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

## TL;DR

This study shows that commonly used neural progenitor cells have built-in developmental biases, which affect how they become nerve or glial cells, and these biases are linked to DNA methylation patterns.

## Contribution

The study reveals inherent lineage biases in ReNcell neural progenitor cell lines through DNA methylation analysis and signaling pathway differences.

## Key findings

- ReNcell CX cells showed methylation patterns resembling more mature neurons, while ReNcell VM cells retained a progenitor-like state.
- DNA methylation differences were observed at genes linked to neuronal and glial identity, influencing signaling pathways like Notch.
- Knockdown of SDC4 had opposing effects on Notch signaling in the two cell lines, highlighting their distinct regulatory mechanisms.

## Abstract

Neural progenitor cells are early brain cells that can develop into nerve cells or support cells called glia. These cells are widely used in research to study human brain development and disease, yet it is often assumed that they are neutral starting points that can be directed equally toward different cell fates. In this study, we asked whether two commonly used human neural progenitor cell models already show built-in differences that influence how they develop. We found that both models showed chemical marks on their DNA that were more similar to glial cells than to nerve cells. Cells derived from the cerebral cortex appeared more developmentally advanced, while cells derived from the midbrain retained a more flexible, progenitor-like state. These differences were reflected in epigenetic changes at genes linked to nerve or glial identity and in signaling systems that control cell fate decisions. We also found that altering a cell-surface molecule involved in organising signaling proteins affected these pathways differently depending on the cell model. Together, these findings show that commonly used neural progenitor cells have inherent developmental biases that can be leveraged, rather than resisted, when modelling human brain development and neurological disease, improving experimental design and interpretation.

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 (DCX, ENO2, MAP2), whereas RVM cultures showed consistent GFAP 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 NOTCH2 and JAG ligands, consistent with active lateral induction and a developmentally advanced state. In contrast, RVM cells exhibited higher DLL1 and NOTCH1 expression, supporting lateral inhibition and cellular heterogeneity. Knockdown of syndecan-4 (SDC4) revealed opposing effects on Notch activity. Together, these findings established DNA methylation as a determinant of lineage-specific signaling in human NPCs.