CN111269875B - Method for directionally differentiating into islet cells by using autoimmune cells - Google Patents
Method for directionally differentiating into islet cells by using autoimmune cells Download PDFInfo
- Publication number
- CN111269875B CN111269875B CN202010212266.8A CN202010212266A CN111269875B CN 111269875 B CN111269875 B CN 111269875B CN 202010212266 A CN202010212266 A CN 202010212266A CN 111269875 B CN111269875 B CN 111269875B
- Authority
- CN
- China
- Prior art keywords
- cells
- culture medium
- islet
- induced
- directionally
- 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
Images
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
- 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
Abstract
The invention provides a method for directionally differentiating islet cells by utilizing autoimmune cells, which is characterized in that the method comprises the steps of obtaining the immune cells from peripheral blood of a patient, carrying out in-vitro dedifferentiation on the immune cells to induce pluripotent stem cells, and then directionally differentiating the cells to generate the islet cells. The method for culturing the islet cells selects the immune cells separated from the peripheral blood of a human body, induces and directionally differentiates the immune cells into the islet cells, and then transplants the islet cells into the same patient to reduce the immunological rejection of the patient. When Induced Pluripotent Stem Cells (iPSCs) are directionally induced and differentiated into islet cells, the purity of the finally induced islet cells is over 62.4% through DTZ staining identification after induced differentiation for 20-28 days.
Description
Technical Field
The invention relates to a method for directionally differentiating islet cells by using autoimmune cells, belonging to the technical field of biology and new medicines.
Background
The pancreas is an exocrine gland that secretes digestive enzymes such as pancreatic lipase, trypsin, elastase, pancreatic amylase, etc., and is also an endocrine gland that secretes pancreatic hormones such as glucagon, insulin, somatostatin, pancreatic polypeptide, etc. The pancreatic hormones are secreted by a population of cells in the pancreas called "islets," which include four types of cells, alpha cells, beta cells, delta cells, pp cells.
Diabetes is a disease caused by insulin deficiency or insufficient work. Once the disease occurs in a patient, it is difficult to completely cure the disease. There are two main types of diabetes, insulin-dependent type I diabetes and insulin-independent type II diabetes. Type II diabetes is a chronic disease that occurs when the body acquires insulin resistance. Type II diabetes is also considered a lifestyle-related disease, resulting from poor lifestyle habits including obesity or stress from excessive diet or lack of exercise. Type II diabetes is common in the elderly population, with many diabetics suffering from type II diabetes.
Type I diabetes is caused by destruction of beta cells or insulin-secreting cells by autoimmune disease or viral infection. Insulin-secreting cells are destroyed and insulin cannot be secreted in vivo. Type I diabetic patients use insulin as symptomatic treatment. In addition, pancreas or islet transplantation is used to allow the patient the ability to automatically control blood glucose levels. Blood sugar level fluctuates frequently, pancreas or islet transplantation can reduce the burden of patients, and the blood sugar of the patients can reach normal level. However, the number of pancreas and islets currently available for transplantation is insufficient and the patient receiving the transplantation must take immunosuppressants for a lifetime, which may cause other side effects.
With the rapid development of stem cells and related research in the field of tissue engineering, a method for inducing pluripotent stem cells to directionally differentiate into pancreatic cells has been developed and applied for patent with the patent number of CN 104726395B, but the method has not yet been applied to clinical application. Furthermore, the pancreatic cells directionally differentiated by using the Embryonic Stem Cells (ESCs) and allogenic Induced Pluripotent Stem Cells (iPSCs) in the patent may cause the immunological rejection of the diabetics, and the application of the embryonic stem cells to the clinic also involves ethical problems. In the prior art, pancreatic cells are obtained from other sources, such as CN109053866A, epidermal stem cells are used for inducing differentiation into pancreatic cells, but the culture method uses fetal bovine serum which is of animal origin and contains animal proteins, so the culture method cannot be directly applied to clinic.
