Method for in vitro induction of human amniotic mesenchymal stem cells (hAMSCs) differentiated into insulin-secreting cells (ISCs)

A technology of insulin secretion and mesenchymal stem cells, applied in the field of insulin secretion cells, can solve problems such as immune rejection and technical difficulty limitations

Inactive Publication Date: 2012-03-28
AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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Problems solved by technology

Although pancreas or islet transplantation is expected to be an ideal method for the treatment of type 1 diabetes,

Method used

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Embodiment

[0013] A method of inducing human amniotic mesenchymal stem cells to differentiate into insulin-secreting cells in vitro of the present invention comprises the following steps:

[0014] (1) Isolation of hAMSCs: Cut the human amniotic membrane into pieces, add 0.05% trypsin digestion solution containing 0.02% EDTA, rotate and digest at 37°C, 200 rpm for 10 minutes, discard the supernatant; 37°C, 200 rpm spinning for 30 minutes, discard the supernatant, keep the undigested amnion fragments, wash twice with D-Hank's solution, add 0.75 mg / ml collagenase containing 0.075 mg / ml DNase I , at 37°C, 200 rpm for about 2 h until the tissue was completely digested, filtered through a 300-mesh steel mesh, collected the cell filtrate, centrifuged at 1500 rpm for 10 minutes, and collected the cell pellet, namely the original hAMSCs;

[0015] (2) Purification of hAMSCs: The isolated original hAMSCs were resuspended in low-glucose-DMEM medium (purchased from Gibco, catalog number: 31600-026; c...

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Abstract

The invention discloses a method for the in vitro induction of hAMSCs differentiated into ISCs. According to the method, the extracted hAMSCs are cultured for 7 days by a serum-free high sugar-DMEM induction medium containing niacinamide of 10mmol/L and an N2 supplement. The hAMSCs induced by the method of the invention express insulin gene mRNA and PDX-1 gene mRNA, and the content of insulin in the supernatant of the culture reaches above 330muIU/ml; and the method of the invention allows the in vitro induction of the hAMSCs differentiated into the ISCs to be realized.

Description

technical field [0001] The invention relates to a method for inducing human amniotic mesenchymal stem cells to differentiate into insulin-secreting cells in vitro. Background technique [0002] Human amniotic mesenchymal stem cells (hAMSCs) have the advantages of multilineage differentiation, low immunogenicity, abundant sources, easy enrichment, strong expansion ability, and no medical ethical issues involved. It has broad application prospects. It has been proved that hAMSCs can differentiate into endothelial and hepatic epithelial cells, cardiomyocyte-like cells, neuron-like cells, osteoblasts, etc. under appropriate induction conditions, but whether they can differentiate into insulin-secreting pancreatic B cells has not been found so far. introduce. [0003] The occurrence of type 1 diabetes is related to genetic factors, environmental factors and immune mechanism, and its fundamental etiology and pathogenesis is insufficient insulin secretion caused by the destructio...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 方宁陈代雄赵玉洁
Owner AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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