350 rub
Journal Technologies of Living Systems №5 for 2009 г.
Article in number:
CHARACTERIZATION OF HUMAN ADIPOSE-DERIVED STEM CELLS PHENO-TYPE BY FLOW CYTOMETRY METHOD
Keywords: adipose tissue mesenchymal human adipose-derived stem cells immunophenotype
Authors:
V.A. Egorova, A.S. Ponomareva, N.B. Bogdanova, V.Yu. Abramov, V.I. Sevastianov
Abstract:
Stem cell biology is currently one of the most exciting areas of biomedical research, especially such aspects as cell-based therapy or tissue engineering. A promising source of adult human mesenchymal stem cells is adipose tissue. Independent groups have identified highly consistent, although not identical, expression profiles of cell-surface proteins on human adipose-derived stem cells (ADSCs). The main goal of this work is to identify human mesenchymal adipose-derived stem cells CD marker profile. The methods of isolation, culture and phenotyping are described. The immunophenotype of undifferentiated ADSCs cultured in vitro has been examined using flow cytometry method. Stem cells were isolated from 4 samples of subcutaneous fat from 4 healthy donors. The main yield of cells was from 70 000 to 300 000 cells per gram of tissue. ADSCs were maintained at 37°C/5% CO2 in no inductive control medium. Cells were passaged repeatedly after achieving a density of about 80% (approximately 14 days in culture) until passage 5. ADSCs expanded easily in vitro and exhibited mostly a fibroblast-like morphology, but some cells had roundish morphology. Early passage cells had spindle shape. Satellite-shaped cells appeared in the 5th passage. They are very few at the beginning but their quantity increases with time. After two weeks in culture with differ-ent density of cells on passage 1 we observed the appearance of colonies. Adipose-derived cells from 1 to 5 passages were examined for expression of monoclonal antigens coupled to either PE or FITC by flow cytometry method. Immunocytofluorimetric analysis showed that ADSCs were positive for the cell-surface markers CD10, CD13, CD29, CD44, CD59, CD73, CD90, CD105, HLA-ABC and negative for the cell-surface markers CD34, CD45, CD71, CD133, and maintained this phenotype during at least five passages. Flow cytometric analysis showed that a population of ADSCs expresses multiple CD marker antigens similar to those observed on mesenchymal stem cells from adipose tissue by different researchers.
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