Abstract
Background
Accumulating preclinical and clinical evidence indicates that human mesenchymal stromal cells (MSCs) are good candidates for cell therapy. For clinical applications of MSCs extensive in vitro expansion is required to obtain an adequate number of cells. It is evident that the pursuit for cell quantity must not affect quality, but it is also a fact that in vitro culture conditions affect MSC phenotype. One possible variable is the degree of cell confluence during expansion.
Methods
We investigate the influence of cell density on homogeneity and differentiation during culture expansion of un-stimulated MSCs isolated from the bone marrow in DMEM and fetal bovine serum (FBS).
MSC morphology, phenotype and differentiation were investigated weekly during 5 weeks culture expansion using electron microscopy, flow cytometry, immunocytochemistry, qualitative RT-PCR and quantitative Q-PCR.
Results
The morphological observation and the phenotypic analyses showed that MSCs after 3 weeks cultivation constituted a phenotypically homogenous MSC cell population, which at low levels expressed genes for different cell lineages, confirming their multilineage plasticity, without actual differentiation. This phenotype persisted independent of increasing cell densities.
Discussion
These data demonstrate that MSC characteristics and plasticity can be maintained during culture expansion from bone marrow mononuclear cells to MSCs and that a homogeneous phenotype of undifferentiated MSCs which persists independent of cell density can be used for clinical therapies.
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References
Haack-Sorensen, M., Friis, T., & Kastrup, J. (2008). Mesenchymal stromal cell and mononuclear cell therapy in heart disease. Future Cardiology, 4(5), 481–494.
Kassem, M., & Abdallah, B. M. (2008). Human bone-marrow-derived mesenchymal stem cells: biological characteristics and potential role in therapy of degenerative diseases. Cell and Tissue Research, 331(1), 157–163.
Mathiasen, A. B., Haack-Sorensen, M., & Kastrup, J. (2009). Mesenchymal stromal cells for cardiovascular repair: current status and future challenges. Future Cardiology, 5(6), 605–617.
Janssens, S. (2010). Stem cells in the treatment of heart disease. Annual Review of Medicine, 61, 287–300.
Kastrup, J. (2011). Stem cell therapy for cardiovascular repair in ischemic heart disease: how to predict and secure optimal outcome? EPMA J. In Press.
Conget, P. A., & Minguell, J. J. (1999). Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. Journal of Cellular Physiology, 181(1), 67–73.
Deans, R. J., & Moseley, A. B. (2000). Mesenchymal stem cells: biology and potential clinical uses. Experimental Hematology, 28(8), 875–884.
Minguell, J. J., Erices, A., & Conget, P. (2001). Mesenchymal stem cells. Experimental Biology and Medicine (Maywood, N.J.), 226(6), 507–520.
Pittenger, M. F., Mackay, A. M., Beck, S. C., et al. (1999). Multilineage potential of adult human mesenchymal stem cells. Science, 284(5411), 143–147.
Bartholomew, A., Sturgeon, C., Siatskas, M., et al. (2002). Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Experimental Hematology, 30(1), 42–48.
Nauta, A. J., & Fibbe, W. E. (2007). Immunomodulatory properties of mesenchymal stromal cells. Blood, 110(10), 3499–3506.
Tyndall, A., Walker, U. A., Cope, A., et al. (2007). Immunomodulatory properties of mesenchymal stem cells: a review based on an interdisciplinary meeting held at the Kennedy Institute of Rheumatology Division, London, UK, 31 October 2005. Arthritis Research & Therapy, 9(1), 301.
Kinnaird, T., Stabile, E., Burnett, M. S., et al. (2004). Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circulation Research, 94(5), 678–685.
Phinney, D. G., & Prockop, D. J. (2007). Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair-current views. Stem Cell, 25(11), 2896–2902.
Resnick, I., Stepensky, P., Elkin, G., et al. (2010). MSC for the improvement of hematopoietic engraftment. Bone Marrow Transplantation, 45(3), 605–606.
Yoshikawa, T., Mitsuno, H., Nonaka, I., et al. (2008). Wound therapy by marrow mesenchymal cell transplantation. Plastic and Reconstructive Surgery, 121(3), 860–877.
Mazzini, L., Ferrero, I., Luparello, V., et al. (2010). Mesenchymal stem cell transplantation in amyotrophic lateral sclerosis: A Phase I clinical trial. Experimental Neurology, 223(1), 229–237.
