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

A technology for pluripotent stem cells and hematopoietic stem cells, which is applied in the field of culture medium for inducing pluripotent stem cells to differentiate into hematopoietic stem cells, can solve the problems of low efficiency of hematopoietic stem cells, pollution, restricting the clinical application of umbilical cord blood hematopoietic stem cells, etc. The effect of solving pollution

Active Publication Date: 2020-04-21
THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the supply of umbilical cord blood provided only through voluntary blood donation from donors is far from meeting the large clinical needs. Because a single umbilical cord blood is small in size, it is difficult to meet the treatment needs of adult patients, which seriously restricts the clinical use of umbilical cord blood hematopoietic stem cells. application
Coupled with the risk of contamination of HIV, HCV and other pathogens in clinical blood, and the impact of unfavorable factors such as transplant failure, graft-versus-host disease (GVHD) and hematopoietic construction delay, more than 50% of patients are permanently disabled or incurable
Therefore, even with hematopoietic stem cell therapy, there is still a serious problem: how can there be enough hematopoietic stem cells for transplantation?
[0006] Although there are various methods of inducing differentiation to obtain hematopoietic stem cells in vitro, the efficiency of traditional methods to induce differentiation of hematopoietic stem cells is generally low

Method used

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

Examples

Experimental program
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Embodiment 1

[0029] This example provides a medium and a method for differentiating induced pluripotent stem cells (iPS) into hematopoietic stem cells.

[0030] The medium used in this embodiment includes the first-stage medium and the second-stage medium:

[0031] In the medium of the first stage, the final concentration ratio of SCF, BMP4, IL-3, VEGF, and Flt3 is 2:1:1:1:1, and the specific formula is: add a final concentration of 10% ( v / v) fetal bovine serum, thioglycerol (MTG) with a final concentration of 100 μmol / L, and bone cells with a final concentration of 40 ng / mL of stem cell factor (SCF) and a final concentration of 20 ng / mL. Morphogenetic protein 4 (BMP4), interleukin-3 (IL-3) at a final concentration of 20 ng / mL, VEGF at a final concentration of 20 ng / mL, and Flt3 at a final concentration of 20 ng / mL.

[0032] The final concentration ratio of SCF, BMP4, IL-3, VEGF, Flt3, and UM171 in the medium of the second stage is 2:1:1:1:1:5, and the specific formula is: add a final co...

Embodiment 2

[0039] This example is a modification of Example 1, and the only difference is that embryonic stem cells (ES) are selected as the type of pluripotent stem cells.

[0040] For comparison, this embodiment also sets up a comparison group. The difference between the comparison group and this embodiment is only that the culture medium of the following formula is only used to induce differentiation once: a-MEM medium is added with a final concentration of 10% (v / v) fetal bovine serum, thioglycerol (MTG) at a final concentration of 100 μmol / L.

Embodiment 3

[0042] This embodiment is a modification example of Embodiment 1, and the change is only in the component contents of the first-stage culture medium and the second-stage culture medium:

[0043] The final concentration ratio of SCF, BMP4, IL-3, VEGF and Flt3 in the medium of the first stage is 2.25:1:1:1:1, and the specific components are: the final concentration of 10% ( v / v) fetal bovine serum, thioglycerol (MTG) at a final concentration of 100 μmol / L, and bone cells with a final concentration of 45 ng / mL of stem cell factor (SCF) and a final concentration of 20 ng / mL. Morphogenetic protein 4 (BMP4), interleukin-3 (IL-3) with a final concentration of 20 ng / mL, VEGF with a final concentration of 20 ng / mL, and Flt3 with a final concentration of 20 ng / mL;

[0044] The final concentration ratio of SCF, BMP4, IL-3, VEGF and Flt3 in the medium of the second stage is 2.25:1:1:1:1:1:4.5, and the specific formula is: add the final concentration of 10% to the a-MEM medium (v / v) fetal...

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Abstract

The invention relates to a culture medium for induced differentiation of pluripotent stem cells into hematopoietic stem cells and a method thereof. The culture medium comprises a first-stage culture medium and a second-stage culture medium. According to the method, the first-stage culture medium and the second-stage culture medium are successively used for two-stage co-culture of pluripotent stemcells and OP9 cells. In comparison with the prior art, the invention has the following beneficial effects: by the use of the culture medium with the above two specific ingredients and concentrations and for induced differentiation of pluripotent stem cells into hematopoietic stem cells, induced differentiation of pluripotent stem cells is successively conducted, and the efficiency of differentiation of pluripotent stem cells into hematopoietic stem cells is raised.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a culture medium and a method for inducing pluripotent stem cells to differentiate into hematopoietic stem cells. Background technique [0002] In recent years, hematopoietic stem cells (HSCs) have been increasingly used clinically to treat hematological malignancies, solid tumors, bone marrow failure diseases, and some congenital diseases. It also shows certain application development prospects in the treatment of diseases and tumors. With the development of alternative sources of stem cell transplantation, including cord blood and homologous donors, and the use of new conditioning therapies, patient outcomes have been greatly enhanced and improved. Hematopoietic stem cells derived from umbilical cord blood have broad application prospects in the clinical treatment of various blood system diseases due to their rich content, strong expansion ability, low immunogenicity, convenient col...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2500/35C12N2500/84C12N2501/125C12N2501/155C12N2501/165C12N2501/2303C12N2501/26C12N2506/45
Inventor 孙筱放冼业星宋兵陈玉嫦欧阳曙明谢玉欢
Owner THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY