CN113046299B - An additive for preparing pancreatic β cells by induced pluripotent stem cell directed differentiation - Google Patents
An additive for preparing pancreatic β cells by induced pluripotent stem cell directed differentiation Download PDFInfo
- Publication number
- CN113046299B CN113046299B CN202110297334.XA CN202110297334A CN113046299B CN 113046299 B CN113046299 B CN 113046299B CN 202110297334 A CN202110297334 A CN 202110297334A CN 113046299 B CN113046299 B CN 113046299B
- Authority
- CN
- China
- Prior art keywords
- cells
- inhibitor
- additive
- differentiation
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0676—Pancreatic cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/113—Acidic fibroblast growth factor (aFGF, FGF-1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The additive for inducing the directional differentiation of the pluripotent stem cells to prepare the islet beta cells comprises a JNK inhibitor, a hedgehog pathway antagonist, an EGF signal factor, an FGF signal factor, a TGF-beta inhibitor, a WNT signal pathway activator, a Notch signal pathway inhibitor, a small molecular compound and trace elements, wherein the mixture ratio of the additive in different stages of cell culture is different, and each component plays a role in a staged directional manner, so that the directional differentiation of the pluripotent stem cells into the islet beta cells can be efficiently induced.
Description
Technical Field
The invention belongs to the technical field of cell engineering, and in particular relates to an additive for preparing islet beta cells by inducing directional differentiation of pluripotent stem cells.
Background
Diabetes mellitus (DiabetesMellitus, DM) is a metabolic disorder disease characterized clinically by elevated blood glucose, and its main pathogenesis is decreased secretion of insulin and its utilization disorder. At present, diabetes mellitus has become the third disease threatening human health and affecting people's quality of life after tumor and cardiovascular and cerebrovascular diseases. Recent reports by the World Health Organization (WHO) in 2016 indicate that 4.22 million people worldwide have diabetes, whereas the number of people with diabetes in china exceeds 1 million people, and is the first worldwide.
There are two types of diabetes: type I diabetes (T1D), accounting for 5-10% of the number of diseases, is an autoimmune disease caused by the selective destruction of islet beta cells; type II diabetes mellitus (T2D), which accounts for more than 90% of the number of diseases, is a disease caused by insulin resistance of peripheral organs including liver, fat and muscle. Diabetes patients manifest a loss of insulin-producing cells, islet beta cells, or a decrease in insulin utilization, and current therapies are all exogenous injections of insulin to control blood glucose balance in vivo. Although this method can effectively control disease progression, prolonged insulin injections do not stably maintain blood glucose physiological balance in the body, resulting in the occurrence of high risk diseases and complications. Vascular lesions caused by complications such as hypoglycemia and hyperglycemia may cause cardiovascular, renal or neurological diseases. Thus, there is a need for a therapeutic strategy for the treatment of diabetes that can reduce or even eliminate long-term complications.
One possible approach is to implant human islets into a patient for treatment of diabetes. The method can well control blood sugar balance in human body, avoid dependence and pain of long-term insulin injection, and further improve overall life quality. However, this is limited by the lack of donor islets, immune rejection of islets between donor-recipient, and variability in islet preparation, making islet transplantation approaches not universally applicable to the treatment of diabetes. Thus, current research is focused on alternatives to the availability of large numbers of insulin-producing secreting cells.
One such method is the use of embryonic stem cells (hESCs) to differentiate directly into insulin-secreting cells. However, due to ethical limitations, the currently available embryonic stem cell lines are limited and are not suitable for future clinical cell therapies. The latter studies have focused mainly on the use of other types of pluripotent stem cells to differentiate into pancreatic endocrine cells, expressed by the combined use of signaling molecules and their related inhibitors/agonists, generally in accordance with 6-7 successive differentiation stages, respectively: definitive Endoderm (DE), primordial embryonic intestinal tube (PrimitiveGutTube), hind foregut (PosteriorForegut), pancreatic Endoderm (PE), endocrine precursor cells (EP) and beta-like early cells, and further differentiate into mature islet beta-like cells. However, such differentiation methods produce still immature beta cells, whose expressed hormones are limited and unstable, of various types and limited insulin content, and cannot be used for transplantation therapy in diabetics.
In conclusion, due to the fact that insulin injection can cause various complications, islet transplantation sources are limited, and the original islet beta cell differentiation scheme is immature, few in number or not provided with GSIS functions and the like, diabetes treatment is greatly limited, and a new islet beta cell differentiation method and a new diabetes treatment strategy are urgently needed to be solved.
Disclosure of Invention
1. Technical problem to be solved by the invention
The invention aims to solve the problems of small quantity and imperfect functions of islet beta cells generated by differentiating the existing islet beta cells.
2. Technical proposal
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
The invention relates to an additive for preparing islet beta cells by inducing multipotent stem cells to directionally differentiate, which comprises a JNK inhibitor, a hedgehog pathway antagonist, an EGF signal factor, an FGF signal factor, a TGF-beta inhibitor, a WNT signal pathway activator, a Notch signal pathway inhibitor, a small molecular compound and trace elements, wherein the proportion of the additive in different stages of cell culture is different.
Preferably, the JNK inhibitor is CC-930, the hedgehog pathway antagonist is N-acetylcysteine or cyclopamine, the EGF signal factor is EGF, the FGF signal factor is FGF10, the TGF-beta superfamily factor is ActivinA, the TGF-beta inhibitor is ALK5iII, the WNT signal pathway activator is BML-284 or WNT3a, and the Notch signal pathway inhibitor is FLI-06 or DAPT.
Preferably, the small molecule compounds include keratin growth factors, protein kinase C activators, ROCK1 inhibitors, sirt1 inhibitors, C-Met inhibitors, thyroid hormones, and retinoic acid (RetinoicAcid).
Preferably, the keratin growth factor is KGF, the protein kinase C activator is TPB, the ROCK1 inhibitor is Y27632, the Sirt1 inhibitor is Nicotinamide, the C-Met inhibitor is BMS-777607, and the thyroid hormone is T3.
