方案中，根据常规程序将药物组合物配制用于向人皮下施用。通常，用于静脉内施用的组合物是在无菌等张水性缓冲剂中的溶液。必要时，药物组合物中还可包含增溶剂和局部麻醉剂(例如利多卡因(lignocamne))以减轻注射部位的疼痛。
在另一优选实施方案中，可以将药物组合物配制成用于通过注射(例如通过推注或连续输注)肠胃外施用。注射用制剂可以以单位剂型存在，例如在具有添加的防腐剂的安瓿或多剂量容器中。药物组合物可以采取诸如在油性或水性载剂中的混悬剂、溶液剂或乳剂的形式，并且可以包含配制剂，例如助悬剂、稳定剂和/或分散剂。
在一些实施方案中，可通过本文中所述的方法或通过本领域普通技术人员已知的任何方法来评估治疗之前、治疗期间和治疗之后的β细胞功能。例如，糖尿病控制和并发症试验(DiabetesControl and Complications Trial，DCCT)研究小组已建立糖基化血红蛋白(HA1和HA1c)百分比监测作为用于评价血糖控制的标准(DCCT,1993,N.Engl.J.Med.329:977-986)。或者，可将对每日胰岛素需求、C-肽水平/响应、低血糖发作和/或FPIR的表征用作β细胞功能的标志物或用于建立治疗指标(分别参见Keymeulen et al.,2005,N.Engl.J.Med.352:2598-2608；Herold et al.,2005,Diabetes 54:1763-1769；美国专利申请公开No.2004/0038867A1；和Greenbaum et al.,2001,Diabetes 50:470-476)。例如，FPIR作为IGTT之后1分钟和3分钟时胰岛素值的总和计算，其根据胰岛细胞抗体注册用户研究方案(IsletCell Antibody Register User's Study protocol)进行(参见，例如Bingley et al.,1996,Diabetes 45:1720-1728和McCulloch et al.,1993,Diabetes Care 16:911-915)。
当在本文中给出范围时，本发明包括了其中包括所述终点的实施方式，其中排除两个终点的实施方式，以及其中包括一个终点并排除另一个终点的实施方式。除非另有指明，否则应该假定两个终点都被包含。此外，应该理解，除非另有指明或从上下文和本领域技术人员的理解明显看出不是如此，否则表述为范围的值在本发明的不同实施方式中可以采用所陈述的范围内的任何特定的值或子范围，直至所述范围的下限的单位的十分之一，除非上下文明确叙述不是如此。还应该理解，当在本文中陈述一系列数值时，本发明包括了类似地涉及任何中间的值或由所述系列中的任两个值定义的范围的实施方式，以及最低值可以被取为最小值并且最高值可以被取为最大值的实施方式。
出于方便的目的，某些权利要求以从属形式提出，但是任何从属性权利要求可以以独立格式重新书写，以包括所述独立权利要求和这个权利要求所从属的任何其他权利要求的限制，并且这种重新书写的权利要求应该被认为在所有方面等同于以独立格式重新书写之前的所述从属权利要求(修改或未修改的)。还应该理解，除非有明确相反的指示，否则在本文要求保护的包括超过一项行动的任何方法中，所述方法的行动的顺序不必定限于所述方法的行动被叙述的顺序，但本发明包括其中顺序受此限制的实施方式。设想了上面描述的所有方面都适用于本发明的所有不同实施方式。还设想了任何上述实施方式可以在适合时与一个或多个其他这种实施方式自由组合。
实施例1 iPSCs诱导分化为胰岛的方法
1、实验材料
本发明实施例中涉及到的实验材料见表1。
表1实验材料
Figure PCTCN2022131004-appb-000001
Figure PCTCN2022131004-appb-000002
2、iPSCs诱导分化为胰岛β细胞的方法
来源于人类的iPSCs(来源于北京呈诺医学科技有限公司，根据本公司在先申请的专利(201910110768.7)中所述的方法制备)聚合度达到70％-80％后用DPBS磷酸盐缓冲液清洗2遍，随后在37℃的条件下，用TrypLE Express消化酶消化3-5min。用DMEM/F12培养基按5:1的比例中和后离心，离心条件为200g，5min。用E8完全培养基+10μM Y-27632(ROCK抑制剂)重悬铺种，密度约为1.8-2.2×10 5cells/cm 2，12孔细胞板提前24-48小时用0.13-0.2mg/mL Matrigel包被。
(1)第一阶段：诱导iPSCs向定型内胚层细胞分化
所述第一阶段的诱导分化培养基由基础培养基MCDB131、碳酸氢钠、谷氨酰胺(GlutaMax)、葡萄糖、脱脂BSA(胎牛血清白蛋白)(BSA)、GDF8和CHIR-99021组成。
在该培养基中，GDF8的浓度为100ng/mL，CHIR-99021的浓度为3μM，胎牛血清白蛋白(BSA)的浓度为0.5％，葡萄糖的浓度为10mM，谷氨酰胺的浓度为2mM，碳酸氢钠的浓度为1.5g/L。
所述第一阶段诱导的时间为3天。其中，在诱导第1天(D1)，培养基中GDF8的浓度为100ng/mL；CHIR-99021的浓度为3μmol/L。在诱导第2-3天(D2-D3)，培养基中GDF8的浓度为100ng/mL。即：诱导第1天使用第一阶段培养基A，所述第一阶段培养基A由基础培养基MCDB131、碳酸氢钠、谷氨酰胺(GlutaMax)、葡萄糖、脱脂BSA(胎牛血清白蛋白)(BSA)、GDF8和CHIR-99021组成；诱导第2-3天，使用第一阶段培养基B，所述第一阶段培养基B由基础培养基MCDB131、碳酸氢钠、谷氨酰胺(GlutaMax)、葡萄糖、脱脂BSA(胎牛血清白蛋白)(BSA)和GDF8组成。每天更换新鲜培养基。
