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RNA-seq reveals tight junction-relevant erythropoietic fate induced by OCT4 in human hair follicle mesenchymal stem cells
Zhijing Liu
Qingdao University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3489-2310
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Stem Cell Research & Therapy.
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.
Keywords
tight junction pathway, human hair follicle mesenchymal stem cells, OCT4, hematopoiesis
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This is a list of supplementary files associated with this preprint. Click to download.
- FigureS1.tif
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.
- Additinalfile2.pdf
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.
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CURRENT STATUS: Published
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Posted 17 Sep, 2020
Published
On 27 Oct, 2020
No community comments so far
Editorial decision: Accept
On 25 Sep, 2020
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Received 24 Sep, 2020
Review #1 received
Received 22 Sep, 2020
Reviewers invited
Invitations sent on 16 Sep, 2020
Reviewer #1 agreed
On 16 Sep, 2020
Reviewer #2 agreed
On 16 Sep, 2020
Editor assigned
On 15 Sep, 2020
Submission checks complete
On 14 Sep, 2020
Editor invited
On 14 Sep, 2020
No comments provided
Review #1 received
Received 13 Aug, 2020
Editorial decision: Major Revision
On 13 Aug, 2020
Review #2 received
Received 10 Aug, 2020
Reviewer #2 agreed
On 31 Jul, 2020
Reviewer #1 agreed
On 29 Jul, 2020
Editor assigned
On 28 Jul, 2020
Reviewers invited
Invitations sent on 28 Jul, 2020
Editor invited
On 27 Jul, 2020
Submission checks complete
On 24 Jul, 2020
First submitted
On 23 Jul, 2020
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Subject Areas
Cellular & Molecular Neuroscience
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This preprint is under consideration at Stem Cell Research & Therapy. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
RNA-seq reveals tight junction-relevant erythropoietic fate induced by OCT4 in human hair follicle mesenchymal stem cells
Zhijing Liu
Qingdao University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3489-2310
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
Version 2
Posted 17 Sep, 2020
Published
On 27 Oct, 2020
No community comments so far
Editorial decision: Accept
On 25 Sep, 2020
Review #2 received
Received 24 Sep, 2020
Review #1 received
Received 22 Sep, 2020
Reviewers invited
Invitations sent on 16 Sep, 2020
Reviewer #1 agreed
On 16 Sep, 2020
Reviewer #2 agreed
On 16 Sep, 2020
Editor assigned
On 15 Sep, 2020
Submission checks complete
On 14 Sep, 2020
Editor invited
On 14 Sep, 2020
No comments provided
Review #1 received
Received 13 Aug, 2020
Editorial decision: Major Revision
On 13 Aug, 2020
Review #2 received
Received 10 Aug, 2020
Reviewer #2 agreed
On 31 Jul, 2020
Reviewer #1 agreed
On 29 Jul, 2020
Editor assigned
On 28 Jul, 2020
Reviewers invited
Invitations sent on 28 Jul, 2020
Editor invited
On 27 Jul, 2020
Submission checks complete
On 24 Jul, 2020
First submitted
On 23 Jul, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Cellular & Molecular Neuroscience
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Stem Cell Research & Therapy.
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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