Material and method for differentiating pluripotent stem cells into hematopoietic stem cells

A technology for pluripotent stem cells and hematopoietic stem cells, which is applied in the field of materials for pluripotent stem cells to differentiate into hematopoietic stem cells, can solve the problems of low cell survival rate, weak cell strength, and high cost, and achieve high differentiation efficiency, reduce differentiation costs, and research and development costs. low effect

Pending Publication Date: 2022-05-24
深圳中旭生物科技有限公司
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Problems solved by technology

[0003] Hematopoietic stem cells are adult stem cells in the blood system. They are a heterogeneous group with the ability of long-term self-renewal and the potential to differentiate into various mature blood cells. It is the longest and most in-depth study of a type of adult stem cells. The study of various stem cells, including tumor stem cells, has important guiding significance, and the purpose of differentiation of pluripotent stem cells into hematopoietic stem cells is to expand and differentiate into hematopoietic stem cells or repair hematopoietic stem cells through pluripotent stem cells, so as to treat blood diseases. Technology is of great significance, but the existing differentiation and expansion methods are not only difficult and costly, but also the survival rate of differentiated cells is low, and the cell strength is weak, and now a material for the differentiation of pluripotent stem cells into hematopoietic stem cells is proposed and methods to solve the above technical problems

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  • Material and method for differentiating pluripotent stem cells into hematopoietic stem cells

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Embodiment 1

[0035] Example 1: A material and method for the differentiation of pluripotent stem cells into hematopoietic stem cells:

[0036] The gel matrix in stem cells is purified from tumor cells to obtain a soluble basement membrane extract. The matrix is ​​polymerized at 20-40°C to form a reconstituted basement membrane. The gel matrix is ​​thawed and diluted in culture medium for refrigeration. Treatment, after thawing, use a pipette to put the basement membrane extract into the medium, and dilute the basement membrane extract with DMEM medium at a dilution ratio of 1:80 to obtain an ECM gel matrix;

[0037] Add a 1:80 dilution of ECM gel matrix to each well of a 6-well plate, rotate the plate to spread the ECM gel matrix evenly on the surface of the plate, and store it at 2-8 °C Refrigerate overnight in medium for at least 2 hours, or wrap the ECM gel matrix with plastic wrap and store at 2-8°C;

[0038] Thaw vitronectin at room temperature and prepare aliquots in sterile polypro...

Embodiment 2

[0042]Embodiment 2: A material and method for the differentiation of pluripotent stem cells into hematopoietic stem cells, which, on the basis of Embodiment 1, replaces the insulin supply in the culture medium with amino acid supplements;

[0043] The gel matrix in stem cells is purified from tumor cells to obtain a soluble basement membrane extract. The matrix is ​​polymerized at 20-40°C to form a reconstituted basement membrane. The gel matrix is ​​thawed and diluted in culture medium for refrigeration. Treatment, after thawing, use a pipette to put the basement membrane extract into the medium, and dilute the basement membrane extract with DMEM medium at a dilution ratio of 1:80 to obtain an ECM gel matrix;

[0044] Add a 1:80 dilution of ECM gel matrix to each well of a 6-well plate, rotate the plate to spread the ECM gel matrix evenly on the surface of the plate, and store it at 2-8 °C Refrigerate overnight in medium for at least 2 hours, or wrap the ECM gel matrix with p...

Embodiment 3

[0049] Embodiment 3: A material and method for the differentiation of pluripotent stem cells into hematopoietic stem cells, which, on the basis of Embodiment 1, replaces the insulin supplement with milk-water protein;

[0050] The gel matrix in stem cells is purified from tumor cells to obtain a soluble basement membrane extract. The matrix is ​​polymerized at 20-40°C to form a reconstituted basement membrane. The gel matrix is ​​thawed and diluted in culture medium for refrigeration. Treatment, after thawing, use a pipette to put the basement membrane extract into the medium, and dilute the basement membrane extract with DMEM medium at a dilution ratio of 1:80 to obtain an ECM gel matrix;

[0051] Add a 1:80 dilution of ECM gel matrix to each well of a 6-well plate, rotate the plate to spread the ECM gel matrix evenly on the surface of the plate, and store it at 2-8 °C Refrigerate overnight in medium for at least 2 hours, or wrap the ECM gel matrix with plastic wrap and store...

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Abstract

The invention discloses a material and a method for differentiating pluripotent stem cells into hematopoietic stem cells, relates to the technical field of biology, and provides the following scheme aiming at the problems of high difficulty in differentiating the pluripotent stem cells into the hematopoietic stem cells, high technical cost and the like: the material comprises a basement membrane extract, vitronectin and a cell culture medium. In the culture medium produced on the basis of different culture solutions, multi-domain induced differentiation of the pluripotent stem cells into the hematopoietic stem cells under the conditions of constant temperature and normal oxygen concentration can be realized, and nutrients are provided for stem cell differentiation by utilizing the basement membrane extract and vitronectin, so that the differentiation cost is reduced, the procedure of adding the culture solutions is reduced, and the differentiation efficiency is improved. Meanwhile, the hematopoietic stem cells are stable in chemical component, low in biological research and development cost, high in safety and high in differentiation efficiency, cells with stable and good quality can be provided for the related fields of biological pharmacy, and the hematopoietic stem cells have high commercial and medicinal values.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a material and method for differentiating pluripotent stem cells into hematopoietic stem cells. Background technique [0002] Pluripotent stem cells refer to all types of stem cells that can develop into the body, but cannot develop into extraembryonic tissue (or placenta). Embryonic stem cells are the inner cell mass of blastocysts after cleavage, and are pluripotent stem cells In general, pluripotent stem cells can be iPSCs (induced pluripotent stem cells) in addition to embryonic stem cells. Multipotent stem cells (also translated into "pluripotent" stem cells) refer to those capable of developing Stem cells are a series of somatic cells but do not have the ability to develop into all somatic cell types. [0003] Hematopoietic stem cells are adult stem cells in the blood system. They are a heterogeneous group with long-term self-renewal ability and the potential to differentiate ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/0789
CPCC12N5/0647C12N2506/45C12N2501/998C12N2500/84C12N2500/32C12N2501/33C12N2509/10
Inventor 冷玉娇刘银笛李丁
Owner 深圳中旭生物科技有限公司
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