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Method for increasing efficiency of inducing pluripotent stem cells to be differentiated into hematopoietic stem cells in vitro

A technology of pluripotent stem cells and hematopoietic stem cells, which is applied in the field of improving the efficiency of in vitro differentiation of induced pluripotent stem cells into hematopoietic stem cells, and can solve problems such as the lack of hematopoietic stem cell guidelines

Inactive Publication Date: 2016-02-03
THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY
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Problems solved by technology

However, it is not strictly reported which method is most efficient
For future medical individualized stem cell therapy, there is a lack of guidelines for in vitro differentiation into hematopoietic stem cells

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  • Method for increasing efficiency of inducing pluripotent stem cells to be differentiated into hematopoietic stem cells in vitro

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Embodiment

[0025] A method for improving the efficiency of in vitro differentiation of induced pluripotent stem cells (iPS cells) into hematopoietic stem cells, comprising the following steps:

[0026] (1) Cultivate OP9 cells (mouse bone marrow stromal cells) in a 60 mm culture dish to an overdense state; the medium used is α-MEM medium with 20% fetal bovine serum added;

[0027] (2) Wash the iPS cells twice with PBS, then add Dispase (dispase) to digest, absorb the Dispase after the border of the clone appears slightly curled, then add 2ml DMEN / F12 medium, blow the clone to into a uniform block, then moved to a 15ml centrifuge tube and waited for it to settle naturally, discarded the supernatant, and added 1ml of differentiation medium (placed in a 35mm culture dish, about 10 6 cells);

[0028] (3) Replace the culture medium of the OP9 cells that have been cultured to an overdense state with a differentiation medium; then inoculate the iPS cells obtained in step (2) into OP9 cells, at ...

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Abstract

The invention discloses a method for increasing the efficiency of inducing pluripotent stem cells to be differentiated into hematopoietic stem cells in vitro. The method comprises the steps of carrying out in-vitro co-culture on OP9 and iPSCs, and meanwhile, adding trophic factors such as SCF, BMP4, IL-3, IL-6 and TPO into a differential medium. By using the method, the double positive rate of CD34+ CD43+ is increased to 15.3%, the differentiation efficiency is remarkably increased, and therefore, iPSCs can be induced to be differentiated into hematopoietic stem cells through adding a positive regulating factor, and the differentiation efficiency can be conveniently and rapidly increased only through a cell factor adding method.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for improving the differentiation efficiency of induced pluripotent stem cells (iPS cells) into hematopoietic stem cells in vitro. Background technique [0002] Hematological diseases are diseases that originate in the hematopoietic system, or affect the hematopoietic system accompanied by abnormal blood changes, which often manifest as anemia, bleeding, fever and other symptoms. [0003] The incidence of malignant cancer in children in my country is on the rise. According to the data as of 2014, among the malignant tumors in children, the incidence of leukemia ranks first, accounting for about one-third. For malignant hematological diseases, the clinical effect of chemotherapy is often not ideal. Since Professor Thomas first carried out hematopoietic stem cell transplantation in the middle of the 20th century, hematopoietic stem cell transplantation has been widely used in t...

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Application Information

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IPC IPC(8): C12N5/0789
Inventor 孙筱放范荻宋兵牛晓华范勇何文茵
Owner THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY
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