The role of Netrin signaling in the regulation of human pluripotency has remained unexplored. Analysis of publicly available RNA-sequencing data from human preimplantation embryos revealed early expression of Netrin-1 (NTN1) and its receptors NEOGENIN (NEO) and UNC5B in the inner cell mass and epiblast. Their salt-and-pepper expression pattern suggests a possible role in regulating the naïve pluripotent state.

To investigate this, we generated human pluripotent stem cells (hPSCs) overexpressing wild-type NTN1 (wtNTN1) and three mutant variants lacking the ability to bind either NEO, UNC5H, or both receptor families. We performed single-cell RNA sequencing (scRNA-seq) on these four lines to characterize the transcriptional effects of each NTN1 form under naïve conditions and during exit from pluripotency. Additionally, we generated a doxycycline-inducible wtNTN1 line to examine short-term responses and a CRISPR-Cas9-generated NTN1-null hPSC line to determine the requirement for endogenous NTN1. Proteomic and phosphoproteomic analyses were also conducted following acute NTN1 induction.

NTN1-null hPSCs exhibited normal colony morphology, robust self-renewal, and unaltered expression of core pluripotency markers under naïve conditions, indicating that NTN1 is not essential for pluripotency maintenance at steady state. However, overexpression of wtNTN1 enhanced expression of naïve markers and suppressed primed marker expression. Upon growth factor withdrawal, wtNTN1-expressing cells retained elevated levels of naïve markers and showed delayed induction of differentiation markers. Phosphoproteomic profiling revealed a global reduction in phosphorylation following NTN1 induction, consistent with a broad dampening of signaling activity, with selective modulation of specific pathways. Surprisingly, mutant NTN1 proteins unable to bind NEO and/or UNC5H elicited transcriptional effects similar to wtNTN1, suggesting the involvement of an as-yet unidentified receptor.

Together, these findings identify Netrin-1 as a modulator of human naïve pluripotency and reveal its unexpected ability to buffer intracellular signaling networks, stabilizing the undifferentiated state through a receptor mechanism that remains to be defined.