Differentiation Potential and Profile of Nuclear Receptor Expression During Expanded Culture of Human Adipose Tissue-Derived Stem Cells Reveals PPAR gamma as an Important Regulator of Oct4 Expression

Abstract

Potential therapeutic use of human adipose tissue-derived stem cells (hADSCs) requires the production of large cell numbers by in vitro expansion. However, long-term in vitro culture is associated with reduced stem cell characteristics and differentiation capability. We investigated the proliferation rate and expression of p16(INK4a) mRNA, surface stem cell markers, and stem cell transcription factors. The proliferation rate decreased significantly as passages increased, and the expression of p16(INK4a) mRNA significantly increased. FACS analysis of CD73, CD90, and CD105 expression showed no significant difference among examined passages; however, the mRNA expression levels of pluripotent markers, Oct4 and Nanog, were significantly decreased at higher passages. At passages 12 and 20, there was decreased differentiation capability into insulin-producing cells, evidenced by significantly decreased expression of insulin and related cell markers. Adipogenic and osteogenic differentiation was also decreased at higher passages. We then analyzed the transcriptional expression profiles of 48 nuclear receptors at four different passages. We found that the expression of peroxisome proliferator-activated receptor (PPAR) and thyroid hormone receptor TR was significantly decreased at higher passages. Treatment with PPAR activators or overexpression of PPAR in hADSCs at passage 20 could recover Oct4 expression levels and increase Oct4 promoter activity. PPAR inactivation by GW9662 inhibited the troglitazone-induced Oct4 mRNA expression. Furthermore, PPAR overexpression in hADSC at passage 20 improved the differentiation potential to insulin-producing cells. In conclusion, we demonstrated that hADSCs undergo characteristic changes and reduction of differentiation capability during expanded culture in vitro, and revealed the role of PPAR as one potential factor in the regulation of Oct4 expression during in vitro aging of hADSCs.