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Regulatory role of apelin receptor signaling in migration and differentiation of mouse embryonic stem cell-derived mesoderm cells and mesenchymal stem/stromal cells

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Abstract

Mesoderm-derived cells, including bone, muscle, and mesenchymal stem/stromal cells (MSCs), constitute various parts of vertebrate body. Cell therapy with mesoderm specification in vitro may be a promising treatment for diseases affecting organs of mesodermal origin. Repair and regeneration of damaged organs with in vitro generation of mesoderm-derived tissues and MSCs hold a great potential for regenerative therapy. Therefore, understanding the signaling pathways involving mesoderm and mesoderm-derived cellular differentiation is important. Previous findings indicated the importance of Apelin receptor (Aplnr) signaling, during embryonic development, in gastrulation, cell migration, and differentiation. Nevertheless, regulatory role of Aplnr pathway in differentiation of mesoderm and mesoderm-derived MSCs remains unclear. In the current study, we tried to elucidate the role of Aplnr signaling during mesoderm cell migration and differentiation from mouse embryonic stem cells (mESCs). By activating and suppressing Aplnr signaling pathway via peptide, small molecule, and genetic modifications including siRNA- and shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout (KO), we revealed that Aplnr signaling not only induces migration of cells during germ layer formation but also enhances mesoderm differentiation through FGF/MAPK pathway. Antibody array and LC/MS protein profiling data demonstrated that Apelin-13 treatment enhanced cell cycle, EGFR, FGF, Wnt, and Integrin signaling pathway proteins. Furthermore, Aplelin-13 treatment improved MSC characteristics, with mesenchymal phenotype and high expression of MSC markers, and silencing Aplnr signaling components resulted in significantly reduced expression of MSC markers. Also, Aplnr signaling activity enhanced proliferation and survival of the cells during MSC derivation from mesoderm.

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Data availability

The datasets generated during and/or analyzed during the current study are not publicly available due to [Because all data were represented in the article. There are no datasets which need to be publicly shared. Raw data can be requested from corresponding author], but are available from the corresponding author on reasonable request.

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Acknowledgements

This study was funded by TÜBİTAK 2232 International Fellowship for Outstanding Researchers Program (Project No: 118C186). The study was also supported by Yeditepe University. Ayşegül Doğan was supported by Turkish Academy of Sciences Outstanding Young Scientists Award (TÜBA-GEBİP 2020). We would like to thank Derya Sağraç for her help during immunofluorescence analysis. We would also like to thank Dr. Murat Kasap from Kocaeli University for his help during LC/MS analysis. Authors declare no conflict of interest.

Funding

This study was funded by TÜBİTAK 2232 International Fellowship for Outstanding Researchers Program (Project No. 118C186). The study was also supported by Yeditepe University. Ayşegül Doğan was supported by Turkish Academy of Sciences Outstanding Young Scientists Award (TÜBA-GEBİP 2020).

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Authors and Affiliations

  1. Faculty of Engineering, Genetics and Bioengineering Department, Yeditepe University, Istanbul, Turkey

    Hatice Burcu Şişli, Selinay Şenkal, Taha Bartu Hayal, Ezgi Bulut & Ayşegül Doğan

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  1. Hatice Burcu Şişli
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  2. Selinay Şenkal
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  3. Taha Bartu Hayal
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Contributions

AD: designed the whole project and experimental plan. Material preparation, data collection, and analysis were performed by HBŞ, SŞ, TBH, EB, and AD. Data interpretation was conducted by HBŞ and checked by AD. The first draft of the manuscript was written by HBŞ and AD. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Ayşegül Doğan.

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Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 Supplementary Video 1. Migration of Wild Type cell (R1)-derived EBs during mesoderm differentiation. (AVI 14753 KB)

Supplementary file2 Supplementary Video 2. Migration of Wild Type cell-derived EBs in the presence of Apelin-13 during mesoderm differentiation. (AVI 21286 KB)

Supplementary file3 Supplementary Video 3. Migration of Apelin KO cell-derived EBs during mesoderm differentiation. (AVI 14886 KB)

Supplementary file4 Supplementary Video 4. Migration of Aplnr KO cell-derived EBs during mesoderm differentiation. (AVI 15084 KB)

Supplementary file5 Supplementary Video 5. Migration of Elabela KO cell-derived EBs during mesoderm differentiation. (AVI 16037 KB)