Based on the above problems, there is an urgent need to develop a method for directionally differentiating islet cells by using autoimmune cells with high efficiency, and the islet cell transplantation has small side effect on patients, low immune rejection and no ethical problem.
The method is characterized in that the method utilizes the autoimmune cells to differentiate into the islet cells, firstly, the autoimmune cells are required to be dedifferentiated to obtain iPSCs, and then the iPSCs are directionally induced and differentiated into the islet cells.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for directionally differentiating the autoimmune cells into the islet cells, which realizes the following purposes:
(1) shortening the cell culture time;
(2) the conversion rate is improved, and the purity of the islet cells induced to differentiate is improved;
(3) the insulin secretion level of the induced islet cells is equal to that of normal pancreatic cells.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for directionally differentiating the self-immune cells into islet cells includes such steps as taking the immune cells from the peripheral blood of patient, in vitro dedifferentiating to induce pluripotent stem cells, and directionally differentiating to obtain islet cells.
The following is a further improvement of the above technical solution:
the purity of the induced pluripotent stem cell is not less than 95%.
The directional differentiation of the islet cells comprises the steps of inducing pluripotent stem cells to directionally differentiate and induce to generate endoderm cells, inducing and differentiate to primitive gut tube cells, inducing and differentiate to pancreatic progenitor cells, inducing and differentiate to pancreatic endothelial layer cells and inducing and differentiate to islet cells.
The induced pluripotent stem cells are directionally differentiated to induce generation of endoderm cells, the induced pluripotent stem cells are inoculated into a culture dish coated with 0.09-0.12% fibronectin, when the cell plating reaches about 60%, the induced pluripotent stem cells are cultured by using an S1 culture medium containing a CHIR99021 compound, after 22-26h of culture, a fresh S1 culture medium is replaced, and the cells are cultured for 48-70h to obtain definitive endoderm cells.
The S1 culture medium is a DMEM low-sugar culture medium, and additionally contains 95-105ng/mL Activin A and 1-3% of B27 by volume percentage; the concentration of the CHIR99021 compound in the S1 culture medium containing the CHIR99021 compound is 13-15 ug/ml.
Inducing and differentiating to primitive gut tube cells, culturing endoderm cells by using an S2 culture medium, and culturing for 68-76h to obtain the primitive gut tube cells; the S2 culture medium is a DMEM low-sugar culture medium, and additionally contains 45-55ng/mL KGF and 1-3% B27 by volume.
The induced differentiation is divided into two stages, the first stage is to induce early pancreatic progenitor cells and culture for 2-3 days, the early pancreatic progenitor cells are firstly cultured in S3 culture medium containing 190-210nM LDN193189 for 22-26h, and the culture medium is replaced with S3 for 24-48 h; in the second stage, early pancreatic progenitor cells are induced into pancreatic progenitor cells, and cultured for 4-6 days using S4 medium.
The S3 culture medium is DMEM/F12 culture medium, and further contains 45-55ng/mL KGF, 0.24-0.26uM SANT1, 490-510nM PdbU, 9.5-10.5uM Y27632, 1.9-2.1uM RA and 1-3% B27 by volume percentage;
the S4 culture medium is DMEM/F12 culture medium, and further contains 45-55ng/mL KGF, 0.24-0.26uM SANT1, 4.5-5.5ng/mL Activin A, 9.5-10.5uM Y27632, 95-105nM RA and 1-3% B27 by volume percentage.
The induced differentiation is carried out on pancreatic endothelial layer cells, pancreatic progenitor cells are cultured in an S5 culture medium for 4-5 days, and the induced differentiation is carried out on the pancreatic endothelial layer cells; the S5 culture medium is DMEM high-sugar culture medium, and further contains 0.24-0.26uM of SANT1, 95-105nM RA, 0.95-1.05uM of XXI, 9.5-10.5uM of SB431542, 0.9-1.1uM of T3, 19-21ng/mL of Betacellulin and 1-3% of B27 by volume percentage.
