Skip to main content

Advertisement

SpringerLink
Log in

Human ESC-derived Neuromesodermal Progenitors (NMPs) Successfully Differentiate into Mesenchymal Stem Cells (MSCs)

  • Published:
Stem Cell Reviews and Reports Aims and scope Submit manuscript

Abstract

Mesenchymal Stem Cells (MSCs), as an adult stem cell type, are used to treat various disorders in clinics. However, derivation of homogenous and adequate amount of MSCs limits the regenerative treatment potential. Although mesoderm is the main source of mesenchymal progenitors during embryonic development, neuromesodermal progenitors (NMPs), reside in the primitive streak during development, is known to differentiate into paraxial mesoderm. In the current study, we generated NMPs from human embryonic stem cells (hESC), subsequently derived MSCs and characterized this cell population in vitro and in vivo. Using a bFGF and CHIR induced NMP formation protocol followed by serum containing culture conditions; here we show that MSCs can be generated from NMPs identified by not only the expression of T/Bra and Sox 2 but also FLK-1/PDGFRα in our study. NMP-derived MSCs were plastic adherent fibroblast like cells with colony forming capacity and trilineage (osteo-, chondro- and adipo-genic) differentiation potential. In the present study, we demonstrate that NMP-derived MSCs have an endothelial tendency which might be related to their FLK-1+/PDGFRα + NMP origin. NMP-derived MSCs displayed a protein expression profile of characterized MSCs. Growth factor and angiogenesis related pathway proteins were similarly expressed in NMP-derived MSCs and characterized MSCs. NMP-derived MSCs keep characteristics after short-term and long-term freeze-thaw cycles and localized into bone marrow followed by tail vein injection into NOD/SCID mice. Together, these data showed that hESC-derived NMPs might be used as a precursor cell population for MSC derivation and could be used for in vitro and in vivo research.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Data Availability

Data is available upon request.

Code Availability

Not applicable.

References

  1. Abdal Dayem, A., Lee, S. B., Kim, K., Lim, K. M., Jeon, T., Seok, J., & Cho, S. G. (2019). Production of mesenchymal stem cells through stem cell reprogramming. International journal of molecular sciences, 20(8), 1922

  2. Ardeshirylajimi, A., Soleimani, M., Hosseinkhani, S., Parivar, K., & Yaghmaei, P. (2014). A comparative study of osteogenic differentiation human induced pluripotent stem cells and adipose tissue derived mesenchymal stem cells. Cell Journal (Yakhteh), 16(3), 235

    Google Scholar 

  3. Attardi, A., Fulton, T., Florescu, M., Shah, G., Muresan, L., Lenz, M. O., Lancaster, C., Huisken, J., van Oudenaarden, A., & Steventon, B. (2018). Neuromesodermal progenitors are a conserved source of spinal cord with divergent growth dynamics. Development, 145(21).

  4. Augustin, M. (2012). Preconditioning methods in cell therapy of the heart, Doctoral dissertation, [online] Available at: https://helda.helsinki.fi/handle/10138/36906. Accessed 15 Oct 2021.

  5. Bernitz, J. M., & Moore, K. A. (2014). Haematopoietic Stem Cells: Uncovering the origins of a niche. eLife, 3, e05041

    PubMed Central  Google Scholar 

  6. Bremer, S., & Hartung, T. (2004). The use of embryonic stem cells for regulatory developmental toxicity testing in vitro-the current status of test development. Current Pharmaceutical Design, 10(22), 2733–2747

    CAS  PubMed  Google Scholar 

  7. Chen, K. G., Mallon, B. S., McKay, R. D., & Robey, P. G. (2014). Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics. Cell Stem Cell, 14(1), 13–26

    CAS  PubMed  PubMed Central  Google Scholar 

  8. ATCC Animal Cell Culture Guide. [online] Available at: https://www.atcc.org/resources/culture-guides/animal-cell-culture-guide. Accessed 15 Oct 2021.

