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ROR2 regulates self-renewal and maintenance of hair follicle stem cells.

ABSTRACT: Hair follicles undergo cycles of regeneration fueled by hair follicle stem cells (HFSCs). While β-catenin-dependent canonical Wnt signaling has been extensively studied and implicated in HFSC activation and fate determination, very little is known about the function of β-catenin-independent Wnt signaling in HFSCs. In this study, we investigate the functional role of ROR2, a Wnt receptor, in HFSCs. By analyzing Ror2-depleted HFSCs, we uncover that ROR2 is not only essential to regulate Wnt-activated signaling that is responsible for HFSC activation and self-renewal, but it is also required to maintain proper ATM/ATR-dependent DNA damage response, which is indispensable for the long-term maintenance of HFSCs. In analyzing HFSCs lacking β-catenin, we identify a compensatory role of ROR2-PKC signaling in protecting β-catenin-null HFSCs from the loss of stem cell pool. Collectively, our study unveils a previously unrecognized role of ROR2 in regulation of stem cell self-renewal and maintenance.

SUBMITTER: Veltri A

PROVIDER: S-EPMC9343661 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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ROR2 regulates self-renewal and maintenance of hair follicle stem cells.

Veltri Anthony A Lang Christopher M R CMR Cangiotti Gaia G Chan Chim Kei CK Lien Wen-Hui WH

Nature communications 20220801 1

Hair follicles undergo cycles of regeneration fueled by hair follicle stem cells (HFSCs). While β-catenin-dependent canonical Wnt signaling has been extensively studied and implicated in HFSC activation and fate determination, very little is known about the function of β-catenin-independent Wnt signaling in HFSCs. In this study, we investigate the functional role of ROR2, a Wnt receptor, in HFSCs. By analyzing Ror2-depleted HFSCs, we uncover that ROR2 is not only essential to regulate Wnt-activa ...[more]

PMID: 35915068

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Project description:Hair follicle regeneration is dependent on reciprocal signaling between epithelial cells and underlying mesenchymal cells within the dermal papilla. Hair follicle dermal stem cells reside within the hair follicle mesenchyme, self-renew in vivo, and function to repopulate the dermal papilla and regenerate the connective tissue sheath with each hair cycle. The identity and temporal pattern of signals that regulate hair follicle dermal stem cell function are not known. Here, we show that platelet-derived growth factor signaling is crucial for hair follicle dermal stem cell function and platelet-derived growth factor deficiency results in a progressive depletion of the hair follicle dermal stem cell pool and their progeny. Using αSMACreERT2:RosaYFP:Pdgfrαflox mice, we ablated Pdgfrα specifically within the adult hair follicle dermal stem cell lineage. This led to significant loss of hair follicle dermal stem cell progeny in connective tissue sheath and dermal papilla of individual follicles, and a progressive reduction in total number of anagen hair follicles containing YFP+ve cells. As well, over successive hair cycles, fewer hair follicle dermal stem cells were retained within each telogen hair follicle suggesting an impact on hair follicle dermal stem cell self-renewal. To further assess this, we grew prospectively isolated hair follicle dermal stem cells (Sox2GFP+ve αSMAdsRed+ve) in the presence or absence of platelet-derived growth factor ligands. Platelet-derived growth factor-BB enhanced proliferation, increased the frequency of Sox2+ve hair follicle dermal stem cell progeny and improved inductive capacity of hair follicle dermal stem cells in an ex vivo hair follicle formation assay. Similar effects on proliferation were observed in adult human SKPs. Our findings impart novel insights into the signals that comprise the adult hair follicle dermal stem cell niche and suggest that platelet-derived growth factor signaling promotes self renewal, is essential to maintain the hair follicle dermal stem cell pool and ultimately their regenerative capacity within the hair follicle.

Project description:BackgroundThe challenge of expanding haematopoietic stem/progenitor cells (HSPCs) in vitro has limited their clinical application. Human hair follicle mesenchymal stem cells (hHFMSCs) can be reprogrammed to generate intermediate stem cells by transducing OCT4 (hHFMSCsOCT4) and pre-inducing with FLT3LG/SCF, and differentiated into erythrocytes. These intermediate cells exhibit gene expression patterns similar to pre-HSCs, making them promising for artificial haematopoiesis. However, further investigation is required to elucidate the in vitro proliferation ability and mechanism underlying the self-renewal of pre-HSCs derived from hHFMSCs.MethodshHFMSCsOCT4 were pre-treated with FLT3LG and SCF cytokines, followed by characterization and isolation of the floating cell subsets for erythroid differentiation through stimulation with hematopoietic cytokines and nutritional factors. Cell adhesion was assessed through disassociation and adhesion assays. OCT4 expression levels were measured using immunofluorescence staining, RT-qPCR, and Western blotting. RNA sequencing and Gene Ontology (GO) enrichment analysis were then conducted to identify proliferation-related biological processes. Proliferative capacity was evaluated through CCK-8, colony formation assays, Ki67 index, and cell cycle analysis. Cytoskeleton was observed through Wright‒Giemsa, Coomassie brilliant blue, and phalloidin staining. Expression of adherens junction (AJ) core members was confirmed through RT‒qPCR, Western blotting, and immunofluorescence staining before and after ZO-1 knockdown. A regulatory network was constructed to determine relationships among cytoskeleton, proliferation, and the AJ pathway. Student's t tests (GraphPad Prism 8.0.2) were used for group comparisons. The results were considered significant at P < 0.05.ResultsPre-treatment of hHFMSCsOCT4 with FLT3LG and SCF leads to the emergence of floating cell subsets exhibiting small, globoid morphology, suspended above adherent cells, forming colonies, and displaying minimal expression of CD45. Excessive OCT4 expression weakens adhesion in floating hHFMSCsOCT4. Floating cells moderately enhanced proliferation and undergo cytoskeleton remodelling, with increased contraction and aggregation of F-actin near the nucleus. The upregulation of ZO-1 could impact the expressions of F-actin, E-cadherin, and β-catenin genes, as well as the nuclear positioning of β-catenin, leading to variations in the cytoskeleton and cell cycle. Finally, a regulatory network revealed that the AJ pathway cored with ZO-1 critically bridges cytoskeletal remodelling and haematopoiesis-related proliferation in a β-catenin-dependent manner.ConclusionsZO-1 improved the self-renewal of pre-HSCs from OCT4-overexpressing hHFMSCs by remodeling the cytoskeleton via the ZO-1-regulated AJ pathway, suggesting floating hHFMSCsOCT4 as the promising seed cells for artificial hematopoiesis.

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ROR2 regulates self-renewal and maintenance of hair follicle stem cells.

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ROR2 regulates self-renewal and maintenance of hair follicle stem cells.

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