ZO-1 remodels the cytoskeleton and boosts self-renewal in OCT4-reprogrammed human hair follicle mesenchymal stem cells through adherens junction pathway

Background: The challenge of expanding haematopoietic stem/progenitor cells (HSPCs) in vitro has limited the clinical application of this technology. Human hair follicle mesenchymal stem cells (hHFMSCs) can be reprogrammed to generate intermediate stem cells by introducing only OCT4 (hHFMSCs ^OCT4 ). Prestimulating these cells with a low concentration of the cytokines FLT3 and SCF leads to the generation of a distinct subset, named floating hHFMSCs ^OCT4 , which can maintain self-renewal in vitro and be induced to undergo erythropoiesis, suggesting that these cells are promising seed cells for artificial haematopoiesis. Methods: Floating cell subsets were isolated from adherent cell subsets using centrifugation. 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 enriched by the upregulated differentially expressed genes (DEGs). Proliferative capacity was assessed using CCK-8 and colony formation assays. 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. Results: Excessive OCT4 expression weakens adhesion and causes floating hHFMSCs ^OCT4 . These cells have moderate proliferation and undergo cytoskeleton remodeling, with increased contraction and aggregation of F-actin near the nucleus. The upregulation of ZO-1 can impact the 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 critically bridges cytoskeletal remodelling and haematopoiesis-related proliferation in a β-catenin-dependent manner. Conclusions: Increasing OCT4 levels can remodel the cytoskeleton through the ZO-1-containing AJ pathway, potentially boosting the self-renewal ability of hHFMSCs to mimic HSPCs, suggesting a plausible mechanism for utilizing novel seed cells in artificial hematopoietic in vitro.