Abstract
BackgroundHuman hair follicle mesenchymal stem cells (hHFMSCs) isolated from hair follicles possess multilineage differentiation potential. OCT4 is a gene critically associated with pluripotency properties. The cell morphology and adhesion of hHFMSCs significantly changed after transduction of OCT4 and two subpopulations emerged, including adherent cells and floating cell. Floating cells cultured in hematopoietic induction medium and stimulated with erythropoetic growth factors could transdifferentiate into mature erythrocytes, whereas adherent cells formed negligible hematopoietic colonies. The aim of this study was to reveal the role of cell morphology and adhesion on erythropoiesis induced by OCT4 in hHFMSCs and to characterize the molecular mechanisms involved.MethodsFloating cell was separated from adherent cell by centrifugation of the upper medium during cell culture. Cell size was observed through flow cytometry and cell adhesion was tested by disassociation and adhesion assays. RNA sequencing was performed to detect genome-wide transcriptomes and identify differentially expressed genes. GO enrichment analysis and KEGG pathway analysis were performed to analysis the functions and pathways enriched by differentially expressed genes. The expression of tight junction core members was verified by qPCR and Western blot. A regulatory network was constructed to figure out the relationship between cell adhesin, cytoskeleton, pluripotency, and hematopoiesis.ResultsThe overexpression of OCT4 influenced the morphology and adhesion of hHFMSCs. Transcripts in floating cells and adherent cells are quite different. Data analysis showed that upregulated genes in floating cells were mainly related to pluripotency, germ layer development (including hematopoiesis lineage development), and downregulated genes were mainly related to cell adhesion, cell junctions, and the cytoskeleton. Most molecules of the tight junction (TJ) pathway were downregulated and molecular homeostasis of the TJ was disturbed, as CLDNs were disrupted, and JAMs and TJPs were upregulated. The dynamic expression of cell adhesion-related gene E-cadherin and cytoskeleton-related gene ACTN2 might cause different morphology and adhesion. Finally, a regulatory network centered to OCT4 was constructed, which elucidated that he TJ pathway critically bridges pluripotency and hematopoiesis in a TJP1-dependent way.ConclusionsRegulations of cell morphology and adhesion via the TJ pathway conducted by OCT4 might modulate hematopoiesis in hHFMSCs, thus developing potential mechanism of erythropoiesis in vitro.
Highlights
Erythropoiesis is a stepwise process through which red blood cells (RBCs, erythrocytes) are generated from hematopoietic stem and progenitor cells (HSPCs) and is controlled by multiple elements [1, 2]
Regulations of cell morphology and adhesion via the tight junctions (TJs) pathway conducted by OCT4 might modulate hematopoiesis in Human hair follicle mesenchymal stem cells (hHFMSCs), developing potential mechanism of erythropoiesis in vitro
When cells proliferated to 70–80%, they were digested with 0.25% trypsinethylene diamine tetra-acetic acid (EDTA) and subsequently subcultured. hHFMSCs were frozen with cryopreservation solution composed of 50% H-DMEM/F12 medium, 40% Fetal bovine serum (FBS) and 10% dimethyl sulfoxide, and stored in liquid nitrogen at passages 0–2
Summary
Erythropoiesis is a stepwise process through which red blood cells (RBCs, erythrocytes) are generated from hematopoietic stem and progenitor cells (HSPCs) and is controlled by multiple elements [1, 2]. A population of small round floating cells with subtle expression of hematopoietic stem cell (HSC) marker CD45, gradually emerged from hHFMSCsOCT4, and could transdifferentiate into mature enucleated RBCs when stimulated with a combination of hematopoietic cytokines [6], while the remaining cells formed negligible hematopoietic colony. This prompted us to consider an association between this particular cell morphology and adhesion with possible erythropoiesis mechanisms, that is, low adhesion and round-like cell morphology conferring higher hematopoietic capacity to hHFMSCsOCT4 when treated with cytokines, promoting transduction of cellular signals and subsequently initiating the process of erythropoiesis. The aim of this study was to reveal the role of cell morphology and adhesion on erythropoiesis induced by OCT4 in hHFMSCs and to characterize the molecular mechanisms involved
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