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A method for inducing mesenchymal stem cells to differentiate into islet-like cells

A technique for islet-like cells and stromal cells, which is used to induce stem cells to differentiate into islet-like cells in vitro. The application of islet-like cells can solve the problem of inability to maintain stable glucose levels, high cost of pancreas/islet separation, and the need for inflammatory responses. Multiple transplantations and other problems to achieve the effect of improving the efficiency of induction and differentiation, reducing the risk of clinical use, and improving the ability of insulin secretion

Active Publication Date: 2019-12-27
天晴干细胞股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pathogenesis of type I diabetes is mainly caused by the autoimmune reaction leading to the destruction of pancreatic β cells and the inability to maintain the stability of glucose levels
Islet transplantation, especially for critically ill patients, is a more direct treatment, but due to the lack of donors, the high cost of pancreas / islet isolation, the inflammatory response to transplantation requiring multiple transplants, and long-term administration of immune rejection drugs, etc. Not widely used clinically

Method used

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  • A method for inducing mesenchymal stem cells to differentiate into islet-like cells
  • A method for inducing mesenchymal stem cells to differentiate into islet-like cells
  • A method for inducing mesenchymal stem cells to differentiate into islet-like cells

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specific Embodiment approach 1

[0021] Specific embodiment one: A method of inducing mesenchymal stem cells to differentiate into islet-like cells according to this embodiment, it is carried out according to the following steps:

[0022] 1. Isolate and obtain mesenchymal stem cells from umbilical cord, placenta or adipose tissue, and subculture;

[0023] 2. Preparation of Differentiation Medium

[0024] The differentiation medium is a brain tissue conditioned medium, a cell differentiation induction medium and a stem cell conditioned medium;

[0025] The preparation process of the brain tissue conditioned medium is as follows: under sterile conditions, take out the brain of a 6-7 day old rat, wash it with PBS or saline, centrifuge at 1000rpm for 6min, discard the supernatant, and use 10% FBS in DMEM Resuspend the pellet in the high-glucose medium, mash the brain tissue into single cells, collect the brain tissue cells and culture them with 10% FBS DMEM high-glucose for 1 day, add cytarabine at a concentrati...

specific Embodiment approach 2

[0029] Embodiment 2: This embodiment is different from Embodiment 1 in that: the specific process of differentiating the umbilical cord, placenta or adipose tissue mesenchymal stem cells into islet-like cells after subculture in Step 1 is as follows:

[0030] The mesenchymal stem cells of the umbilical cord, placenta or adipose tissue subcultured in step 1 to the P3 generation were mixed with 4×10 4 Inoculum / mL inoculum was inoculated in a 6-well plate, and cultured in DMEM / F12 medium containing 10% FBS. After 24 hours of culture, BTCM was replaced for 7 days, and BTCM was replaced every 2 days. After 7 days of culture, cells were used The differentiation induction medium was cultured for 7 days, the induction medium was changed every 2 days, and then the cell differentiation induction medium and SCM were co-cultured at a volume ratio of 1:1 for 14 days to obtain mature islet-like cells. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0031] Specific embodiment three: the difference between this embodiment and specific embodiment one is: the described umbilical cord mesenchymal stem cell isolation and subculture process are as follows:

[0032] Take 15-20cm umbilical cords of full-term fetuses, wash them with normal saline and 75% alcohol by volume, cut them into lengths of 2-3cm, separate the arteries and veins, and separate the Wharton’s jelly in the middle. Cut into 0.5~2mm under sterile conditions 3 The tissue block was inoculated according to 1g / T75 bottle, cultured with 10% FBS medium, and the medium was changed after 5 days. After the cell fusion degree reached more than 80%, it was digested by trypsin with a concentration of 0.125% by volume, and obtained Single cell, according to 8×10 5 Cells / T75 bottle inoculated, cultured with 10% FBS medium, placed in a carbon dioxide incubator, and passaged after the cell fusion degree reached more than 90%, at the same time, the immunolabeling of umbilical co...

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Abstract

The invention relates to a method for inducing mesenchymal stem cells to differentiate into islet-like cells, and aims at providing the method for differentiating the mesenchymal stem cells coming different sources into the islet-like cells to obtain a large number of the islet-like cells. In order to achieve the aim, the method particularly comprises the following steps: 1) carrying out separation and primary culture on the mesenchymal stem cells of umbilical cord, placenta or fat sources; 2) preparing a differentiation medium: preparing BTCM, cell differentiation induced liquid and SCM; 3) inducing the mesenchymal stem cells to differentiate into the islet-like cells in the third stage; 4) obtaining identification of the islet-like cells. The islet-like cells obtained through the method are high in maturity, and less number of the cells can reach a fast hypoglycemic effect, so that the method for inducing the mesenchymal stem cells to differentiate into the islet-like cells has wide application prospects and is applied to the field of biomedicine.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a method for inducing stem cells to differentiate into islet-like cells in vitro and the application of the islet-like cells. Background technique [0002] Diabetes mellitus is a chronic metabolic, multifactorial disease, caused by absolute (type I diabetes) and relative (type II diabetes) insulin deficiency leading to hyperglycemia. There are no obvious symptoms in the early stage, and there will be more than three and one less in the symptomatic stage, which is often accompanied by damage to multiple body systems, seriously injuring the body, and even threatening life. The pathogenesis of type I diabetes is mainly due to the autoimmune reaction leading to the destruction of pancreatic β cells, which cannot maintain the stability of glucose level. Islet transplantation, especially for critically ill patients, is a more direct treatment, but it is limited due to the lack of donors, th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/0775
CPCC12N5/0613C12N2500/38C12N2501/06C12N2506/1384C12N2506/1392
Inventor 孟庆雪张怡刘艳青
Owner 天晴干细胞股份有限公司
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