(2)第二阶段：诱导定型内胚层细胞向原始肠管细胞分化
所述第二阶段的诱导分化培养基由基础培养基MCDB131、碳酸氢钠、谷氨酰胺(GlutaMax)、葡萄糖、脱脂BSA(胎牛血清白蛋白)(BSA)、抗坏血酸和成纤维细胞生长因子7(FGF-7)组成。
在该培养基中，抗坏血酸的浓度为0.25mM，成纤维细胞生长因子7(FGF-7)的浓度为50ng/mL，胎牛血清白蛋白(BSA)的浓度为0.5％，葡萄糖的浓度为10mM，谷氨酰胺的浓度为2mM，碳酸氢钠的浓 度为1.5g/L。
所述第二阶段诱导的时间为2天。其中，在诱导第4-5天(D4-D5)，培养基中抗坏血酸的浓度为0.25mM；成纤维细胞生长因子7(FGF-7)的浓度为50ng/mL；即：诱导第4-5天使用所述第二阶段的诱导分化培养基。每天更换新鲜培养基。
(3)第三阶段：诱导原始肠管细胞向后前肠细胞分化
所述第三阶段的诱导分化培养基由基础培养基MCDB131、碳酸氢钠、谷氨酰胺(GlutaMax)、葡萄糖、脱脂BSA(胎牛血清白蛋白)(BSA)、抗坏血酸、成纤维细胞生长因子7(FGF-7)、SANT-1、视黄酸、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂和蛋白激酶C活化物TPPB组成。
在该培养基中，抗坏血酸的浓度为0.25mM，成纤维细胞生长因子7(FGF-7)的浓度为50ng/mL，SANT-1的浓度为0.25μM，视黄酸的浓度为1μM，LDN193189的浓度为100nM，胰岛素-转铁蛋白-硒-乙醇胺添加剂的浓度为0.5％，蛋白激酶C活化物TPPB的浓度为200nM，胎牛血清白蛋白的浓度为2％，葡萄糖的浓度为10mM，谷氨酰胺的浓度为2mM，碳酸氢钠的浓度为2.5g/L。
所述第三阶段诱导的时间为2天，在诱导第6-7天(D6-D7)，培养基中抗坏血酸的浓度为0.25mM；成纤维细胞生长因子7(FGF-7)的浓度为50ng/mL；SANT-1的浓度为0.25μM；视黄酸的浓度为1μM；LDN193189的浓度为100nM；胰岛素-转铁蛋白-硒-乙醇胺添加剂的浓度为0.5％，蛋白激酶C活化物TPPB的浓度为200nM，即：诱导第6-7天使用第三阶段的诱导分化培养基，每天更换新鲜培养基。
(4)第四阶段：诱导后前肠细胞向胰腺祖细胞分化
所述第四阶段的诱导分化培养基由基础培养基MCDB131、碳酸氢钠、谷氨酰胺(GlutaMax)、葡萄糖、脱脂BSA(胎牛血清白蛋白)(BSA)、抗坏血酸、成纤维细胞生长因子7(FGF-7)、SANT-1、视黄酸、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂和蛋白激酶C活化物TPPB组成。
在该培养基中，抗坏血酸的浓度为0.25mM，成纤维细胞生长因子7(FGF-7)的浓度为2ng/mL，SANT-1的浓度为0.25μM，视黄酸的浓度为1μM，LDN193189的浓度为100nM，胰岛素-转铁蛋白-硒-乙醇胺添加剂的浓度为0.5％，蛋白激酶C活化物TPPB的浓度为200nM，胎牛血清白蛋白的浓度为2％，葡萄糖的浓度为10mM，谷氨酰胺的浓度为2mM，碳酸氢钠的浓度为2.5g/L。
所述第四阶段诱导的时间为3天，在诱导第8-10天(D8-D10)，培养基中抗坏血酸的浓度为0.25mM；成纤维细胞生长因子7(FGF-7)的浓度为2ng/mL；SANT-1的浓度为0.25μM；视黄酸的浓度为1μM；LDN193189的浓度为100nM；胰岛素-转铁蛋白-硒-乙醇胺添加剂的浓度为0.5％；蛋白激酶C活化物TPPB的浓度为200nM，即：诱导第8-10天使用所述第四阶段的诱导分化培养基，每天更换新鲜培养基。
(5)第五阶段：诱导胰腺祖细胞向内分泌祖细胞分化
所述第五阶段的诱导分化培养基由基础培养基MCDB131、碳酸氢钠、谷氨酰胺(GlutaMax)、葡萄糖、脱脂BSA(胎牛血清白蛋白)(BSA)、SANT-1、视黄酸、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、三碘甲状腺原氨酸、ALK5抑制剂ALK5i II、硫酸锌和肝素组成。
在该培养基中，SANT-1的浓度为0.25μM，视黄酸的浓度为0.1μM，LDN193189的浓度为100nM，胰岛素-转铁蛋白-硒-乙醇胺添加剂的浓度为0.5％，三碘甲状腺原氨酸的浓度为1μM，ALK5抑制剂ALK5i II的浓度为10μM，硫酸锌的浓度为10μM，肝素的浓度为10μg/mL，碳酸氢钠的浓度为1.5g/L，谷氨酰胺的浓度为2mM，葡萄糖的浓度为20mM，胎牛血清白蛋白的浓度为2％。
所述第五阶段诱导的时间为3天，在诱导第11天(D11)，采用细胞刮将细胞铺种到低亲附性培养板中，在诱导第11-13天(D11-D13)，培养基中SANT-1的浓度为0.25μM；视黄酸的浓度为0.1μM；LDN193189的浓度为100nM；胰岛素-转铁蛋白-硒-乙醇胺添加剂的浓度为0.5％；三碘甲状腺原氨酸的浓度为1μM； ALK5抑制剂ALK5i II的浓度为10μM；硫酸锌的浓度为10μM；肝素的浓度为10μg/mL，即：诱导第11-13天使用所述第五阶段的诱导分化培养基，每天更换新鲜培养基。
(6)第六阶段：诱导内分泌祖细胞向未成熟胰岛β细胞分化
