ZO-1 boosts the in vitro self-renewal of pre-haematopoietic stem cells from OCT4-reprogrammed human hair follicle mesenchymal stem cells through cytoskeleton remodeling.

BACKGROUND

The 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 (hHFMSCs) 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.

METHODS

hHFMSCs 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.

RESULTS

Pre-treatment of hHFMSCs 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 hHFMSCs. 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.

CONCLUSIONS

ZO-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 hHFMSCs as the promising seed cells for artificial hematopoiesis.