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
Abstract 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 (hHFMSCsOCT4) and pre-inducing...
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BMC
2024-12-01
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| Series: | Stem Cell Research & Therapy |
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| Online Access: | https://doi.org/10.1186/s13287-024-04080-w |
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| author | Yingchun Ruan Xingang Huang Pengpeng Sun Xiaozhen Yu Xiaohua Tan Yaolin Song Hua Chen Zhijing Liu |
| author_facet | Yingchun Ruan Xingang Huang Pengpeng Sun Xiaozhen Yu Xiaohua Tan Yaolin Song Hua Chen Zhijing Liu |
| author_sort | Yingchun Ruan |
| collection | DOAJ |
| description | Abstract 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 (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. Methods hHFMSCsOCT4 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 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. 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 hHFMSCsOCT4 as the promising seed cells for artificial hematopoiesis. |
| format | Article |
| id | doaj-art-3ee8a2dcb1ee42b889b6215e2124b4e1 |
| institution | OA Journals |
| issn | 1757-6512 |
| language | English |
| publishDate | 2024-12-01 |
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| series | Stem Cell Research & Therapy |
| spelling | doaj-art-3ee8a2dcb1ee42b889b6215e2124b4e12025-08-20T01:57:51ZengBMCStem Cell Research & Therapy1757-65122024-12-0115112010.1186/s13287-024-04080-wZO-1 boosts the in vitro self-renewal of pre-haematopoietic stem cells from OCT4-reprogrammed human hair follicle mesenchymal stem cells through cytoskeleton remodelingYingchun Ruan0Xingang Huang1Pengpeng Sun2Xiaozhen Yu3Xiaohua Tan4Yaolin Song5Hua Chen6Zhijing Liu7Department of Pathology, Qingdao Municipal Hospital GroupDepartment of Pathology, Qingdao Municipal Hospital GroupDepartment of Critical Care Medicine, Qingdao Central Hospital, University of Health and Rehabilitation Sciences (Qingdao Central Hospital)Department of Pathology, Qingdao Municipal Hospital GroupDepartment of Pathology, College of Basic Medical Sciences, Qingdao UniversityDepartment of Pathology, The Affiliated Hospital of Qingdao UniversityDepartment of Pathology, Qingdao Municipal Hospital GroupDepartment of Pathology, Qingdao Municipal Hospital GroupAbstract 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 (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. Methods hHFMSCsOCT4 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 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. 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 hHFMSCsOCT4 as the promising seed cells for artificial hematopoiesis.https://doi.org/10.1186/s13287-024-04080-wAdherens junctionSelf-renewalHuman hair follicle mesenchymal stem cellsReprogrammingPre-haematopoietic stem cells |
| spellingShingle | Yingchun Ruan Xingang Huang Pengpeng Sun Xiaozhen Yu Xiaohua Tan Yaolin Song Hua Chen Zhijing Liu 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 Stem Cell Research & Therapy Adherens junction Self-renewal Human hair follicle mesenchymal stem cells Reprogramming Pre-haematopoietic stem cells |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | Adherens junction Self-renewal Human hair follicle mesenchymal stem cells Reprogramming Pre-haematopoietic stem cells |
| url | https://doi.org/10.1186/s13287-024-04080-w |
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