所述第六阶段的诱导分化培养基由基础培养基MCDB131、碳酸氢钠、谷氨酰胺(GlutaMax)、葡萄糖、脱脂BSA(胎牛血清白蛋白)(BSA)、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、三碘甲状腺原氨酸、ALK5抑制剂ALK5i II、硫酸锌、γ-分泌酶抑制剂GSi XX和肝素组成。
在该培养基中，LDN193189的浓度为100nM，胰岛素-转铁蛋白-硒-乙醇胺添加剂的浓度为0.5％，三碘甲状腺原氨酸的浓度为1μM，ALK5抑制剂ALK5i II的浓度为10μM，硫酸锌的浓度为10μM，γ-分泌酶抑制剂GSi XX的浓度为100nM，肝素的浓度为10μg/mL，碳酸氢钠的浓度为1.5g/L，谷氨酰胺的浓度为2mM，葡萄糖的浓度为20mM，胎牛血清白蛋白的浓度为2％。
所述第六阶段诱导的时间为14天，在诱导第14-20天(D14-D20)，培养基中LDN193189的浓度为100nM；胰岛素-转铁蛋白-硒-乙醇胺添加剂的浓度为0.5％；三碘甲状腺原氨酸的浓度为1μM；ALK5抑制剂ALK5i II的浓度为10μM；硫酸锌的浓度为10μM；γ-分泌酶抑制剂GSi XX为100nM，肝素的浓度为10μg/mL。在诱导第21-27(D21-D27)天，培养基中LDN193189的浓度为100nM；胰岛素-转铁蛋白-硒-乙醇胺添加剂的浓度为0.5％；三碘甲状腺原氨酸的浓度为1μM；ALK5抑制剂ALK5i II的浓度为10μM；硫酸锌的浓度为10μM；肝素的浓度为10μg/mL，即：诱导第14-20天，使用第六阶段培养基A，所述第六阶段培养基A由基础培养基MCDB131、碳酸氢钠、谷氨酰胺(GlutaMax)、葡萄糖、脱脂BSA(胎牛血清白蛋白)(BSA)、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、三碘甲状腺原氨酸、ALK5抑制剂ALK5i II、硫酸锌、γ-分泌酶抑制剂GSi XX和肝素组成；诱导第21-27天使用第六阶段培养基B，所述第六阶段培养基B由基础培养基MCDB131、碳酸氢钠、谷氨酰胺(GlutaMax)、葡萄糖、脱脂BSA(胎牛血清白蛋白)(BSA)、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、三碘甲状腺原氨酸、ALK5抑制剂ALK5i II、硫酸锌和肝素组成。每天更换新鲜培养基。
(7)第七阶段：诱导未成熟胰岛β细胞向成熟胰岛β细胞分化
所述第七阶段的诱导分化培养基由基础培养基MCDB131、碳酸氢钠、谷氨酰胺(GlutaMax)、葡萄糖、脱脂BSA(胎牛血清白蛋白)(BSA)、胰岛素-转铁蛋白-硒-乙醇胺添加剂、三碘甲状腺原氨酸、ALK5抑制剂ALK5i II、硫酸锌、肝素、乙酰-l-半胱氨酸(N-Cys)、水溶性维生素E和Axl抑制剂R428组成。
在该培养基中，胰岛素-转铁蛋白-硒-乙醇胺添加剂的浓度为0.5％，三碘甲状腺原氨酸的浓度为1μM，ALK5抑制剂ALK5i II的浓度为10μM，硫酸锌的浓度为10μM，肝素的浓度为10μg/mL，乙酰-l-半胱氨酸(N-Cys)的浓度为1mM，水溶性维生素E的浓度为10μM，Axl抑制剂R428的浓度为2μM，碳酸氢钠的浓度为1.5g/L，谷氨酰胺的浓度为2mM，葡萄糖的浓度为20mM，胎牛血清白蛋白的浓度为2％。
所述第七阶段诱导的时间为13天，在诱导第28-34天(D28-D34)，培养基中胰岛素-转铁蛋白-硒-乙醇胺添加剂的浓度为0.5％；三碘甲状腺原氨酸的浓度为1μM；ALK5抑制剂ALK5i II的浓度为10μM；硫酸锌的浓度为10μM；肝素的浓度为10μg/mL；乙酰-l-半胱氨酸(N-Cys)的浓度为1mM；水溶性维生素E的浓度为10μM；Axl抑制剂R428的浓度为2μM，即：诱导第28-34天使用第七阶段的诱导分化培养基。每天更换新鲜培养基。
(8)第八阶段：进一步诱导胰岛β细胞的成熟
所述第八阶段的诱导分化培养基使用了胰岛a细胞转β细胞培养基，该培养基中包括50％Ham’s F-12medium、50％medium 199、谷氨酰胺、二水氯化钙、N-2羟乙基哌嗪-N-2-乙烷磺酸、脱脂BSA(胎牛血清白蛋白)(BSA)、胰岛素-转铁蛋白-硒-乙醇胺添加剂、水溶性维生素E、烟酰胺、肝素、脱氧核糖核酸酶Ⅰ、 坏死性凋亡抑制剂Necrostatin-1、丝氨酸蛋白酶抑制剂Pefabloc组成，蒿甲醚、GABAA激活剂NS11394为额外添加的小分子。
在该培养基中，胰岛素-转铁蛋白-硒-乙醇胺添加剂的浓度为0.5％，水溶性维生素E的浓度为10μM，烟酰胺的浓度为10mM，肝素的浓度为10μg/mL，脱氧核糖核酸酶Ⅰ的浓度为1U/mL，坏死性凋亡抑制剂Necrostatin-1的浓度为100μM，丝氨酸蛋白酶抑制剂Pefabloc的浓度为0.1μM，谷氨酰胺的浓度为2mM，二水氯化钙为2.5mM，N-2羟乙基哌嗪-N-2-乙烷磺酸的浓度为10mM，胎牛血清白蛋白的浓度为2％，蒿甲醚的浓度为10μM，GABAA激活剂NS11394的浓度为10μM。
所述第八阶段诱导的时间6天，在诱导第35-40天(D35-D40)，培养基中胰岛素-转铁蛋白-硒-乙醇胺添加剂的浓度为0.5％；水溶性维生素E的浓度为10μM；烟酰胺的浓度为10mM；肝素的浓度为10μg/mL；脱氧核糖核酸酶Ⅰ的浓度为1U/mL；坏死性凋亡抑制剂Necrostatin-1的浓度为100μM；丝氨酸蛋白酶抑制剂Pefabloc的浓度为0.1μM，额外添加的小分子蒿甲醚的浓度为10μM；GABAA激活剂NS11394的浓度为10μM；即：诱导第35-40天使用第八阶段的诱导分化培养基。每天更换新鲜培养基。