Supplementary file6 Supplementary Video 6. Migration of R1 cell-derived EBs after Control shRNA-mediated silencing during mesoderm differentiation. (AVI 21995 KB)

Supplementary file7 Supplementary Video 7. Migration of R1 cell-derived EBs after shRNA-mediated silencing of Apelin during mesoderm differentiation. (AVI 18157 KB)

Supplementary file8 Supplementary Video 8. Migration of R1 cell-derived EBs after shRNA-mediated silencing of Aplnr during mesoderm differentiation. (AVI 24308 KB)

Supplementary file9 Supplementary Video 9. Migration of R1 cell-derived EBs after Control siRNA-mediated silencing during mesoderm differentiation. (AVI 14500 KB)

Supplementary file10 Supplementary Video 10. Migration of R1 cell-derived EBs after siRNA-mediated silencing of Apelin during mesoderm differentiation. (AVI 16868 KB)

Supplementary file11 Supplementary Video 11. Migration of R1 cell-derived EBs after siRNA-mediated silencing of Aplnr during mesoderm differentiation. (AVI 14156 KB)

13577_2023_861_MOESM12_ESM.tif

Supplementary file12 Supplementary Figure 1. Effect of knocking out Apelin, Aplnr and Elabela on migration of EBs during mesoderm differentiation of mESCs. (A) CRISPR constructs, carrying the gRNA scaffolds of Aplnr signaling components, to generate Apelin KO, Aplnr KO and Elabela KO cell lines. (B) The protocol for mesoderm differentiation of mESC. EBs, formed from mESCs, were transferred to Matrigel–Gelatin coated surface at Day2 and migration of the EBs were observed until Day 6. Apelin-13 or ML233 was optionally added at Day 2 to activate Aplnr signaling. (C) Morphological pictures of the EBs were taken at Day2, Day3 and Day6 for wild type cells. (D) Total area, core area and migration area (total area-core area) as well as core intensity and migration intensity were measured at Day 2, Day 3 and Day 6 and visualized as a heat map for Control, Apelin-13 and ML233 treated wild type EBs and (E) scattered line graphs of migration area and migration intensity as well as total migration area and total migration intensity between D6 and D2 were drawn. (F) Morphological pictures of the EBs were taken at Day2, Day3 and Day6 for Apelin KO cells. (G) Total area, core area and migration area (total area-core area) as well as core intensity and migration intensity were measured at Day 2, Day 3 and Day 6 and visualized as a heat map for Control, Apelin-13 and ML233 treated Apelin KO EBs and (H) scattered line graphs of migration area and migration intensity as well as total migration area and total migration intensity between D6 and D2 were drawn. (I) Morphological pictures of the EBs were taken at Day2, Day3 and Day6 for Elabela KO cells. (J) Total area, core area and migration area (total area-core area) as well as core intensity and migration intensity were measured at Day 2, Day 3 and Day 6 and visualized as a heat map for Control, Apelin-13 and ML233 treated Elabela KO EBs and (K) scattered line graphs of migration area and migration intensity as well as total migration area and total migration intensity between D6 and D2 were drawn. (L) Morphological pictures of the EBs were taken at Day2, Day3 and Day6 for Aplnr KO cells. (M) Total area, core area and migration area (total area-core area) as well as core intensity and migration intensity were measured at Day 2, Day 3 and Day 6 and visualized as a heat map for Control, Apelin-13 and ML233 treated Aplnr KO EBs and (N) scattered line graphs of migration area and migration intensity as well as total migration area and total migration intensity between D6 and D2 were drawn. (O) Bar graphs of total migration area (Day 6 Area - Day 2 Area) and (P) total migration intensity (Day 6 Intensity - Day 2 Intensity) compared to the ones of R1 were drawn. Scale bar: 50µm, 100µm. *P<0.05 (TIF 2260 KB)

