Comparable characterization of human hair follicle cell types and differentiation states is currently lacking as profiling of human hair follicles is difficult due to their relative scarcity within the skin compared to mice. In the current study, we gained access to follicular-enriched fractions of human skin that were discarded from hair transplant procedures and subjected them to high throughput sc-RNA procedures to gain single cell transcriptomes for many of the cells associated with human follicles. differentially expressed genes between clusters were defined as logFC>1 and adjusted p-value<0.05). Only enriched categories with an adjusted p-value <0.05 are shown. Overrepresentation analysis and visualization performed using ClusterProfiler R package. NIHMS1542173-supplement-5.tif (83M) GUID:?4DEA929D-F94A-4A4D-A88D-49C838FE5E26 6. NIHMS1542173-supplement-6.docx (17K) GUID:?FEA9C97A-E215-4413-A3FD-1B842DCFA4B2 S4: Figure S4: Immunohistochemical confirmation of expression of proteins predicted by sc-RNA-seq. A, immunofluorescence for the same markers shown in Figure 3, but at lower magnification (5). B, Immunostains were derived from the Human Protein Atlas (Uhlen, Fagerberg et al., 2015). NIHMS1542173-supplement-S4.jpg (1.4M) GUID:?1D05340F-257E-40A8-8CB6-EE76E38E1890 Data Availability StatementData Availability The scRNA-seq data are in NIH-GEO (“type”:”entrez-geo”,”attrs”:”text”:”GSE129611″,”term_id”:”129611″GSE129611). The averaged data for all cell clusters are available in TableS2. The raw data are available through Figshare: https://figshare.com/s/74a818b57f08b0873a8c Abstract The epidermis and its appendage, the hair follicle, represent an elegant developmental system in which cells are replenished with regularity because of controlled proliferation, lineage specification, and terminal differentiation. While transcriptome data exists for human epidermal and dermal cells, the hair follicle remains poorly characterized. Through single-cell resolution profiling of the epidermis and anagen hair follicle, we characterized the anatomical, transcriptional, functional, and pathological profiles of distinct epidermal, hair follicle, and hair follicle-associated cell subpopulations including melanocytes, endothelial cells, and immune cells. We additionally traced the differentiation trajectory of interfollicular and matrix cell progenitors and explored association of specific cell subpopulations to known molecular signatures of common skin conditions. These data simultaneously corroborate prior preliminary murine and human investigations while offering new insights into epidermal and hair follicle differentiation and pathogenesis. INTRODUCTION Skin has been the subject of extensive characterization at the pathological and molecular level in humans and mice for decades (Fuchs, 1998). During that time, numerous cell types within the epidermis and its appendages, the hair follicle, and sweat glands have been identified via immunohistochemical and array-based bulk gene expression analyses in each species. The characterization of epidermal, dermal, and hair follicles cells at a single cell resolution has lagged compared to other tissues, largely owing to limited sample sizes and the limitations of early single cell technologies. Previous single cell transcriptional methods (e.g. Fluidigm) required labor-intensive manual sorting of cells for array hybridization or sequencing, which necessitated significant sample sizes to overcome processing losses for reliable downstream application. Newer methods (e.g., Drop-seq, 10X Genomics), however, employ multiplex barcoding and microfluidics that enable the rapid parallel processing of thousands of cells at a lower cost with significantly improved sensitivity (Klein and Macosko, 2017; Picelli, 2017; Ziegenhain, Vieth et al., 2017). The first single cell mapping of murine epidermal and hair follicle cells yielded 1422 unique transcriptome profiles, clustering into 25 unique subpopulations MRPS31 (Acosta, Joost et al., 2017; Joost, Zeisel et al., 2016). These human studies focused primarily on elucidation of interfollicular epidermal and dermal cell types and differential transcriptional programs activated by pathological processes such as psoriasis and wound healing (Cheng, Sedgewick et al., 2018; Philippeos, Telerman et al., 2018). Comparable characterization of human hair follicle cell types and differentiation states is currently lacking as profiling Ilaprazole of human hair follicles is difficult due to their relative scarcity within the skin compared to mice. In the current study, we gained access to follicular-enriched fractions of human skin that were discarded from hair transplant procedures and subjected them to high throughput sc-RNA Ilaprazole procedures to gain single cell transcriptomes for many of the cells associated with human follicles. This allowed for the generation of single cell transcriptomes of numerous cell states within the follicle as well as epidermal keratinocytes, endothelial cells, mesenchymal populations, immune cells, and melanocytes. With these data, cell fate trajectories can be generated, and patterns of gene expression in skin diseases can be probed to find individual cell types that are targets of disease. RESULTS Single cell profiling of follicle-enriched human skin grafts Prior human single cell investigations used fractions from total epidermis, resulting in a high ratio of epidermal to follicular cells (Cheng, Sedgewick et al., 2018). To improve single cell resolution of follicular cell types, anagen hair follicles were obtained from discarded human scalp micrografts collected for transplantation (Figure 1A). Micrografts are composed of several hair follicles, the hair bulbs, and Ilaprazole variable amounts of surrounding tissues, including interfollicular epidermis, dermis, sebaceous/apocrine, among others. Micrografts were collected from five patients for single cell analysis and immunohistochemistry. Anagen phase was confirmed by H&E-labeled sections of adjacent graft samples. Following enzymatic and Ilaprazole physical dissociation,.