实施例2qPCR检测胰岛成熟标志基因mRNA的表达情况
1、实验方法
iPSCs经诱导后，用Insulin(INS)、Glucagon(GCG)、MAFA作为胰岛标记物，检测经iPSCs诱导分化得到的胰岛细胞中胰岛标记物mRNA的表达情况。
其中，Insulin(INS)是指胰岛素，由胰岛β细胞分泌的一种α和β双链构成的多肽激素，由51个氨基酸组成，分子量约5800Da。胰岛素是机体内唯一降低血糖的激素。为胰岛β细胞的标志基因；
Glucagon(GCG)是指胰高血糖素，由胰岛α细胞分泌的一种由29个氨基酸组成的直链多肽激素，分子量为3485Da。在体内具有升高血糖的作用；
MAFA是指肌腱膜纤维肉瘤癌基因同源物A基因(v-maf musculoaponeurotic fibrosarcoma oncogene homologue A)，是一种具有亮氨酸拉链结构的转录因子。MAFA是目前发现的唯一一种胰岛β细胞胰岛素基因活化转录因子。胰腺β细胞的成熟和功能的维持都有赖于MAFA蛋白的正常表达。因此，MAFA为胰岛β细胞的成熟标志基因。
具体实验方法如下：
1.1总RNA提取及反转
(1)细胞培养皿中细胞样品用PBS洗两次后，用1mL枪将PBS吸干净，加入1mL Trizol(Invitrogen)溶液，吹打混匀，并吸至1.5mL RNase free EP管中使细胞充分裂解，室温静置5分钟；
(2)每1mL Trizol加0.2mL氯仿。盖紧样品管盖，剧烈振荡15秒并将其在室温下孵育3分钟；
(3)于4℃12,000rpm离心10分钟，样品会分成三层：下层有机相，中间层和上层无色的水相，RNA存在于水相中。水相层的容量大约为所加RL体积的60％，把水相转移到新管中，进行下一步操作；
(4)加入1倍体积70％乙醇(请先检查是否已加入无水乙醇！)，颠倒混匀(此时可能会出现沉淀)。得到的溶液和可能沉淀一起转入吸附柱RA中10,000rpm离心45秒，弃掉废液，将吸附柱重新套回收集管；
(5)加500μL去蛋白液RE，12,000rpm离心45秒，弃掉废液；
(6)加入700μL漂洗液RW(请先检查是否已加入无水乙醇！)，12,000rpm离心60秒，弃掉废液；
(7)加入500μL漂洗液RW，12,000rpm离心60秒，弃掉废液；
(8)将吸附柱RA放回空收集管中，12,000rpm离心2分钟，尽量除去漂洗液，以免漂洗液中残留乙醇 抑制下游反应；
(9)取出吸附柱RA，放入一个RNase free离心管中，根据预期RNA产量在吸附膜的中间部位加50-80μL RNase free water，事先在65-70℃水浴中加热效果更好；
(10)室温放置2分钟，12,000rpm离心1分钟。如果需要较多RNA，可将得到的溶液重新加入离心吸附柱中，离心1分钟，或者另外再加30μL RNase free water，离心1分钟，合并两次洗脱液；
(11)洗脱体积越大，洗脱效率越高，如果需要RNA浓度较高，可以适当减少洗脱体积，但是最小体积最好不少于30μL，体积过小降低RNA洗脱效率，减少RNA产量。
1.2总RNA纯度和完整性检测
(1)纯度检测：取1μL RNA样品，在核酸蛋白检测仪上测定OD值，OD260/OD280的比值为2，说明制备的RNA较纯，无蛋白质污染；
(2)总RNA完整性检测：取RNA样品6μL，1％琼脂糖凝胶电泳150V×10min，用凝胶成像系统观察并拍照，总RNA的5s rRNA，18s rRNA和28s rRNA条带，三条条带完整的话即可证明总RNA抽提比较完整。
1.3RNA反转
(1)使用分光光度计测量RNA浓度并进行计算，按500ng浓度进行反转；
(2)按照如下体系将试剂与总RNA进行预混：Total RNA 500ng，Oligo dT 1μL，2XTS Reaction Mix 10μL，RI/RT Enzyme MiX 1μL，gDNA Remover 1μL，RNase-free Water调整Total volume为20μL；
(3)将预混液转移至PCR仪中，使用cDNA模板进行反应(42℃孵育30min，85℃孵育5s)；
(4)迅速将反转完成的cDNA转移置冰上1min进行降温；
(5)-20℃保存，使用前可按所需浓度进行稀释。
1.4荧光定量PCR(qPCR)实验方法
qPCR检测基因INS、GCG、MAFA的引物序列信息见下表2；
表2 qPCR检测基因INS、GCG、MAFA的引物序列信息
Figure PCTCN2022131004-appb-000003
模板如下：Forward Primer(10μM)0.4μL，Reverse Primer(10μM)0.4μL，2xTransStar Top/Tip Green qPCR SuperMix 10μL，Nuclease-free Water 8.2μL，cDNA 1μL，Total volume 20μL。
(1)首先将试剂与引物按如上模板进行预混，加入八连管中，每孔18μL；
(2)迅速在八连管中加入cDNA 2μL，盖上盖子进行瞬离；
(3)放入Light cycler仪器中按照3步法进行反应，循环数为40；
(4)模板如下Temp Time 94℃30s 94℃5s 45cycles 55℃15s 72℃10s。
1.5具体分组方式如下：
