Differentiation Of Human Stromal Cells Research Articles

Stability of Reference Genes during Tri-Lineage Differentiation of Human Adipose-Derived Stromal Cells.

Quantitative real-time PCR can detect variations in gene expression. The identification of the stable reference genes (RGs) is necessary to evaluate the expression of specific genes of interest under various conditions in many cell types, including human adipose-derived stromal cells (hASCs). In this study, we used the algorithms BestKeeper, NormFinder, geNorm, and RefFinder to investigate the stability of 15 potential RGs (B2M, eEF1A1, GAPDH, H2AFZ, HMBS, HPRT1, PGK1, PPIA, RPL5, SDHA, TBP, TKT, TRFC, TUBB, and UBC) in hASCs during control, adipo-, chondro-, and osteogenic differentiation for 28 days. RPL5, GAPDH, H2AFZ, and HPRT1 were the most stable RGs, while B2M and UBC were the least stable RGs for the majority of group analyses (tri-lineage differentiation and control analyzed combined or individually; each lineage combined with the control). These RGs were used to normalize adipo- (FABP4, LPL, and PPARG), chondro- (COMP and SOX9), and osteogenic gene expression markers (BMP4, COL1A1, and RUNX2). Each marker showed a similar expression when normalized by H2AFZ, HPRT1, or RPL5, confirming that these RGs exhibit stable expression. However, GAPDH, B2M, and UBC exhibited high standard deviation (SD), down-regulated and/or up-regulated differentiation gene expression markers when compared with stable RGs, demonstrating that these RGs are unstable.

Proliferation and Differentiation of Human Stromal Cells Derived from Fat Tissue on Different Culture Substrates

The objective of this study is to investigate the proliferation and differentiation of stromal cells derived from human fat tissues cultured on substrates with different surface properties. In addition, the similar investigation was performed for the cells proliferated in different concentrations of basic fibroblast growth factor (bFGF). The culture substrates include several polymer films with different water wettabilities, glass or a cell culture plate, and that coated with collagen type I or IV, gelatin, and bFGF. The proliferation profiles of cells were influenced by the type of culture substrates and the growth factor concentration. A larger number of cells proliferated was observed for substrates with the water wettability around 80o, while the cell number was significantly larger for every protein-coated substrate. The rate of cell proliferation became maximum in a bFGF concentration of 1,000 ng/mL. The bFGF concentration used for cell proliferation affected the differentiation profile of cells proliferated. The stromal cells proliferated in 1 ng/mL bFGF were osteogenically differentiated to the strongest and fastest extent among those in other growth factor doses. The alkaline phosphatase (ALP) activity of cells increased with the increased cell number although the activity per cells was indentical, irrespective of the substrates type. The strongest adipogenic differentiation was observed for cells proliferated in 1,000 ng/mL bFGF and the differentiation induction was maintained for a long time period. No clear dependence of the cell number on adipogenesis was observed. For chondrogenic differentiation, the bFGF concentration had no influence on the glycosaminoglycans (GAG) amount. These findings indicate that the proliferation and differentiation of human fat tissue-derived stromal cells are influenced by the culture substrate and the concentration of bFGF used for proliferation.