Preferably, the kit further comprises an ALK4/5/7 inhibitor, a CDK5 inhibitor, an L-type calcium channel (LTCC) activator and a Hippo signal pathway effector inhibitor, wherein the ALK4/5/7 inhibitor is A83-01, the CDK5 inhibitor is AT7519, the L-type calcium channel (LTCC) activator is BayK8644, and the Hippo signal pathway effector inhibitor is Super-TDU1-31.
Preferably, the proportion of the additive in different stages of cell culture is different, and the additive can be specifically divided into an additive A, an additive B, an additive C and an additive D, wherein the additive A is used for inducing the differentiation of the multifunctional cell sphere into a definitive endoderm cell stage, the additive B is used for differentiating the definitive endoderm cell into a pancreatic precursor cell stage, the additive C is used for differentiating the pancreatic precursor cell into a pancreatic endocrine progenitor cell stage, and the additive D is used for differentiating the pancreatic endocrine progenitor cell into a pancreatic islet beta cell stage.
Preferably, the additive A comprises TGF-beta factor, WNT signal pathway activator and JNK signal inhibitor, wherein the concentration of TGF-beta factor is 30-100ng/mL; the concentration of WNT signal pathway activator is 1-5. Mu.M; JNK signal inhibitor concentration is 0.5-5. Mu.M.
Preferably, the additive B comprises EGF signal factor, retinoic acid, keratin growth factor, protein kinase C activator and Sirt1 inhibitor, wherein the concentration of EGF signal factor is 2-20ng/mL; retinoic acid (RetinoicAcid) at a concentration of 1-5 μm; the concentration of the keratin growth factor is 20-40ng/mL; the concentration of the protein kinase C activator is 100-300nM; the concentration of Sirt1 inhibitor is 10-40. Mu.M.
Preferably, the additive C comprises TGF- β inhibitors, hedgehog signaling pathway inhibitors, notch signaling pathway inhibitors, FGF signaling factors, and thyroid hormones; wherein the concentration of TGF-beta inhibitor is 5-20 mu M; the concentration of the hedgehog signaling pathway inhibitor is 10-50ng/mL; the concentration of Notch signaling pathway inhibitor is 0.2-1. Mu.M; FGF signal factor concentration is 5-50ng/ml; the concentration of thyroid hormone is 0.1-1. Mu.M.
Preferably, the additive D comprises a TGF-beta inhibitor, a C-Met inhibitor and trace elements, wherein the concentration of the TGF-beta inhibitor is 5-20 mu M; the concentration of the C-Met inhibitor is 20-100nM.
The concentration of the additive factors in the additives A-D is in a concentration range which can successfully induce and differentiate the cell types in the stage, and factors lower or higher than the concentration range can not successfully obtain or obtain final cells with poor quality (such as low induction efficiency, small quantity of final products, incomplete functions and the like).
The application method of the additive for preparing islet beta cells by inducing the directional differentiation of pluripotent stem cells comprises the following steps:
S100, preparing a culture medium, and respectively adding an additive A, an additive B, an additive C and an additive D into the same culture medium to obtain a culture medium A, a culture medium B, a culture medium C and a culture medium D;
s200, preparing an induced multifunctional cell sphere;
S300, primary differentiation, namely adding a culture medium A into the induced multifunctional cell sphere and performing directional differentiation culture to obtain definitive endoderm cells;
s400, performing secondary differentiation, namely adding a culture medium B into the definitive endoderm cells and performing induced differentiation to obtain pancreatic precursor cells;
S500, performing three-time differentiation, namely adding a culture medium C into pancreatic precursor cells and performing induced differentiation to obtain pancreatic endocrine progenitor cells;
And S600, four times of differentiation, adding a culture medium D into the pancreatic endocrine progenitor cells, and performing induced differentiation to obtain the required islet beta cells.
Preferably, the preparation of the induced multifunctional cytoball in the step S200 is specifically that after the induced multifunctional stem cells subjected to the adherence culture are digested into small cell blocks, mTESR-1 complete culture medium containing a ROCK1 inhibitor is used for resuspension, the cells are inoculated into an ultralow adsorption six-hole plate at the density of 0.2x10 6/cm2, and are cultured for 24 hours under the condition of 5% oxygen at the temperature of 37 ℃ until the cells become regular pellets, and then 3D suspension culture is carried out for 2-3 days, so that the induced multifunctional cytoball is obtained.
Preferably, the step S300 is specifically to add the culture medium A to the induction multifunctional cell pellet, and continuously culture the cell pellet at 37 ℃ under the condition of 5% CO 2 for 3 days to obtain the definitive endoderm cell, wherein the culture medium A is replaced every 1 day during the culture period.
Preferably, the step S400 is specifically to add the culture medium B to the definitive endoderm cells and further culture the definitive endoderm cells at 37℃under 5% CO 2 for 5 days to obtain pancreatic precursor cells, and the culture medium B is replaced every 1 day during the culture.
Preferably, the step S500 is specifically to add the culture medium C to the pancreatic precursor cells, and further culture the cells at 37℃under 5% CO 2 for 7 days to obtain pancreatic endocrine progenitor cells, and the culture medium C is replaced every 1 day during the culture.
Preferably, the step S600 is specifically to add the culture medium D to the pancreatic endocrine progenitor cells, and culture the pancreatic islet beta cells at 37 ℃ and 5% CO 2 for 7 days to obtain the pancreatic islet beta cells, and continuously changing the culture medium D every 2-4 days during the culture period.
3. Advantageous effects
Compared with the prior art, the technical scheme provided by the invention has the following beneficial effects:
The additive for inducing the directional differentiation of the pluripotent stem cells to prepare the islet beta cells comprises a JNK inhibitor, a hedgehog pathway antagonist, an EGF signal factor, an FGF signal factor, a TGF-beta inhibitor, a WNT signal pathway activator, a Notch signal pathway inhibitor, a small molecular compound and trace elements, wherein the mixture ratio of the additive in different stages of cell culture is different, and each component plays a role in a staged directional manner, so that the directional differentiation of the pluripotent stem cells into the islet beta cells can be efficiently induced.