13577_2023_861_MOESM13_ESM.tif

Supplementary file13 Supplementary Figure 2. Effect of shRNA and siRNA mediated silencing of Aplnr signaling on migration of EBs during mesoderm differentiation of mESCs. (A) Protocol for mesoderm differentiation of mESCs after lentiviral transduction of shApelin, shAplnr and shControl lentiviruses. EBs, formed from mESCs, were transferred to Matrigel–Gelatin coated surface at Day2 and incubated until Day 6. (B) Bar graph representation of qPCR analysis indicates relative gene expression of Apelin and Aplnr in the cells administered with the shRNA viruses. (C) Morphological pictures of the EBs were taken at Day2, Day3 and Day6. (D) Total area, core area and migration area (total area-core area) as well as core intensity and migration intensity were measured at Day 2, Day 3 and Day 6 and visualized as a heat map. (E) Scattered line graphs of migration area and migration intensity at Day 2, Day3 and Day6 as well as (F) bar graphs of total migration area and total migration intensity between D6 and D2 were drawn. (G) Protocol for mesoderm differentiation of mESCs after siRNA treatment. (H) Bar graph representation of qPCR analysis indicates relative gene expression of Apelin and Aplnr in the cells treated with Apelin siRNA, Aplnr siRNA and Control siRNA. (I) Morphological pictures of the EBs were taken at Day2, Day3 and Day6. (J) Total area, core area and migration area (total area-core area) as well as core intensity and migration intensity were measured at Day 2, Day 3 and Day 6 and visualized as a heat map. (K) Scattered line graphs of migration area and migration intensity at Day 2, Day3 and Day6 as well as (L) bar graphs of total migration area and total migration intensity between D6 and D2 were drawn for the siRNA treated EBs. Scale bar: 50µm, 100µm. *P<0.05 (TIF 1823 KB)

13577_2023_861_MOESM14_ESM.tif

Supplementary file14 Supplementary Figure 3. Effect of medium ingredients on Aplnr expression and migration of mESCs-derived EBs during mesoderm differentiation. (A) Medium supplements for each media options and timeline for mesoderm differentiation of R1 mESCs were shown. mESCs were used at Day0 to form EBs. The EBs were transferred to Matrigel–Gelatin coated surface at Day2 and their migration were observed until Day 6. (B) Morphological pictures of the EBs were taken at Day2, Day3 and Day6. (C) Total area, core area and migration area (total area-core area) as well as core intensity and migration intensity were measured at Day 2, Day 3 and Day 6 and visualized as a heat map. (D) Scattered line graphs of migration area and migration intensity at Day 2, Day3 and Day6 as well as bar graphs of total migration area and total migration intensity between D6 and D2 were drawn. (E) Heatmap of relative gene expression for neural rosette markers. ND: not detected. (F) Detection of Aplnr expression by flow cytometry at Day 2, Day 3 and Day 6 EBs. (G) Percentage of Aplnr positive cells in the populations were shown in the graph. Scale bar: 50µm, 100µm. *P<0.05 (TIF 2020 KB)

13577_2023_861_MOESM15_ESM.tif

Supplementary file15 Supplementary Figure 4. Effect of CRISPR-Cas9 mediated KO of Aplnr signaling components on mesoderm commitment of mESC-derived EBs during mesoderm differentiation. (A) The EBs were fixed using 4% paraformaldehyde at the end of Day3 and they were analyzed for protein expression of the Apelin, Aplnr, ectoderm marker protein, Sox2, the related pathway proteins, Akt and mTOR, MAPK pathway proteins involved in mesoderm commitment, Mek and Erk1/2, Pdgfrα, which guides migration of mesoderm cells, and mesoderm markers, Flk1 and MyoD. DAPI was used to stain nuclei of the cells. (B) IF analysis and the intensity of the expression as a heat map were shown. (C) Heatmap of relative gene expressions. Scale bar: 50µm. (TIF 5192 KB)

13577_2023_861_MOESM16_ESM.tif

Supplementary file16 Supplementary Figure 5. Effect of silencing Aplnr signaling components, via shRNA delivery on mesoderm commitment of mESC-derived EBs during mesoderm differentiation. (A) The EBs were fixed using 4% paraformaldehyde at the end of Day3 and they were analyzed for protein expression of the Apelin, Aplnr, ectoderm marker protein, Sox2, the related pathway proteins, Akt and mTOR, MAPK pathway proteins involved in mesoderm commitment, Mek and Erk1/2, Pdgfrα, which guides migration of mesoderm cells, and mesoderm markers, Flk1 and MyoD. DAPI was used to stain nuclei of the cells. (B) IF analysis and the intensity of the expression as a heat map were shown. (C) Heatmap of relative gene expressions. ND: not detected. Scale bar: 50µm. (TIF 3342 KB)

