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A method for promoting directed differentiation of human pluripotent stem cells into endothelial cells

A technology for human pluripotent stem cells and endothelial cells, which is applied in the field of promoting the directional differentiation of human pluripotent stem cells into endothelial cells, can solve the problems of low differentiation efficiency, unsuitable for mass culture and differentiation of endothelial cells, and high cost, and eliminates various factors. The effect of the interference, the differentiation process is convenient and fast, and the differentiation results are reliable.

Active Publication Date: 2021-06-01
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, StemDiff APEL, 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

Method used

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  • A method for promoting directed differentiation of human pluripotent stem cells into endothelial cells
  • A method for promoting directed differentiation of human pluripotent stem cells into endothelial cells
  • A method for promoting directed differentiation of human pluripotent 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 the Matrigel-coated culture dish, use PSeasy-E8 medium to culture human embryonic stem cell H1 line. When the confluence of the cells reaches 80% or above, suck out the medium, add DPBS to wash once; add EDTA, and stabilize at 37°C. Incubate in the 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 well . 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 cell line H1 was photographed under an inverted fluorescence ...

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 pluripotent stem cells into endothelial cells. The method of the invention comprises: using CDM3 differentiation medium added with 4-8 μM GSK3I on the 0th to 1st day to increase the cell density to more than 95%. Human pluripotent stem cells were induced to differentiate; on the second day, the cells after the previous induction culture were further induced to differentiate by using CDM3 differentiation medium supplemented with 40-60 ng / ml bFGF; The CDM3 differentiation medium of ml VEGF and 20-30ng / ml BMP4 further induces the differentiation of the cells after the induction culture in the previous step; digest the cells after the induction culture in the previous step on the 6th day, and then use the endothelial cell culture medium to continue the culture for 3~ 4 days. The method for differentiating endothelial cells of the present invention is more economical and effective, and the differentiation process is more convenient and fast; no serum is used in the differentiation process, the interference of various factors is eliminated, and the differentiation result is more reliable; and the addition of RA can promote the efficiency of endothelial differentiation, and one-time differentiation More endothelial cells can be obtained.

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 Patents(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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