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A New Methodological Sequence to Expand and Transdifferentiate Human Umbilical Cord Blood Derived CD133+ Cells into a Cardiomyocyte-like Phenotype

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Abstract

Transplantation of antigenic-separated stem cells for human cardiovascular diseases such as myocardial infarction needs to be supported by experimental studies that allow refinement of the procedure. In this study we investigated optimising a protocol for the expansion and subsequent differentiation of human umbilical cord blood (HUCB) derived CD133+ stem cells into a cardiomyocyte-like lineage. CD133+ cells from HUCB were selected first by immunomagnetic separation and their purity was confirmed by flow cytometry analysis. For expansion and differentiation we developed a novel culture medium recipe that involves sequential signalling factors. Briefly, CD133+ cells were expanded for 6 days under optimal serum-free conditions in combination with fibronectin and assessed by microscopy and AlamarBlue proliferation assay. Expanded CD133+ cells were then plated in a cardiac differentiation promoting medium and cultured up to 4 weeks. With this protocol HUCB-CD133+ cells can be regularly expanded in serum-free medium to obtain recovery and growth in vitro up to 6 folds. The addition of recombinant human thrombopoietin to the remaining factors of the expanding medium was associated with larger cell expansion. Expanded UCB CD133+ cells showed a cardiomyocyte-like phenotype following differentiation in vitro through expressing intracellular cardiac specific markers including cardiac-specific α-actin, myosin heavy chain and troponin I. This change in phenotype was associated with the expression of cardiac-specific transcription factors Gata-4 and MEF2C. In addition, the change in phenotype was associated with an upregulation of nuclear receptor transcription factors including PPAR α, PPARγ, RXR α and RXRβ. We believe our protocol represents a significant advancement and overcome the technical hurdle of deriving cardiomyogenic-like cells from HUCB CD133+ stem cells. In addition, it has the required attributes of simplicity and consistency. This will permit more robust manipulation of these cells towards better engraftment and repair in patients with myocardial infarction.

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References

  1. Kocher, A. A., Schuster, M. D., Szabolcs, M. J., et al. (2001). Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nature Medicine, 7, 430–436.

    Article  CAS  PubMed  Google Scholar 

  2. Orlic, D., Kajstura, J., Chimenti, S., et al. (2001). Bone marrow cells regenerate infarcted myocardium. Nature, 410, 701–705.

    Article  CAS  PubMed  Google Scholar 

  3. Assmus, B., Schachinger, V., Teupe, C., et al. (2002). Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation, 106, 3009–3017.

    Article  PubMed  Google Scholar 

  4. Britten, M. B., Abolmaali, N. D., Assmus, B., et al. (2003). Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation, 108, 2212–2218.

    Article  CAS  PubMed  Google Scholar 

  5. Schächinger, V., Erbs, S., Elsässer, A., et al. (2006). Intracoronary bone marrow–derived progenitor cells in acute myocardial infarction. The New England Journal of Medicine, 355, 1210–1221.

    Article  PubMed  Google Scholar 

  6. Assmus, B., Rolf, A., Erbs, S., et al. (2010). Clinical outcome 2 years after intracoronary administration of bone marrow–derived progenitor cells in acute myocardial infarction CLINICAL PERSPECTIVE. Circulation. Heart Failure, 3, 89.

    Article  PubMed  Google Scholar 

  7. Leistner, D. M., Fischer-Rasokat, U., Honold, J., et al. (2011). Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI): final 5-year results suggest long-term safety and efficacy. Clinical Research in Cardiology, 1–10.

  8. Weigmann, A., Corbeil, D., Hellwig, A., & Huttner, W. B. (1997). Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells. Proceedings of the National Academy of Sciences of the United States of America, 94, 12425–12430.

    Article  CAS  PubMed  Google Scholar 

  9. Corbeil, D., Roper, K., Hellwig, A., et al. (2000). The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions. Journal of Biological Chemistry, 275, 5512–5520.

    Article  CAS  PubMed  Google Scholar 

  10. Richardson, G. D., Robson, C. N., Lang, S. H., Neal, D. E., Maitland, N. J., & Collins, A. T. (2004). CD133, a novel marker for human prostatic epithelial stem cells. Journal of Cell Science, 117, 3539–3545.

    Article  CAS  PubMed  Google Scholar 

  11. Torrente, Y., Belicchi, M., Sampaolesi, M., et al. (2004). Human circulating AC133(+) stem cells restore dystrophin expression and ameliorate function in dystrophic skeletal muscle. The Journal of Clinical Investigation, 114, 182–195.

    CAS  PubMed  Google Scholar 

  12. Maw, M. A., Corbeil, D., Koch, J., et al. (2000). A frameshift mutation in prominin (mouse)-like 1 causes human retinal degeneration. Human Molecular Genetics, 9, 27–34.

    Article  CAS  PubMed  Google Scholar 

  13. Singh, S. K., Hawkins, C., Clarke, I. D., et al. (2004). Identification of human brain tumour initiating cells. Nature, 432, 396–401.

