{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T05:17:54Z","timestamp":1788585474077,"version":"build-2803163510"},"reference-count":45,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T00:00:00Z","timestamp":1788134400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"The Seventh Affiliated Hospital of Sun Yat-sen University","award":["ZSQY202573534"],"award-info":[{"award-number":["ZSQY202573534"]}]},{"award":["ZSQY202573534"],"award-info":[{"award-number":["ZSQY202573534"]}],"id":[{"id":"https:\/\/ror.org\/00rfd5b88","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003785","name":"Health Commission of Guandgong Province","doi-asserted-by":"publisher","award":["B2025503"],"award-info":[{"award-number":["B2025503"]}],"id":[{"id":"10.13039\/501100003785","id-type":"DOI","asserted-by":"publisher"}]},{"award":["B2025503"],"award-info":[{"award-number":["B2025503"]}],"id":[{"id":"https:\/\/ror.org\/05bxtha84","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Background: Secondary hair germ (SHG)-associated epithelial cells are a key progenitor population that contribute to hair follicle regeneration during early anagen. Although SHG-associated cells become activated during early anagen and contribute to the formation of regenerating follicular structures, the morphologically recognizable SHG compartment becomes less apparent as lower hair follicle structures develop during mid-anagen. However, the transcriptional changes accompanying the transition from SHG-associated states toward lower hair follicle-associated states remain poorly characterized. This study aimed to characterize the transcriptional changes associated with this transition and identify a proliferative intermediate transcriptional state during early anagen. By integrating single-cell transcriptomic analysis with regulatory and cell\u2013cell communication analyses, we sought to improve the understanding of cellular state transitions and microenvironmental changes during early hair follicle regeneration. Methods: Hematoxylin and eosin staining (H&amp;E) was performed to examine histological changes in mouse dorsal skin on days 0, 5, 10, 15, 20, 25, and 30 of the hair cycle. In parallel, previously published single-cell RNA sequencing data from mouse dorsal skin during early anagen were reanalyzed. Hair follicle epithelial cells were extracted and reclustered to identify cell subpopulations with distinct transcriptional characteristics. Cell types were annotated based on canonical marker genes and subpopulation-specific expression patterns. Differential expression analysis, functional enrichment analysis, cell-cycle analysis, developmental potential assessment, trajectory inference, transcription factor regulon analysis, and cell\u2013cell communication analysis were further performed to characterize the relationships among hair germ cells, proliferative lower hair follicle-like cells (PR-loHF), and lower hair follicle cells. Results: Proliferative lower hair follicle-like cells (PR-loHF) were identified as a transcriptionally defined intermediate state associated with the transition from secondary hair germ (SHG)-associated cells toward lower hair follicle-associated cells. PR-loHF exhibited enrichment of biological programs associated with cell-cycle activity, epithelial remodeling, extracellular matrix organization, and developmental regulation. Moreover, compared with SHG-associated and lower hair follicle-associated states, PR-loHF exhibited distinct transcription factor regulon activity and cell\u2013cell communication patterns, further supporting its characterization as a transcriptionally distinguishable intermediate state. Conclusions: PR-loHF represents a proliferative intermediate transcriptional state emerging during the transition from secondary hair germ (SHG)-associated cells toward lower hair follicle-associated states, providing new insights into transcriptional transitions and microenvironmental regulation during early hair follicle regeneration.<\/jats:p>","DOI":"10.3390\/genes17091058","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T01:05:26Z","timestamp":1788224726000},"page":"1058","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Single-Cell Transcriptomic Identification of a Proliferative Intermediate State During Early Anagen Hair Follicle Regeneration"],"prefix":"10.3390","volume":"17","author":[{"given":"Yueyan","family":"Liu","sequence":"first","affiliation":[{"name":"School of Pharmaceutical Sciences, Sun Yat-sen University, Shenzhen 518107, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0997-8705","authenticated-orcid":false,"given":"Xusheng","family":"Wang","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Sun Yat-sen University, Shenzhen 518107, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Wen","sequence":"additional","affiliation":[{"name":"The Seventh Affiliated Hospital, Sun Yat-sen University, Shenzhen 518107, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1046\/j.0022-202x.2001.01377.x","article-title":"A Comprehensive Guide for the Accurate Classification of Murine Hair Follicles in Distinct Hair Cycle Stages","volume":"117","author":"Handjiski","year":"2001","journal-title":"J. Investig. Dermatol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"R132","DOI":"10.1016\/j.cub.2008.12.005","article-title":"The Hair Follicle as a Dynamic Miniorgan","volume":"19","author":"Schneider","year":"2009","journal-title":"Curr. Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1329","DOI":"10.1016\/0092-8674(90)90696-C","article-title":"Label-Retaining Cells Reside in the Bulge Area of Pilosebaceous Unit: Implications for Follicular Stem Cells, Hair Cycle, and Skin Carcinogenesis","volume":"61","author":"Cotsarelis","year":"1990","journal-title":"Cell"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1126\/science.1092436","article-title":"Defining the Epithelial Stem Cell Niche in Skin","volume":"303","author":"Tumbar","year":"2004","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/j.stem.2008.12.009","article-title":"A Two-Step Mechanism for Stem Cell Activation during Hair Regeneration","volume":"4","author":"Greco","year":"2009","journal-title":"Cell Stem Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.cell.2010.11.049","article-title":"Dynamics between Stem Cells, Niche, and Progeny in the Hair Follicle","volume":"144","author":"Hsu","year":"2011","journal-title":"Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"496","DOI":"10.1038\/nature11218","article-title":"Live Imaging of Stem Cell and Progeny Behaviour in Physiological Hair-Follicle Regeneration","volume":"487","author":"Rompolas","year":"2012","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1291","DOI":"10.1038\/ng.239","article-title":"Lgr5 Marks Cycling, yet Long-Lived, Hair Follicle Stem Cells","volume":"40","author":"Jaks","year":"2008","journal-title":"Nat. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"340","DOI":"10.1038\/nature06457","article-title":"Cyclic Dermal BMP Signalling Regulates Stem Cell Activation during Hair Regeneration","volume":"451","author":"Plikus","year":"2008","journal-title":"Nature"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.stem.2011.11.005","article-title":"Paracrine TGF-\u03b2 Signaling Counterbalances BMP-Mediated Repression in Hair Follicle Stem Cell Activation","volume":"10","author":"Oshimori","year":"2012","journal-title":"Cell Stem Cell"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3001","DOI":"10.1016\/j.celrep.2016.02.078","article-title":"Signaling Networks among Stem Cell Precursors, Transit-Amplifying Progenitors, and Their Niche in Developing Hair Follicles","volume":"14","author":"Rezza","year":"2016","journal-title":"Cell Rep."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/j.cels.2016.08.010","article-title":"Single-Cell Transcriptomics Reveals That Differentiation and Spatial Signatures Shape Epidermal and Hair Follicle Heterogeneity","volume":"3","author":"Joost","year":"2016","journal-title":"Cell Syst."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1016\/j.stem.2020.01.012","article-title":"The Molecular Anatomy of Mouse Skin during Hair Growth and Rest","volume":"26","author":"Joost","year":"2020","journal-title":"Cell Stem Cell"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1016\/j.cell.2017.03.038","article-title":"Epithelial-Mesenchymal Micro-Niches Govern Stem Cell Lineage Choices","volume":"169","author":"Yang","year":"2017","journal-title":"Cell"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e70460","DOI":"10.1096\/fj.202403343R","article-title":"Intensive Stress Impedes Hair Follicle Growth through Triggering Cell Cycle Arrest of Hair Follicle Stem Cells","volume":"39","author":"Wang","year":"2025","journal-title":"FASEB J."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1038\/nbt.4096","article-title":"Integrating Single-Cell Transcriptomic Data across Different Conditions, Technologies, and Species","volume":"36","author":"Butler","year":"2018","journal-title":"Nat. Biotechnol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1016\/j.cels.2019.03.003","article-title":"DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors","volume":"8","author":"McGinnis","year":"2019","journal-title":"Cell Syst."},{"key":"ref_18","first-page":"2.1","article-title":"Multilevel Local Search Algorithms for Modularity Clustering","volume":"16","author":"Rotta","year":"2011","journal-title":"J. Exp. Algorithmics JEA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1038\/nbt.4314","article-title":"Dimensionality Reduction for Visualizing Single-Cell Data Using UMAP","volume":"37","author":"Becht","year":"2018","journal-title":"Nat. Biotechnol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1186\/s13059-019-1663-x","article-title":"PAGA: Graph Abstraction Reconciles Clustering with Trajectory Inference through a Topology Preserving Map of Single Cells","volume":"20","author":"Wolf","year":"2019","journal-title":"Genome Biol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Street, K., Risso, D., Fletcher, R.B., Das, D., Ngai, J., Yosef, N., Purdom, E., and Dudoit, S. (2018). Slingshot: Cell Lineage and Pseudotime Inference for Single-Cell Transcriptomics. BMC Genom., 19.","DOI":"10.1186\/s12864-018-4772-0"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"496","DOI":"10.1038\/s41586-019-0969-x","article-title":"The Single-Cell Transcriptional Landscape of Mammalian Organogenesis","volume":"566","author":"Cao","year":"2019","journal-title":"Nature"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1186\/s13059-023-02988-9","article-title":"scTour: A Deep Learning Architecture for Robust Inference and Accurate Prediction of Cellular Dynamics","volume":"24","author":"Li","year":"2023","journal-title":"Genome Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1126\/science.aax0249","article-title":"Single-Cell Transcriptional Diversity Is a Hallmark of Developmental Potential","volume":"367","author":"Gulati","year":"2020","journal-title":"Science"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1202","DOI":"10.1016\/j.cell.2015.05.002","article-title":"Highly Parallel Genome-Wide Expression Profiling of Individual Cells Using