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.
Similar content being viewed by others
References
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.
Orlic, D., Kajstura, J., Chimenti, S., et al. (2001). Bone marrow cells regenerate infarcted myocardium. Nature, 410, 701–705.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Singh, S. K., Hawkins, C., Clarke, I. D., et al. (2004). Identification of human brain tumour initiating cells. Nature, 432, 396–401.
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.
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.
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.
Rafii, S., & Lyden, D. (2003). Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nature Medicine, 9, 702–712.
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.
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.
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.
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.
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.
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.
Wang, X., & Seed, B. (2003). A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Research, 31, e154.
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.
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.
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.
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.
Disclosures
The authors indicate no potential conflicts of interest.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
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
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12015-011-9316-9