the way scientists are studying hair follicle (HF) functions at a molecular level with single-cell resolution. Recent studies are now allowing a better understanding of the diverse cell subpopu­lations residing in the HF, their transcriptional proles at different stages of the hair cycle, and the potential interactions between them. Moreover, clues are emerging as to how specic cell subpopulations in the HF are replenished, as well as the molecular mechanisms allowing their fate selection and terminal differentiation. Elucidating the complex intercellular communi­cation underlying the hair cycle will help us understand common hair pathologies and the cell subpopulations involved.

40]. Instead, other protocols using dispase,

microdissection, or explant cultures have been proposed [36, 41–43].

A pioneering study performed by Joost, and colleagues was the rst to comprehensively address murine telogen epidermis and HF cellu­lar heterogeneity [44]. Through sequencing 1422 single-cell transcriptomes, authors were able to identify 25 different subpopulations of interfol­licular and follicular epidermal cells. Unsupervised clustering and afnity propagation using high variance genes conrmed the exis­tence of 13 previously known epidermal subpop­ulations: sebaceous gland (SG cells), outer bulge and inner bulge keratinocytes, IFE basal cells, two stages of IFE differentiated cells, two termi­nally differentiated IFE keratinized layer cells, three distinct groups of upper HF cells, and two immune cell populations (Langerhans and resi­dent T cells). Interestingly, subclustering of outer bulge, inner bulge, upper HF, and basal IFE pop­ulations derived in novel subpopulations which were conrmed by immunohistochemistry and single-molecule mRNA uorescence in situ hybridization (smRNA-FISH). Analysis of each subpopulation’s gene proles led to the observa­tion that most heterogeneity in the HF was a result of a combination of recurring gene signa­tures. Thus, authors performed pseudotemporal ordering of IFE cells and identied 1627 genes

unbiased systematic assessment of gene expression proles and spatial location of all cell subpopulations, now using murine full-thickness skin during growth and rest [45]. Samples obtained from dorsal mouse skin during anagen and telogen were processed and 5767 and 7601 single cells were sequenced consisting of testing and validation datasets respectively. According to their expression proles, cells were grouped into 7 main clusters: keratinocytes from the perma­nent epidermis (IFE, SG, non-cycling HF), kera­tinocytes from the anagen HF, broblast-like cells, immune cells (T-cells, macrophages, den­dritic cells, Langerhans cells), vascular cells, neural crest-derived cells (melanocytes and Schwann cells), and others (skeletal muscle and red blood cells). These clusters were further strat­ied into 56 transcriptionally distinct cell types and cell states and were validated through smRNA-FISH.Interestingly, anagen-specic HF cells comprised 20 different subpopulations, sug­gesting that hair shaft growth is the result of a complex interplay between numerous subpopula­tions with multiple cell states. Given that anagen HF keratinocytes and broblast-like subpopula­tions undergo substantial remodeling through the hair cycle, authors focused on them to perform a more in-depth analysis. Interestingly, authors uncovered six distinct subpopulations of kerati­nocytes in the outer layer (OL) which were broadly classied into basal and suprabasal. The basal OL was composed of two different basal ORS populations (ORS B1 and ORS B2) and a lower proximal cup (LPC) group. Likewise, the suprabasal OL cluster was subdivided into a suprabasal ORS (ORS SB), a mid-part compan­ion layer (mCP), and an upper companion layer (uCP) subpopulation. Basal and suprabasal OL cells were found to be intermingling even though they are transcriptionally distinct and depict no evidence of transitions between them. Moreover, authors revealed a trajectory from mCP to uCP cells, conrming that the companion layer is formed through upward dynamics from the hair bulb to the bulge [45–47]. Keratinocytes of the matrix and inner layers of the HF were grouped in a cluster that displayed a peculiar branching topology. RNA velocity analysis and an unsuper-

Currently, the pathogenesis of hair patholo­gies, for example, androgenetic alopecia are still unclear. Using scRNA-seq and spatial transcrip­tomics will provide means for cell subpopulation identication, pseudo-time analysis, cell-cell ligand-receptor interactions, and gene regulatory network reconstruction, tools which will allow a better understanding of hair pathologies and deri­vation of novel therapeutic targets.