Nanoliter Droplets","volume":"161","author":"Macosko","year":"2015","journal-title":"Cell"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1186\/s13059-015-0844-5","article-title":"MAST: A Flexible Statistical Framework for Assessing Transcriptional Changes and Characterizing Heterogeneity in Single-Cell RNA Sequencing Data","volume":"16","author":"Finak","year":"2015","journal-title":"Genome Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1888","DOI":"10.1016\/j.cell.2019.05.031","article-title":"Comprehensive Integration of Single-Cell Data","volume":"177","author":"Stuart","year":"2019","journal-title":"Cell"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1038\/75556","article-title":"Gene Ontology: Tool for the Unification of Biology. The Gene Ontology Consortium","volume":"25","author":"Ashburner","year":"2000","journal-title":"Nat. Genet."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1093\/nar\/28.1.27","article-title":"KEGG: Kyoto Encyclopedia of Genes and Genomes","volume":"28","author":"Kanehisa","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"D649","DOI":"10.1093\/nar\/gkx1132","article-title":"The Reactome Pathway Knowledgebase","volume":"46","author":"Fabregat","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1083","DOI":"10.1038\/nmeth.4463","article-title":"SCENIC: Single-Cell Regulatory Network Inference and Clustering","volume":"14","author":"Aibar","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Huynh-Thu, V.A., Irrthum, A., Wehenkel, L., and Geurts, P. (2010). Inferring Regulatory Networks from Expression Data Using Tree-Based Methods. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0012776"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2498","DOI":"10.1101\/gr.1239303","article-title":"Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks","volume":"13","author":"Shannon","year":"2003","journal-title":"Genome Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1038\/s41596-024-01045-4","article-title":"CellChat for Systematic Analysis of Cell-Cell Communication from Single-Cell Transcriptomics","volume":"20","author":"Jin","year":"2025","journal-title":"Nat. Protoc."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1016\/j.cell.2014.02.057","article-title":"Transit-Amplifying Cells Orchestrate Stem Cell Activity and Tissue Regeneration","volume":"157","author":"Hsu","year":"2014","journal-title":"Cell"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1946","DOI":"10.1126\/science.1128004","article-title":"Lhx2 Maintains Stem Cell Character in Hair Follicles","volume":"312","author":"Rhee","year":"2006","journal-title":"Science"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/j.stem.2008.05.009","article-title":"Hair Follicle Stem Cells Are Specified and Function in Early Skin Morphogenesis","volume":"3","author":"Nowak","year":"2008","journal-title":"Cell Stem Cell"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"aad4395","DOI":"10.1126\/science.aad4395","article-title":"Hair Follicle Aging Is Driven by Transepidermal Elimination of Stem Cells via COL17A1 Proteolysis","volume":"351","author":"Matsumura","year":"2016","journal-title":"Science"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2111","DOI":"10.1101\/gad.1689908","article-title":"Laminin-511 Is an Epithelial Message Promoting Dermal Papilla Development and Function during Early Hair Morphogenesis","volume":"22","author":"Gao","year":"2008","journal-title":"Genes Dev."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"3990","DOI":"10.1093\/emboj\/19.15.3990","article-title":"Skin and Hair Follicle Integrity Is Crucially Dependent on Beta 1 Integrin Expression on Keratinocytes","volume":"19","author":"Brakebusch","year":"2000","journal-title":"EMBO J."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1596","DOI":"10.1101\/gad.1324905","article-title":"Defining the Impact of Beta-Catenin\/Tcf Transactivation on Epithelial Stem Cells","volume":"19","author":"Lowry","year":"2005","journal-title":"Genes Dev."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"720","DOI":"10.1016\/j.stem.2013.10.003","article-title":"Distinct Functions for Wnt\/\u03b2-Catenin in Hair Follicle Stem Cell Proliferation and Survival and Interfollicular Epidermal Homeostasis","volume":"13","author":"Choi","year":"2013","journal-title":"Cell Stem Cell"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"731","DOI":"10.1016\/j.devcel.2004.09.014","article-title":"Gamma-Secretase Functions through Notch Signaling to Maintain Skin Appendages but Is Not Required for Their Patterning or Initial Morphogenesis","volume":"7","author":"Pan","year":"2004","journal-title":"Dev. Cell"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Rendl, M., Lewis, L., and Fuchs, E. (2005). Molecular Dissection of Mesenchymal\u2013Epithelial Interactions in the Hair Follicle. PLoS Biol., 3.","DOI":"10.1371\/journal.pbio.0030331"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1038\/nature11847","article-title":"NFIB Is a Governor of Epithelial-Melanocyte Stem Cell Behaviour in a Shared Niche","volume":"495","author":"Chang","year":"2013","journal-title":"Nature"}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/9\/1058\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T04:54:16Z","timestamp":1788584056000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/9\/1058"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,31]]},"references-count":45,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["genes17091058"],"URL":"https:\/\/doi.org\/10.3390\/genes17091058","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,31]]}}}