Chen, S. L., Fang, W. W., Ye, F., et al. (2004). Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. The American Journal of Cardiology, 94(1), 92–95.
Friis, T., Haack-Sørensen, M., Mathiasen, A. B., et al. (2011). Mesenchymal stromal cell derived endothelial progenitor cell treatment in patients with severe refractory angina. Scandinavian Cardiovascular Journal, 45(3), 161–168.
Haack-Sørensen, M., Friis, T., Mathiasen, A.B. et al. (2012). Mesenchymal stromal therapy in patients with severe refractory angina - one year follow-up. Cell Transplant, In Press.
Katritsis, D. G., Sotiropoulou, P. A., Karvouni, E., et al. (2005). Transcoronary transplantation of autologous mesenchymal stem cells and endothelial progenitors into infarcted human myocardium. Catheterization and Cardiovascular Interventions, 65(3), 321–329.
Katritsis, D. G., Sotiropoulou, P., Giazitzoglou, E., Karvouni, E., & Papamichail, M. (2007). Electrophysiological effects of intracoronary transplantation of autologous mesenchymal and endothelial progenitor cells. Europace, 9(3), 167–171.
Mohyeddin-Bonab, M., Mohamad-Hassani, M. R., Alimoghaddam, K., et al. (2007). Autologous in vitro expanded mesenchymal stem cell therapy for human old myocardial infarction. Archives of Iranian Medicine, 10(4), 467–473.
Bruder, S. P., Fink, D. J., & Caplan, A. I. (1994). Mesenchymal stem cells in bone development, bone repair, and skeletal regeneration therapy. Journal of Cellular Biochemistry, 56(3), 283–294.
Caplan, A. I. (2007). Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. Journal of Cellular Physiology, 213(2), 341–347.
Sotiropoulou, P. A., Perez, S. A., Salagianni, M., Baxevanis, C. N., & Papamichail, M. (2006). Cell culture medium composition and translational adult bone marrow-derived stem cell research. Stem Cells, 24(5), 1409–1410.
Neuhuber, B., Swanger, S. A., Howard, L., Mackay, A., & Fischer, I. (2008). Effects of plating density and culture time on bone marrow stromal cell characteristics. Experimental Hematology, 36(9), 1176–1185.
Wagner, W., Horn, P., Castoldi, M., et al. (2008). Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS One, 3(5), e2213.
World Medical Association Declaration of Helsinki. (2007). Recommendations guiding physicians in biomedical research involving human subjects. Cardiovascular Research, 35(1), 2–3.
Gaster, M., Kristensen, S. R., Beck-Nielsen, H., & Schroder, H. D. (2001). A cellular model system of differentiated human myotubes. APMIS, 109(11), 735–744.
Pasquinelli, G., Tazzari, P., Ricci, F., et al. (2007). Ultrastructural characteristics of human mesenchymal stromal (stem) cells derived from bone marrow and term placenta. Ultrastructural Pathology, 31(1), 23–31.
Steinert, A., Weber, M., Dimmler, A., et al. (2003). Chondrogenic differentiation of mesenchymal progenitor cells encapsulated in ultrahigh-viscosity alginate. Journal of Orthopaedic Research, 21(6), 1090–1097.
DiGirolamo, C. M., Stokes, D., Colter, D., Phinney, D. G., Class, R., & Prockop, D. J. (1999). Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. British Journal of Haematology, 107(2), 275–281.
Colter, D. C., Class, R., DiGirolamo, C. M., & Prockop, D. J. (2000). Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proceedings of the National Academy of Sciences of the United States of America, 97(7), 3213–3218.
Prockop, D. J., Brenner, M., Fibbe, W. E., et al. (2010). Defining the risks of mesenchymal stromal cell therapy. Cytotherapy, 12(5), 576–578.
Pieri, L., Urbani, S., Mazzanti, B., et al. (2011). Human mesenchymal stromal cells preserve their stem features better when cultured in the Dulbecco’s modified Eagle medium. Cytotherapy, 13(5), 539–548.
Dominici, M., Le, B. K., Mueller, I., et al. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8(4), 315–317.