  9. Demirci, S., Doğan, A., Apdik, H., Tuysuz, E. C., Gulluoglu, S., Bayrak, O. F., & Şahin, F. (2018). Cytoglobin inhibits migration through PI3K/AKT/mTOR pathway in fibroblast cells. Molecular and Cellular Biochemistry, 437(1), 133–142

    CAS  PubMed  Google Scholar 

  10. Demirci, S., Doğan, A., Karakuş, E., Halıcı, Z., Topçu, A., Demirci, E., & Sahin, F. (2015). Boron and poloxamer (F68 and F127) containing hydrogel formulation for burn wound healing. Biological Trace Element Research, 168(1), 169–180

    CAS  PubMed  Google Scholar 

  11. Demirci, S., Doğan, A., Şişli, B., & Sahin, F. (2014). Boron increases the cell viability of mesenchymal stem cells after long-term cryopreservation. Cryobiology, 68(1), 139–146

    CAS  PubMed  Google Scholar 

  12. Demirci, S., Kaya, M. S., Doğan, A., Kalay, Å, ALTIN, N. Ã, & Şahin, Y. A. R. A. T. A. (2015). Antibacterial and cytotoxic properties of boron-containing dental composite. Turkish Journal of Biology, 39(3), 417–426

    CAS  Google Scholar 

  13. Ding, G., Tanaka, Y., Hayashi, M., Nishikawa, S. I., & Kataoka, H. (2013). PDGF receptor alpha+ mesoderm contributes to endothelial and hematopoietic cells in mice. Developmental Dynamics, 242(3), 254–268

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Doğan, A. (2018). Embryonic stem cells in development and regenerative medicine. Cell Biology and Translational Medicine, 1, 1–15

    Google Scholar 

  15. Doğan, A., Demirci, S., Apdik, H., Apdik, E. A., & Şahin, F. (2017). Dental pulp stem cells (DPSCs) increase prostate cancer cell proliferation and migration under in vitro conditions. Tissue and Cell, 49(6), 711–718

    PubMed  Google Scholar 

  16. Doğan, A., Yalvaç, M. E., Şahin, F., Kabanov, A. V., Palotás, A., & Rizvanov, A. A. (2012). Differentiation of human stem cells is promoted by amphiphilic pluronic block copolymers. International Journal of Nanomedicine, 7, 4849

    PubMed  PubMed Central  Google Scholar 

  17. Dou, Z., Ghosh, K., Vizioli, M. G., Zhu, J., Sen, P., Wangensteen, K. J., & Zhou, Z. (2017). Cytoplasmic chromatin triggers inflammation in senescence and cancer. Nature, 550(7676), 402–406

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Doyle, E. C., Wragg, N. M., & Wilson, S. L. (2020). Intraarticular injection of bone marrow-derived mesenchymal stem cells enhances regeneration in knee osteoarthritis. Knee Surgery, Sports Traumatology, Arthroscopy, 28, 3827–3842

  19. Du, Z. W., Hu, B. Y., Ayala, M., Sauer, B., & Zhang, S. C. (2009). Cre recombination-mediated cassette exchange for building versatile transgenic human embryonic stem cells lines. Stem Cells, 27(5), 1032–1041

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Eirin, A., & Lerman, L. O. (2014). Mesenchymal stem cell treatment for chronic renal failure. Stem Cell Research & Therapy, 5(4), 1–8

    Google Scholar 

  21. Farahani, R. M., & Xaymardan, M. (2015). Platelet-derived growth factor receptor alpha as a marker of mesenchymal stem cells in development and stem cell biology. Stem Cells International, 2015, 1–8

  22. Fujiki, Y., Tao, K., Bianchi, D. W., Giel-Moloney, M., Leiter, A. B., & Johnson, K. L. (2008). Quantification of green fluorescent protein by in vivo imaging, PCR, and flow cytometry: comparison of transgenic strains and relevance for fetal cell microchimerism. Cytometry Part A: The Journal of the International Society for Analytical Cytology, 73(2), 11–118

    Google Scholar 

  23. Galipeau, J. (2013). The mesenchymal stromal cells dilemma—does a negative phase III trial of random donor mesenchymal stromal cells in steroid-resistant graft-versus-host disease represent a death knell or a bump in the road? Cytotherapy, 15(1), 2–8

    PubMed  Google Scholar 

  24. Gouti, M., Tsakiridis, A., Wymeersch, F. J., Huang, Y., Kleinjung, J., Wilson, V., & Briscoe, J. (2014). In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity. PLoS Biology, 12(8), e1001937

