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Culture medium, kit and application thereof

A culture medium and kit technology, applied in the field of regeneration of early mesoderm progenitor cells and hematopoietic endothelial cells, can solve problems that need to be improved, and achieve the effect of effective preparation

Active Publication Date: 2013-10-23
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, current methods for inducing hESCs / iPSCs to differentiate into early mesodermal progenitor cells or hematopoietic endothelial cells in vitro still need to be improved.

Method used

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  • Culture medium, kit and application thereof
  • Culture medium, kit and application thereof
  • Culture medium, kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Culture of human embryonic stem cell lines

[0032] The inventor introduced the American Wicell Research Center to establish internationally recognized human embryonic stem cell lines H1 and H9. On the basis of referring to relevant literature at home and abroad, a human embryonic stem cell culture system suitable for the actual working environment and conditions was established, including the co-culture culture system using fibroblasts isolated from E13.5 day ICR strain mouse embryos as the feeder layer and the use of Feeder-free culture system based on Matrigel and defined mTeSR medium. The expression of pluripotent marker proteins such as SSEA4, Oct4, Nanog, Sox2, etc. were identified and detected by flow cytometry and immunofluorescence techniques in the cultured cells, and more than 90% of the cells were guaranteed to be positive cells, alkaline phosphatase (AP) staining was positive, and in vitro After the results of the three-germ layer induction differentiat...

Embodiment 2

[0047] Example 2. Identification of early mesoderm progenitor cells obtained by induction of differentiation

[0048] The early mesoderm progenitor cells induced in Example 1 were collected and identified.

[0049] 1. Identification method

[0050] 1. RT-Real Time PCR identification

[0051] The early mesoderm progenitor cells (i.e. cells to be tested) obtained in Example 1 were respectively identified for the expression level of cell marker genes (i.e. target genes), wherein the target genes selected in this example included the totipotency marker genes Nanog, Oct4, early Mesoderm progenitor cell marker genes Brachyury, Mixl-1, EOMES, PDGFRα, WNT3, CDX2, CXCR4 and GSC, the specific steps are as follows:

[0052] First, use TRIZOL (invitrogen, 15596-018) reagent to extract the total RNA of the cells to be tested according to the method provided in the product manual.

[0053] Next, the mRNA in the total RNA of the cells to be tested was reverse-transcribed using a PCR instr...

Embodiment 3

[0087] Example 3 Effects of Different Concentrations of PGE2 on the Differentiation of Human Embryonic Stem Cells to Mesoderm Progenitor Cells and Hematopoietic Endothelial Cells

[0088] The early mesoderm progenitor cells and hematopoietic endothelial cells (i.e. cells to be tested) induced in Example 1 were collected and subjected to the following experiments:

[0089] 1. RT-Real Time PCR identification

[0090] Experimental method is the same as embodiment 2.

[0091] 2. Flow cytometry detection

[0092] Will 1×10 6 The cells to be tested were digested into single cells with trypsin, washed once with cold PBS, resuspended in 100 μl of PBS and mixed with corresponding antibodies carrying fluorescent labels, incubated at 4°C for 45 minutes in the dark, and then washed with cold PBS Two times to remove unbound antibodies, and then the cells were treated with 7-AAD labeling and resuspended in 400 μl PBS, and detected on a flow cytometer (BD, FACSCalibur). Among them, the f...

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Abstract

The invention discloses a culture medium, a kit, a method for preparing early stage mesoderm ancestral cells and a method for preparing hematogenesis endothelial cells by using the kit, wherein the culture medium contains a basic culture medium namely an SFDM (serum-free differentiation medium), PGE2 (prostaglandin E2) and BMP4 (bone morphogenetic protein 4). By using the culture medium disclosed by the invention, a large amount of early stage mesoderm ancestral cells can be prepared efficiently and quickly.

Description

technical field [0001] The invention relates to the technical field of regeneration of early mesoderm progenitor cells and hematopoietic endothelial cells, in particular to culture medium, kit and application thereof. More specifically, the present invention relates to a culture medium, a kit, a method for preparing early mesoderm progenitor cells and a method for preparing hematopoietic endothelial cells using the kit of the present invention. Background technique [0002] Blood cell transfusion and hematopoietic stem cell transplantation are common methods of cell therapy, but their application has long been restricted by the shortage of donor sources, which greatly limits the promotion and application of this treatment. Therefore, finding new sources of hematopoietic stem cells and mature blood cells has become one of the most watched research directions in the field of regenerative medicine. [0003] Human embryonic stem cells (hESCs) or induced pluripotent stem cells (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078C12N5/071
Inventor 裴雪涛李艳华张博文何丽娟习佳飞岳文姚海雷
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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