The aim of this study was to isolate and characterize rat Adipose Derived Mesenchymal Stem Cells (AD-MSCs) in order to evaluate the stemness markers gene expression levels, together with the assessment of phenotypic expression of the CD90 and CD45 markers. MSCs were obtained from subcutaneous adipose tissue of 16 Wistar rats. Microbiological controls were performed to exclude the presence of bacteria, fungi and viruses in tissue. Adipose tissue was mechanically and enzimatically fragmented and stromal cell fraction was seeded in adherent culture flasks in DMEM 20% FBS. After 48h, the medium was replaced. Cells were characterized by evaluating their ability to adhere to the plastic; the clonogenic potential by Colony Forming Unit (CFU) assay and their ability to differentiate adipocytes and osteocytes. AD-MSCs were analysed for the expression of three stem cell master genes (OCT-4, SOX-2 and NANOG) at different cell subcultures by Sybr Green Real-Time PCR. Statistical analysis, performed with REST software, showed that the expression of the genes is maintained from subculture 0 to 5. Moreover, flow cytometry analysis confirmed the mesenchymal nature of cells isolated from adipose tissue as they were positive for CD90, which is a MSCs surface marker, and negative for CD45 (typical hematopoietic marker).
Gene Expression Analysis of Rat Adipose Tissue-Derived Stem Cells
PICCIONE, Giuseppe;
2015-01-01
Abstract
The aim of this study was to isolate and characterize rat Adipose Derived Mesenchymal Stem Cells (AD-MSCs) in order to evaluate the stemness markers gene expression levels, together with the assessment of phenotypic expression of the CD90 and CD45 markers. MSCs were obtained from subcutaneous adipose tissue of 16 Wistar rats. Microbiological controls were performed to exclude the presence of bacteria, fungi and viruses in tissue. Adipose tissue was mechanically and enzimatically fragmented and stromal cell fraction was seeded in adherent culture flasks in DMEM 20% FBS. After 48h, the medium was replaced. Cells were characterized by evaluating their ability to adhere to the plastic; the clonogenic potential by Colony Forming Unit (CFU) assay and their ability to differentiate adipocytes and osteocytes. AD-MSCs were analysed for the expression of three stem cell master genes (OCT-4, SOX-2 and NANOG) at different cell subcultures by Sybr Green Real-Time PCR. Statistical analysis, performed with REST software, showed that the expression of the genes is maintained from subculture 0 to 5. Moreover, flow cytometry analysis confirmed the mesenchymal nature of cells isolated from adipose tissue as they were positive for CD90, which is a MSCs surface marker, and negative for CD45 (typical hematopoietic marker).Pubblicazioni consigliate
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