    PubMed  PubMed Central  Google Scholar 

  25. Henrique, D., Abranches, E., Verrier, L., & Storey, K. G. (2015). Neuromesodermal progenitors and the making of the spinal cord. Development, 142(17), 2864–2875

    CAS  PubMed  Google Scholar 

  26. Herberts, C. A., Kwa, M. S., & Hermsen, H. P. (2011). Risk factors in the development of stem cell therapy. Journal of Translational Medicine, 9(1), 1–14

    Google Scholar 

  27. Hirvonen, T. (2014). Glycan binding proteins in therapeutic mesenchymal stem cell research. Doctoral dissertation, [online] Available at: https://helda.helsinki.fi/handle/10138/135978. Accessed 15 Oct 2021.

  28. Hwang, J. H., Shim, S. S., Seok, O. S., Lee, H. Y., Woo, S. K., Kim, B. H., & Park, Y. K. (2009). Comparison of cytokine expression in mesenchymal stem cells from human placenta, cord blood, and bone marrow. Journal of Korean Medical Science, 24(4), 547

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Kiani, A. A., Kazemi, A., Halabian, R., Mohammadipour, M., Jahanian-Najafabadi, A., & Roudkenar, M. H. (2013). HIF-1α confers resistance to induced stress in bone marrow-derived mesenchymal stem cells. Archives of Medical Research, 44(3), 185–193

    CAS  PubMed  Google Scholar 

  30. Kyurkchiev, D., Bochev, I., Ivanova-Todorova, E., Mourdjeva, M., Oreshkova, T., Belemezova, K., & Kyurkchiev, S. (2014). Secretion of immunoregulatory cytokines by mesenchymal stem cells. World Journal of Stem Cells, 6(5), 552

    PubMed  PubMed Central  Google Scholar 

  31. Ludwig, T. E., Bergendahl, V., Levenstein, M. E., Yu, J., Probasco, M. D., & Thomson, J. A. (2006). Feeder-independent culture of human embryonic stem cells. Nature Methods, 3(8), 637–646. https://doi.org/10.1038/nmeth902

    Article  CAS  PubMed  Google Scholar 

  32. Lukomska, B., Stanaszek, L., Zuba-Surma, E., Legosz, P., Sarzynska, S., & Drela, K. (2019). Challenges and controversies in human mesenchymal stem cell therapy. Stem Cells International, 2019, 1–10.

  33. Metsalu, T., & Vilo, J. (2015). ClustVis: a web tool for visualizing clustering of multivariate data using Principal Component Analysis and heatmap. Nucleic Acids Research, 43(W1), W566–W570

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Peng, K. Y., Lee, Y. W., Hsu, P. J., Wang, H. H., Wang, Y., Liou, J. Y., & Yen, B. L. (2016). Human pluripotent stem cell (PSC)-derived mesenchymal stem cells (MSCs) show potent neurogenic capacity which is enhanced with cytoskeletal rearrangement. Oncotarget, 7(28), 43949

    PubMed  PubMed Central  Google Scholar 

  35. Phinney, D. G. (2012). Functional heterogeneity of mesenchymal stem cells: implications for cell therapy. Journal of Cellular Biochemistry, 113(9), 2806–2812

    CAS  PubMed  Google Scholar 

  36. Pittenger, M. F., Discher, D. E., Péault, B. M., Phinney, D. G., Hare, J. M., & Caplan, A. I. (2019). Mesenchymal stem cell perspective: cell biology to clinical progress. NPJ Regenerative Medicine, 4(1), 1–15

    CAS  Google Scholar 

  37. Pourquié, O. (2001). Vertebrate somitogenesis. Annual Review of Cell and Developmental Biology, 17(1), 311–350

    PubMed  Google Scholar 

  38. Ripoll, C. B. (2010). Adult stem cell therapy in the twitcher mouse model of Krabbe’s disease utilizing mesenchymal lineage stem cells. Tulane University

  39. Rolletschek, A., Blyszczuk, P., & Wobus, A. M. (2004). Embryonic stem cell-derived cardiac, neuronal and pancreatic cells as model systems to study toxicological effects. Toxicology Letters, 149(1–3), 361–369