Haack-Sorensen, M., Bindslev, L., Mortensen, S., Friis, T., & Kastrup, J. (2007). The influence of freezing and storage on the characteristics and functions of human mesenchymal stromal cells isolated for clinical use. Cytotherapy, 9(4), 328–337.
Kassem, M., Kristiansen, M., & Abdallah, B. M. (2004). Mesenchymal stem cells: cell biology and potential use in therapy. Basic & Clinical Pharmacology & Toxicology, 95(5), 209–214.
Friis, T., Haack-Sorensen, M., Hansen, S. K., Hansen, L., Bindslev, L., & Kastrup, J. (2011). Comparison of mesenchymal stromal cells from young healthy donors and patients with severe chronic coronary artery disease. Scandinavian Journal of Clinical and Laboratory Investigation, 71(3), 193–202.
Sekiya, I., Larson, B. L., Smith, J. R., Pochampally, R., Cui, J. G., & Prockop, D. J. (2002). Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells, 20(6), 530–541.
Hung, S. C., Cheng, H., Pan, C. Y., Tsai, M. J., Kao, L. S., & Ma, H. L. (2002). In vitro differentiation of size-sieved stem cells into electrically active neural cells. Stem Cells, 20(6), 522–529.
Sanchez-Ramos, J., Song, S., Cardozo-Pelaez, F., et al. (2000). Adult bone marrow stromal cells differentiate into neural cells in vitro. Experimental Neurology, 164(2), 247–256.
Makino, S., Fukuda, K., Miyoshi, S., et al. (1999). Cardiomyocytes can be generated from marrow stromal cells in vitro. The Journal of Clinical Investigation, 103(5), 697–705.
Reyes, M., Dudek, A., Jahagirdar, B., Koodie, L., Marker, P. H., & Verfaillie, C. M. (2002). Origin of endothelial progenitors in human postnatal bone marrow. The Journal of Clinical Investigation, 109(3), 337–346.
Kang, X. Q., Zang, W. J., Song, T. S., et al. (2005). Rat bone marrow mesenchymal stem cells differentiate into hepatocytes in vitro. World Journal of Gastroenterology, 11(22), 3479–3484.
Denizot, Y., Trimoreau, F., & Praloran, V. (1998). Effects of lipid mediators on the synthesis of leukaemia inhibitory factor and interleukin 6 by human bone marrow stromal cells. Cytokine, 10(10), 781–785.
Rougier, F., Cornu, E., Praloran, V., & Denizot, Y. (1998). IL-6 and IL-8 production by human bone marrow stromal cells. Cytokine, 10(2), 93–97.
Shahdadfar, A., Fronsdal, K., Haug, T., Reinholt, F. P., & Brinchmann, J. E. (2005). In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem Cells, 23(9), 1357–1366.
Gnecchi, M., He, H., Liang, O. D., et al. (2005). Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nature Medicine, 11(4), 367–368.
Hegner, B., Weber, M., Dragun, D., & Schulze-Lohoff, E. (2005). Differential regulation of smooth muscle markers in human bone marrow-derived mesenchymal stem cells. Journal of Hypertension, 23(6), 1191–1202.
Chichester, C. O., Fernandez, M., & Minguell, J. J. (1993). Extracellular matrix gene expression by human bone marrow stroma and by marrow fibroblasts. Cell Adhesion and Communication, 1(2), 93–99.
Sensebe, L., Bourin, P., & Tarte, K. (2011). Good Manufacturing Practices Production of Mesenchymal Stem/Stromal Cells. Human Gene Therapy, 22(1), 19–26.
Gregory, C. A., Prockop, D. J., & Spees, J. L. (2005). Non-hematopoietic bone marrow stem cells: molecular control of expansion and differentiation. Experimental Cell Research, 306(2), 330–335.
Fink, T., Rasmussen, J. G., Lund, P., Pilgaard, L., Soballe, K., & Zachar, V. (2011). Isolation and expansion of adipose-derived stem cells for tissue engineering. Frontiers in Bioscience (Elite Edition), 3, 256–263.
Lee, J. W., Kim, Y. H., Park, K. D., Jee, K. S., Shin, J. W., & Hahn, S. B. (2004). Importance of integrin beta1-mediated cell adhesion on biodegradable polymers under serum depletion in mesenchymal stem cells and chondrocytes. Biomaterials, 25(10), 1901–1909.
