Study On Mechanisms Of Hematopoiesis In OCT4-induced Human Hair Follicle Mesenchymal Stem Cells Through The Tight Junction Pathway

Human hair follicle mesenchymal stem cells(hHFMSCs)isolated from human hair follicles possess multilineage differentiation potential towards adipocytes,chondrocytes and osteoblasts.Recently,the cell morphology and adhesion of hHFMSCs significantly changed after transduction of OCT4.Two subpopulations emerged,including adherent cells and floating cell,whereas floating cells cultured in hematopoietic induction medium and stimulated with erythropoetic growth factors could transdifferentiate into mature erythrocytes.Although many researches on the mechanism of erythropoiesis in hematopoietic cells,the effect of morphology and adhesion on the production of erythrocytes in OCT4-reprogrammed hHFMSCs remains to be explored.Objective:To reveal the correlation between cell morphology,cell adhesion,pluripotency and hematopoiesis in OCT4-reprogrammed hHFMSCs,we experimented to measure the relative cell size,cell-cell adhesion and cell-cell extracellular matrix adhesion,and detected the expression of related genes.We also analyzed the expression and function of target genes for OCT4.Remarkably,study on molecular alterations and relationship between morphological changes and hematopoietic capacity would help to elucidate the mechanisms of erythrocytes production in OCT4-reprogrammed hHFMSCs and further exploration of erythropoiesis in hematopoietic cells.Methods:?Floating cell were separated from adherent cell by centrifugation of the upper medium during cell culture.?We first observed relative cell size and adhesion through flow cytometry and cell adhesion assay.?We performed RNA sequencing to detect genome-wide transcriptomes using Illumina HiSeq X Ten.In-depth analysis was conducted to compare the transcripts and the expression of pluripotent genes,such as LEFTY2,KLF4 and MYC.?In addition,we identified target genes for OCT4 and further analyzed the function.?GO enrichment analysis and KEGG pathway analysis were performed in upregulated genes and downregulated genes,respectively.The tight junction(TJ)pathway was annotated with differential expressed genes according to the KEGG database.?Significant genes were validated by qPCR and Western blot.?Finally,we visualized these genes using STRING database and constructed a regulatory network by Cytoscape software.Results:?A subpopulation of cells emerged in OCT4-reprogrammed hHFMSCs and floated over the adherent cells,which display small and round morphology,and low adhesion.?OCT4 transduction conferred pluripotency to OCT4-reprogrammed hHFMSCs as the upregulation of pluripotent genes,but floating cell lost some of it,while some pluripotent genes were downregulated relative to adherent cells.?Analysis of target genes for OCT4 revealed that the expression of pluripotency-and hematopoiesis-related target genes was upregulated.GO enrichment analysis indicated erythroid lineage differentiation tendency in floating cells.Genes related to cell junction,adhesion and cytoskeleton,as well as TJ members,were mainly downregulated in floating cells.?Western blot and qPCR verified the gene expression of TJ pathway members and found CLDNs were disrupted,JAMs and TJPs were upregulated,thus the molecular homeostasis of the TJ pathway was disturbed.This might induce dynamic expression of cell adhesion-related gene E-cadherin and cytoskeleton-related gene ACTN2,causing different morphology and adhesion.?A regulatory network centered to OCT4 was constructed,which elucidated that the TJ pathway critically bridges pluripotency and hematopoiesis in a TJP1-dependent way through KLF4 and RUNX1,thus developing potential mechanisms of erythropoiesis in vitro.Conclusion:Accordingly,OCT4-reprogrammed hHFMSCs modulate pluripotency and hematopoietic capacity through regulating the morphology and adhesion via TJ pathway,as well as regulating pluripotent and hematopoietic target genes for OCT4.Significance:This study provides an experimental basis for hHFMSCs serving as a new source for erythrocytes production in vitro for patient-specific treatment by elucidating the molecular mechanisms of erythropoiesis in OCT4-programmed hHFMSCs.