DNA Methylation Landscape of ReNcell Common Neural Progenitor Cell Lines Reveals Distinct Lineage Bias

Neural progenitor cell (NPC) fate decisions are governed by transcriptional and signaling programmes, yet the epigenetic mechanisms stabilising early neuronal versus glial lineage trajectories remain unresolved. Here, DNA methylation landscapes in two widely used human NPC models-ReNcell VM (RVM) and ReNcell CX (RCX)-were examined under several different culture conditions to define regulatory pathways shaping lineage specification. Exploratory analyses revealed that the ReNcell lines exhibited methylation similar to primary glial populations rather than neuronal subtypes, with RCX cells positioned further along a maturation trajectory and RVM cells retaining a multipotent state. RCX cultures displayed hypomethylation of neuronal markers (<i>DCX</i>, <i>ENO2</i>, <i>MAP2</i>), whereas RVM cultures showed consistent <i>GFAP</i> hypomethylation, indicative of glial or early progenitor identity. Signaling pathways regulating lineage commitment were highlighted, including TGFβ, Wnt, and Notch signaling. Within the Notch pathway, RCX cells exhibited higher gene expression of <i>NOTCH2</i> and <i>JAG</i> ligands, consistent with active lateral induction and a developmentally advanced state. In contrast, RVM cells exhibited higher <i>DLL1</i> and <i>NOTCH1</i> expression, supporting lateral inhibition and cellular heterogeneity. Knockdown of syndecan-4 (<i>SDC4</i>) revealed opposing effects on Notch activity. Together, these findings established DNA methylation as a determinant of lineage-specific signaling in human NPCs.