Single keratinocyte analysis uncovers Foxm1 as a Yap-dependent regulator of human epidermal stem cells (scRNA-Seq)

Autologous epidermal cultures can permanently restore a functional epidermis on severely burned patients. Transgenic epidermal grafts do so also in genetic skin diseases as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells. To date, such cells can be only partially distinguished from the other transient amplifying clonogenic keratinocytes and cannot be prospectively isolated. Here we show that genome-wide single-cell transcriptome analysis performed on primary human epidermal keratinocyte cultures identified categories of genes clearly distinguishing the different clonal types, unveiled that holoclone-forming cells are enriched in genes regulating cell cycle, DNA repair (including telomerase), chromosome segregation and spindle organization, confirmed that human epidermal keratinocytes are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells and uncovered the role of FOXM1 as a YAP-dependent key regulator of normal and adhesion-defective epidermal stem cells. The purpose of this study is to define, at single-cell levels, transcriptional profile of keratinocytes stem cells and transient amplifying progenitors. Single-cell RNA seq data from two human primary keratinocyte cultures (K82 and K86) harvest at early passsages (t1, profiled 3.367 and 3.978 cells respectively) and after serial cultivation (t2, profiled 4292 and 3576 cells respectively) were obtained with 10X Genomics Chromium Technology.

自体表皮培养物可对重度烧伤患者实现功能性表皮的永久性修复。转基因表皮移植物亦可在交界型大疱性表皮松解症（Junctional Epidermolysis Bullosa）这类遗传性皮肤病中达成上述修复效果。临床成功严格依赖于足量的表皮干细胞，这类细胞可被鉴定为全克隆形成细胞（holoclone-forming cells）。迄今为止，此类细胞仅能与其他瞬时扩增性克隆形成角质形成细胞进行部分区分，且无法通过前瞻性方法分离获取。本研究通过对原代人表皮角质形成细胞培养物开展全基因组范围的单细胞转录组分析，成功鉴定出可明确区分不同克隆类型的基因类别；研究揭示全克隆形成细胞显著富集于调控细胞周期、DNA修复（包括端粒酶）、染色体分离及纺锤体组装的基因集合中；证实人表皮角质形成细胞沿一条连续且近似线性的轨迹呈现层级组织结构，即干细胞依次分化为祖细胞并最终产生终末分化细胞；同时阐明了FOXM1作为依赖于YAP的关键调控因子，在正常及黏附缺陷型表皮干细胞中的作用。本研究的核心目的是在单细胞层面解析角质形成干细胞与瞬时扩增祖细胞的转录组特征。本研究采用10X Genomics Chromium技术获取了两株原代人表皮角质形成细胞（K82与K86）的单细胞RNA测序数据：分别在早期传代阶段（t1，分别对3367和3978个细胞进行转录组分析）及连续传代培养后（t2，分别对4292和3576个细胞进行转录组分析）采集样本。