Drawings
FIG. 1 is a photomicrograph of a suspension culture of Induced Pluripotent Stem Cells (iPSCs) of the invention; wherein FIG. 1-A is a picture of normal iPSCs clone (adherent culture), and FIGS. 1-B, C and D are cell sphere morphology (10 Xmirror picture) after 2h, 24h and 72h of suspension culture, respectively.
FIG. 2 is a photomicrograph of islet beta cells obtained by using and culturing the additive of the present invention; wherein FIGS. 2-A, B, C and D are the morphology of the cell pellet of the four stages (DE stage, PP stage, EN stage and islet beta cell stage) cultivated using the method of example 3, respectively; while figures 2-a '-D' show the morphology of the cell spheres (4 x mirror images) at four stages of culture using the method of example 4 (addition of marker molecules).
FIG. 3 is an identification of protein expression characteristic of DE cells at a key stage in the use of the additives of the present invention to direct differentiation of islet beta cells, including immunofluorescence microscopy and flow cell identification; FIGS. 3-A' are the immunofluorescence pictures of the cell ball in the DE stage, including DE Marker-FOXA2 and SOX17 and the close-up; FIGS. 3-B' are single cell immunofluorescence pictures of DE stage; FIGS. 3-C' show the results of the DE stage cytometry identification, including FOXA2 and SOX17 markers (immunofluorescence images were taken with 20 Xmirror).
FIG. 4 is an immunofluorescence identification of protein expression characteristic of islet precursor cells (PP) at a critical stage in the use of the additive of the invention to direct differentiation of islet beta cells; wherein, the figures 4-A 'and 4-B' are respectively immunofluorescence staining pictures of the cell ball and single cell in the PP stage, including PDX1, NKX6.1 and the combined pictures (the immunofluorescence pictures are all taken by a 20X mirror).
FIG. 5 is an identification of the characteristic protein expression and insulin release function assay of additives of the present invention using directionally differentiated islet beta cells; wherein FIG. 5-A is an immunofluorescence identification picture (20 Xmirror photograph) of islet beta cell Marker (NKX 6.1, C-peptide, insulin and MAFA) and islet alpha cell Marker (Glucagon); FIG. 5-B shows the results of an Elisa assay for insulin content (or secretion function) of islet beta cells (experiments Exp-1 and Exp-2); FIG. 5-C shows the results of an insulin secretion function (GSIS) Elisa assay after stimulation of islet beta cells with varying concentrations of glucose.
Detailed Description
In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which, however, the invention may be embodied in many different forms and are not limited to the embodiments described herein, but are instead provided for the purpose of providing a more thorough and complete disclosure of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention; the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Example 1
The additive for inducing the directional differentiation of the pluripotent stem cells to prepare the islet beta cells comprises a JNK inhibitor, a hedgehog pathway antagonist, an EGF signal factor, an FGF signal factor, a TGF-beta inhibitor, a WNT signal pathway activator, a Notch signal pathway inhibitor, a small molecular compound and a trace element, and the proportion of the additive in different stages of cell culture is different.
The JNK inhibitor is CC-930, the hedgehog pathway antagonist is N-acetylcysteine or cyclopamine, the EGF signal factor is EGF, the FGF signal factor is FGF10, the TGF-beta superfamily factor is ActivinA, the TGF-beta inhibitor is ALK5iII, the WNT signal pathway activator is BML-284 or WNT3a, and the Notch signal pathway inhibitor is FLI-06 or DAPT.
The small molecule compounds include keratin growth factors, protein kinase C activators, ROCK1 inhibitors, sirt1 inhibitors, C-Met inhibitors, thyroid hormones, and retinoic acid (RetinoicAcid).
The keratin growth factor is KGF, the protein kinase C activator is TPB, the ROCK1 inhibitor is Y27632, the Sirt1 inhibitor is Nicotinamide, the C-Met inhibitor is BMS-777607, and the thyroid hormone is T3.
Also included are ALK4/5/7 inhibitors, CDK5 inhibitors, L-type calcium channel (LTCC) activators, and Hippo signal pathway effector inhibitors, wherein the ALK4/5/7 inhibitor is A83-01, the CDK5 inhibitor is AT7519, the L-type calcium channel (LTCC) activator is BayK8644, and the Hippo signal pathway effector inhibitor is Super-TDU1-31.
Wherein the TGF-beta superfamily factor is Activin A, the WNT signal pathway activator is BML-284 or WNT3a, and is a key factor for specific differentiation of endodermal lineage cells; the JNK signal pathway inhibitor CC-930 can enhance the actions of the Activin A and the WNT3a and can obviously promote the efficient differentiation of pluripotent stem cells into endoderm; retinoic Acid (RA) may promote differentiation into PDX1 + pancreatic precursor cells; the Sirt1 inhibitor is Nicotinamide, and can promote differentiation to NKX6.1 + pancreatic precursor cells; the combined use of epidermal growth factor and keratin growth factor can promote differentiation of PDX1 + and NKX6.1 + bi-positive pancreatic precursor cells; the protein kinase C activator is TPB, can promote the specific differentiation to cells of a pancreatic lineage, has a synergistic effect with RA, and can remarkably enhance the differentiation of PDX1 + cells; the hedgehog pathway antagonist is N-acetylcysteine or cyclopamine, and can promote differentiation of the inwardly secreted precursor cells; FGF10 can promote the survival and proliferation of pancreatic endocrine progenitor cells, improves the differentiation efficiency and stability at the stage, and has great synergy on the final pancreatic beta yield; meanwhile, the combination of the hedgehog pathway antagonist and FGF10 can promote differentiation to PDX1 + endocrine precursor cells; the TGF-beta inhibitor is ALK5 iII, can promote differentiation and maturation of islet beta cells, and can increase the release of insulin in islet beta cells; the combined use of TGF-beta inhibitors and thyroid hormones may promote endocrine precursor cell expansion of NGN3 +; the Notch signaling pathway inhibitor is FLI-06 or DAPT, and can increase the number of endocrine precursor cells of NGN3 +; the C-Met inhibitor is BMS-777607, and can promote the maturation of islet beta cells.
