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.
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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