Characterization of structural variants in KOLF2.1J iPSCs and their potential impact on forebrain organoid differentiation.

Abstract

The KOLF2.1J human induced pluripotent stem cell (iPSC) line was created as a reference for neurological disease modeling. Previous genotyping efforts identified several large structural variants (SVs), raising questions about their potential impact on neuronal differentiation. Here, we identified 37 additional SVs using long-read sequencing and optical genome mapping. Additionally, we found that one of the known SVs contained a previously undetected overlap with RYBP, a neurodevelopmental gene. To assess the functional consequences of these SVs, we performed single-cell RNA sequencing of day 90 forebrain organoids derived from KOLF2.1J and two independent control iPSC lines. While cell-type composition was preserved, multiple SV-affected genes were differentially expressed and cell-cell adhesion and synapse-related processes were enriched in KOLF2.1J organoids. These findings highlight the importance of robust genetic characterization and multi-line validation for reliable iPSC-based modeling.