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In-vitro efficient preparation method and application of mesenchymal stem cells derived from human inducible pluripotent stem cells

A technology of pluripotent stem cells and mesenchymal stem cells, applied in the field of high-efficiency preparation of mesenchymal stem cells in vitro, can solve the problems of long differentiation cycle, low efficiency and complicated differentiation operation of mesenchymal stem cells

Active Publication Date: 2020-10-27
云南东森生物科技有限公司
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0007] Aiming at the technical defects of human induced pluripotent stem cell-derived mesenchymal stem cells with long differentiation cycle, complicated differentiation operation and low production efficiency in vitro in the prior art, the present invention provides the following solutions

Method used

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  • In-vitro efficient preparation method and application of mesenchymal stem cells derived from human inducible pluripotent stem cells
  • In-vitro efficient preparation method and application of mesenchymal stem cells derived from human inducible pluripotent stem cells
  • In-vitro efficient preparation method and application of mesenchymal stem cells derived from human inducible pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Culture of human induced pluripotent stem cells.

[0029] 1. GFR matrigel plating: according to the GFR matrigel (Corning) instruction manual, after diluting with DMEM-F12 medium, transfer to a six-well cell culture plate (1mL / well) with a pipette, and transfer to 37°C cell culture box incubate for 1 hour for later use;

[0030] 2. Digestion of human induced pluripotent stem cells: According to the Dispase enzyme product manual (StemCell Technology), human induced pluripotent stem cells (hiPSCs) were digested at 37°C, 5% CO 2 Incubate and digest in a cell incubator for 5 minutes to single cells, resuspend in 5 mL of DMEM-F12 medium and pipette to single cells with a 10 mL pipette, then centrifuge (at room temperature, 300g for 5 minutes), then maintain with 3 mL of human pluripotent stem cells The above hiPSCs were resuspended in medium (Gibco E8 medium, with a final concentration of 10 nM Y-27632 added) and counted for later use.

[0031] Inoculation of hu...

Embodiment 2

[0032] Example 2: Differentiation of human induced pluripotent stem cells into mesenchymal stem cells.

[0033] 1. Preparation of hiPSCs-to-MSCs-inducing differentiation medium: prepare in advance the induction-differentiation basal medium (3% fetal bovine serum, DMEM-F12 medium, 5nM Y-27632), in which the experimental group adds corresponding small chemical molecules (PCI- 24781, EPZ011989, J147, UNC1999, TG101348, CUDC-101, MS-275, BI 2536, LLY-507, Ginkgolide C, BI-7273, CPI-637, OTX015, UNC1215, EPZ6438, LBH589, GSK126, UNC0379, SP2 (+)-JQ-1, PFI-3, UNC669, CPI-455, OICR-9429, MS023, AZD5153, EED226), the final concentration was 10nM / mL, and the control group was the basal medium for inducing differentiation (without adding chemical small molecular).

[0034] 2. Cell treatment: Observe the morphology and density of hiPSCs under a microscope. When the cells grow into small clones, add the original old human pluripotent stem cell maintenance medium (Gibco E8 medium, and add...

Embodiment 3

[0040] Example 3: In vitro functional identification of mesenchymal stem cells (hiPSC-MSCs) produced by differentiation of human induced pluripotent stem cells.

[0041] 1. Adipogenic, osteogenic and chondrogenic differentiation of hiPSC-MSCs in vitro: according to 6×10 4 Cells / well were seeded in six-well cell-adherent culture plates (Corning), and when hiPSC-MSCs grew adherently until the cell confluence reached 70%-80%, the hiPSC-MSCs maintenance medium was discarded and the in vitro directional induction and differentiation were performed as follows.

[0042] (1) In vitro induced adipogenic differentiation culture system: After discarding the mesenchymal stem cell maintenance medium (DMEM-F12 medium, adding 10% fetal bovine serum, 10ng / ml bFGF, 4ng / ml EGF), replace Stem Cell company The MensenCult adipogenic differentiation medium was replaced with a fresh above-mentioned adipogenic differentiation medium every 3.5 days, and the culture continued until the 18th day. The d...

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Abstract

The invention discloses an in-vitro efficient preparation method and application of mesenchymal stem cells derived from human inducible pluripotent stem cells. The preparation method comprises the following steps: 1) culturing and amplifying human inducible pluripotent stem cells (hiPSCs); 2) through the strategy of library screening and combination optimization of chemical small molecules, jointly treating the hiPSCs for nine days by utilizing two chemical small molecules, namely LLY-507 and AZD5153 so as to obtain hiPSC-derived mesenchymal precursor cells with a CD73 positive mesenchymal stem cell proportion of 80% or above; 3) subculturing the mesenchymal precursor cells twice to obtain further mature CD73<+>CD105<+> hiPSC-derived mesenchymal stem cells (hiPSC-MSCs); and 4) identifyingthe prepared CD73<+>CD105<+> hiPSC-derived cells as mesenchymal stem cells on the basis of immunological means and cytobiological analysis. Experimental results prove that through usage of the chemical small molecule combination treatment strategy, the efficiency of producing the mesenchymal stem cells by differentiation of the hiPSCs can be substantially improved; and low-expression pluripotencyrelated marker molecules of the hiPSC-MSCs have the typical functions of adipogenesis, osteogenesis and chondrogenesis differentiation and low immunogenicity, and can be used for treating immune related diseases.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, is related to cell therapy technology and products, and specifically relates to a high-efficiency preparation method and application of mesenchymal stem cells derived from human induced pluripotent stem cells in vitro. Background technique [0002] Mesenchymal stem cells (mesenchymal stem / stromal cells, MSCs) have unique biological properties of hematopoietic support and immune regulation, and can be directed to adipogenic, osteogenic and chondrogenic differentiation in vitro, and are widely involved in various tissue repair and reconstruction. Therefore, it has superior regenerative medical value. MSCs were first isolated from bone marrow in the 1960s, and were subsequently isolated and identified from dental pulp, umbilical cord, fat and other tissues. Existing preclinical and clinical studies have shown that MSCs play a therapeutic and improving role in a variety of blood and immune-relat...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61K35/28A61P37/00
CPCC12N5/0662A61K35/28A61P37/00C12N2506/45C12N2501/999
Inventor 张磊升权海宗
Owner 云南东森生物科技有限公司
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