A组(对照组)：在诱导第35-40天使用第七阶段的诱导分化培养基；
B组：在在诱导第35-40天使用a转β培养基；
C组：在诱导第35-40天使用a转β培养基，同时添加蒿甲醚和GABAA激活剂NS11394，其中，蒿甲醚的浓度为10μM，GABAA激活剂NS11394的浓度为10μM。
2、实验结果
qPCR检测结果见图1，结果显示，C组INS、GCG、MAFA基因的mRNA表达水平显著高于其它组(p<0.05)。C组经诱导后获得的胰岛细胞INS基因的mRNA表达水平较A组增加27.76倍，GCG基因的mRNA表达水平较A组增加14.82倍，MAFA基因的mRNA表达水平较A组增加4.34倍，表明了在胰岛成熟阶段，采用α细胞转β细胞培养基联合蒿甲醚和GABAA激活剂NS11394对iPSCs进行诱导，可促进β细胞成熟，获得更多数量的胰岛β细胞，即诱导效率更高。
胰岛β细胞的分化路径为iPSC—胰腺祖细胞—幼稚型α细胞—β细胞，向胰岛β细胞的分化过程是整体进行的，在成熟β细胞生成的同时也会有成熟α细胞的生成，且成熟的α细胞是不能向β细胞转化的，图1中间图中采用C组的培养基+蒿甲醚+GABAA激活剂后，在促进幼稚型α细胞向β细胞转化的同时也会促进幼稚型α细胞转化为成熟型α细胞，因此，图1中间图的结果(C组GCG mRNA的表达水平显著高于A组和B组)表明了C组所述的培养基+蒿甲醚+GABAA激活剂在显著促进胰岛α细胞转化为胰岛β细胞的同时也能促进幼稚型α细胞转化为成熟型α细胞；图1左图的结果(C组INS mRNA的表达水平显著高于A组和B组)表明了C组所述的培养基+蒿甲醚+GABAA激活剂能够显著促进胰岛α细胞转化为胰岛β细胞，即采用α细胞转β细胞培养基联合蒿甲醚和GABAA激活剂NS11394对iPSCs进行诱导，可显著促进β细胞成熟，获得更多数量的功能性胰岛β细胞，即诱导效率显著更高。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。

Claims (67)

  1. 一种诱导多能干细胞分化为功能性胰岛β细胞的诱导分化剂，其特征在于，所述诱导分化剂包括第一阶段诱导分化剂、第二阶段诱导分化剂、第三阶段诱导分化剂、第四阶段诱导分化剂、第五阶段诱导分化剂、第六阶段诱导分化剂、第七阶段诱导分化剂、第八阶段诱导分化剂；
    所述第一阶段诱导分化剂包括第一阶段诱导分化剂A、第一阶段诱导分化剂B；
    所述第一阶段诱导分化剂A包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、GDF8、CHIR-99021；
    所述第一阶段诱导分化剂B包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、GDF8；
    所述第二阶段诱导分化剂包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、抗坏血酸、FGF-7；
    所述第三阶段诱导分化剂包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、抗坏血酸、FGF-7、SANT-1、视黄酸、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、TPPB；
    所述第四阶段诱导分化剂包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、抗坏血酸、FGF-7、SANT-1、视黄酸、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、TPPB；
    所述第五阶段诱导分化剂包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、SANT-1、视黄酸、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、三碘甲状腺原氨酸、ALK5i II、硫酸锌、肝素；
    所述第六阶段诱导分化剂包括第六阶段诱导分化剂A、第六阶段诱导分化剂B；
    所述第六阶段诱导分化剂A包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、三碘甲状腺原氨酸、ALK5i II、硫酸锌、GSi XX、肝素；
    所述第六阶段诱导分化剂B包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、LDN193189、胰岛素-转铁蛋白-硒-乙醇胺添加剂、三碘甲状腺原氨酸、ALK5i II、硫酸锌、肝素；
    所述第七阶段诱导分化剂包含碳酸氢钠、谷氨酰胺、葡萄糖、胎牛血清白蛋白、胰岛素-转铁蛋白-硒-乙醇胺添加剂、三碘甲状腺原氨酸、ALK5i II、硫酸锌、肝素、乙酰-l-半胱氨酸、水溶性维生素E、R428；
    所述第八阶段诱导分化剂包含蒿甲醚和NS11394；
    所述第八阶段诱导分化剂还包含谷氨酰胺、二水氯化钙、N-2羟乙基哌嗪-N-2-乙烷磺酸、胎牛血清白蛋白、胰岛素-转铁蛋白-硒-乙醇胺添加剂、水溶性维生素E、烟酰胺、肝素、脱氧核糖核酸酶Ⅰ、Necrostatin-1、Pefabloc。
  2. 根据权利要求1所述的诱导分化剂，其特征在于，所述第一阶段诱导分化剂中各成分的浓度分别为：(10-500)ng/mL GDF8、(0.5-20)μM CHIR-99021、(0.01-5)％胎牛血清白蛋白、(1-50)mM葡萄糖、(0.05-10)mM谷氨酰胺、(0.05-10)g/L碳酸氢钠。