    CAS  PubMed  Google Scholar 

  40. Saldaña, L., Bensiamar, F., Vallés, G., Mancebo, F. J., García-Rey, E., & Vilaboa, N. (2019). Immunoregulatory potential of mesenchymal stem cells following activation by macrophage-derived soluble factors. Stem Cell Research & Therapy, 10(1), 1–15

    Google Scholar 

  41. Sarugaser, R., Hanoun, L., Keating, A., Stanford, W. L., & Davies, J. E. (2009). Human mesenchymal stem cells self-renew and differentiate according to a deterministic hierarchy. PloS One, 4(8), e6498

  42. Sheng, G. (2015). The developmental basis of mesenchymal stem/stromal cells (MSCs). BMC Developmental Biology, 15(1), 1–8

    Google Scholar 

  43. Şişli, H. B., Hayal, T. B., Seçkin, S., Şenkal, S., Kıratlı, B., Şahin, F., & Doğan, A. (2019). Gene editing in human pluripotent stem cells: recent advances for clinical therapies. Cell Biology and Translational Medicine, 7, 17–28

    Google Scholar 

  44. Steinemann, D., Göhring, G., & Schlegelberger, B. (2013). Genetic instability of modified stem cells-a first step towards malignant transformation? American Journal of Stem Cells, 2(1), 39

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Steventon, B., & Arias, A. M. (2017). Evo-engineering and the cellular and molecular origins of the vertebrate spinal cord. Developmental Biology, 432(1), 3–13

    CAS  PubMed  Google Scholar 

  46. Su, W., Zhou, M., Zheng, Y., Fan, Y., Wang, L., Han, Z., & Xiang, R. (2011). Bioluminescence reporter gene imaging characterize human embryonic stem cell-derived teratoma formation. Journal of Cellular Biochemistry, 112(3), 840–848

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Sweetman, D., Wagstaff, L., Cooper, O., Weijer, C., & Münsterberg, A. (2008). The migration of paraxial and lateral plate mesoderm cells emerging from the late primitive streak is controlled by different Wnt signals. BMC Developmental Biology, 8(1), 1–15

    Google Scholar 

  48. Taşlı, P. N., Doğan, A., Demirci, S., & Şahin, F. (2016). Myogenic and neurogenic differentiation of human tooth germ stem cells (hTGSCs) are regulated by pluronic block copolymers. Cytotechnology, 68(2), 319–329

    PubMed  Google Scholar 

  49. Truong, M. D., Choi, B., Kim, Y., Kim, M., & Min, B. H. (2017). Granulocyte macrophage–colony stimulating factor (GM-CSF) significantly enhances articular cartilage repair potential by microfracture. Osteoarthritis and Cartilage, 25(8), 1345–1352

    PubMed  Google Scholar 

  50. van Poll, D., Parekkadan, B., Rinkes, I. B., Tilles, A. W., & Yarmush, M. L. (2008). Mesenchymal stem cell therapy for protection and repair of injured vital organs. Cellular and Molecular Bioengineering, 1(1), 42–50

    Google Scholar 

  51. Vanderlaan, R. D., Oudit, G. Y., & Backx, P. H. (2003). Electrophysiological profiling of cardiomyocytes in embryonic bodies derived from human embryonic stem cells: Therapeutic implications. American Heart Association

  52. Vodyanik, M. A., Yu, J., Zhang, X., Tian, S., Stewart, R., Thomson, J. A., & Slukvin, I. I. (2010). A mesoderm-derived precursor for mesenchymal stem and endothelial cells. Cell Stem Cell, 7(6), 718–729

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Wang, H., Li, D., Zhai, Z., Zhang, X., Huang, W., Chen, X., & Zou, Z. (2019). Characterization and therapeutic application of mesenchymal stem cells with neuromesodermal origin from human pluripotent stem cells. Theranostics, 9(6), 1683

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Wei, X., Yang, X., Han, Z., Qu, F., Shao, L., & Shi, Y. (2013). Mesenchymal stem cells: a new trend for cell therapy. Acta Pharmacologica Sinica, 34(6), 747–754

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Xu, M., Shaw, G., Murphy, M., & Barry, F. (2019). Induced pluripotent stem cell-derived mesenchymal stromal cells are functionally and genetically different from bone marrow‐derived mesenchymal stromal cells. Stem cells, 37(6), 754–765

