Method for obtaining endothelial cells from human pluripotent stem cells by efficient differentiation

A technology of human pluripotent stem cells and endothelial cells, applied in the field of biopharmaceuticals, can solve the problems of high cost, long time-consuming embryoid bodies, and low efficiency

Active Publication Date: 2020-07-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the differentiation of embryoid bodies often takes a long time, resulting in high cost and low efficiency of this method.
Relatively speaking, the monolayer cell differentiation method has high efficiency, but still needs further improvement to better understand the complex factors

Method used

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  • Method for obtaining endothelial cells from human pluripotent stem cells by efficient differentiation
  • Method for obtaining endothelial cells from human pluripotent stem cells by efficient differentiation
  • Method for obtaining endothelial cells from human pluripotent stem cells by efficient differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Differentiation, purification and culture of endothelial progenitor cells

[0084] When human ESCs / iPSCs grew to 80%-90%, they were isolated with Accutase (Gibco#A11105-01) and 3×10 4 Cells / well were cultured in vitronectin (Cauliscell Inc. #500125)-bottomed 12-well cell culture plates supplemented with 25 ng / mL Actin A (R&D, Cat# 338-AC), 10 μM Y27632 and 10 ng / mL ESC / iPSC were differentiated into mesodermal endothelial cells by cultured in E8 medium (Gibco A1516901) with mL bone morphogenetic protein 4 (R&D, catalog number 314-BP) for 3 days. Mesodermal endothelial cells were incubated with 100 ng / ml FGF2 (R&D, Cat #233-FB), 50 ng / ml VEGF (R&D, Cat #293-VE), 50 ng / ml BMP4, 5 μM SB431542 (Sigma-Aldrich, CAS 301836-41 -9-Calbiochem) in E6 medium (Gibco A1516401) for 4 days to produce EPCs, and the whole differentiation process lasted for 7 days. Finally, the cell suspension was prepared, counted, and prepared for EPCs isolation. EPCs were sorted by magnetic beads usi...

Embodiment 2

[0087] Endothelial progenitor cells from adult peripheral blood samples

[0088] EPCs were isolated from human peripheral blood (PB). Fresh human peripheral blood (20mL) was obtained under full ethical approval. Mononuclear cells (MNCs) were isolated from PB by density gradient centrifugation and cultured in EGM2 (Lonza) medium supplemented with 16% fetal bovine serum.

Embodiment 3

[0090] Human pluripotent stem cell culture

[0091] ESC / iPSC requires E8 medium or mTeSR TM 1Complete medium (Cat#85850), or hPSC-CDM (Cauliscell Inc.#400105) medium containing hPSC-CDM supplement factor (Cauliscell Inc.#600301) on Matrigel (BD Bioscences#356230) Cultured in a six-well cell culture plate, digested with 500μM EDTA for 3-5min for passage. hPSC-EPCs were maintained in EGM2+16% fetal bovine serum (HyClone) medium.

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Abstract

The invention relates to a method for obtaining endothelial cells from human pluripotent stem cells by efficient differentiation. The method comprises following steps: step 1, the human pluripotent stem cells are cultured in a culture medium containing Actin A, BMP4 and Y27632 for 3 days, and are differentiated into mesoderm endothelial precursor cells; and step 2, the culture medium is changed into a culture medium containing FGF2, VEGF, BMP4 and SB431542 on the fourth day, the human pluripotent stem cells are cultured for 4 days, and endothelial progenitor cells are obtained.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals. Specifically, it provides an efficient and economical method for culturing endothelial cells, and the obtained endothelial progenitor cells can be used for cell therapy and drug screening of cardiopulmonary diseases including pulmonary hypertension. Background technique [0002] Pulmonary hypertension (PH) is a disease with poor prognosis, high disability and mortality. In the early stage of PH, the pulmonary vascular resistance is progressively increased, causing a persistent increase in pulmonary artery pressure, which eventually leads to right heart failure and death. Pulmonary hypertension is a chronic persistent complex disease involving many associated pathologies. Epidemiological surveys in Europe and the United States have shown that idiopathic pulmonary hypertension is most common in women, and familial pulmonary hypertension is associated with genes. [0003] The survival t...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/0735C12N5/10
CPCC12N5/0692C12N2501/155C12N2501/998C12N2501/115C12N2501/165C12N2501/727C12N2501/15C12N2506/02C12N2506/45C12N2510/00
Inventor 杨隽秦克周王雍刘洪宪
Owner ZHEJIANG UNIV
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