The induced differentiation into islet cells was performed using CMRL1066 medium containing 9.5-10.5uM of SB431542 and 0.9-1.1uM of T3 for 4-7 days.
The self-body cell of the invention is immune cell separated from peripheral blood, induced pluripotent stem cell is obtained through in vitro dedifferentiation, and then the induced pluripotent stem cell is directionally induced and differentiated into islet cell to form functional islet cell.
In the invention, the method for obtaining the induced pluripotent stem cells by utilizing the in-vitro dedifferentiation of the immune cells refers to the method in the patent CN 108642014A, and on the basis of the method, four kinds of lentiviruses are combined into one lentivirus, so that the transfection efficiency is improved.
The pattern of inducing directed differentiation of pluripotent stem cells into islet cells is: inducing pluripotent stem cells-definitive endoderm cells-primitive gut tube stage-pancreatic progenitor cells-pancreatic endoderm cells-pancreatic islet cells, and performing directional induction to the pancreatic islet cells by combining small molecule compounds and cytokines.
In the invention, the immune cells obtained by separating the autologous peripheral blood are used for inducing and directionally differentiating into the islet cells, and then the islet cells are transplanted to the same patient, so that the immunological rejection of the patient is reduced. In the process of culturing the islet cells, factors with definite chemical components are added into the culture medium, and animal-derived proteins are not present.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method for culturing the islet cells selects the immune cells separated from the peripheral blood of a human body, induces and directionally differentiates the immune cells into the islet cells, and then transplants the islet cells into the same patient to reduce the immunological rejection of the patient.
(2) When Induced Pluripotent Stem Cells (iPSCs) are directionally induced and differentiated into islet cells, the purity of the finally induced islet cells reaches 62.4% through DTZ staining identification after induced differentiation for 20-28 days.
(3) The islet cells induced by the scheme of the invention have the function of secreting insulin, and the level of the islet cells is equivalent to that of normal pancreatic cells.
Drawings
FIG. 1 is a microscopic view of induced pluripotent stem cells induced by immune cells of the present invention;
FIG. 2 is a microscopic image of pancreatic progenitor cells directionally induced by induced pluripotent stem cells of the present invention;
FIG. 3 is a microscopic image of islet cells after induction according to the present invention;
FIG. 4 is a bar graph of RT-qPCR detection of definitive endoderm cell marker expression;
FIG. 5 is a bar graph of RT-qPCR detection of pancreatic progenitor marker expression;
FIG. 6 is a bar graph of RT-qPCR detection of islet cell marker expression;
FIG. 7 is a bar graph of the amount of insulin in cells measured by ELISA;
FIG. 8 is a microscopic view of DTZ staining results of islet cells.
Detailed Description
The invention is further illustrated by the following specific examples. The use and purpose of these exemplary embodiments are to illustrate the present invention, not to limit the actual scope of the present invention in any way, and not to limit the scope of the present invention in any way.
Example 1 in vitro dedifferentiation of immune cells obtained from peripheral blood of a patient into induced pluripotent stem cells
The methods of this example refer to the methods of inducing pluripotent stem cells in the patent CN 108642014A. Mixing peripheral blood 50mL and physiological saline according to the proportion of 1: dilution at a ratio of 1. The diluted blood was carefully added to the same volume of lymphocyte separation medium to form distinct layers, and centrifuged horizontally at room temperature for 900g for 25 min. At this time, 4 layers were formed from top to bottom in the centrifuge tube, and the second white film layer was taken out and taken out as completely as possible. Adding 2 times of physiological saline, washing cells for 2 times, centrifuging, removing supernatant, and collecting immune cells.
After the immune cells are cultured by RPMI culture medium overnight, the prepared lentiviruses containing four genes of OCT4, SOX2, KLF4 and C-MYC infect the immune cells, and the induced pluripotent stem cells (shown in figure 1) are obtained by culturing, screening and inducing, wherein the purity is more than 95%.