The additive A comprises TGF-beta factor, WNT signal pathway activator and JNK signal inhibitor, wherein the concentration of the TGF-beta factor is 30-100ng/mL, preferably 50ng/mL; the concentration of WNT signaling pathway activator is 1-5. Mu.M, preferably 3. Mu.M; the concentration of the JNK signal inhibitor is 0.5-5. Mu.M, preferably 1. Mu.M.
The additive B comprises EGF signal factor, retinoic acid, keratin growth factor, protein kinase C activator and Sirt1 inhibitor, wherein the concentration of EGF signal factor is 2-20ng/mL, preferably 10ng/mL; retinoic acid (RetinoicAcid) at a concentration of 1-5 μm; the concentration of keratin growth factor is 20-40ng/mL, preferably 30ng/mL; the concentration of the protein kinase C activator is 100-300nM, preferably 200nM; the concentration of Sirt1 inhibitor is 10-40. Mu.M, preferably 20. Mu.M.
Additive B also includes an ALK4/5/7 inhibitor, the concentration of ALK4/5/7 inhibitor being 10-50 nM, preferably 20nM. The ALK4/5/7 inhibitor is A83-01, can selectively inhibit TGF-beta activin receptor ALK4, I type receptor ALK5 and node receptor ALK7, can inhibit transformation of epithelial cells into mesenchymal cells, and has the functions of promoting specific differentiation of pancreatic lineage cells and expansion of pancreatic precursor cells.
The additive C comprises TGF-beta inhibitor, hedgehog signaling pathway inhibitor, notch signaling pathway inhibitor, FGF signaling factor and thyroid hormone; wherein the concentration of TGF-beta inhibitor is 5-20. Mu.M, preferably 10. Mu.M; the concentration of hedgehog signaling pathway inhibitor is 10-50ng/mL, preferably 30ng/mL; the concentration of Notch signaling pathway inhibitor is 0.2-1. Mu.M, preferably 0.5. Mu.M; FGF signal factor concentration is 5-50ng/ml, preferably 10ng/ml; the concentration of thyroid hormone is 0.1-1. Mu.M, preferably 0.5. Mu.M.
Additive C also includes CDK5 inhibitors, with CDK5 inhibitors at a concentration of 5 to 30nM, preferably 15nM. The CDK5 inhibitor is AT7519, which has dual efficacy: firstly, the activators P35 and P39 of CDK5 are expressed in pancreas cells and play a role in regulating the maturation of islet beta cells, and secondly, AT7519 also has GSK3 beta inhibition function, can activate WNT signaling pathway to promote cell differentiation, and in addition, the action of CDK5 inhibitor can be enhanced under the premise of the existence of Notch signaling inhibitor.
The additive D comprises a TGF-beta inhibitor, a C-Met inhibitor and trace elements, wherein the concentration of the TGF-beta inhibitor is 5-20 mu M, preferably 10nM; the concentration of the C-Met inhibitor is 20-100nM, preferably 50nM.
Additive D also includes L-type calcium channel (LTCC) activators and Hippo signal pathway effector inhibitors; wherein, the concentration of the L-type calcium ion channel (LTCC) activator is 10-30 nM, preferably 20nM; the concentration of the inhibitor of the Hippo signal pathway effector is 0.2 to 0.6. Mu.M, preferably 0.35. Mu.M. The L-type calcium ion channel (LTCC) activator is BayK8644, which can activate Ras signals, strengthen cell cycle and has the function of promoting proliferation of islet beta cells; the inhibitor of the Hippo signal pathway effector YAP is Super-TDU1-31, which can break the interaction between YAP and TEADs transcription factors and has important roles in promoting the differentiation of endocrine cells and inhibiting proliferative precursor cells.
The culture medium of the embodiment adopts ADVANCED DMEM/F12 culture medium, and is added with key components such as glucose, sodium bicarbonate, human Serum Albumin (HSA), glutamine, vitamin C and the like which are necessary for maintaining the growth of cells, and the added additives comprise agonists or antagonists of JNK, hedgehog, EGF, FGF, TGF-beta, WNT and other signal paths, necessary small molecular compounds, trace elements and the like, and each component plays roles in a staged and directional manner, so that the pluripotent stem cells can be efficiently induced to be directionally differentiated into islet beta cells. By the method, the induced multifunctional stem cells can be directionally differentiated and cultured in a short time to obtain the islet beta cells. Firstly, the number of raw materials is not limited, the induced multifunctional stem cells can be infinitely amplified, and the ethical dispute of the use aspect of the embryonic stem cells can be avoided; secondly, the period of culturing islet beta cells is about three weeks, and compared with the existing differentiation technology, the preparation speed is greatly improved, and the production cost is reduced; thirdly, the islet beta cells are cultured in a 3D suspension mode, and compared with the existing adherence (2D) culture technology, the yield of differentiated cells and the loss caused by reagent replacement in the culture process are greatly improved; fourth, the specific signal pathway inhibitor and small molecular compound are adopted in the process of culturing the islet beta cells, so that the purity and the yield of the islet beta cells can be greatly improved; fifth, the cultured islet beta cells selectively adopt cell sorting or biological material wrapping technology, so that the potential safety hazard caused by poor treatment effect due to lower cell purity or immune rejection reaction after cell transplantation can be avoided.
The application method of the additive for preparing islet beta cells by inducing the directional differentiation of pluripotent stem cells comprises the following steps:
S100, preparing a culture medium, and respectively adding an additive A, an additive B, an additive C and an additive D into the same culture medium to obtain a culture medium A, a culture medium B, a culture medium C and a culture medium D;
s200, preparing an induced multifunctional cell sphere;
S300, primary differentiation, namely adding a culture medium A into the induced multifunctional cell sphere and performing directional differentiation culture to obtain definitive endoderm cells;
s400, performing secondary differentiation, namely adding a culture medium B into the definitive endoderm cells and performing induced differentiation to obtain pancreatic precursor cells;
S500, performing three-time differentiation, namely adding a culture medium C into pancreatic precursor cells and performing induced differentiation to obtain pancreatic endocrine progenitor cells;
And S600, four times of differentiation, adding a culture medium D into the pancreatic endocrine progenitor cells, and performing induced differentiation to obtain the required islet beta cells.
The preparation of the induced multifunctional cytoball in step S200 of this example specifically comprises digesting the induced multifunctional stem cells subjected to the adherent culture into small cell blocks, re-suspending the small cell blocks with a complete mTeSR-1 culture medium containing a ROCK1 inhibitor, inoculating the small cell blocks into an ultra-low adsorption six-well plate at a density of 0.2x10 6/cm2, culturing the small cell blocks for 24 hours at 37 ℃ under the condition of 5% oxygen until the small cell blocks become regular small cell blocks, and then performing 3D suspension culture for 2-3 days to obtain the induced multifunctional cytoball.
Step S300 of this example is specifically to add medium A to the induction multifunctional pellet and continue culturing at 37℃under 5% CO 2 for 3 days to obtain definitive endoderm cells, with medium A being changed every 1 day during the culturing period.
Step S400 of this example is specifically to add Medium B to definitive endoderm cells and continue culturing at 37℃under 5% CO 2 for 5 days to obtain pancreatic precursor cells, with Medium B being changed every 1 day during the culturing.
Step S500 of this example is specifically to add medium C to pancreatic precursor cells and continue culturing at 37℃under 5% CO 2 for 7 days to obtain pancreatic endocrine progenitor cells, with medium C being changed every 1 day during the culturing period.
Step S600 of this example is specifically to add medium D to pancreatic endocrine progenitor cells and culture it at 37℃under 5% CO 2 for 7 days to obtain islet beta cells, and to replace medium D every 2-4 days during continued culture.
Example 2
The content of the embodiment is mainly the preparation of islet beta cell differentiation medium, and the specific steps are as follows:
The islet beta cell differentiation medium is divided into four stages, namely a stage one (DE induction medium composed of an additive A), a stage two (PP induction medium composed of an additive B), a stage three (EN induction medium composed of an additive C) and a stage four (islet beta cell induction medium composed of an additive D), wherein the culture medium components consist of a basic culture medium component (basal medium) and an additive component (supplement) in the following table 1-4:
TABLE 1 DE composition and ratio of Induction Medium
TABLE 2 PP composition and ratio of Induction Medium
Remarks: the addition of A83-01 can significantly improve the cell differentiation efficiency.
TABLE 3 EN composition and ratio of Induction Medium
Remarks: the addition of AT7519 can significantly increase cell differentiation efficiency.
TABLE 4 composition and ratio of islet beta cell induction Medium
Remarks: the addition of BayK8644 and Super-TDU1-31 can significantly increase the differentiation efficiency of cells.
Example 3
The main content of the embodiment is to culture and induce the multifunctional stem cells by using a 3D suspension culture mode, which is as follows:
1. Required reagent
The culture medium used for inducing the culture of the multifunctional stem cells (iPSCs) is mTESR-1 complete culture medium, the digestive enzyme used for amplification and passage is EDTA, the reagent used for cell rinsing and balancing is DPBS, and the inhibitor used for promoting cell survival is Y27632.
2. Culture process
After removing the culture medium from the iPSCs cultivated in the way of adhering to 100mm-dish and using 2mL of DPBS for rinsing, adding 4mL of EDTA digestive enzyme, standing for 3-4 min at 37 ℃, discarding the digestive enzyme after the intercellular separation, adding 12mL of mTESR-1 complete culture medium with the final concentration of 10 mu M Y7632, blowing and separating the cells into cell small blocks, and aggregating 10-30 cells of each cell small block.
The isolated cell pellet is transferred on average to an ultra-low adsorption 6-well plate, cultured at 37℃under 5% oxygen for 24 hours until the cells become regular pellets, then transferred to 75 or 125mL spinner Flask, and subjected to 3D suspension culture at 70-120 rpm, preferably 100 rpm.
3. Cell sphere morphology observation
100Mm-dis was placed on a sterile console, the suspended cell pellet cultured for 1 day or 3 days was transferred to the above-mentioned dis using a 10mL pipette, the dis was gently shaken to uniformly distribute the cell pellet, and observed under a EVOS microscope and photographed.
The results are shown in FIG. 1: the method can obtain a large number of cell balls with uniform morphology.
Example 4
The main content of the embodiment is to utilize induced multifunctional stem cells to directionally differentiate and culture islet beta cells, and the method is as follows:
1. Culture of undifferentiated cells
Inducing multifunctional Stem cells 3D suspension cell pellets were obtained according to the method of example 2, cultured in 125mL SPINNER FLASK, passaged as single cells every 3-4 days using Acceutase digestive enzyme, resuspended in complete medium with mTESR-1 containing 10 μ M Y7632 inhibitor, and cultured in suspension at 37℃in a incubator of 5% CO 2 at 70-100 rpm.
In preparation for differentiation of islet beta cells, the cells digested with Ackutase are seeded at a density of 2 to 8X 10 5 cells/mL, preferably 6X 10 5 cells/mL in SPINNER FLASK described above, and after 3 days of culture, they are replaced with differentiation medium.
2. Directional differentiation culture of islet beta cells
The 3 day old undifferentiated iPSCs pellet was allowed to settle naturally for 3-5 min, the top mTESR-1 medium and dead cells were gently discarded using a 10mL pipette, and the induction medium (no. Sign additive) was added sequentially (30 mL/Flask, medium preferably ready to use, no more than 7 days if stored at 4℃and equilibrated to room temperature for 30 minutes before use) according to the differentiation stage induction medium (no. Sign additive) described in example 1, placed on a magnetic stirrer in a incubator at 37℃with 5% CO 2, the rotational speed was adjusted to 70rpm, and the time for the corresponding stage was allowed to incubate. Wherein the culture medium is replaced every 2 days for 3 days in the first stage, 5 days in the second stage, 7 days in the third stage and more than 7 days in the fourth stage.
