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 (hHFMSCs^OCT4) 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^OCT4 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^OCT4 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^OCT4. 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^OCT4 as the promising seed cells for artificial hematopoiesis.

Keywords: Adherens junction; Human hair follicle mesenchymal stem cells; Pre-haematopoietic stem cells; Reprogramming; Self-renewal.

Fig. 1

FLT3LG and SCF caused floating cells to appear from hHFMSCs^OCT4. (a) Schema showing the transformation of floating cells derived from hHFMSCs^OCT4 into erythroblasts. (b) Cell morphology changes and floating cells observed under immunofluorescence microscope, with a floating cell indicated by a yellow arrow and a floating cell colony outlined by a dashed circle. (c) Phase contrast microscope used to observe floating cells’ morphology. Floating cell indicated by yellow arrow. (d) Collecting and re-cultivating suspended cells. (e and f). Floating cell colonies were selected to identify CD45 expression, with green indicating EGFR, red indicating PE-CD45, and blue indicating Hoechst. Peripheral blood cells served as a positive control

Fig. 2

Floating hHFMSCs^OCT4 differentiated into erythrocytes. (a) Erythroblast differentiation and cell morphology change in response to hematopoietic cytokines. Expansion D7 refers to erythroid cells expanded for 7 days in erythroid cell medium. (b) Erythroblast growth and maturation in erythroid expansion medium with nutrients. Green arrow: promyelocyte and mesmyelocyte. Orange arrow: metarubricyte. Red arrows: enucleated erythrocyte. (c) Erythrocytes were enucleated in a culture medium without cytokines and the enucleation efficiency was measured

Fig. 3

The difference in OCT4 overexpression between adherent hHFMSCs^OCT4 and floating hHFMSCs^OCT4. (a) IF analysis of OCT4 expression and localization in hHFMSCs, adherent hHFMSCs^OCT4, and floating hHFMSCs^OCT4. NC, negative control. Red represents OCT4, blue represents DAPI, and green represents EGFP, (n = 3). (b) and (c) Histograms were generated to validate the expression of OCT4 by RT‒qPCR (n = 3) and (d) Western blots (n = 1), respectively. Full-length blots/gels are presented in Supplementary Figure S3. **P˂0.01, ***P˂0.001, ****P˂0.0001

Fig. 4

The difference in self-renewal and proliferative ability between the adherent hHFMSCs^OCT4 and floating hHFMSCs^OCT4. (a) Cell proliferation curve of hHFMSCs, adherent hHFMSCs^OCT4 and floating hHFMSCs^OCT4 obtained from the CCK-8 assay (n = 3). (b) Colony formation assay of hHFMSCs, adherent hHFMSCs^OCT4, and floating hHFMSCs^OCT4; the enlarged views show the difference between the three cell clones (n = 3). The histogram is the rate of colony formation for each cell. (c) ICC of proliferation-associated protein Ki67 expression in three cell groups, and Ki67 protein was localized in the nucleus (n = 3). *P˂0.05, **P˂0.01, ****P˂0.0001. (d) Discrete cutoffs for distinct cell cycle phases are visually selected to calculate the percentages of cells in G1, S, and G2/M phases using R software

Fig. 5

Remodelling of the cytoskeleton in floating hHFMSCs^OCT4. (a) The cytoplasm and nucleus were stained with Wright‒Giemsa stain in three groups of cells (n = 3). (b) Cytoskeletal microfilaments in the three groups were stained with Coomassie brilliant blue (n = 3). (c) F-actin morphology and distribution were detected by phalloidin staining (n = 3). These images were all taken from randomly selected fields. *P˂0.05, **P˂0.01

Fig. 6

Expression of AJ core genes and target genes in floating hHFMSCs^OCT4. (a) RT‒qPCR analysis of the expression of ZO-1, ACTN2, E-cadherin, β-catenin, and Cyclin D1 (n = 3). (b) IF staining images showing the location of the β-catenin protein in hHFMSCs, adherent hHFMSCs^OCT4, and floating hHFMSCs^OCT4. (c) Fluorescence intensity of the merged colour in the nucleus was measured in three groups using ImageJ. *P˂0.05, **P˂0.01, ***P˂0.001

Fig. 7

Changes in AJ gene expression and F-actin arrangement in floating hHFMSCs^OCT4 after ZO-1 knockdown. (a) RT‒qPCR and Western blot showing the expression of ZO-1 after RNAi (n = 2). Full-length blots/gels are presented in Supplementary Figure S4. (b) RT‒qPCR and detection of the mRNA expression of ACTN2, E-cadherin, β-catenin, and Cyclin D1 (n = 3). The error bars represent the standard deviations of measurements in three separate sample runs. (c) Western blots showing the protein expression levels of β-catenin (n = 1). Full-length blots/gels are presented in Supplementary Figure S5, n = 1. (d) Phalloidin staining showing F-actin morphology and distribution (n = 3). (e and f) Fluorescence microscopy (Olympus) captured DAPI-stained cell images, and CellProfiler software detected nuclei and calculated their integrated intensity. R software then used visually determined cutoffs to calculate the percentages of cells in G1, S, and G2/M phases. NC, negative control. si-ZO-1, ZO-1-knockdown. *P˂0.05, **P˂0.01, ***P˂0.001, ****P˂0.0001

Fig. 8

Network analysis. A gene network regulates haematopoiesis-related proliferation of floating hHFMSCs^OCT4. The solid line shows gene relationships within the group, while the dashed line shows relationships between genes in different groups