Example 2 Induction of directed differentiation of pluripotent Stem cells into islet cells
Inducing pluripotent stem cells to directionally differentiate and induce endoderm cells, comprising the following steps: the induced pluripotent stem cells after induction were separated with a separation medium, the cell mass was blown off with a pipette, and the cells were seeded on a petri dish coated with 0.1% fibronectin, and when the cell plating reached about 60%, the cells were cultured in S1 medium, and 14ug/ml of CHIR99021 compound was added on the first day to induce the cells. The next day, the culture medium was changed with fresh S1 medium (without adding CHIR99021 compound), and cultured for 2 days to obtain definitive endoderm cells. Cells were collected and expression of definitive endoderm marker genes SOX17, FOXA2 were identified using RT-qPCR.
And continuously differentiating and inducing the endoderm cells into a primitive gut tube, replacing the culture medium with an S2 culture medium, changing the culture medium every other day, and culturing for 3 days to obtain the primitive gut tube cells.
Differentiation into pancreatic progenitor cells was induced by the gastrula cells, and the cells were cultured in two stages using S3 and S4 media. The first stage is the induction of early pancreatic progenitor cells, which requires three days of culture, the first day with replacement of S3 medium with 200nM LDN193189 for one day, and the second day with replacement of fresh S3 medium (without LDN 193189) for 2 days. In the second stage, early pancreatic progenitor cells are induced to become pancreatic progenitor cells, the S4 medium is replaced, the culture is carried out for 5 days, and the medium is replaced every two days. Pancreatic progenitor cells were induced at this stage (see FIG. 2).
The pancreatic progenitor cells are induced to differentiate into pancreatic endothelial cells, which takes 4 days, and at this stage, culture is performed using S5 medium, and fluid exchange is required every other day.
The pancreatic endothelial layer cells are induced to differentiate into islet cells finally, the culture medium used at this stage is CMRL1066 culture medium containing SB431542 (10 uM) and T3 (1 uM), and the fresh culture medium and factors are replaced every other day, and the islet cells are identified by detecting protein markers after culturing for about 4 days (see figure 3).
The induction time for example 2 was 22 days.
In the invention, as a preferred technical scheme, the components of each culture medium are as follows:
the S1 culture medium is DMEM low-sugar medium, and contains 100ng/mL Activin A and 2% by volume of B27.
The S2 medium was DMEM low-sugar medium containing 50ng/mL of KGF and 2% by volume of B27.
The S3 medium was DMEM/F12 medium, which further contained 50ng/mL KGF, 0.25uM SANT1, 500nM PdbU, 10uM Y27632, 2uM RA, 2% by volume of B27.
The S4 medium was DMEM/F12 medium, which further contained 50ng/mL KGF, 0.25uM SANT1, 5ng/mL Activin A, 10uM Y27632, 100nM RA, and 2% by volume B27.
The S5 medium was DMEM high-glucose medium, and further contained 0.25uM of SANT1, 100nM RA, 1uM of XXI, 10uM of SB431542, 1uM of T3, 20ng/mL of Betacellulin, and 2% by volume of B27.
The B27 is a 50-fold concentrated mother liquor.
Example 3 RT-qPCR method for detecting expression of cell-specific markers at various stages
Collecting cells from the last day of S1, S4 and S6 stages, extracting total RNA, synthesizing cDNA, and amplifying target genes by q-PCR by using a conventional method, wherein specific markers for detection comprise SOX17, FOXA2, PDX1, NKX6-1, INS, GCG and SST, and the results are shown in the attached figures 4-6. The primer sequence was synthesized by Beijing Bomaide Biotechnology GmbH, and the primer sequence and annealing temperature of the gene are shown in the following table.
