The OCT4 gene, a POU homeodomain transcription factor, has been shown to be expressed in embryonic stein cells (ESC) as well as hUCB-MSCs. In this study, the roles played by OCT4A in hUCB-MSCs were determined by stably inhibiting OCT4A with lenti-viral vector-based small hairpin RNA (shRNA). A decreased rate of cell proliferation was observed in OCT4-inhibited hUCB-MSCs. Down-regulation of CCNA2 expression in OCT4-inhibited hUCB-MSCs was confirmed by RT-PCR and real-time RT-PCR analysis in three genetically independent hUCB-MSC clones. Adipogenic differentiation was also suppressed in OCT4-inhibited hUCB-MSCs. The up-regulation of DTX1 and down-regulation of HDAC1, 2, and 4 expressions may be related to this differentiation deformity. The expression of other transcription factors, including SOX2, RFX1 and c-MYC, was also affected by OCT4 inhibition in hUCB-MSCs. In conclusion, these finding suggest that OCT4A performs functionally conserved roles in hUCB-MSCs, making its expression biologically important for ex vivo culture of hUCB-MSCs. (c) 2009 Elsevier Inc. All rights reserved.