Method, probe and kit for identifying human embryo bone marrow-derived mesenchymal stem cells and application of method, probe and kit

A bone marrow-derived, mesenchymal stem cell technology, applied in the biological field, can solve the problems of DNA methylation differences in cells and MSCs that cannot truly reflect the functions in vivo

Pending Publication Date: 2022-07-15
PEKING UNIV SCHOOL OF STOMATOLOGY
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, after the primary MSCs are expanded in vitro, the DNA methylation of the cells will also be significantly different 15
All these studies clearly show that the in vitro characteristics of MSCs do not truly reflect their in vivo functions

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method, probe and kit for identifying human embryo bone marrow-derived mesenchymal stem cells and application of method, probe and kit
  • Method, probe and kit for identifying human embryo bone marrow-derived mesenchymal stem cells and application of method, probe and kit
  • Method, probe and kit for identifying human embryo bone marrow-derived mesenchymal stem cells and application of method, probe and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 - Human Embryonic Bone Marrow Stromal Cell Expression Profile

[0073] To explore the diversity of human embryonic BM stromal cells, the inventors performed single-cell transcriptome analysis of BMNCs using 10X genomics and scRNA-seq technology.

[0074] Since the BM is full of red blood cells, the inventors used ice-cold sterile water to lyse the red blood cells 18 . To test the feasibility of the experimental protocol, 2,634 quality-controlled single cells derived from two embryos (ie: 20 and 21 weeks old) were obtained. The nucleated cells derived from human embryonic bone marrow used in the present invention were obtained from the Third Hospital of Peking University.

[0075] The results show that the inventors identified 12 clusters with batch effect correction in Harmony and unsupervised clusters in Seurat ( Figure 7 A) 19，20 . These clusters are annotated according to classical marker genes: two erythrocyte subsets (specifically expressing GYPA ...

Embodiment 2

[0078] Example 2 - Heterogeneity of MSCs in Human Embryonic BM

[0079] A recent study elucidated that perinatal chondrocytes form the majority of new osteoblasts and gradually decrease with age 22 . Therefore, the inventors carried out an experimental test on the chondrocyte population by inoculating the sorted single cells in 96-well plates respectively, and then transplanting the expanded colonies subcutaneously on the dorsal side of nude mice with β-TCP vector ( figure 2 a).

[0080] like figure 2 As shown in b, the chondrocyte population specifically expresses the surface markers CD44, NT5E and TM4SF1. Subsequently, the inventors harvested CD44 by FACS sorting + CD73 + TM4SF1 + / CD45 - CD31 - CD235a - cell( Figure 8 a). STRT analysis further confirmed the accuracy of FACS ( Figure 8 b).

[0081] It was found that a single CD44 + CD73 + TM4SF1 + Cell-expanded colonies are able to efficiently form bone tissue ( figure 2 c). Therefore, the findings o...

Embodiment 3

[0086] Example 3 - Differences between early and late stages of MSC development

[0087] To trace the origin of human embryonic MSCs, the inventors attempted to capture mesenchymal cells at an earlier developmental stage (6-9 weeks). However, due to the limited number of cells obtained early in development, the inventors did not use 10X genomics scRNA-seq technology, but collected cells by a random selection method and sequenced the cells using STRT scRNA-seq technology. The inventors obtained a total of 2,989 high-quality single-cell transcriptomes from cells derived from 17 embryos aged 6-24 weeks.

[0088] The inventors identified 3 main groups in the STRT dataset, namely, endothelial cells (specifically expressing CDH5), mesenchymal cells (highly expressing CTHRC1, collagen triple helical repeat protein-1) and hematopoietic cells (highly expressing PTPRC) and GYPA)( image 3 b, Figure 9 a-b). Notably, most of the sequenced cells at later stages of development (after...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a method for identifying and screening human embryo bone marrow-derived mesenchymal stem cells, a related detection agent combination, a kit and related application. According to the invention, human embryo BM karyocytes (BMNC) are comprehensively screened by single cell resolution, scRNA-seq analysis is carried out by using two complementary strategies (STRT and 10X genomics), and LIFR + PDGFRB + is identified as a specific marker of MSC. The invention also finds that the hematopoietic microenvironment can be effectively reconstructed in vivo by using the LIFR, the PDGFRB and the CD45-CD31-CD235a-MSC. The invention also identifies a monopotent bone progenitor cell subset for expressing the TM4SF1 + CD44 + CD73 + CD45-CD31-CD235a-, and the monopotent bone progenitor cell subset has osteogenic potential. Therefore, the invention identifies two types of human embryo bone marrow MSCs, and the invention deepens the understanding of the human embryo bone marrow derived MSCs and further promotes the understanding of the clinical application of the MSCs.

Description

technical field [0001] The present invention relates to the field of biology, and particularly relates to a method for molecularly and functionally identifying human embryonic bone marrow-derived mesenchymal stem cells at single-cell resolution through single-cell transcriptomics and functional analysis. Background technique [0002] Bone marrow (BM)-derived mesenchymal stem cells (MSCs) have long been described as a cell population that can self-renew and produce stroma, bone, cartilage and fat 1-5 . However, the previous understanding of MSCs has mainly focused on in vitro studies, and their true identity in vivo has not yet been fully clarified. Several studies using gene-edited mice showed that LepR in mouse BM + , Nestin + and Grem1 + Stromal cells, in addition to having MSC activity, are also an important component of the HSC microenvironment of hematopoietic stem cells and have functions to support HSCs 6-8 . Although our knowledge of MSCs has been expanded thro...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888A61K35/28A61P19/08
CPCC12Q1/6888A61K35/28A61P19/08C12Q2600/158
Inventor 周永胜张萍乔杰汤富酬董骥
Owner PEKING UNIV SCHOOL OF STOMATOLOGY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap