Stem cell originated from blood capillary, use of same, and method for producing same
A capillary and stem cell technology, applied in the field of stem cells from capillaries, its use and its preparation, can solve the problem of not yet establishing the isolation of peripheral stem cells, and achieve the effects of ethical problems, low risk of rejection, and low risk of canceration
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[0081] 2. Preparation method of CapSCs
[0082] The cell population of the present invention can be prepared by isolating an EphA7-positive cell population from adherent cells derived from tissues containing capillaries.
[0083] (1) Separation of cells
[0084] First, cells are isolated from tissue containing capillaries. Examples of "tissues including capillaries" include subcutaneous fat tissue, skeletal muscle tissue, heart tissue, kidney tissue, visceral fat tissue, and the like, as described above. Cells can be isolated by treating these tissues with enzymes (collagenase, protease, etc.) or a commercially available cell dispersion buffer or the like according to a conventional method.
[0085] (2) Cell culture
[0086] The isolated cells are inoculated into a medium generally used for culturing mesenchymal cells, and subcultured for 3 to 4 days (preferably at least 2 generations or more). The obtained adherent cells are treated with trypsin or trypsin / EDTA, and separ...
Embodiment 1
[0119] Example 1: Array analysis of CapSCs cell lines
[0120] From the clonal pericyte strains (see WO2013 / 118786) of 10 strains from peripheral tissue capillaries established by temperature-sensitive SV40T antigen-expressing mice, three cell strains with different degrees of differentiation ability were selected ( CapSCs#7 (highly differentiated), CapSCs#9 (poorly differentiated), and CapSCs#3 (moderately differentiated), using the RNA expression array (Toray 3D-Gene array) to conduct a comprehensive array comparative analysis of the expressed genes.
[0121] Firstly, the gene clusters whose RNA expression levels increase or decrease in the order of #7-3-9 are selected. Next, select 850 factors whose change ratio is more than 1.5 times the threshold. Referring to the results of cluster analysis (gene homology, gene function, intracellular localization) and pathway analysis based on Gene Ontology (Gene Ontology, GO), etc., the candidate factors were reduced to 125. Furthe...
Embodiment 2
[0123] Example 2: Isolation and Analysis of Mouse CapSCs
[0124] 1. Using specific cell markers to isolate mouse CapSCs ( figure 1 )
[0125] The cells were isolated from mouse subcutaneous adipose tissue (wet weight: 0.5 g) by treatment with type I / II collagenase and Accumax, and cultured in DMEM medium containing 10% FBS for 2-3 days. The medium was replaced, cultured for another 3 to 4 days, and adherent cells (adipogenic stromal cells: ASC) were collected by trypsin treatment.
[0126] From the recovered cells, NG2-positive cells were separated by magnetic cell sorting (MACS) using microbeads immobilized with anti-NG2 antibodies. Furthermore, EphA7-positive cells were isolated from NG2-positive cells by flow cytometry (FACS) using an anti-EphA7 antibody. Finally, NG2-positive EphA7-positive cells accounting for 10-15% of adipose stromal cells were obtained. Cell preparation steps are shown in figure 1 .
[0127] In the analysis of surface marker expression by FACS...
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