As can be seen from FIGS. 4-6, the endoderm-specific marker SOX17 was strongly expressed in the definitive endoderm stage, FOXA2 was also highly expressed in the definitive endoderm stage, while the pancreatic development-related transcription factor PDX1 and pancreatic endoderm-related transcription factor NKX6-1 were highly expressed in the stage of primitive gut tube cells induced into pancreatic progenitor cells. Following activation of these transcription factors, INS, a marker for beta cells in islets, is also activated to begin expression and continues to be maintained at higher levels, expressing the genes for GCG and SST, two other endocrine cell markers for alpha cells and delta cells in islets in addition to the INS gene. The cells express the corresponding genes according to the law of pancreas development.
Example 4 glucose stimulated insulin Release assay
(1) Grouping experiments: islet cell group, iPSC group, human pancreatic cell group.
(2) Cell preparation: culture supernatants of the islet cell group, the human pancreatic cell group and the iPSC group to be detected are removed, 200uL of sugar-free KRB buffer is added, and the culture is carried out for 1 h.
(3) The supernatant was removed and 200uL of KRB buffer containing 2mmol/l (low sugar) or 22mmol/l (high sugar) glucose was added and after further incubation for 1h, the supernatant was collected into an EP tube.
(4) A human insulin ELISA kit is used for detecting the content of insulin in the supernatant, and the human insulin ELISA kit is purchased from Beijing Solaibao Biotech limited company.
The group of mature-induced islet cells was compared to human pancreatic cells and the insulin content of the cell supernatant was determined by ELISA assay, the results are shown in fig. 7. Under low-sugar and high-sugar stimulation, the insulin secretion level of the induced islet cells is equivalent to that of normal islet cells.
Example 5 identification of islet cells by DTZ staining
(1) After the induction is finished, cell induction liquid in the culture flask is discarded, PBS is added to the culture flask, and the culture flask is gently washed for 3 times and discarded.
(2) Adding DTZ working solution, and incubating for 20min at 37 ℃ in the dark.
(3) Discard the solution, add PBS and wash gently for 3 times, discard the supernatant.
(4) Adding PBS, observing under an inverted microscope and taking a picture, wherein scarlet cells are insulin positive cells.
iPSC was induced to differentiate for 20 days and stained with DTZ, and the results are shown in FIG. 8. The differentiated islet cells are compact mass-like or ball-like tissues, and the DTZ staining is scarlet, which indicates that the induced cytoplasm contains a large amount of zinc ions, and the cells which are not successfully differentiated or immature in differentiation (see background) are light pink or yellowish brown. It can be seen that ipscs were induced to differentiate into islet cells. The induction rate of the islet cells is calculated under a microscope, namely the purity reaches 62.4%.
Claims (2)
1. A method for directionally differentiating into islet cells by utilizing autoimmune cells is characterized in that: the method comprises the steps of obtaining immune cells from peripheral blood of a patient, dedifferentiating the immune cells in vitro into induced pluripotent stem cells, and then directionally differentiating the cells to generate islet cells;
the directional differentiation is used for generating islet cells, and comprises the steps of inducing pluripotent stem cells to directionally differentiate and induce to generate endoderm cells, inducing and differentiate to be primitive gut tube cells, inducing and differentiate to be pancreatic progenitor cells, inducing and differentiate to be pancreatic endothelial layer cells and inducing and differentiate to be islet cells;
the induced pluripotent stem cells are directionally differentiated to induce and generate endoderm cells, cultured by an S1 culture medium containing a CHIR99021 compound, replaced by a fresh S1 culture medium after being cultured for 22-26h, and cultured for 48-70h to obtain definitive endoderm cells;