3. Morphology observation of islet beta cell spheres
100Mm-dish was placed on a sterile console, the islet beta cell pellet cultured for about three weeks was transferred into dish using a 10mL pipette, the dish was gently shaken to uniformly distribute the cell pellet, and observed under a EVOS microscope.
FIGS. 2-A-D show islet beta cell pellets (including pellet morphology at each stage) cultured by the differentiation method of example 4, and it is found that islet beta cell pellets having a preferable morphology can be obtained by this method.
Example 5
The main content of this example is the islet β cell differentiation method after adding small molecular compound (to increase differentiation efficiency), specifically as follows:
Using the islet beta cell differentiation method of example 4, the morphology and number of islet beta cells obtained after three weeks of culture were compared with small molecule compounds A83-01, AT7519, bayK8644, and Super-TDU1-31 (labeled with a number) in the induction medium of the differentiated PP stage, EN stage, and islet beta cell stage.
As shown in the results of FIG. 2-A '-D', the induction medium after adding the small molecular compound can obtain more islet beta cells, and the morphology of islet beta cell spheres is more uniform.
Example 6
The main content of this example is the identification of the expression of proteins characteristic of cells in the DE phase by immunofluorescence and flow cytometry, as follows:
The directional differentiation of islet beta cells was performed using the procedure of example 5, in which stage one (DE cells) induction was the initiation and key of differentiation, and DE cell identification was performed using immunofluorescence and flow cytometry as follows.
A, immunofluorescence method:
placing the cells cultured to the first stage in an ultra-low adsorption 24-pore plate, and standing for 1min to enable the cell spheres to sink into the bottom of the pore plate; after the upper layer of the culture solution was carefully aspirated, 1 XPBS (pH 7.4, the same shall apply hereinafter, 1 mL/well) was slowly added along the 24-well plate wall and washed 2 times; then, 4% PFA (paraformaldehyde, 0.5 mL/well) preheated at 37℃was slowly added along the plate wall, and the cells were fixed by standing at room temperature for 18 minutes, the PFA was gently aspirated, and 1 XPBS was added for 3 washes (1 mL/well/time).
0.3% Triton X100 (0.5 mL/well) was added and incubated at 37℃for 30min; then 5% BSA (0.5 mL/well) was added for blocking and incubated at 37℃for 30min; next, primary antibodies (SOX 17 and FOXA 2) were directly added to the blocking solution in an amount of 10. Mu.L/well, incubated at 37℃for 1 hour, and washed 3 times (1 mL/well/time) with 1 XPBS.
Adding secondary antibody diluted by 1% BSA (dilution ratio 1:1000), wherein the addition amount of the secondary antibody is 0.5 mL/hole, and after incubation at 37 ℃ in dark for 60 minutes, the secondary antibody is sucked, and the secondary antibody is washed 3 times (1 mL/hole/time) by adding 1 XPBS for 5 minutes each time; DAPI (0.5 mL/well) formulated with 1 XPBS at a final concentration of 1 μg/mL was then added, stained for 5 minutes, blotted off, and washed 2 times (1 mL/well/time) with 1 XPBS; finally, 1 XPBS (0.5 mL/well) was added to resuspend the pellet, and the pellet was observed under a microscope and photographed.
Remarks: in addition to performing the immunofluorescent staining of the cell spheres as described above, the cell spheres were also subjected to immunofluorescent staining (better looking at the expression of each marker in the cells of the list) using Accutase to single cell plating in 24-well plates.
B, flow cytometry:
Placing the cell balls which are cultured to the first stage in a 15mL centrifuge tube, naturally settling for 1-3 min, discarding the culture solution, and using 1mL DPBS for one time and discarding; adding 1-2 mL of Ackutase or TrypLE digestive juice into the cell ball, placing at 37 ℃ for 3-5 minutes, and separating the cell ball into single cells; then adding 2-3 times of DPBS to dilute the digestion solution, centrifuging at 200-300 g for 3 minutes, and discarding the digestion solution and the DPBS.
1ML of 4% Paraformaldehyde (PFA) was added to the cells, and the cells were fixed at 4℃for 30 minutes and discarded; then 0.3% Triton X100 (1 mL) or other stabilizing buffer was added and blocked and permeabilized at 4deg.C for 30 minutes and discarded; next, 1mL of the primary antibody (SOX 17 and FOXA 2) was added at 4℃overnight.
Discarding the primary antibody in the next day, adding 1mL of secondary antibody prepared by the sealing solution, and incubating at 4 ℃ in a dark place for 2 hours; and finally, performing flow cytometry analysis.
FIGS. 3-A-B "and FIGS. 3-C' show immunofluorescence and flow cytometry identification, respectively, of DE cells cultured in example 5, showing that the cultured DE cells have high expression of marker molecules.
Example 7
The main content of this example is the identification of the expression of proteins characteristic of cells in PP phase using immunofluorescence, in particular as follows:
cells (PP cells) cultured to stage two were removed, and the expression of the critical proteins (PDX 1 and NKX 6.1) in the PP stage was examined according to the immunofluorescence procedure of example 6.
FIG. 4 shows the results of immunofluorescence identification of Marker (PDX 1 and NKX 6.1) of PP cells cultured in example 5, showing that PP cells cultured have high expression of Marker molecules.
Example 8
The main content of the embodiment is the identification of the expression of the islet beta cell characteristic protein by using an immunofluorescence method, which is specifically as follows:
Cells cultured to stage four (islet beta cells) were removed, and the expression of islet beta cell key proteins (NKX 6.1, C-peptide, insulin) was examined according to the immunofluorescence procedure of example 6.
FIG. 5-A shows the immunofluorescence assay results of islet beta cells obtained by culturing in example 5, which shows that islet beta cells obtained by culturing have high expression of marker molecules (NKX 6.1, C-peptide, insulin and MAFA).
Example 9
The main content of the embodiment is to use an Elisa kit to detect insulin secretion function of islet beta cells, and the method is as follows:
Islet beta cell pellets (20-30) cultured for 7 days in the stage four are collected in a 15mL centrifuge tube by using a 5mL pipette, and the cell pellets naturally settle for 1-3 minutes and then the upper culture solution is discarded. Then 1mL of 70% ethanol containing 1.5% hydrochloric acid (HCL) was added thereto, and the mixture was left at-20℃for 24 hours. After 24 hours the pellet was gently shaken and left for a further 24 hours.