  3. 根据权利要求2所述的诱导分化剂，其特征在于，所述第一阶段诱导分化剂中各成分的浓度分别为：100ng/mL GDF8、3μM CHIR-99021、0.5％胎牛血清白蛋白、10mM葡萄糖、2mM谷氨酰胺、1.5g/L碳酸氢钠。
  4. 根据权利要求3所述的诱导分化剂，其特征在于，所述第二阶段诱导分化剂中各成分的浓度分别为：(0.01-5)mM抗坏血酸、(10-100)ng/mL FGF-7、(0.01-5)％胎牛血清白蛋白、(1-50)mM葡萄糖、(0.05-10)mM谷氨酰胺、(0.05-10)g/L碳酸氢钠。
  5. 根据权利要求4所述的诱导分化剂，其特征在于，所述第二阶段诱导分化剂中各成分的浓度分别为：0.25mM抗坏血酸、50ng/mL FGF-7、0.5％胎牛血清白蛋白、10mM葡萄糖、2mM谷氨酰胺、1.5g/L碳酸氢钠。
  6. 根据权利要求5所述的诱导分化剂，其特征在于，所述第三阶段诱导分化剂中各成分的浓度分别为： (0.01-5)mM抗坏血酸、(10-100)ng/mL FGF-7、(0.01-5)μM SANT-1、(0.01-10)μM视黄酸、(10-500)nM LDN193189、(0.01-5)％胰岛素-转铁蛋白-硒-乙醇胺添加剂、(50-500)nM TPPB、(0.05-10)％胎牛血清白蛋白、(1-50)mM葡萄糖、(0.05-10)mM谷氨酰胺、(0.05-10)g/L碳酸氢钠。
  7. 根据权利要求6所述的诱导分化剂，其特征在于，所述第三阶段诱导分化剂中各成分的浓度分别为：0.25mM抗坏血酸、50ng/mL FGF-7、0.25μM SANT-1、1μM视黄酸、100nM LDN193189、0.5％胰岛素-转铁蛋白-硒-乙醇胺添加剂、200nM TPPB、2％胎牛血清白蛋白、10mM葡萄糖、2mM谷氨酰胺、2.5g/L碳酸氢钠。
  8. 根据权利要求7所述的诱导分化剂，其特征在于，所述第四阶段诱导分化剂中各成分的浓度分别为：(0.01-5)mM抗坏血酸、(0.01-10)ng/mL FGF-7、(0.01-5)μM SANT-1、(0.01-10)μM视黄酸、(10-500)nM LDN193189、(0.01-5)％胰岛素-转铁蛋白-硒-乙醇胺添加剂、(50-500)nM TPPB、(0.05-10)％胎牛血清白蛋白、(1-50)mM葡萄糖、(0.05-10)mM谷氨酰胺、(0.05-10)g/L碳酸氢钠。
  9. 根据权利要求8所述的诱导分化剂，其特征在于，所述第四阶段诱导分化剂中各成分的浓度分别为：0.25mM抗坏血酸、2ng/mL FGF-7、0.25μM SANT-1、1μM视黄酸、100nM LDN193189、0.5％胰岛素-转铁蛋白-硒-乙醇胺添加剂、200nM TPPB、2％胎牛血清白蛋白、10mM葡萄糖、2mM谷氨酰胺、2.5g/L碳酸氢钠。
  10. 根据权利要求9所述的诱导分化剂，其特征在于，所述第五阶段诱导分化剂中各成分的浓度分别为：(0.01-5)μM SANT-1、(0.01-10)μM视黄酸、(10-500)nM LDN193189、(0.01-5)％胰岛素-转铁蛋白-硒-乙醇胺添加剂、(0.01-10)μM三碘甲状腺原氨酸、(0.01-50)μM ALK5i II、(1-50)μM硫酸锌、(1-50)μg/mL肝素、(0.05-10)g/L碳酸氢钠、(0.05-10)mM谷氨酰胺、(1-50)mM葡萄糖、(0.05-10)％胎牛血清白蛋白。
  11. 根据权利要求10所述的诱导分化剂，其特征在于，所述第五阶段诱导分化剂中各成分的浓度分别为：0.25μM SANT-1、0.1μM视黄酸、100nM LDN193189、0.5％胰岛素-转铁蛋白-硒-乙醇胺添加剂、1μM三碘甲状腺原氨酸、10μM ALK5i II、10μM硫酸锌、10μg/mL肝素、1.5g/L碳酸氢钠、2mM谷氨酰胺、20mM葡萄糖、2％胎牛血清白蛋白。
  12. 根据权利要求11所述的诱导分化剂，其特征在于，所述第六阶段诱导分化剂中各成分的浓度分别为：(10-500)nM LDN193189、(0.01-5)％胰岛素-转铁蛋白-硒-乙醇胺添加剂、(0.01-10)μM三碘甲状腺原氨酸、(0.01-50)μM ALK5i II、(1-50)μM硫酸锌、(10-500)nM GSi XX、(1-50)μg/mL肝素、(0.05-10)g/L碳酸氢钠、(0.05-10)mM谷氨酰胺、(1-50)mM葡萄糖、(0.05-10)％胎牛血清白蛋白。
  13. 根据权利要求12所述的诱导分化剂，其特征在于，所述第六阶段诱导分化剂中各成分的浓度分别为：100nM LDN193189、0.5％胰岛素-转铁蛋白-硒-乙醇胺添加剂、1μM三碘甲状腺原氨酸、10μM ALK5i II、10μM硫酸锌、100nM GSi XX、10μg/mL肝素、1.5g/L碳酸氢钠、2mM谷氨酰胺、20mM葡萄糖、2％胎牛血清白蛋白。
  14. 根据权利要求13所述的诱导分化剂，其特征在于，所述第七阶段诱导分化剂中各成分的浓度分别为：(0.01-5)％胰岛素-转铁蛋白-硒-乙醇胺添加剂、(0.01-10)μM三碘甲状腺原氨酸、(0.01-50)μM ALK5i II、(1-50)μM硫酸锌、(1-50)μg/mL肝素、(0.01-10)mM乙酰-l-半胱氨酸、(1-50)μM水溶性维生素E、(1-10)μM R428、(0.05-10)g/L碳酸氢钠、(0.05-10)mM谷氨酰胺、(1-50)mM葡萄糖、(0.05-10)％胎牛血清白蛋白。
