RNA-seq reveals tight junction-relevant erythropoietic fate induced by OCT4 in human hair follicle mesenchymal stem cells
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.
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Additional file 1: Figure S1. Comparisons of the transcripts. (a) Correlation coefficient between samples displayed in a heatmap. The closer the correlation coefficient is to 1, the higher the sample similarity is. (b) Hierarchical clustering of correlation of the three groups of cells. The closer the sample clustering distance is, the higher the sample similarity is.
Additional file 2: Table S1. Pluripotency related genes expression in hHFMSCs, adherent hHFMSCsOCT4 and floating hHFMSCsOCT4. Table S2. Differentiation and development relative GO terms enriched of upregulated DEGs in adherent hHFMSCsOCT4 versus hHFMSCs. Table S3. Differentiation and development relative GO terms enriched of upregulated DEGs in floating hHFMSCsOCT4 versus adherent hHFMSCsOCT4.
Posted 17 Sep, 2020
On 27 Oct, 2020
On 25 Sep, 2020
Received 24 Sep, 2020
Received 22 Sep, 2020
Invitations sent on 16 Sep, 2020
On 16 Sep, 2020
On 16 Sep, 2020
On 15 Sep, 2020
On 14 Sep, 2020
On 14 Sep, 2020
Received 13 Aug, 2020
On 13 Aug, 2020
Received 10 Aug, 2020
On 31 Jul, 2020
On 29 Jul, 2020
On 28 Jul, 2020
Invitations sent on 28 Jul, 2020
On 27 Jul, 2020
On 24 Jul, 2020
On 23 Jul, 2020
RNA-seq reveals tight junction-relevant erythropoietic fate induced by OCT4 in human hair follicle mesenchymal stem cells
Posted 17 Sep, 2020
On 27 Oct, 2020
On 25 Sep, 2020
Received 24 Sep, 2020
Received 22 Sep, 2020
Invitations sent on 16 Sep, 2020
On 16 Sep, 2020
On 16 Sep, 2020
On 15 Sep, 2020
On 14 Sep, 2020
On 14 Sep, 2020
Received 13 Aug, 2020
On 13 Aug, 2020
Received 10 Aug, 2020
On 31 Jul, 2020
On 29 Jul, 2020
On 28 Jul, 2020
Invitations sent on 28 Jul, 2020
On 27 Jul, 2020
On 24 Jul, 2020
On 23 Jul, 2020
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6