the S1 culture medium is a DMEM low-sugar culture medium, and additionally contains 95-105ng/mL Activin A and 1-3% of B27 by volume percentage; the concentration of the CHIR99021 compound in the S1 culture medium containing the CHIR99021 compound is 13-15 ug/ml;
inducing and differentiating to primitive gut tube cells, culturing endoderm cells by using an S2 culture medium, and culturing for 68-76h to obtain the primitive gut tube cells; the S2 culture medium is a DMEM low-sugar culture medium, and additionally contains 45-55ng/mL KGF and 1-3% B27 by volume;
the induced differentiation into pancreatic progenitor cells is carried out, the induced differentiation into pancreatic progenitor cells is continued from the primitive gut tube cells, the first stage is to induce early pancreatic progenitor cells, the early pancreatic progenitor cells are firstly cultured in S3 culture medium containing 190-210nM LDN193189 for 22-26h, and the culture medium is replaced with S3 for 24-48 h; in the second stage, early pancreatic progenitor cells are induced into pancreatic progenitor cells, and are cultured for 4-6 days by using an S4 culture medium;
the S3 culture medium is DMEM/F12 culture medium, and further contains 45-55ng/mL KGF, 0.24-0.26uM SANT1, 490-510nM PdbU, 9.5-10.5uM Y27632, 1.9-2.1uM RA and 1-3% B27 by volume percentage;
the S4 culture medium is DMEM/F12 culture medium, and additionally contains 45-55ng/mL KGF, 0.24-0.26uM SANT1, 4.5-5.5ng/mL Activin A, 9.5-10.5uM Y27632, 95-105nM RA and 1-3% B27 by volume percentage;
the induced differentiation is carried out on pancreatic endothelial layer cells, pancreatic progenitor cells are cultured in an S5 culture medium for 4-5 days, and the induced differentiation is carried out on the pancreatic endothelial layer cells; the S5 culture medium is a DMEM high-sugar culture medium, and further comprises 0.24-0.26uM of SANT1, 95-105nM RA, 0.95-1.05uM of XXI, 9.5-10.5uM of SB431542, 0.9-1.1uM of T3, 19-21ng/mL of Betacellulin and 1-3% of B27 by volume percentage;
the induced differentiation into islet cells was performed using CMRL1066 medium containing 9.5-10.5uM of SB431542 and 0.9-1.1uM of T3 for 4-7 days.
2. The method for the directed differentiation of the autoimmune cells into the islet cells according to claim 1, wherein: the purity of the induced pluripotent stem cell is not less than 95%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010212266.8A CN111269875B (en) | 2020-03-24 | 2020-03-24 | Method for directionally differentiating into islet cells by using autoimmune cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010212266.8A CN111269875B (en) | 2020-03-24 | 2020-03-24 | Method for directionally differentiating into islet cells by using autoimmune cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111269875A CN111269875A (en) | 2020-06-12 |
| CN111269875B true CN111269875B (en) | 2022-04-08 |
Family
ID=70995886
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010212266.8A Active CN111269875B (en) | 2020-03-24 | 2020-03-24 | Method for directionally differentiating into islet cells by using autoimmune cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111269875B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112680405B (en) * | 2021-01-19 | 2022-05-10 | 中山大学 | Human endoderm differentiation medium and culture method |
| CN113046299A (en) * | 2021-03-19 | 2021-06-29 | 上海爱萨尔生物科技有限公司 | Additive for preparing islet beta cells by inducing pluripotent stem cells to differentiate directionally |
| CN112961823B (en) * | 2021-03-19 | 2024-01-23 | 上海爱萨尔生物科技有限公司 | Culture solution for preparing islet beta cells by inducing directional differentiation of pluripotent stem cells |
| CN114807013B (en) * | 2022-04-25 | 2023-11-17 | 宫念樵 | Method for differentiating peripheral blood stem cells into endoderm cells and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139573A (en) * | 2007-08-22 | 2008-03-12 | 中日友好医院 | Method for forming pancreatic islet like structure by external evoked stem cell differentiation pancreatic islet cell |