Tao, H., Rao, R., & Ma, D. D. (2005). Cytokine-induced stable neuronal differentiation of human bone marrow mesenchymal stem cells in a serum/feeder cell-free condition. Development, Growth & Differentiation, 47(6), 423–433.
Solmesky, L., Lefler, S., Jacob-Hirsch, J., Bulvik, S., Rechavi, G., & Weil, M. (2010). Serum free cultured bone marrow mesenchymal stem cells as a platform to characterize the effects of specific molecules. PLoS One, 5(9), e12689. pii.
Kume, T., Taguchi, R., Katsuki, H., et al. (2006). Serofendic acid, a neuroprotective substance derived from fetal calf serum, inhibits mitochondrial membrane depolarization and caspase-3 activation. European Journal of Pharmacology, 542(1–3), 69–76.
Le, B. K., Samuelsson, H., Gustafsson, B., et al. (2007). Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells. Leukemia, 21(8), 1733–1738.
Bernardo, M. E., Zaffaroni, N., Novara, F., et al. (2007). Human bone marrow derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms. Cancer Research, 67(19), 9142–9149.
Dahl, J. A., Duggal, S., Coulston, N., et al. (2008). Genetic and epigenetic instability of human bone marrow mesenchymal stem cells expanded in autologous serum or fetal bovine serum. International Journal of Developmental Biology, 52(8), 1033–1042.
Tarte, K., Gaillard, J., Lataillade, J. J., et al. (2010). Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation. Blood, 115(8), 1549–1553.
Zhang, Z. X., Guan, L. X., Zhang, K., et al. (2007). Cytogenetic analysis of human bone marrow-derived mesenchymal stem cells passaged in vitro. Cell Biology International, 31(6), 645–648.
Doerr, H. W., Cinatl, J., Sturmer, M., & Rabenau, H. F. (2003). Prions and orthopedic surgery. Infection, 31(3), 163–171.
Haack-Sorensen, M., Friis, T., Bindslev, L., Mortensen, S., Johnsen, H. E., & Kastrup, J. (2008). Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy. Scandinavian Journal of Clinical and Laboratory Investigation, 68(3), 192–203.
Aldahmash, A., Haack-Sorensen, M., Al-Nbaheen, M., Harkness, L., Abdallah, B. M., & Kassem, M. (2011). Human serum is as efficient as fetal bovine serum in supporting proliferation and differentiation of human multipotent stromal (mesenchymal) stem cells in vitro and in vivo. Stem Cell Reviews, 7(4), 860–868.
Horwitz, E. M., Gordon, P. L., Koo, W. K., et al. (2002). Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proceedings of the National Academy of Sciences of the United States of America, 99(13), 8932–8937.
Lazarus, H. M., Koc, O. N., Devine, S. M., et al. (2005). Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biology of Blood and Marrow Transplantation, 11(5), 389–398.
Le, B. K., Rasmusson, I., Sundberg, B., et al. (2004). Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet, 363(9419), 1439–1441.
Le Ricousse-Roussanne, S., Larghero, J., Zini, J. M., et al. (2007). Ex vivo generation of mature and functional human smooth muscle cells differentiated from skeletal myoblasts. Experimental Cell Research, 313(7), 1337–1346.
Abdallah, B. M., Haack-Sorensen, M., Burns, J. S., et al. (2005). Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite [corrected] extensive proliferation. Biochemical and Biophysical Research Communications, 326(3), 527–538.
Sekiya, I., Vuoristo, J. T., Larson, B. L., & Prockop, D. J. (2002). In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proceedings of the National Academy of Sciences of the United States of America, 99(7), 4397–4402.
Acknowledgements
We thank Irene Lynfort, Louise Degn Neilsen, Robert Burdorf and Hanne Holm for their technical support. The study was supported by research grants from the Aase and Ejnar Danielsen Foundation, the Research Foundation at Rigshospitalet, FP7 Health-2007-2.4.2.-5, grand no. 222995and the Danish Medical Research Council.
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The authors declare no competing financial interests.
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Haack-Sørensen, M., Hansen, S.K., Hansen, L. et al. Mesenchymal Stromal Cell Phenotype is not Influenced by Confluence during Culture Expansion. Stem Cell Rev and Rep 9, 44–58 (2013). https://doi.org/10.1007/s12015-012-9386-3
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DOI: https://doi.org/10.1007/s12015-012-9386-3