Nuclear Factor I genes drive chondrogenic cell-fate commitment

Human induced pluripotent stem cells (hiPSCs) offer a powerful platform to model chondrogenesis and enable regenerative strategies, yet regulation of cell-fate commitment remains elusive. Here, we systematically mapped cell-fate trajectories from 7 time points during a 49-day chondrogenic hiPSC differentiation protocol using single-nucleus multimodal transcriptomic and chromatin accessibility profiling (scRNA-seq and scATAC-seq). Integrative analysis of dynamics revealed branching differentiation trajectories with clear bifurcation points and distinct cell-fates. Notably, the chondrogenic trajectory originated at day 6 as a neurogenic development and branched off at day 21 to a chondrogenic cell-fate. Through transcription factor activity analysis (TFAA) and cis-co-accessibility networks, we found that NFIA and NFIB drove chondrogenic distinction, exhibited in both modalities as directly targeting chondrogenic genes such as COMP , FIBIN , VIM. This was then confirmed by experimental validation as modulation of NFIA expression at this point further enhanced chondrocyte formation. Together, our study provides a high-resolution multimodal atlas of chondrogenic differentiation and identified critical transcriptional regulators that can now be leveraged to implement regenerative cartilage therapies from hiPSCs.