| CN105579577A (en) * | 2013-06-11 | 2016-05-11 | 哈佛学院校长同事会 | SC-beta cells and compositions and methods for generating the same |
| CN108642014A (en) * | 2018-05-17 | 2018-10-12 | 山东兴瑞生物科技有限公司 | The method for preparing retinal pigment epithelium thin slice using autoimmune cell |
-
2020
- 2020-03-24 CN CN202010212266.8A patent/CN111269875B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101139573A (en) * | 2007-08-22 | 2008-03-12 | 中日友好医院 | Method for forming pancreatic islet like structure by external evoked stem cell differentiation pancreatic islet cell |
| CN105579577A (en) * | 2013-06-11 | 2016-05-11 | 哈佛学院校长同事会 | SC-beta cells and compositions and methods for generating the same |
| CN108642014A (en) * | 2018-05-17 | 2018-10-12 | 山东兴瑞生物科技有限公司 | The method for preparing retinal pigment epithelium thin slice using autoimmune cell |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111269875A (en) | 2020-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111269875B (en) | Method for directionally differentiating into islet cells by using autoimmune cells | |
| CN109749986B (en) | Method for obtaining pancreatic precursor cells and pancreatic beta cells by differentiation of human pluripotent stem cells | |
| Wang et al. | Conversion of human gastric epithelial cells to multipotent endodermal progenitors using defined small molecules | |
| CN102307991B (en) | Multipotential stem cell culture on microcarrier | |
| JP4336821B2 (en) | Induction of cardiomyocytes using mammalian bone marrow cells or cord blood-derived cells and adipose tissue | |
| CN113151157B (en) | Passage method and application of endoderm stem/progenitor cells obtained by reprogramming epithelial cells derived from digestive tract | |
| CN106795487A (en) | The enrichment procedure of pancreatic progenitor cell | |
| JPWO2008066199A1 (en) | Method for inducing differentiation of embryonic stem cells into insulin-secreting cells, insulin-secreting cells induced by the method, and use thereof | |
| CN101724602B (en) | Method for the differentiation of human adult stem cells into insulin-secreting cells | |
| CN114807015B (en) | Induction method for promoting islet alpha cells to be converted into beta cells and application thereof | |
| CN117264892B (en) | In vitro preparation method of medial ganglion bulge progenitor cells and GABA (gamma-amino acid-gamma-amino acid) interneurons | |
| CN111575227B (en) | Method for establishing human-derived diabetic cardiomyopathy model | |
| CN114395523B (en) | Kit for inducing stem cells to differentiate into hepatocytes and application thereof | |
| CN115975914A (en) | Method for inducing pluripotent stem cells by reprogramming of chemical small molecule drugs | |
| CN103881962A (en) | Method of establishing rat pancreatic duct epithelioid stem cell system | |
| CN109486770B (en) | Method for promoting directional differentiation of human iPS into islet beta cells by using microRNA-181c-5p | |
| RU2663118C1 (en) | Cell product of insulin-producing mammalian cells and use thereof for diabetes mellitus therapy | |
| CN116144584B (en) | Pancreatic progenitor cells differentiated from human induced pluripotent stem cells, and preparation method and application thereof | |
| CN115011544B (en) | Method for obtaining islet delta cells by in vitro induction and application thereof | |
| CN110592007A (en) | Mesenchymal stem cell and preparation method and application thereof | |
| CN116240163B (en) | Culture solution for inducing differentiation of mesenchymal stem cells to islet-like cells and application thereof | |
| CN115044534B (en) | Method for producing islet beta cells in vitro by utilizing BMP7 factor, obtained islet beta cells and application | |
| CN116426468A (en) | Method for inducing mesenchymal stem cells to differentiate into islet-like cell masses | |
| CN1609198A (en) | Method of differentiating human bone marrow matrix stem cell to form insulin cell | |
| CN116333971A (en) | Method for improving differentiation efficiency of human induced pluripotent stem cells to insulin-producing cells by co-culturing human mature islet cells and application |
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 |