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Summary: The conjunctival epithelium consists of conjunctival epithelial cells and goblet cells derived from conjunctival epithelial stem/progenitor cells. However, the source of these cells is not well known because no specific markers for conjunctival epithelial stem/progenitor cells have been discovered. Therefore, to identify conjunctival epithelial stem/progenitor cell markers, we performed single-cell RNA sequencing of a conjunctival epithelial cell population derived from human-induced pluripotent stem cells (hiPSCs). The following conjunctival epithelial markers were identified: BST2, SLC2A3, AGR2, TMEM54, OLR1, and TRIM29. Notably, BST2 was strongly positive in the basal conjunctival epithelium, which is thought to be rich in stem/progenitor cells. Moreover, BST2 was able to sort conjunctival epithelial stem/progenitor cells from hiPSC-derived ocular surface epithelial cell populations. BST2-positive cells were highly proliferative and capable of successfully generating conjunctival epithelial sheets containing goblet cells. In conclusion, BST2 has been identified as a specific marker of conjunctival epithelial stem/progenitor cells.

Summary: The extracellular matrix plays a key role in stem cell maintenance, expansion, and differentiation. Laminin, a basement membrane protein, is a widely used substrate for cell culture including the growth of human induced pluripotent stem cells (hiPSCs). Here, we show that different isoforms of laminin lead to the selective differentiation of hiPSCs into different eye-like tissues. Specifically, the 211 isoform of the E8 fragment of laminin (LN211E8) promotes differentiation into neural crest cells via Wnt activation, whereas LN332E8 promotes differentiation into corneal epithelial cells. The immunohistochemical distributions of these laminin isoforms in the developing mouse eye mirrors the hiPSC type that was induced in vitro. Moreover, LN511E8 enables generation of dense hiPSC colonies due to actomyosin contraction, which in turn led to cell density-dependent YAP inactivation and subsequent retinal differentiation in colony centers. Thus, distinct laminin isoforms determine the fate of expanded hiPSCs into eye-like tissues. : Shibata et al. report that laminin isoforms differentially regulate the ocular cell differentiation from hiPSCs. The binding affinity of laminin and integrins determines the nature of expanded hiPSC colonies in terms of cell motility, cell-cell interactions, and cell density, with the involvement of Wnt and YAP signals. Keywords: human induced pluripotent stem cells, hiPSCs, laminin isoforms, YAP, Wnt, ocular cell differentiation, self-formed ectodermal autonomous multi-zone, SEAM