Human pluripotent stem cells (hPSCs) have key defining features including the ability to proliferate extensively in vitro, and the ability to be differentiated into any cell type. These properties confer hPSCs with great potential for investigating normal and disease biology, developing transplantation technologies, and discovering novel drug targets and candidates. A study by O’Connor et al., 2011 identified the retinoblastoma (RB)-binding protein 9 (RBBP9) as a novel pluripotency regulator via siRNA screening. Specifically, decreased RBBP9 protein levels in hPSCs led to decreased expression of pluripotency and cell cycle genes, and increased expression of differentiation genes.
Literature reports show RBBP9 has two potential mechanisms of action: i) the ability to bind RB protein and thus influence the RB/E2F pathway, and ii) to act as a serine hydrolase. To determine whether loss of this serine hydrolase activity can phenocopy the effects of RBBP9 siRNA, we cultured hPSCs in the presence of a newly identified RBBP9 serine hydrolase inhibitor, ML114.
hPSCs treated with 100 μM ML114 retained recognized pluripotency phenotypes and functions including alkaline phosphatase activity, pluripotency antigens, colony formation, and teratoma formation. However, ML114-treated cells displayed a reduced population growth rate, with more cells in G0/G1 and fewer cells in S-phase compared to control-treated cells. Affymetrix profiling revealed 2918 genes downregulated by ML114 treatment with no genes statistically significantly increased in expression. Taken together, these data indicate that RBBP9 serine hydrolase inhibition does not phenocopy RBBP9 protein loss, suggesting that both RBBP9 activities play distinct roles in pluripotency maintenance. Interestingly, promoter analysis of the ML114-downregulated genes identified binding motifs for the transcription factors, NF-YA and DEAF1, suggesting they may be targets of RBBP9 serine hydrolase activity. Further characterisation of the two RBBP9 activities will uncover new molecular mechanisms of hPSC maintenance, which may enable more efficient hPSC generation and maintenance.