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Method for promoting directional differentiation of human multipotent stem cells into endothelial cells

A technology of human pluripotent stem cells and endothelial cells, which is applied in the field of promoting directional differentiation of human pluripotent stem cells into endothelial cells, which can solve the problems of low differentiation efficiency, unsuitability for large-scale cultivation and differentiation of endothelial cells, and high cost, so as to eliminate various factors interference, convenient and fast differentiation process, and reliable differentiation results

Active Publication Date: 2019-05-24
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are the following defects in the differentiation of endothelial cells: the cost is too high, the price of mTeSR medium, StemDiffAPEL, and ECGM-MV2 medium is too high, and it is not suitable for mass culture and differentiation of endothelial cells; the existing technology has low differentiation efficiency and low yield

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  • Method for promoting directional differentiation of human multipotent stem cells into endothelial cells
  • Method for promoting directional differentiation of human multipotent stem cells into endothelial cells
  • Method for promoting directional differentiation of human multipotent stem cells into endothelial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Inducers of Directed Differentiation and Small Molecular Compounds

[0033] Induced factors: CHIR99021, the effect concentration is 6μM; basic fibroblast growth factor (bFGF), the effect concentration is 100ng / μl; vascular endothelial cell growth factor (VEGF), the effect concentration is 50ng / μl; bone morphogenic protein 4 (BMP4), the effect Concentration 25ng / μl;

[0034] Small molecular compound: retinoic acid (RA), the concentration of action is 1 μM.

Embodiment 2

[0035] Example 2: Culture and passage of embryonic stem cells

[0036] On Matrigel-coated culture dishes, use PSeasy-E8 medium to culture human embryonic stem cells (hESCs). When the confluence of the cells reaches 80% and above, remove the medium, add DPBS to wash once; Incubate in a stable incubator for 3-5 minutes; suck off the digestion solution, immediately add fresh PSeasy-E8 medium, fan the bottom of the culture dish with a pipette gun to make the cell colony attached to the bottom of the dish fall off, gently and slowly blow and mix uniform. Then transfer the cells to a new Matrigel-coated cell culture plate at a ratio of 1:8 or 1:10, and use Pseasy-E8 medium for subculture, and add 10 μM ROCK to the medium for the first 24 hours after subculture. -I(Y27632), then replace with new culture medium every day until the next passage.

[0037] The undifferentiated human embryonic stem cells were photographed under an inverted fluorescence microscope, and the results were a...

Embodiment 3

[0038] Example 3: In vitro directed differentiation of endothelial cells

[0039] Continue culturing with PSeasy-E8 medium until the density is 95%-100%, then replace the differentiation medium for induction. The differentiation medium is CDM3 (containing RPMI Medium 1640 basal medium, double antibody, bovine serum albumin and ascorbic acid).

[0040] Retinoic acid treatment group (RA group): Day 0 was recorded from the time of induction, and 6 μM CHIR99021 was added during the first two days of differentiation (day 0-day 1). On day 2, the medium was replaced with CDM3 and 50 ng / ml bFGF and 1 μM RA were added. From the 3rd day to the 5th day, the culture medium was changed to CDM3 supplemented with 50ng / ml VEGF and 25ng / ml BMP4. On the 6th day, it was digested with 0.1% trypsin or Accutase, and then inoculated into Gelatin-coated 60mm culture dishes for 3-4 days, and the culture medium was replaced with ECM.

[0041] Control group (DMSO group): the culture process was the sam...

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Abstract

The invention discloses a method for promoting directional differentiation of human multipotent stem cells into endothelial cells. The method includes: from day 0 to day 1, performing induced differentiation on human multipotent stem cells with cell density reaching 95% or above by use of a CDM3 differential medium containing 4-8micron of GSK3I; on the second day, performing further induced differentiation on the cells subjected to induced culture in the last step by use of a CDM3 differential medium containing 40-60ng / ml of bFGF; from day 3 to day 5, performing further induced differentiationon the cells subjected to induced culture in the last step by use of a CDM3 differential medium containing 40-60ng / ml of VEGF and 20-30ng / ml of BMP4; on the sixth day, digesting the cells subjected to induced culture in the last step, and adopting an endothelial cell culture medium to continue culture for 3-4 days. The method is economical and effective, and convenience and quickness in a differentiation process are achieved; serum consumption is avoided in a differentiation process, interferences of various factors are eliminated, and differentiation results are high in reliability; by adding of RA, endothelial differentiation efficiency can be promoted, and more endothelial cells can be obtained in once differentiation.

Description

technical field [0001] The invention relates to a method for promoting directional differentiation of human pluripotent stem cells into endothelial cells, belonging to the technical field of cell culture. Background technique [0002] Vascular disease affects the health of millions of people. Severe vascular disease leads to amputations and life-threatening strokes. Endothelial cells line the lumen of all blood vessels and play a crucial role in regulating vascular permeability, angiogenesis and tissue regeneration. Under physiological conditions, blood vessels can be generated by angiogenesis and angiogenesis. Angiogenesis refers to the formation of new blood vessels from progenitor cells whereas angiogenesis refers to the formation of new blood vessels from existing blood vessels through the migration and proliferation of existing blood vessel cells. However, these processes are impaired in patients with stroke, diabetes or Alzheimer's disease. In vascular diseases, ab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 胡士军雷伟杨壮壮赵振奥
Owner SUZHOU UNIV
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