Adipocyte Differentiation in Human Embryonic Stem Cells Transduced With Oct4 shRNA Lentivirus

Abstract

Oct4 is one of the master pluripotency genes that controls differentiation of human embryonic stem cells (hESCs). We generated HES2 and HES3 hESC lines stably transduced with lentivirus carrying Oct4 short hairpin RNA (shRNA) that display 80-90% reduction of Oct4 expression. Analysis of pluripotency marker expression shows that these Oct4 shRNA-transduced hESCs display normal wild-type expression levels of the pluripotency marker CD9 but an absence of GCTM2 expression. These hESC-derived adipocyte precursor cells display a characteristic morphology and can be propagated and cryopreserved as a standard stem cell line. Interestingly, Oct4 shRNA-transduced hESCs display a remarkably high lineage-specific spontaneous differentiation toward adipocytes. After two weeks of spontaneous differentiation under feeder-free conditions, 60-70% of cells display a mature adipocyte morphology as well as the expression of multiple adipocyte-specific mRNAs as assessed by RT-PCR. The upregulation of trophoblast, mesoderm, and endoderm transcripts is, however, also detected in these spontaneously differentiating cultures. These Oct4 shRNA hESCs will be an interesting model system to study human fetal adipogenesis and constitutes a renewable resource for obesity drug screening purposes.