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Supplementary file17 Supplementary Figure 6. Effect of silencing Aplnr signaling components, via siRNA delivery on mesoderm commitment of mESC-derived EBs during mesoderm differentiation. (A) The EBs were fixed using 4% paraformaldehyde at the end of Day3 and they were analyzed for protein expression of the Apelin, Aplnr, ectoderm marker protein, Sox2, the related pathway proteins, Akt and mTOR, MAPK pathway proteins involved in mesoderm commitment, Mek and Erk1/2, Pdgfrα, which guides migration of mesoderm cells, and mesoderm markers, Flk1 and MyoD. DAPI was used to stain nuclei of the cells. (B) IF analysis and the intensity of the expression as a heat map were shown. (C) Heatmap of relative gene expressions. Scale bar: 50µm. (TIF 2506 KB)

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Supplementary file18 Supplementary Figure 7. Antibody array profile of mESC derived EBs treated with Apelin-13. (A) Membranes of the antibody array. Mesoderm related proteins were selected with black boxes on the membranes. (B) Hierarchical cluster analysis and heat map representation of proteins from Control and Apelin-13-treated EBs at Day 6. (C) Correlation coefficient matrix of the proteins. (D) Principal component analysis (PCA) of the cells. (E) A scattered graph indicating relative dot intensity for Control and Apelin-13-treated groups. (TIF 1632 KB)

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Supplementary file19 Supplementary Figure 8. Antibody array profile of mESC derived EBs when Aplnr was silenced. (A) Membranes of the antibody array. Mesoderm related proteins were selected with black boxes on the membranes. (B) Hierarchical cluster analysis and heat map representation of proteins from shControl and shAplnr EBs at Day 6. (C) Correlation coefficient matrix of the proteins. (D) Principal component analysis (PCA) of the cells. (E) A scattered graph indicating relative dot intensity for shControl and shAplnr groups. (TIF 1395 KB)

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Supplementary file20 Supplementary Figure 9. Optimization of MSC differentiation protocol from mESCs. (A) Protocols and timeline for MSC differentiation from mESCs. Standard protocol, mESC protocol and FCS protocol were applied. (B) Morphological pictures of R1 EBs at Day 2, Day 3, Day 6, Day 8 and Day 10. (C) Heatmap of relative gene expression analysis. Scale Bar: 200µm. (TIF 1243 KB)

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Supplementary file21 Supplementary Figure 10. Optimization of Apelin-13 addition to the MSC differentiation media. (A) Protocols and timelines for Apelin-13 addition to MSC differentiation protocol. (B) Morphological pictures of R1 EBs treated with the media options at Day 2, Day 3, Day 6, Day 8 and Day 10. (C) Flow cytometry analysis of R1 EBs at Day 10 for MSC markers and (D) its graphic representation indicating the positive cells for the markers. (E) A heat map representation of relative gene expressions for R1 EBs at Day 10. Scale Bar: 200µm. (TIF 2159 KB)

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Supplementary file22 Supplementary Figure 11. Effect of shRNA mediated silencing of Aplnr signaling components on MSC differentiation of R1 cells. (A) MSC differentiation protocol for Control, shApelin and shAplnr cells. (B) Morphological pictures of the cells taken at Day 2, Day 3, Day 6, Day 8 and Day 10 during MSC differentiation. (C) A heat map representation of relative gene expressions for the cells at Day 10. Scale Bar: 200µm. (TIF 1696 KB)

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Supplementary file23 Supplementary Figure 12. Effect of CRISPR-Cas9 mediated KO of Aplnr signaling components on MSC differentiation of R1 cells. (A) MSC differentiation protocol for wild type, Apelin KO, Aplnr KO and Elabela KO cells. (B) Morphological pictures of the cells taken at Day 2, Day 3, Day 6, Day 8 and Day 10 during MSC differentiation. (C) A heat map representation of relative gene expressions for the cells at Day 10. Scale Bar: 200µm. (TIF 2439 KB)

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Supplementary file24 Supplementary Figure 13. Effect of siRNA mediated silencing of Aplnr signaling components on MSC differentiation of R1 cells. (A) MSC differentiation protocol for Control, siApelin and siAplnr cells. (B) Morphological pictures of the cells taken at Day 2, Day 3, Day 6, Day 8 and Day 10 during MSC differentiation. (C) A heat map representation of relative gene expressions for the cells at Day 10. Scale Bar: 200µm. (TIF 1703 KB)

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Şişli, H.B., Şenkal, S., Hayal, T.B. et al. Regulatory role of apelin receptor signaling in migration and differentiation of mouse embryonic stem cell-derived mesoderm cells and mesenchymal stem/stromal cells. Human Cell 36, 612–630 (2023). https://doi.org/10.1007/s13577-023-00861-2

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