Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells

A technology of hematopoietic precursor cells and differentiation medium, applied in the direction of artificially induced pluripotent cells, cell culture active agents, cell culture support/coating, etc., can solve poor operability, low differentiation efficiency and yield, differentiation Long cycle and other issues, to achieve the effect of increasing instability and safety, improving differentiation efficiency, and low differentiation efficiency

Active Publication Date: 2021-06-29
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some progress has been made in this field, from the perspective of current clinical application, the research on hematopoietic differentiation of human pluripotent stem cells in vitro still faces great challenges, such as the hematopoietic endothelial precursors induced by human pluripotent stem cells The number of cells, hematopoietic stem cells and functional cells is not enough to meet the needs of one input; the differentiated hematopoietic stem cells induced by human pluripotent stem cells do not have the ability to transplant in vivo; the conditions for in vitro culture include exogenous sources such as serum and feeder cells Substances, these problems will greatly limit the current clinical application of human pluripotent stem cells in vitro hematopoietic differentiation
[0003] Due to the complex culture conditions of these current methods, the differentiation cycle is relatively long, the differentiation efficiency and yield are low, and the operability is poor.

Method used

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  • Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells
  • Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells
  • Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Embodiment 1: Preparation of serum-free culture system medium

[0095] (1) Preparation of IF9S medium:

[0096] This application uses IMDM medium, F12 medium, polyvinyl alcohol (PVA), Lipids (100X), ITS-X (100X), monothioglycerol (αMTG), AA2P, GlutaMax TM (100X), non-essential amino acids (NEAA, 100X) to prepare IF9S medium.

[0097] Wherein, each component of culture medium was purchased from:

[0098] IMDM medium was purchased from Gibco Company, the article number is 21056-023;

[0099] F12 medium was purchased from Gibco, the product number is 31765-027;

[0100] Polyvinyl alcohol (PVA) is purchased from Sigma company, and article number is P8136;

[0101] Lipids (100X) is purchased from Gibco company, and article number is 11905031;

[0102] ITS-X (100X) was purchased from Gibco Company, the article number is 51500-056;

[0103] Monothioglycerol (αMTG) was purchased from Sigma Company, the product number is M6145;

[0104] AA2P was purchased from Sigma Com...

Embodiment 2

[0120] Example 2: Preparation of hematopoietic endothelial precursor cells derived from human pluripotent stem cells

[0121] The human pluripotent stem cells used in this example are human embryonic stem cell lines (H1) (derived from the University of Wisconsin, USA).

[0122] a. First, the dish of cultured cells is coated with Matrigel, an extracellular matrix of embryonic stem cell level, and then human embryonic stem cells are inoculated in the coated dish and the commercial medium TeSR with defined chemical composition is used. TM -E8 TM Cultures were performed with fresh medium replaced every 24 hours. Human embryonic stem cells grow in the form of clones, and when the cell clone grows to a density of 70-80%, the cells need to be digested and passaged or subsequently induced and differentiated. The cycle of cell subculture is generally 3-4 days.

[0123] b. Digest the human embryonic stem cell clone grown in the above a. with ReLeSRTM digestive enzyme, and digest at...

Embodiment 3

[0129] Example 3: Detection of hematopoietic endothelial precursor cells

[0130] The vascular / hematopoietic endothelial precursor cells produced in Example 2 were collected for cell immunofluorescence staining identification.

[0131] specifically:

[0132] a. Discard the hematopoietic endothelial precursor cell induction medium in the culture flask, and wash the cells twice with PBS to remove dead cells;

[0133] b. Add 4% paraformaldehyde for fixation, and let it stand at room temperature for 20 minutes;

[0134] c. Discard 4% paraformaldehyde, add PBS to wash 2-3 times;

[0135] d. Add 5% donkey serum for blocking, and block for 1 hour at room temperature;

[0136] e. Add fluorescently labeled mouse anti-human PE-CD31 antibody (Cat. No.: 560983, BD Company), fluorescently labeled mouse anti-human FITC-CD34 antibody (Cat. No.: 560942, BD Company) and 0.1 μg / mL Hoechst33342, antibody The dilution factor is recommended to dilute with blocking solution according to the i...

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Abstract

The invention relates to a culture medium and a method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells. Specifically, the method comprises the following steps: 1) preparing hematopoietic endothelial precursor cells by a method of differentiating human pluripotent stem cells to generate the hematopoietic endothelial precursor cells; and 2) preparing the hematopoietic precursor cells by a method for promoting the hematopoietic endothelial precursor cells to differentiate into the hematopoietic precursor cells. In the preparation process, the culture medium is adopted, a random rotation mode is adopted for culture, the excellent effective efficiency is obtained, and components of the culture medium are definite.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for inducing differentiation of pluripotent stem cells to hematopoietic precursor cells. Background technique [0002] Pluripotent stem cells include embryonic stem cells derived from embryos and induced pluripotent stem cells derived from in vitro reprogramming induction. Pluripotent stem cells can maintain self-renewal ability in long-term culture in vitro, and have the potential of multidirectional differentiation, including differentiation into almost all functional blood cells. Hematopoietic progenitor cells are precursor cells that have the ability to self-renew and differentiate into various blood cells, eventually producing various blood cell components, including red blood cells, white blood cells, and platelets, which can also differentiate into various other cells. In the clinic, hematopoietic stem / progenitor cells and various mature blood cells can serve the impor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789C12N5/071
CPCC12N5/0647C12N2501/155C12N2501/16C12N2501/405C12N2500/50C12N2500/38C12N2501/15C12N2501/165C12N2501/115C12N2501/125C12N2500/36C12N2500/25C12N2501/999C12N2501/2303C12N2501/2306C12N2501/145C12N2533/90C12N2506/45
Inventor 雷晓华张键马驰原赵华山汪宝蓓李梦霞李荣荣
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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