After 48 hours of placement, the tube was centrifuged at 2100rcf for 15 minutes, 1mL of supernatant was collected in a fresh 15mL centrifuge tube, and 1mL of 1M TRIS (pH 7.5) was added for neutralization. The neutralized liquid was tested for insulin release using the human insulin Elisa kit.
FIG. 5-B shows the results of insulin release assays of islet beta cells obtained by culturing in example 5, which shows that the islet beta cells obtained by culturing have a high amount of insulin release.
Example 10
The main content of this example is to use the Elisa kit to detect GSIS function of islet β cells, specifically as follows:
Reagent preparation: krb buffer was prepared from 128mM NaCl, 5mM KCl, 2.7mM CaCl2, 1.2mM MgCl2, 1mM Na2HPO4, 1.2mM KH2PO4, 5mM NaHCO3, 10mM HEPES and 0.1% BSA using deionized water; low concentration glucose (2 mM) and high concentration glucose (20 mM) were prepared from Krb buffer; 30mM KCl was formulated from 1M KCl in water and 20mM high concentration glucose solution; all reagents were filter sterilized with a 0.22 μm filter membrane after formulation.
GSIS functional verification: first, islet beta cell pellets (20) cultured for 14 days in stage four were collected in a 15mL centrifuge tube using a 5mL pipette, and the pellets were allowed to settle naturally for 1 to 3 minutes, and then the upper culture solution was discarded. Then, 1mL of Krb buffer was added for rinsing, and pre-incubation was performed for 1 hour using 200 μl of a low concentration (2 mM) glucose Krb solution for removing residual insulin. After washing again with 1mL of Krb buffer 2 times, the cell pellet was incubated with 200 μl of low concentration (2 mM), high concentration (2 mM) glucose Krb solution and KCl solution, respectively, for 1 hour (each time the solutions of different concentrations were changed, krb equilibration solution was used for washing, and supernatants were collected at the end of incubation). Finally, 200 μl of glucose and KCl-stimulated supernatants were collected and insulin release (GSIS) levels of islet β cells stimulated with different concentrations of glucose were detected using the insulin Elisa kit.
FIG. 5-C shows the GSIS test results of islet beta cells obtained by culturing in example 4, showing that the islet beta cells obtained by culturing have different degrees of insulin release for different concentrations of glucose stimulation, the insulin content released by the stimulated beta cells increases with increasing glucose content, and the content reaches the highest after KCl stimulation.
From the results of the identification in examples 4 to 10, it can be seen that the cells obtained by the directional differentiation culture of induced multifunctional stem cells have not only the expression of proteins characteristic of islet beta cells such as PDX1, NKX6.1, C-Peptide and instulin, but also high yield and purity, and have glucose stimulation-Insulin release (GSIS) function, indicating that they are mature and functional islet beta cells, using the additives and methods of use of the present invention.
The additive of the invention leads the induced multifunctional stem cells subjected to 3D suspension culture to be directionally differentiated into islet beta cells, and has the following advantages compared with other differentiation methods: firstly, the number of raw materials is not limited, and induced multifunctional stem cells can be infinitely amplified; secondly, the source of the raw materials is not limited, the induced multifunctional stem cells can be obtained by reprogramming adult cells of healthy people such as peripheral blood mononuclear cells, and the use of embryonic stem cells has ethical limitation; thirdly, the differentiation efficiency is high, and mature and functional islet beta cells can be obtained by using suspension culture and specific signal channel protein factors and small molecular compounds; fourthly, the obtained yield is high, and mature islet beta cells with huge quantity and higher purity can be obtained by adding specific small molecular compounds; fifthly, the time consumption is short, the culture period is 22-30 days, and the higher cell yield can be obtained by prolonging the amplification time, so that the cost loss is reduced; sixth, safe and reliable, use methods such as cell sorting or biomaterial encapsulation, etc., can improve the purity of the cell obtained or reduce even have no immune rejection reaction of the cell to host.
In addition, the use of small molecule compounds such as ALK4/5/7 inhibitors (A83-01), CDK5 inhibitors (AT 7519), LTCC activators (BayK 8644) and Hippo signal pathway effector inhibitors (Super-TDU 1-31) promotes the directed differentiation of islet beta cells, manifesting in a massive expansion of intermediates (pancreatic precursor cells) during differentiation, and maturation and high expansion efficiency of differentiation products (islet beta cells). The islet beta cells obtained by the additive and the application method have the advantages of large quantity, high maturity and perfect functions, and a new method is provided for treating future diabetes.
