Application of the RBBP9 Serine Hydrolase Inhibitor, ML114, Decouples Human Pluripotent Stem Cell Proliferation and...

Area of Science:

• Stem cell biology
• Molecular genetics
• Biochemistry

Background:

• Retinoblastoma binding protein 9 (RBBP9) maintains pluripotency and cell cycle gene expression in human pluripotent stem cells (hPSCs).
• RBBP9 possesses serine hydrolase (SH) activity in non-pluripotent cells, with its role in hPSCs and development being unclear.
• Previous studies linked RBBP9 to cell cycle regulation via the RB/E2F pathway in hPSCs.

Purpose of the Study:

• To investigate the role of RBBP9's serine hydrolase (SH) activity in maintaining human pluripotent stem cells (hPSCs).
• To determine if inhibiting RBBP9 SH activity impacts hPSC proliferation and differentiation.

Main Methods:

• Human pluripotent stem cells (hPSCs) were treated with ML114, a selective inhibitor of RBBP9 SH activity.
• Cell population growth rate, colony size, cell cycle progression, morphology, and pluripotency antigen expression were assessed.
• Teratoma formation assays were used to evaluate differentiation capacity.
• Microarray and Western blot analyses were performed to identify potential RBBP9 SH activity effectors.

Main Results:

• ML114 treatment significantly reduced hPSC proliferation, colony size, and cell cycle progression.
• No changes in cell morphology or pluripotency markers were observed, indicating no initiation of differentiation.
• hPSCs treated with ML114 retained their capacity for tri-lineage differentiation.
• Nuclear transcription factor Y subunit A (NFYA) emerged as a potential target of RBBP9 SH activity in hPSCs.

Conclusions:

• RBBP9 regulates hPSC proliferation independently of differentiation.
• Inhibition of RBBP9 SH activity decreases hPSC proliferation without inducing differentiation.
• NFYA is identified as a potential effector of RBBP9 SH activity in hPSCs, linking proliferation control to differentiation potential.