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bioRxiv · 10.64898/2026.09.16.751974

Mapping heterogeneity drivers in human PSC-derived limbal stem cell differentiation through single-cell multi-modal analysis

Arts, J. A.·Lima Cunha, D.·Harjuntausta, S.·Vattulainen, M.·Smits, J. G.·Bonthuis, I.·Fragoso Bento, D.·Bocci, F.·Skottman, H.·Zhou, H.
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Abstract

Understanding the molecular underpinnings of stem cell differentiation is pivotal for generating appropriate cell types in cell-based therapies. Differentiation of human pluripotent stem cells (PSC) into corneal limbal stem cells offers a promising avenue to regenerate the corneal epithelium. However, current differentiation strategies remain inconsistent in efficiency and yield heterogeneous cell populations that incompletely recapitulate the regenerative properties of donor-derived limbal stem cells. Here, we mapped the molecular landscape of cell states throughout the differentiation process. First, we construct PSC differentiation paths from single-cell RNA sequencing (scRNA-seq) data using the computational framework of optimal transport, identifying on- and off-track cell states toward the limbal stem cell state. Single cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq) was performed to profile accessible genomic regions, and subsequently integrated with scRNA-seq data through gene regulatory network analysis to identify key drivers governing the diverse cell states. We showed that genomic enhancers play a major role in cell state determinations. Through this single-cell multi-modal approach, we identified potential transcription factors driving limbal epithelial lineage specification and off-track cell populations. Our findings provide a framework for rational optimization of PSC-derived limbal stem cell generation to advance the development of corneal cell therapies.

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Arts, J. A., Lima Cunha, D., Harjuntausta, S., Vattulainen, M., Smits, J. G., Bonthuis, I., Fragoso Bento, D., Bocci, F., Skottman, H., Zhou, H.. 2026-09-17. Mapping heterogeneity drivers in human PSC-derived limbal stem cell differentiation through single-cell multi-modal analysis. https://doi.org/10.64898/2026.09.16.751974

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