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

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. To explore whether holoclones, meroclones and paraclones activate different transcriptional programs, we performed genome-wide microarray gene expression profiling of 62 clones (22 holoclones, 29 meroclones and 11 paraclones) isolated from 6 different primary cultures (K5, K18, K22, K38, K42 and K49). Sub-confluent epidermal cultures were trypsinized, serially diluted and plated in 96-well plates (0.5-1 cells per well). After 7 days of cultivation, single clones were identified under an inverted microscope and trypsinized. One-quarter of the clone was cultured for 12 days onto an indicator dish, which was then fixed and stained with rhodamine B for the classification of clonal type 12-d after. The remaining three-quarters of the clone was cultivated on 6-well plates for 4–5 days and then used to collect mRNA.