Method for inducing and differentiating human embryonic stem cells into mesenchymal stem cells

A technology for inducing differentiation and cells, applied in embryonic cells, biochemical equipment and methods, germ cells, etc., can solve the problems of difficulty in obtaining MSCs with uniform quality, incapable of precise quality control in the intermediate process, and achieve low production cost and simple operation Effect

Active Publication Date: 2020-10-30
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The current methods for inducing hESCs to differentiate into MSCs, due to the lack of understanding of the intermediate process, all use the same medium for extensive culture, and it is impossible to accurately control the intermediate process, making it difficult to obtain MSCs with uniform quality

Method used

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  • Method for inducing and differentiating human embryonic stem cells into mesenchymal stem cells
  • Method for inducing and differentiating human embryonic stem cells into mesenchymal stem cells
  • Method for inducing and differentiating human embryonic stem cells into mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 PS induction stage, obtaining PS

[0067] The hESC line used in this example is WA09, which was purchased from WiCell Research Center and signed relevant regulations.

[0068] (1) Dissociate hESCs into single cells with Accutase, inoculate 6-well plates coated with Matrigel at a ratio of 1:10, and each well contains 2 ml of mTeSR1 medium (added with ROCK inhibitor Y-27632) .

[0069] (2) After culturing overnight, discard the original medium, wash the hESC briefly with DMEM / F12, and add PS induction medium (mTeSR1 no selected factors (Promega Stemcell, Cat. No. 05896)) as the basic medium, supplemented with 35 ng / mL Activin A (R&D Systems, Cat. No. 338-AC)+4μM CHIR99021 (Selleckchem, Cat. No.: S1263)+19ng / mL bFGF (Peprotech, Cat. No.: 100-18B)+60nM PIK90 (Selleckchem, Cat. No.: S1187 )) were incubated for 24 hours.

[0070](3) The positive expression of OCT4 is a sign that hESC cells maintain their pluripotency, and the positive expression of T is a sign of...

Embodiment 2

[0071] Example 2 Lateral Mesoderm Induction Stage, Obtaining Lateral Mesoderm Cells

[0072] (1) Take the PS obtained in Example 1, discard the original medium, wash the hESC briefly with DMEM / F12, and add the lateral mesoderm induction medium (mTeSR1 no selected factors (Promega Stemcell, Cat. No. 05896)) as the basic culture base, added with 1.2 μM A-83-01 (Selleckchem Company, catalog number: S7692) + 260 nM LDN-193189 (Selleckchem Company, catalog number: S2618) + 16 ng / mL BMP4 (Peprotech Company, catalog number: 120-05) + 3 μM CHIR99021 (Selleckchem company, product number: S1263) + 21ng / mL bFGF (Peprotech product number: 100-18B)), cultured for 2 days. The culture medium was changed every day, and the cells were subcultured when the growth rate reached more than 90%.

[0073] (2) The positive expression of HAND1 is a sign of lateral mesoderm cells. After the induction of the lateral mesoderm, on the 4th day, immunofluorescence staining was performed, rinsed with 0.01M ...

Embodiment 3

[0074] Example 3 MSCs induction stage, obtaining mature MSCs

[0075] (1) Take the lateral mesoderm cells obtained in Example 2, and pass the lateral mesoderm cells 1:6 into Matrigel-coated 6-well plates, each well containing 2ml of lateral mesoderm medium. After overnight, discard the original medium, wash the wells with D-PBS (no calcium and magnesium ions), add MSCs induction medium (low sugar DMEM (GIBCO company, product number C11885500BT) + 10% FBS + 1% penicillin-strand Mycin solution + 4mM glutamine + 8ng / ml bFGF (Peprotech product number: 100-18B) + 6ng / ml PDGF-AB (Peprotech product number: 100-00AB) + 11ng / ml EGF (Peprotech product number: 100-61) + 35 μg / ml ascorbic acid), cultivated for 3-6 days, and replaced half of the fresh medium every day. Subculture was performed when the cells grew to 80-90%.

[0076] (2) Subculture the cells 1:3 into a gelatin-coated 6-well plate, each well containing 2ml of MSCs induction medium, after overnight, discard the original med...

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Abstract

The invention provides a method for inducing and differentiating human embryonic stem cells (hESCs) into mesenchymal stem cells (MSCs). The method mainly relates to three stages, including induction of primitive streak (PS) cells, differentiation to outer mesoderm cells and differentiation of the MSCs. According to the method, a staged differentiation method is adopted, the whole differentiation process can be tracked and subjected to quality control, the cost is low, and operation is easy. An intermediate product of hESCs induced and differentiated into MSCs is obtained and comprises PS cells, outer mesoderm cells and MSCs, more MSCs can be efficiently generated, the multiplication time is shorter, the MSCs can be repeated, and mature MSCs can be obtained in 17 days at the shortest time,wherein the differentiation induction culture media in the first two stages are serum-free culture media with clear components, so that differentiated MSCs are prevented from being polluted by exogenous substances, and the uniformity and clinical applicability of the differentiated MSCs are guaranteed.

Description

technical field [0001] The invention relates to the technical field of biology and medicine, in particular, the invention relates to a method for inducing differentiation of human embryonic stem cells (hESCs) into mesenchymal stem cells (MSCs). Background technique [0002] Mesenchymal stem cells (MSCs) have attracted extensive attention due to their unique biological characteristics, multiple functions and potential therapeutic value. Due to their regenerative capacity and immunomodulatory properties (Le et al., 2012), MSCs have been used to treat various degenerative and inflammatory diseases (Shi et al., 2018), such as articular cartilage damage, graft-versus-host disease and myocardial ischemia. MSCs are found in almost all connective tissues, such as umbilical cord and blood, amniotic membrane and body fluids, placenta, bone marrow, fat and dental pulp. MSCs have high proliferative capacity and plasticity, can differentiate into many somatic cell lineages, and can mig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/0735
CPCC12N5/0662C12N2506/02C12N2501/115C12N2501/16C12N2501/727C12N2501/155C12N2501/15C12N2500/32C12N2501/135C12N2501/11C12N2500/38
Inventor 柳华周珂
Owner ZHEJIANG UNIV
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