NOTCH1 signaling promotes human iPSC-to-PGCLC-like differentiation

Background Research into human infertility is limited by incomplete knowledge of germline specification and the absence of reproducible human models for investigating signaling pathways. This study examines whether NOTCH signaling, particularly the DLL4–NOTCH1 axis, contributes to PGCLC-like differentiation from human induced pluripotent stem cells (iPSCs). Methods A single commercially purchased human iPSC line was subjected to staged PGCLC-like induction, with sampling at D0, D3, and D7. Temporal pathway activity and lineage status were assessed using RT-qPCR (pluripotency genes and NOTCH1 targets), immunoblotting (NICD, HES1, and HEY1), and flow cytometry (CD38 and intracellular DDX4). Pathway involvement was examined by continuous DLL4 stimulation, γ-secretase inhibition with DAPT, and siRNA-mediated NOTCH1 knockdown. Results NOTCH pathway readouts were highest at the earliest sampled differentiation point (D3) and remained above D0 at D7, coinciding with reduced pluripotency markers and increased PGCLC-associated markers. Pharmacological activation with DLL4 increased pathway readouts and the frequency of CD38+/DDX4+ cells, whereas γ-secretase inhibition with DAPT reduced both. At D7, the representative CD38+/DDX4+ fractions were 7.45% in control cells, 19.1% with DLL4, and 3.21% with DAPT. NOTCH1 knockdown attenuated pathway activity and PGCLC-associated readouts. In the NOTCH1-silenced background, DLL4 restored the measured readouts toward baseline but did not reproduce the enhancement observed with intact NOTCH1, a pattern consistent with a substantial contribution of NOTCH1 while not excluding residual NOTCH1 activity or other NOTCH receptors. In the genetic series, representative CD38+/DDX4+ fractions were 8.39% for si-NC, 0.642% for si-NOTCH1, and 7.98% for DLL4 plus si-NOTCH1. Conclusion NOTCH pathway readouts peaked at D3 and remained elevated at D7, while DLL4 stimulation, DAPT inhibition, and NOTCH1 knockdown produced directionally consistent changes in PGCLC-associated readouts. These findings support a contribution of sustained NOTCH signaling and NOTCH1 to PGCLC-like differentiation.