Human pluripotent stem cell-derived limbal stem cells (hPSC-derived LSCs) provide a promising cell source for corneal transplants and ocular surface reconstruction. Although recent efforts in the identification of LSC markers have increased our understanding of the biology of LSCs, much more remains to be characterized in the developmental origin, cell fate determination, and identity of human LSCs. The lack of knowledge hindered the establishment of efficient differentiation protocols for generating hPSC-derived LSCs and held back their clinical application. Here, we performed a time-course single-cell RNA-seq to investigate transcriptional heterogeneity and expression changes of LSCs derived from human embryonic stem cells (hESCs). Based on current protocol, expression heterogeneity of reported LSC markers were identified in subpopulations of differentiated cells. EMT has been shown to occur during differentiation process, which could possibly result in generation of untargeted cells. Pseudotime trajectory analysis revealed transcriptional changes and signatures of commitment of hESCs-derived LSCs and their progeny—the transit amplifying cells. Single-cell RNA-seq revealed time-course expression changes and significant transcriptional heterogeneity during hESC-derived LSC differentiation in vitro. Our results demonstrated candidate developmental trajectory and several new candidate markers for LSCs, which could facilitate elucidating the identity and developmental origin of human LSCs in vivo.

中文翻译：

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时程单细胞RNA测序揭示了源自人多能干细胞的角膜缘干细胞的转录动力学和异质性

人多能干细胞衍生的角膜缘干细胞（hPSC衍生的LSC）为角膜移植和眼表重建提供了有希望的细胞来源。尽管最近在鉴定LSC标记物方面的努力增加了我们对LSC生物学的理解，但在人类LSC的发育起源，细胞命运确定和身份鉴定方面还有更多的特征。知识的缺乏阻碍了建立有效的分化协议以产生hPSC衍生的LSC，并阻碍了其临床应用。在这里，我们进行了时程单细胞RNA测序，以研究人类胚胎干细胞（hESCs）衍生的LSC的转录异质性和表达变化。根据当前协议，报告的LSC标记的表达异质性已在分化细胞亚群中鉴定。已经表明EMT在分化过程中发生，这可能导致未靶向细胞的产生。伪时轨迹分析揭示了hESCs衍生的LSCs及其子代-转运扩增细胞的转录变化和承诺的特征。单细胞RNA序列揭示了在hESC衍生的LSC体外分化过程中时程表达的变化和明显的转录异质性。我们的研究结果证明了LSCs的候选发展轨迹和几种新的候选标记，这可能有助于阐明人LSCs在体内的身份和发育起源。这可能会导致产生非靶向细胞。伪时轨迹分析揭示了hESCs衍生的LSCs及其子代-转运扩增细胞的转录变化和承诺的特征。单细胞RNA序列揭示了在hESC衍生的LSC体外分化过程中时程表达的变化和明显的转录异质性。我们的研究结果证明了LSCs的候选发展轨迹和几种新的候选标记，这可能有助于阐明人LSCs在体内的身份和发育起源。这可能会导致产生非靶向细胞。伪时轨迹分析揭示了hESCs衍生的LSCs及其子代-转运扩增细胞的转录变化和承诺的特征。单细胞RNA序列揭示了在hESC衍生的LSC体外分化过程中时程表达的变化和明显的转录异质性。我们的研究结果证明了LSCs的候选发展轨迹和几种新的候选标记，这可能有助于阐明人LSCs在体内的身份和发育起源。单细胞RNA序列揭示了在hESC衍生的LSC体外分化过程中时程表达的变化和明显的转录异质性。我们的研究结果证明了LSCs的候选发展轨迹和几种新的候选标记，这可能有助于阐明人LSCs在体内的身份和发育起源。单细胞RNA序列揭示了在hESC衍生的LSC体外分化过程中时程表达的变化和明显的转录异质性。我们的研究结果证明了LSCs的候选发展轨迹和几种新的候选标记，这可能有助于阐明人LSCs在体内的身份和发育起源。