No abstract

SummarymiR-184 is a highly evolutionary conserved microRNA (miRNA) from fly to human. The importance of miR-184 was underscored by the discovery that point mutations in miR-184 gene led to corneal/lens blinding disease. However, miR-184-related function in vivo remained unclear. Here, we report that the miR-184 knockout mouse model displayed increased p63 expression in line with epidermal hyperplasia, while forced expression of miR-184 by stem/progenitor cells enhanced the Notch pathway and induced epidermal hypoplasia. In line, miR-184 reduced clonogenicity and accelerated differentiation of human epidermal cells. We showed that by directly repressing cytokeratin 15 (K15) and FIH1, miR-184 induces Notch activation and epidermal differentiation. The disease-causing miR-184C57U mutant failed to repress K15 and FIH1 and to induce Notch activation, suggesting a loss-of-function mechanism. Altogether, we propose that, by targeting K15 and FIH1, miR-184 regulates the transition from proliferation to early differentiation, while mis-expression or mutation in miR-184 results in impaired homeostasis.

Mutations in key transcription factors SOX2 and P63 were linked with developmental defects and postnatal abnormalities such as corneal opacification, neovascularization, and blindness. The latter phenotypes suggest that SOX2 and P63 may be involved in corneal epithelial regeneration. Although P63 has been shown to be a key regulator of limbal stem cells, the expression pattern and function of SOX2 in the adult cornea remained unclear. Here, we show that SOX2 regulates P63 to control corneal epithelial stem/progenitor cell function. SOX2 and P63 were co‐expressed in the stem/progenitor cell compartments of the murine cornea in vivo and in undifferentiated human limbal epithelial stem/progenitor cells in vitro. In line, a new consensus site that allows SOX2‐mediated regulation of P63 enhancer was identified while repression of SOX2 reduced P63 expression, suggesting that SOX2 is upstream to P63. Importantly, knockdown of SOX2 significantly attenuated cell proliferation, long‐term colony‐forming potential of stem/progenitor cells, and induced robust cell differentiation. However, this effect was reverted by forced expression of P63, suggesting that SOX2 acts, at least in part, through P63. Finally, miR‐450b was identified as a direct repressor of SOX2 that was required for SOX2/P63 downregulation and cell differentiation. Altogether, we propose that SOX2/P63 pathway is an essential regulator of corneal stem/progenitor cells while mutations in SOX2 or P63 may disrupt epithelial regeneration, leading to loss of corneal transparency and blindness. Stem Cells 2019;37:417–429

SummaryThe transition from naive to primed state of pluripotent stem cells is hallmarked by epithelial-mesenchymal transition, metabolic switch from oxidative phosphorylation to aerobic glycolysis, and changes in the epigenetic landscape. Since these changes are also seen as putative hallmarks of neoplastic cell transformation, we hypothesized that oncogenic pathways may be involved in this process. We report that the activity of RAS is repressed in the naive state of mouse embryonic stem cells (ESCs) and that all three RAS isoforms are significantly activated upon early differentiation induced by LIF withdrawal, embryoid body formation, or transition to the primed state. Forced expression of active RAS and RAS inhibition have shown that RAS regulates glycolysis, CADHERIN expression, and the expression of repressive epigenetic marks in pluripotent stem cells. Altogether, this study indicates that RAS is located at a key junction of early ESC differentiation controlling key processes in priming of naive cells.

AbstractStem cells (SCs) are traditionally viewed as rare, slow-cycling cells that follow deterministic rules dictating their self-renewal or differentiation. It was several decades ago, when limbal epithelial SCs (LSCs) that regenerate the corneal epithelium were one of the first sporadic, quiescent SCs ever discovered. However, LSC dynamics, heterogeneity and genetic signature are largely unknown. Moreover, recent accumulating evidence strongly suggested that epithelial SCs are actually abundant, frequently dividing cells that display stochastic behavior.In this work, we performed an in-depth analysis of the murine limbal epithelium by single-cell RNA sequencing and quantitative lineage tracing. The generated data provided an atlas of cell states of the corneal epithelial lineage, and particularly, revealed the co-existence of two novel LSC populations that reside in separate and well-defined sub-compartments. In the “outer” limbus, we identified a primitive widespread population of quiescent LSCs (qLSCs) that uniformly express Krt15/Gpha2/Ifitm3/Cd63 proteins, while the “inner” limbus host prevalent active LSCs (aLSCs) co-expressing Krt15-GFP/Atf3/Mt1-2/Socs3. Analysis of LSC population dynamics suggests that while qLSCs and aLSCs possess different proliferation rates, they both follow similar stochastic rules that dictate their self-renewal and differentiation. Finally, T cells were distributed in close proximity to qLSCs. Indeed, their absence or inhibition resulted in the loss of quiescence and delayed wound healing. Taken together, we propose that divergent regenerative strategies are tailored to properly support tissue-specific physiological constraints. The present study suggests that in the case of the cornea, quiescent epithelial SCs are abundant, follow stochastic rules and neutral drift dynamics.