    Article  CAS  PubMed  Google Scholar 

  14. Forraz, N., Pettengell, R., Deglesne, P. A., & McGuckin, C. P. (2002). AC133+ umbilical cord blood progenitors demonstrate rapid self-renewal and low apoptosis. British Journal of Haematology, 119, 516–524.

    Article  CAS  PubMed  Google Scholar 

  15. Zulehner, G., Mikula, M., Schneller, D., et al. (2010). Nuclear beta-catenin induces an early liver progenitor phenotype in hepatocellular carcinoma and promotes tumor recurrence. American Journal of Pathology, 176, 472–481.

    Article  CAS  PubMed  Google Scholar 

  16. Friedrich, E. B., Walenta, K., Scharlau, J., Nickenig, G., & Werner, N. (2006). CD34-/CD133+/VEGFR-2+ endothelial progenitor cell subpopulation with potent vasoregenerative capacities. Circulation Research, 98, e20–e25.

    Article  CAS  PubMed  Google Scholar 

  17. Rafii, S., & Lyden, D. (2003). Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nature Medicine, 9, 702–712.

    Article  CAS  PubMed  Google Scholar 

  18. Agbulut, O., Mazo, M., Bressolle, C., et al. (2006). Can bone marrow-derived multipotent adult progenitor cells regenerate infarcted myocardium? Cardiovascular Research, 72, 175–183.

    Article  CAS  PubMed  Google Scholar 

  19. Bartunek, J., Vanderheyden, M., Vandekerckhove, B., et al. (2005). Intracoronary injection of CD133-positive enriched bone marrow progenitor cells promotes cardiac recovery after recent myocardial infarction: feasibility and safety. Circulation, 112, I178–I183.

    PubMed  Google Scholar 

  20. Perin, E. C., & Silva, G. V. (2009). Autologous cell-based therapy for ischemic heart disease: clinical evidence, proposed mechanisms of action, and current limitations. Catheterization and Cardiovascular Interventions, 73, 281–288.

    Article  PubMed  Google Scholar 

  21. Martin-Rendon, E., Brunskill, S. J., Hyde, C. J., Stanworth, S. J., Mathur, A., & Watt, S. M. (2008). Autologous bone marrow stem cells to treat acute myocardial infarction: a systematic review. European Heart Journal, 29, 1807–1818.

    Article  CAS  PubMed  Google Scholar 

  22. Stamm, C., Kleine, H. D., Choi, Y. H., et al. (2007). Intramyocardial delivery of CD133+ bone marrow cells and coronary artery bypass grafting for chronic ischemic heart disease: safety and efficacy studies. The Journal of Thoracic and Cardiovascular Surgery, 133, 717–725. e5.

    Article  PubMed  Google Scholar 

  23. Yerebakan, C., Kaminski, A., Westphal, B., et al. (2011). Impact of preoperative left ventricular function and time from infarction on the long-term benefits after intramyocardial CD133+ bone marrow stem cell transplant. The Journal of Thoracic and Cardiovascular Surgery.

  24. Wang, X., & Seed, B. (2003). A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Research, 31, e154.

    Article  PubMed  Google Scholar 

  25. Singh, K., Srivastava, A., Mathur, N., et al. (2009). Evaluation of four methods for processing human cord blood and subsequent study of the expansion of progenitor stem cells isolated using the best method. Cytotherapy, 1–10.

  26. Summers, Y. J., Heyworth, C. M., de Wynter, E. A., Hart, C. A., Chang, J., & Testa, N. G. (2004). AC133+ G0 cells from cord blood show a high incidence of long-term culture-initiating cells and a capacity for more than 100 million-fold amplification of colony-forming cells in vitro. Stem Cells (Dayton, Ohio), 22, 704–715.

    Article  Google Scholar 

  27. Koehl, U., Zimmermann, S., Esser, R., et al. (2002). Autologous transplantation of CD133 selected hematopoietic progenitor cells in a pediatric patient with relapsed leukemia. Bone Marrow Transplantation, 29, 927–930.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This study was funded by the National Institute for Health Research Biomedical Research Unit in Cardiovascular Medicine and by the British Heart Foundation. We thank NHS Blood and Transplant (NHSBT) for providing the human UCB samples and for all related arrangements. Finally we would like to thank Mrs Lin Hua for her technical support for the lab work.

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The authors indicate no potential conflicts of interest.

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Authors and Affiliations

  1. Bristol Heart Institute, University of Bristol, Level 7, Bristol Royal Infirmary, Upper Maudlin Street, Bristol, BS2 8HW, UK

    Yu-Xin Cui, Wael Kafienah, M-S Suleiman & Raimondo Ascione

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  1. Yu-Xin Cui
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  2. Wael Kafienah
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  3. M-S Suleiman
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  4. Raimondo Ascione
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Correspondence to Raimondo Ascione.

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Cui, YX., Kafienah, W., Suleiman, MS. et al. A New Methodological Sequence to Expand and Transdifferentiate Human Umbilical Cord Blood Derived CD133+ Cells into a Cardiomyocyte-like Phenotype. Stem Cell Rev and Rep 9, 350–359 (2013). https://doi.org/10.1007/s12015-011-9316-9

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  • DOI: https://doi.org/10.1007/s12015-011-9316-9

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