The foregoing examples merely illustrate certain embodiments of the invention and are described in more detail and are not to be construed as limiting the scope of the invention; it should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the concept of the invention, all of which fall within the scope of protection of the invention; accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (8)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110297334.XA CN113046299B (en) | 2021-03-19 | 2021-03-19 | An additive for preparing pancreatic β cells by induced pluripotent stem cell directed differentiation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110297334.XA CN113046299B (en) | 2021-03-19 | 2021-03-19 | An additive for preparing pancreatic β cells by induced pluripotent stem cell directed differentiation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113046299A CN113046299A (en) | 2021-06-29 |
| CN113046299B true CN113046299B (en) | 2024-06-28 |
Family
ID=76514277
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110297334.XA Active CN113046299B (en) | 2021-03-19 | 2021-03-19 | An additive for preparing pancreatic β cells by induced pluripotent stem cell directed differentiation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113046299B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20250006874A (en) * | 2022-04-25 | 2025-01-13 | 오리즈루 세라퓨틱스 가부시키가이샤 | Maturation agent with ALK5 inhibitory activity and CDK8/19 inhibitory activity |
| CN114634904B (en) * | 2022-05-17 | 2022-09-13 | 天津外泌体科技有限公司 | Method for producing high-purity pancreatic progenitor cells |
| CN117645970A (en) * | 2023-11-17 | 2024-03-05 | 广州百康细胞生命科技有限公司 | Efficient and rapid acquisition of human pluripotent stem cells by using chemical re-editing method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109749986A (en) * | 2019-03-13 | 2019-05-14 | 武汉大学 | A method for obtaining pancreatic precursor cells and pancreatic islet beta cells differentiated from human pluripotent stem cells |
| CN112226402A (en) * | 2013-06-11 | 2021-01-15 | 哈佛学院校长同事会 | SC-beta cells and compositions and methods for producing the same |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB201216796D0 (en) * | 2012-09-20 | 2012-11-07 | Cambridge Entpr Ltd | In vitro pancreatic differentiation |
| SG10201709338RA (en) * | 2012-12-31 | 2017-12-28 | Janssen Biotech Inc | Culturing of human embryonic stem cells at the air-liquid interface for differentiation into pancreatic endocrine cells |
| US10941384B2 (en) * | 2013-03-15 | 2021-03-09 | President And Fellows Of Harvard College | Compositions and methods for promoting the generation of endocrine cells |
| US11332716B2 (en) * | 2015-07-27 | 2022-05-17 | The Regents Of The University Of California | Methods and compositions for producing pancreatic beta cells |
| MX2018013175A (en) * | 2016-04-28 | 2019-02-21 | Takeda Pharmaceuticals Co | PURIFICATION METHOD FOR PANCREATIC PRECURSOR CELLS DERIVED FROM PLURIPOTENT MOTHER CELLS AND AMPLIFICATION METHOD FOR THEM. |
| WO2018136005A1 (en) * | 2017-01-17 | 2018-07-26 | Agency For Science, Technology And Research | Maintenance and expansion of pancreatic progenitor cells |
| CN110317790B (en) * | 2019-07-22 | 2021-05-25 | 中山大学孙逸仙纪念医院 | Method for separating and culturing human pancreatic cancer tissue organoid in vitro |
| CN111269875B (en) * | 2020-03-24 | 2022-04-08 | 山东兴瑞生物科技有限公司 | Method for directionally differentiating into islet cells by using autoimmune cells |
| CN111440761A (en) * | 2020-04-09 | 2020-07-24 | 上海赛尔维医疗科技有限公司 | Method for expanding and differentiating pancreatic cells and uses thereof |
| CN112251396B (en) * | 2020-10-09 | 2022-08-16 | 北京呈诺医学科技有限公司 | Culture medium and application thereof, and method for differentiating induced pluripotent stem cells into pancreatic islets |
-
2021
- 2021-03-19 CN CN202110297334.XA patent/CN113046299B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112226402A (en) * | 2013-06-11 | 2021-01-15 | 哈佛学院校长同事会 | SC-beta cells and compositions and methods for producing the same |
| CN109749986A (en) * | 2019-03-13 | 2019-05-14 | 武汉大学 | A method for obtaining pancreatic precursor cells and pancreatic islet beta cells differentiated from human pluripotent stem cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113046299A (en) | 2021-06-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112980774B (en) | Culture method for preparing islet beta cells by inducing pluripotent stem cells to differentiate directionally | |
| CN113046299B (en) | An additive for preparing pancreatic β cells by induced pluripotent stem cell directed differentiation | |
| JP7023820B2 (en) | Cortical interneurons and other neuronal cells generated by directing the differentiation of pluripotent cells and pluripotent cells | |
| JP7178264B2 (en) | Compositions and methods for organoid production and disease modeling | |
| CN112961823B (en) | A culture medium for preparing pancreatic islet beta cells through directed differentiation of induced pluripotent stem cells | |
| Nakayama et al. | Astrocyte-derived factors instruct differentiation of embryonic stem cells into neurons | |
| US20040132183A1 (en) | Methods and compositions for expanding and differentiating insulin-producing cells | |
| EP1507552A2 (en) | Methods, compositions, and growth and differentiation factors for insulin-producing cells | |
| US20050074880A1 (en) | Generation of multipotent central nervous system stem cells | |
| JP6685327B2 (en) | Improved method for islet transplantation | |
| CN112980771A (en) | Method for preparing pancreatic beta cells and application thereof | |
| Montanucci et al. | Treatment of diabetes mellitus with microencapsulated fetal human liver (FH-B-TPN) engineered cells | |
| WO2016101010A1 (en) | Method of inducing beta cells from urine-derived cells using small molecules | |
| CN101724602B (en) | Method for the differentiation of human adult stem cells into insulin-secreting cells | |
| WO2018119155A1 (en) | Methods of pancreatic differentiation of stem cells | |
| WO2014179198A1 (en) | Methods for producing insulin-secreting beta cells from human pluripotent stem cells | |
| KR101574659B1 (en) | Method for producing endocrine aggregate of insulin-producing beta cells differentiated from human pluripotent stem cells | |
| CN117384824A (en) | Human vascularized lung organoid based on iPSC and construction method thereof | |
| CN114807015B (en) | Induction method for promoting islet alpha cells to be converted into beta cells and application thereof | |
| Koblas et al. | Differentiation of CD133-positive pancreatic cells into insulin-producing islet-like cell clusters | |
| WO2019144968A1 (en) | Cell induction method | |
| SG177238A1 (en) | A method of producing neurons from stem cells, the neurons and uses thereof | |
| KR20240146656A (en) | Method for generating functional islets from pluripotent stem cells | |
| CN113015537A (en) | Compositions and methods for proliferating insulin-producing islet cells and therapeutic uses thereof | |
| KR20160077720A (en) | Method for inducing differentiation of pluripotent stem cell into insulin producing cells using co-culture with mature islet cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address |
Address after: 201200 Shanghai Pudong New Area China (Shanghai) Pilot Free Trade Zone No. 151 Libing Road Building 6 Building 6 Patentee after: Shanghai Aisar Biopharmaceutical Co.,Ltd. Country or region after: China Address before: 200000 Shanghai Pudong New Area, No. 781 Cailun Road, Room 802 Patentee before: IXCELL BIOTECHNOLOGY Co.,Ltd. Country or region before: China |
|
| CP03 | Change of name, title or address |