Optimization of Human Adipose-derived Stem Cell (ADSC) Culture for Growth, Multi-lineage Differentiation and Regenerative Potential

Abstract

Adipose tissue is rich with stem cells and harvesting these cells is minimally invasive to donors, making it an ideal tissue to work with. Stem cells have the capacity to maintain prolonged self-renewal, as well as the ability to differentiate into cells with more specialized functions. Given their unique regenerative abilities, adipose-derived mesenchymal stem cells (ADSCs) offer new potentials for use in clinical therapy and regenerative medicine. Optimizing ADSC growth for therapeutic use involves both rapid expansion of cells and cellular characterization including the growth factor/ cytokine secretome that may be crucial to promote healing. The first part of this study was to optimize stem cell growth using xeno-free medium. We grew cells in DMEM/F12 with 1% human serum (HS), 2.5% HS 10% fetal bovine serum (FBS) with and without FGF-2, with or without ITS, on uncoated polysterile or pre-coated fibronectin plates. FGF-2 was essential for growth in low-serum conditions while pre-coated fibronectin plates provided minimal increases. Doubling times on uncoated plates were 26.7 hours for 1%HS+FGF, 25.1 hours for 1%HS+FGF+ITS, and 26.7 hours for 2.5%HS+FGF and 17.9 hours for 10%FBS+FGF. Additionally we evaluated the regenerative potential of ADSCs culture-expanded under different conditions by comparing the ratio of gene expression of hepatic growth factor (HGF) to the expression of transforming growth factor (TGF-beta) by quantitation with RT-qPCR. The HGF/TGFB1 ratio was 20.5 for 1%HS+FGF, 23.8 for 1%HS+FGF+ITS, 2.0 for 10%FBS+FGF and 0.1 for 10%FBS noFGF. Further evaluation of gene expression, of ADSCs culture-expanded in 1%HS+FGF, 1%HS+FGF +ITS, 10%FBS+FGF, and 10%FBS noFGF, was done using an Affymetrix microarray. Significant differences in pathway-dependent gene expression between 10% FBS and 1%HS conditions were reflected in the ability of the stem cells to undergo differentiation into osteogenic, chondrogenic, adipogenic and myogenic lineages. 1%HS+FGF produced the best osteogenic differentiation whereas 10%FBS noFGF produced the best chondrogenic differentiation. The variation in gene expression observed between the growth conditions may help to predict the most efficient conditions to build tissue models for future genetic disease research and/or clinical drug screening and development.