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Yamaguchi, T. P., Dumont, D. J., Conlon, R. A., Breitman, M. L., & Rossant, J. (1993). flk-1, an flt-related receptor tyrosine kinase is an early marker for endothelial cell precursors. Development, 118(2), 489–498

    CAS  PubMed  Google Scholar 

  57. Yuan, Z., Lourenco, S. D. S., Sage, E. K., Kolluri, K. K., Lowdell, M. W., & Janes, S. M. (2016). Cryopreservation of human mesenchymal stromal cells expressing TRAIL for human anti-cancer therapy. Cytotherapy, 18(7), 860–869

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Zhu, Y., Zhang, P., Gu, R. L., Liu, Y. S., & Zhou, Y. S. (2018). Origin and clinical applications of neural crest-derived dental stem cells. The Chinese Journal of Dental Research, 21(2), 89–100

    PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported by Yeditepe University and Turkish Academy of Sciences Outstanding Young Scientists Award (TÜBA-GEBİP 2020). Albert A Rizvanov was supported by KFU state assignment 0671-2020-0058.

Funding

This study was supported by Yeditepe University and Outstanding Young Scientists Award (TÜBA-GEBİP 2020). Albert A Rizvanov was supported by KFU state assignment 0671-2020-0058.

Author information

Author notes

  1. Selinay Şenkal and Ayşegül Doğan contributed equally to this work.

Authors and Affiliations

  1. Faculty of Engineering, Genetics and Bioengineering Department, Yeditepe University, İstanbul, 34755, Turkey

    Selinay Şenkal, Taha Bartu Hayal, Derya Sağraç, Hatice Burcu Şişli, Ayla Burçin Asutay, Binnur Kıratlı, Fikrettin Şahin & Ayşegül Doğan

  2. Faculty of Pharmacy, Pharmaceutical Toxicology Department, Yeditepe University, 34755, İstanbul, Turkey

    Engin Sümer

  3. Institute of Fundamental Medicine and Biology, Kazan Federal University, 420008, Kazan, Russia

    Albert A. Rizvanov

Authors
  1. Selinay Şenkal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Taha Bartu Hayal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Derya Sağraç
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hatice Burcu Şişli
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ayla Burçin Asutay
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Binnur Kıratlı
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Engin Sümer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Albert A. Rizvanov
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Fikrettin Şahin
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ayşegül Doğan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Ayşegül Doğan, Fikrettin Şahin, Albert A. Rizvanov and Selinay Şenkal contributed to the study conception and design. Material preparation, data collection and analysis were performed by Taha Bartu Hayal and Selinay Şenkal. In vitro experiments were conducted by Ayşegül Doğan, Taha Bartu Hayal, Selinay Şenkal and Derya Sağraç. In vivo experiments were conducted by Hatice Burcu Şişli, Engin Sümer and Fikrettin Şahin. Flow cytometry analysis and Immunocytochemistry experiments were performed by Ayla Burçin Asutay and Binnur Kıratlı. The first draft of the manuscript was written by Ayşegül Doğan, revised by Albert A. Rizvanov and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ayşegül Doğan.

Ethics declarations

Conflicts of Interest/Competing Interests

The authors have no conflicts of interest to declare that are relevant to the content of this article.

Ethics Approval

Not applicable.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

ESM 1

(DOCX 22 kb)

Supplementary Figure 1

Characterization of NMPs. (a) T/Bra and Sox2 staining of cells at different time points (D3, D5, D8, D10 and D24). (b) T/Bra, Sox2, Wnt3, Nkx1-2 gene expression at different time points (D3, D5, D8, D10 and D24) of differentiation protocol. (c) OCT3/4, Nanog, c-MYC gene expression at different time points. *P<0.05. (PNG 5829 kb)

High Resolution Image (TIF 50698 kb)

Supplementary Figure 2

Characterization of NMP-derived MSC population. (a) CD73 and CD45 immunostaining of cells during differentiation (b) Heat map representation of CD73 and CD45 immunostaining during MSC derivation (c) CD73, CD90 and CD105 gene expression analysis during differentiation protocol. *P<0.05. (PNG 6445 kb)

