RNA-seq reveals tight junction-relevant erythropoietic fate induced by OCT4 in human hair follicle mesenchymal stem cells

Abstract

Background: Human 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.Methods: Floating cell were 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.Results: The 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.Conclusions: Regulations 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.

ZO-1 remodels the cytoskeleton and boosts self-renewal in OCT4-reprogrammed human hair follicle mesenchymal stem cells through adherens junction pathway

“…Cell-cell adhesion was assayed through a dispase disassociation assay as previously described [5] . Cells were seeded on Matrigel-coated 24-well plates at a density of 2×10 5 cells/well and cultured overnight until the cells reached con uence.…”

“…Cell-extracellular matrix adhesion was assayed as previously reported with minor modi cations [5] . The cells were plated in 24-well plates at a density of 2×10 5 cells/well and cultured until the hHFMSCs adhered to the plates and fully expanded.…”

“…As a result, this process eliminates the need for sorting HSCs and maintaining their status. Signi cantly, a unique subset of suspended cells displaying distinct cellular morphology characteristics was identi ed, characterized by a small volume, round shape, and low adhesion, and subsequently designated as oating cells derived from hHFMSC OCT4 ( oating hHFMSCs OCT4 ) [4,5] . Nevertheless, the correlation between alterations in morphology and adhesion of reprogrammed cells and their capacity for self-renewal remained ambiguous.…”

“…In a previous study, we found that OCT4-induced reprogramming of hHFMSCs resulted in changes in the expression of cytoskeleton-and adhesion-related genes, leading to the loss of hair follicle and skin development potential. Interestingly, the reprogrammed hHFMSCs showed upregulation of pluripotent and haematopoietic genes, with enrichment of Gene Ontology (GO) terms related to haematopoietic lineage differentiation [5] . Based on these ndings, we hypothesize that cytoskeleton genes and adhesion molecules may play a role in regulating the dedifferentiation and self-renewal of oating hHFMSCs OCT4 .…”

“…Our previous studies found a notable rise in ZO-1 expression in oating hHFMSCs OCT4 . Additionally, the expression of the cytoskeleton gene ACTN2 and the AJ molecule Ecadherin signi cantly changed [5] . Currently, AJs are believed to regulate cell proliferation via the nuclear transport of β-catenin [11] .…”