  15. 根据权利要求14所述的诱导分化剂，其特征在于，所述第七阶段诱导分化剂中各成分的浓度分别为：0.5％胰岛素-转铁蛋白-硒-乙醇胺添加剂、1μM三碘甲状腺原氨酸、10μM ALK5i II、10μM硫酸锌、10μg/mL肝素、1mM乙酰-l-半胱氨酸、10μM水溶性维生素E、2μM R428、1.5g/L碳酸氢钠、2mM谷氨酰胺、20mM葡萄糖、2％胎牛血清白蛋白。
  16. 根据权利要求15所述的诱导分化剂，其特征在于，所述第八阶段诱导分化剂中各成分的浓度分别 为：(0.01-5)％胰岛素-转铁蛋白-硒-乙醇胺添加剂、(1-50)μM水溶性维生素E、(1-50)mM烟酰胺、(1-50)μg/mL肝素、(0.01-10)U/mL脱氧核糖核酸酶Ⅰ、(10-500)μM Necrostatin-1、(0.01-5)μM Pefabloc、(0.01-10)mM谷氨酰胺、(0.01-10)mM二水氯化钙、(0.1-50)mM N-2羟乙基哌嗪-N-2-乙烷磺酸、(0.05-10)％胎牛血清白蛋白、(1-50)μM蒿甲醚、(1-50)μM NS11394。
  17. 根据权利要求16所述的诱导分化剂，其特征在于，所述第八阶段诱导分化培养剂中各成分的浓度分别为：0.5％胰岛素-转铁蛋白-硒-乙醇胺添加剂、10μM水溶性维生素E、10mM烟酰胺、10μg/mL肝素、1U/mL脱氧核糖核酸酶Ⅰ、100μM Necrostatin-1、0.1μM Pefabloc、2mM谷氨酰胺、2.5mM二水氯化钙、10mM N-2羟乙基哌嗪-N-2-乙烷磺酸、2％胎牛血清白蛋白、10μM蒿甲醚、10μM NS11394。
  18. 一种诱导多能干细胞分化为功能性胰岛β细胞的诱导分化培养基，其特征在于，所述诱导分化培养基包括第一阶段诱导分化培养基、第二阶段诱导分化培养基、第三阶段诱导分化培养基、第四阶段诱导分化培养基、第五阶段诱导分化培养基、第六阶段诱导分化培养基、第七阶段诱导分化培养基、第八阶段诱导分化培养基；
    所述第一阶段诱导分化培养基包括基础培养基MCDB131、权利要求1中所述的第一阶段诱导分化剂；
    所述第二阶段诱导分化培养基包括基础培养基MCDB131、权利要求1中所述的第二阶段诱导分化剂；
    所述第三阶段诱导分化培养基包括基础培养基MCDB131、权利要求1中所述的第三阶段诱导分化剂；
    所述第四阶段诱导分化培养基包括基础培养基MCDB131、权利要求1中所述的第四阶段诱导分化剂；
    所述第五阶段诱导分化培养基包括基础培养基MCDB131、权利要求1中所述的第五阶段诱导分化剂；
    所述第六阶段诱导分化培养基包括基础培养基MCDB131、权利要求1中所述的第六阶段诱导分化剂；
    所述第七阶段诱导分化培养基包括基础培养基MCDB131、权利要求1中所述的第七阶段诱导分化剂；
    所述第八阶段诱导分化培养基包括50％Ham’s F-12medium、50％medium 199、权利要求1中所述的第八阶段诱导分化剂。
  19. 一种诱导多能干细胞分化为功能性胰岛β细胞的诱导分化方法，其特征在于，所述方法包括如下步骤：
    (1)提供诱导多能干细胞并在完全培养基中进行培养；
    (2)第一阶段诱导分化，采用权利要求18中所述的第一阶段诱导分化培养基诱导步骤(1)得到的诱导多能干细胞向定型内胚层细胞分化；
    (3)第二阶段诱导分化，采用权利要求18中所述的第二阶段诱导分化培养基诱导定型内胚层细胞向原始肠管细胞分化；
    (4)第三阶段诱导分化，采用权利要求18中所述的第三阶段诱导分化培养基诱导原始肠管细胞向后前肠细胞分化；
    (5)第四阶段诱导分化，采用权利要求18中所述的第四阶段诱导分化培养基诱导后前肠细胞向胰腺祖细胞分化；
    (6)第五阶段诱导分化，采用权利要求18中所述的第五阶段诱导分化培养基诱导胰腺祖细胞向内分泌祖细胞分化；
    (7)第六阶段诱导分化，采用权利要求18中所述的第六阶段诱导分化培养基诱导内分泌祖细胞向未成熟胰岛β细胞分化；
    (8)第七阶段诱导分化，采用权利要求18中所述的第七阶段诱导分化培养基诱导未成熟胰岛β细胞向成熟胰岛β细胞分化；
    (9)第八阶段诱导分化，采用权利要求18中所述的第八阶段诱导分化培养基进一步诱导胰岛β细胞的成熟，得到功能性胰岛β细胞。
  20. 根据权利要求19所述的方法，其特征在于，步骤(1)中所述的完全培养基为E8完全培养基。
  21. 根据权利要求20所述的方法，其特征在于，所述E8完全培养基中含有ROCK抑制剂。
  22. 根据权利要求21所述的方法，其特征在于，所述ROCK抑制剂包括Y-27632、GSK429286A、RKI-1447、Y-33075dihydrochloride、Thiazovivin、K-115、SLx-2119、Chroman1、SAR407899和/或SR-3677。
  23. 根据权利要求22所述的方法，其特征在于，所述ROCK抑制剂为Y-27632。
  24. 根据权利要求23所述的方法，其特征在于，所述Y-27632的浓度为0.001-100μM。
  25. 根据权利要求24所述的方法，其特征在于，所述Y-27632的浓度为10μM。