High Resolution Image (TIF 8183 kb)

Supplementary Figure 3

Three lineage differentiation analyses of NMP-derived MSCs. Osteo-, chondro- and adipo-genic differentiation of (a) MSC-II, (b) MSC-I, (c) ASC telo s and (d) DPSCs. (PNG 15537 kb)

High Resolution Image (TIF 20239 kb)

Supplementary Figure 4

Gene expression analyses after three lineage differentiation of NMP-derived MSCs. Osteo-, chondro- and adipo-genic differentiation related gene expression analyses of MSC II, MSC I, ASC telo and DPSCs. Osteocalcin, Aggrecan and Adiponectin gene expression were calculated. *P<0.05. (PNG 138 kb)

High Resolution Image (TIF 11845 kb)

Supplementary Figure 5

Repeated short-term freeze-thaw cycles of MSC II cell population. (a) Experimental design of four repeated short-term freeze-thaw cycles. (b) Morphological images of selected MSC II colonies after each freeze-thaw. (c) CD73 and CD45 immunostaining of cells short-term freeze-thaw cycles (d) Viable cell number of MSC II cells after four repeated short-term freeze-thaw cycles. (e) Three lineage differentiation analyses of NMP-derived MSCs (MSC II) after short-term freeze-thaw cycles. (PNG 1153 kb)

High Resolution Image (TIF 7209 kb)

Supplementary Figure 6

Gene expression analyses after three lineage differentiation of cryopreserved MSC II cells. Osteo-, chondro- and adipo-genic differentiation related gene expression analyses of MSC II followed by short-term and long-term cryopreservation analyses. Osteocalcin, Aggrecan and Adiponectin gene expression were calculated. *P<0.05. (PNG 141 kb)

High Resolution Image (TIF 10544 kb)

Supplementary Figure 7

Characterization of MSC II cells after long-term cryopreservation. (a) Crystal violet staining and morphological analyses. (b) Diameter and (c) Number of MSC II cell colonies after long-term cryopreservation. (d) Viable cell number of MSC II cells after long-term cryopreservation (e) CD73 and CD45 immunostaining of cells after long-term cryopreservation (f) Three lineage differentiation analyses of NMP-derived MSCs (MSC II) after long-term cryopreservation. (PNG 22023 kb)

High Resolution Image (TIF 100587 kb)

Supplementary Figure 8

Immunocytochemistry analyses of (a) FLK-1 and (b) PDGFRα during differentiation protocol. (PNG 1714 kb)

High Resolution Image (TIF 10294 kb)

Supplementary Figure 9

Heat map representation of (a) FLK-1 and PDGFRα, (b) PECAM1, VE-Cadherin, VCAM-1 and VEGF immunostaining during NMP-derived MSC derivation. (PNG 59 kb)

High Resolution Image (TIF 4138 kb)

Supplementary Figure. 10

Immunocytochemistry analyses of VE-Cadherin and VCAM-1 during NMP-derived MSC derivation. (PNG 6186 kb)

High Resolution Image (TIF 25010 kb)

Supplementary Figure 11

Immunocytochemistry analyses of PECAM1 (CD31) and VEGF immunostaining during MSC derivation. (PNG 5033 kb)

High Resolution Image (TIF 25061 kb)

Supplementary Figure 12

Protein membrane array analysis of differentiated cells (a) Cytokine, Growth Factor and Angiogenesis arrays of cells at different time points of the differentiation protocol. (b) Selected differentially expressed proteins Cytokine, Growth Factor and Angiogenesis arrays. (PNG 3566 kb)

High Resolution Image (TIF 40496 kb)

Supplementary Figure 13

Flow cytometry analyses of GFP+ differentiated cells in various tissues and percentage of tissue distribution during differentiation. (PNG 2558 kb)

High Resolution Image (TIF 41558 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Şenkal, S., Hayal, T.B., Sağraç, D. et al. Human ESC-derived Neuromesodermal Progenitors (NMPs) Successfully Differentiate into Mesenchymal Stem Cells (MSCs). Stem Cell Rev and Rep 18, 278–293 (2022). https://doi.org/10.1007/s12015-021-10281-0

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12015-021-10281-0

Keywords

Search

Navigation