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Application of CD61 as hematopoietic endothelial cell marker

A CD61, hematopoietic endothelium technology, applied in the biological field, can solve the problems of distinguishing between cells without hematopoietic function, unable to judge CD73-, hematopoietic endothelium and non-hematopoietic endothelium, etc., so as to improve the sensitivity, accuracy and accuracy. and the effect of increased sensitivity

Active Publication Date: 2016-12-14
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] It is generally believed that HE exists in CD34+CD31+ / CD144+ endothelial cells, but it is difficult to distinguish hematopoietic endothelium from non-hematopoietic endothelium; there is only one case report, and it is believed that CD73 has a distinguishing function, and CD73+ cells do not have hematopoietic function
However, it is not yet possible to determine whether all CD73- cells are HE

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1 Constructing a human embryonic stem cell line in which the GATA2 / eGFP gene is knocked in

[0091] The inventors used the TALEN-mediated gene knock-in method to replace the stop codon with the eGFP reporter gene containing the flag-2A sequence after the last exon of the GATA2 gene in the H1 genome. The specific construction process is as follows: figure 1 As shown in A, the build process is as follows:

[0092] (1) Construction of targeting vectors: Genomic DNA of human embryonic stem cell lines was extracted, and using the genomic DNA of human embryonic stem cell lines as a template, a homologous left arm and a homologous right arm of about 1 kb each were amplified from the upstream and downstream of the GATA2 stop codon. arm, then connect the homologous left arm and the homologous right arm into the pUC57 vector, and then insert the flag-2A-eGFP-loxP-PGK-puro-loxP sequence in the middle of the left and right homologous arms, which is the targeting vector (tar...

Embodiment 2

[0100] Example 2 GATA2 / eGFP expression can trace HE (hematopoietic endothelium) and HPC (hematopoietic precursor cells)

[0101] The inventors analyzed the expression of CD34, CD31, CD43 and eGFP in the process of hematopoietic differentiation and found that the expression of eGFP is related to the temporal expression of CD34, CD31 and CD43, suggesting that GATA2 may be related to the generation of endothelial and hematopoietic precursor cells. The results Such as figure 2 As shown in A, figure 2 A shows H1-GATA2 w / eGFP Co-cultured with OP9 (a mouse bone marrow mesenchymal cell that secretes hematopoietic differentiation factors to support hematopoietic differentiation) for hematopoietic differentiation, at different time points, the expression of CD34, CD31, CD43 and eGFP were related.

[0102] On day 8 of hematopoietic differentiation, the inventors found that CD34 + CD31 + CD43 - cells containing GATA2 / eGPF + and GATA2 / eGFP - Two populations of cells with only GATA...

Embodiment 3

[0106] Example 3 Transcriptome comparative analysis of hematopoietic endothelium and non-hematopoietic endothelium

[0107] Based on the findings of Example 1 and Example 2, the inventors used the RNA-Seq method to analyze CD34 + CD31 + CD43 - In cells, GATA2 / eGPF + Hematopoietic endothelium (HE) and GATA2 / eGFP - Non-hematopoietic endothelium, differences at the transcriptome level. The result is as image 3 As shown in A, image 3 A shows GATA2 / eGPF + and GATA2 / eGFP - MA profiling of cellular RNA-Seq results; and GATA2 / eGPF + and GATA2 / eGFP - The results of GO analysis of highly expressed genes in the cells, image 3 A results show GATA2 / eGPF + The highly expressed genes in the cells are enriched in biological processes related to blood development; while GATA2 / eGPF - The highly expressed genes in the cells are enriched in the biological process of endothelial and vascular development.

[0108] The inventor further analyzed the classic blood and endothelial relat...

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Abstract

The invention provides application of CD61 as a hematopoietic endothelial cell marker as well as a kit and a method for detecting hematopoietic endothelial cells. The kit comprises a reagent for specifically recognizing the CD61. In a hematopoietic differentiation process, the endothelial cells of CD61<+> have potency of generating hematopoietic precursor cells, namely the endothelial cells of the CD61<+> are hematopoietic endothelial cells. The kit and the method, for detecting the hematopoietic endothelial cells, provided by the invention have the effect of detecting the hematopoietic endothelial cells in the endothelial cells in the hematopoietic differentiation process quickly, sensitively and accurately.

Description

technical field [0001] The present invention relates to the field of biology, specifically, the present invention relates to biomarkers, and more specifically, the present invention relates to the use of CD61 as a marker of hematopoietic endothelial cells, a kit for detecting hematopoietic endothelial cells, and a method for identifying hematopoietic endothelial cells. Background technique [0002] Differentiate human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), to obtain hematopoietic stem / precursor cells progenitor cells; HS / PCs), has very important application prospects. However, currently, HPCs differentiated in vitro do not have hematopoietic reconstitution ability. Therefore, it is necessary to analyze the generation mechanism of HPCs in detail, so as to facilitate the differentiation research from PSCs to HPCs in vitro. [0003] During embryonic development, hematopoietic stem cells are transformed ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569C07K14/705
CPCC07K14/70557G01N33/56966G01N33/68G01N2333/70557
Inventor 潘光锦黄可杜鹃刘兵高娇廖宝剑
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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