  26. 根据权利要求25所述的方法，其特征在于，步骤(1)中所述的诱导多能干细胞的细胞密度为0.1-10.0×10 5cells/cm 2。
  27. 根据权利要求26所述的方法，其特征在于，步骤(1)中所述的诱导多能干细胞的细胞密度为1.8-2.2×10 5cells/cm 2。
  28. 根据权利要求27所述的方法，其特征在于，步骤(1)中所述的诱导多能干细胞来源于哺乳动物。
  29. 根据权利要求28所述的方法，其特征在于，步骤(1)中所述的诱导多能干细胞来源于人类、小鼠、大鼠、山羊、绵羊、猪、猫、兔、狗、狼、马、或牛。
  30. 根据权利要求29所述的方法，其特征在于，步骤(1)中所述的诱导多能干细胞来源于人类。
  31. 根据权利要求30所述的方法，其特征在于，所述第一阶段诱导分化的时间为3天。
  32. 根据权利要求31所述的方法，其特征在于，在诱导第1天，采用第一阶段诱导分化培养基A进行诱导分化。
  33. 根据权利要求32所述的方法，其特征在于，在诱导第2-3天，采用第一阶段诱导分化培养基B进行诱导分化。
  34. 根据权利要求33所述的方法，其特征在于，所述第二阶段诱导分化的时间为2天。
  35. 根据权利要求34所述的方法，其特征在于，在诱导第4-5天，采用所述第二阶段诱导分化培养基进行诱导分化。
  36. 根据权利要求35所述的方法，其特征在于，所述第三阶段诱导分化的时间为2天。
  37. 根据权利要求36所述的方法，其特征在于，在诱导第6-7天，采用所述第三阶段诱导分化培养基进行诱导分化。
  38. 根据权利要求37所述的方法，其特征在于，所述第四阶段诱导分化的时间为3天。
  39. 根据权利要求38所述的方法，其特征在于，在诱导第8-10天，采用所述第四阶段诱导分化培养基进行诱导分化。
  40. 根据权利要求39所述的方法，其特征在于，所述第五阶段诱导分化的时间为3天。
  41. 根据权利要求40所述的方法，其特征在于，在诱导第11天，将细胞铺种到低亲附性培养板中，在诱导第11-13天，采用所述第五阶段诱导分化培养基进行诱导分化。
  42. 根据权利要求41所述的方法，其特征在于，所述第六阶段诱导分化的时间为14天。
  43. 根据权利要求42所述的方法，其特征在于，在诱导第14-20天，采用第六阶段诱导分化培养基A进行诱导分化。
  44. 根据权利要求43所述的方法，其特征在于，在诱导第21-27天，采用第六阶段诱导分化培养基B进行诱导分化。
  45. 根据权利要求44所述的方法，其特征在于，所述第七阶段诱导分化的时间为13天。
  46. 根据权利要求45所述的方法，其特征在于，在诱导第28-34天，采用所述第七阶段诱导分化培养基进行诱导分化。
  47. 根据权利要求46所述的方法，其特征在于，所述第八阶段诱导分化的时间为6天。
  48. 根据权利要求47所述的方法，其特征在于，在诱导第35-40天，采用所述第八阶段诱导分化培养基进行诱导分化。
  49. 一种诱导多能干细胞来源的功能性胰岛β细胞或细胞群体，其特征在于，所述功能性胰岛β细胞或细胞群体为采用权利要求19-48中任一项所述的方法诱导分化得到的。
  50. 根据权利要求49所述的功能性胰岛β细胞或细胞群体，其特征在于，所述功能性胰岛β细胞或细胞群体为功能性的、稳定的胰岛β细胞或细胞群体。
  51. 一种用于治疗和/或预防糖尿病的药物组合物，其特征在于，所述药物组合物包含权利要求50所述的功能性胰岛β细胞或细胞群体。
  52. 根据权利要求51所述的药物组合物，其特征在于，所述药物组合物还包含药学上可接受的载体和/或辅料。
  53. 根据权利要求51所述的药物组合物，其特征在于，所述糖尿病包括1型糖尿病、2型糖尿病、妊娠糖尿病、单基因糖尿病。
  54. 根据权利要求53所述的药物组合物，其特征在于，所述糖尿病为1型糖尿病。
  55. 一种用于治疗和/或预防糖尿病的方法，其特征在于，所述方法包括给有需要的受试者施用有效量的权利要求49所述的细胞或细胞群体和/或权利要求51所述的药物组合物。
  56. 根据权利要求55所述的方法，其特征在于，所述糖尿病包括1型糖尿病、2型糖尿病、妊娠糖尿病、单基因糖尿病。
  57. 根据权利要求56所述的方法，其特征在于，所述糖尿病为1型糖尿病。
  58. 蒿甲醚和NS11394联合在促进胰岛α细胞转化为胰岛β细胞中的应用。
  59. 蒿甲醚和NS11394联合在诱导多能干细胞分化为功能性胰岛β细胞中的应用。
  60. 权利要求1-17中任一项所述的诱导分化剂在制备诱导多能干细胞分化为功能性胰岛β细胞的诱导分化培养基中的应用。
  61. 权利要求1-17中任一项所述的诱导分化剂在诱导多能干细胞分化为功能性胰岛β细胞中的应用。
  62. 权利要求18所述的诱导分化培养基在诱导多能干细胞分化为功能性胰岛β细胞中的应用。
  63. 权利要求49所述的细胞或细胞群体在制备治疗和/或预防糖尿病的药物中的应用。
  64. 权利要求49所述的细胞或细胞群体在治疗和/或预防糖尿病中的应用。
  65. 权利要求51所述的药物组合物在治疗和/或预防糖尿病中的应用。
  66. 根据权利要求63-65中任一项所述的应用，其特征在于，所述糖尿病包括1型糖尿病、2型糖尿病、妊娠糖尿病、单基因糖尿病。
  67. 根据权利要求63所述的应用，其特征在于，所述糖尿病为1型糖尿病。
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