Microarray based global hierarchical clustering clearly sepa

Microarray-based global hierarchical clustering clearly separated all the directly isolated keratinocyte populations. In the first round of analysis () where we focused on known and well characterized populations the Plet population clearly separated from the rest of the populations, which is probably explained by Plet being suprabasal. Subsequently, the cluster analysis separated Bulge followed by IFE. PletA6 and PletA6Sca are the two populations that most resemble each other and this may be explained by the Sca-1 expression profiles that define the two overlapping populations (A). However, a greater similarity was seen between PletA6Sca and IFE compared to PletA6 and IFE (A and C; Table S3) and again may suggest PletA6Sca as the progeny of PletA6 that has initiated migration to the infundibulum where abscisic acid become Sca-1pos. It is debated to what extent the characteristics of hair follicle stem cells are determined by their niches (Hsu et al., 2014; Mesa et al., 2015). In contrast to the directly isolated cells, we can show that cultured cells from each of the populations acquire similar global gene-expression profiles that cannot be distinguished from each other (A and B) after 14days in culture (also see Table S4). This indicates that the cells are either highly affected by extrinsic influences from nearby environment or that a specific phenotype is selected for during culturing. Clearly the phenotype regarding CFE is not changed by the environment suggesting that intrinsic factors that we are unable to measure in the array analysis are playing a important role. The most notable pathways to be affected was the Wnt pathway which was markedly down regulated in cultured cells primarily from the hair follicle populations located in the basal layer and Wnt signalling is a well known key factor in hair follicle morphogenesis and maintenance (Huelsken et al., 2001). Trp63 is also downregulated in cultured keratinocytes and in human keratinocytes Trp63 has previously been linked to stemness (Pellegrini et al., 2001).
Conclusion We show that PletA6Sca is a highly clonogenic and proliferating population while PletA6 most likely represents the Lgr6pos population described by Snippert et al. (2010). By using gene-expression profiling we show that the populations Plet and IRK express several suprabasal differentiation markers and most likely constituting cells of the inner root sheath within the central and lower hair follicle respectively. Our data show that the different keratinocyte populations acquire similar gene expression profiles without much resemblance to the parent population when subjected to culture conditions, and this also highlights the limitations in gene expression studies on cultured keratinocytes. Finely our results raises some concerns regarding studies where specific promoters are used in lineage tracing experiments as our flow cytometry data suggest a more complex expression pattern of markers such as Plet-1 and LRIG1 being shared by more than one population and in more than one cell layer of the hair follicle. The following are the supplementary data related to this article.
Acknowledgements This work was supported by Karen Elise Jensen Fund (UBJ), Agnes og Poul Friis fond (UBJ), Jeppe Juhl og Hustru Ovita Juhls Mindelegat, (UBJ) and Aage Bang Fund (UBJ). Region Midts Forskningsfond supported AG. The proficient technical assistance by Anette Thomsen and Christina Sønderskov is highly appreciated. Jian Li thanks the support from the Natural Science Foundation of Jiangsu Province, China (Project No. BK20151403). Flow cytometry/cell sorting was performed at the FACS Core Facility, Aarhus University, Denmark.
Resource table
Resource details RCe021-A (RC-17) was received as day 3 embryo that was surplus to requirement or unsuitable for clinical use and was cultivated to the blastocyst stage in medium containing GMP grade granulocyte-macrophage colony-stimulating factor (GM-CSF) to improve survival of the inner cell mass (Sjöblom et al. 1999). Human embryonic stem cell (hESC) isolation, expansion and qualification were performed in a facilities whose specification, operation and monitoring complied with GMP standards enabling; i) a fully traceable procurement procedure with informed ethical consent which includes provision for commercial use, ii) detailed medical history and blood borne virus (BBV) screening of donors, and iii) compilation of a cell line history providing details on hESC manufacturing process and quality control testing regime.