Mesenchymal stem cells (MSCs) hold great promise for the treatment of numerous diseases. A major problem for MSC therapeutic use is represented by the very low amount of MSCs which can be isolated from different tissues; thus ex vivo expansion is indispensable. Long-term culture, however, is associated with extensive morphological and functional changes of MSCs. In addition, the concern that they may accumulate stochastic mutations which lead the risk of malignant transformation still remains. Overall, the genome of human MSCs (hMSCs) appears to be apparently stable throughout culture, though transient clonal aneuploidies have been detected. Particular attention should be given to the use of low-oxygen environment in order to increase the proliferative capacity of hMSCs, since data on the effect of hypoxic culture conditions on genomic stability are few and contradictory. Furthermore, specific and reproducible epigenetic changes were acquired by hMSCs during ex vivo expansion, which may be connected and trigger all the biological changes observed. In this review we address current issues on long-term culture of hMSCs with a 360-degree view, starting from the genomic profiles and back, looking for an epigenetic interpretation of their genetic stability. 1. Properties of Mesenchymal Stem Cells Mesenchymal stem cells (MSCs) are multipotent adult stem cells with a great therapeutic potential in tissue engineering, regenerative medicine, autoimmune diseases, and pathologies characterized by chronic inflammatory processes [1, 2]. MSCs from bone marrow (BM-MSCs) are the best characterized adult stem cells but MSC-like populations have been isolated from several tissues such as adipose tissue, umbilical cord blood, skin, skeletal muscle, and also from dental tissues as dental pulp, exfoliated deciduous teeth, and periodontal ligament [3, 4]. Compared with other stem cell types, such as embryonic stem cells (ESCs) and neural stem cells, MSCs have several advantages and no ethical concerns limit their use. MSCs can be easily isolated, have a capacity for extensive proliferation and self-renewal, present a low risk of tumorigenicity, and can be used autologously. Moreover MSCs are considered immunoprivileged because they express low level of MHC-I molecules but not MHC-II and costimulatory molecules CD80, CD86, and CD40 [5]. The therapeutic effect of MSCs is mainly based on some key properties: (1) MSCs are able to differentiate not only into mesodermal lineages (osteogenic, adipogenic, and chondrogenic lineages) but also towards endodermal or ectodermal
References
[1]
M. E. Bernardo, D. Pagliara, and F. Locatelli, “Mesenchymal stromal cell therapy: a revolution in Regenerative Medicine,” Bone Marrow Transplantation, vol. 47, no. 2, pp. 164–171, 2012.
[2]
Y. L. Si, Y. L. Zhao, H. J. Hao, X. B. Fu, and W. D. Han, “MSCs: biological characteristics, clinical applications and their outstanding concerns,” Ageing Research Reviews, vol. 10, no. 1, pp. 93–103, 2011.
[3]
M. E. Bernardo, A. M. Cometa, D. Pagliara et al., “Ex vivo expansion of mesenchymal stromal cells,” Best Practice and Research: Clinical Haematology, vol. 24, no. 1, pp. 73–81, 2011.
[4]
G. T. J. Huang, S. Gronthos, and S. Shi, “Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in Regenerative Medicine,” Journal of Dental Research, vol. 88, no. 9, pp. 792–806, 2009.
[5]
P. M. Chen, M. L. Yen, K. J. Liu, H. K. Sytwu, and B. L. Yen, “Immunomodulatory properties of human adult and fetal multipotent mesenchymal stem cells,” Journal of Biomedical Science, vol. 18, no. 1, article 49, 2011.
[6]
S. Wang, X. Qu, and R. C. Zhao, “Clinical applications of mesenchymal stem cells,” Journal of Hematology & Oncology, vol. 5, article 19, 2012.
[7]
M. Shi, Z. W. Liu, and F. S. Wang, “Immunomodulatory properties and therapeutic application of mesenchymal stem cells,” Clinical and Experimental Immunology, vol. 164, no. 1, pp. 1–8, 2011.
[8]
E. Fossett and W. S. Khan, “Optimising human mesenchymal stem cell numbers for clinical application: a literature review,” Stem Cells International, vol. 2012, Article ID 465259, 5 pages, 2012.
[9]
L. Zhang, L. P. Peng, N. Wu, and L. P. Li, “Development of bone marrow mesenchymal stem cell culture in vitro,” Chinese Medical Journal, vol. 125, no. 9, pp. 1650–1655, 2012.
[10]
S. Carrancio, N. López-Holgado, F. M. Sánchez-Guijo et al., “Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification,” Experimental Hematology, vol. 36, no. 8, pp. 1014–1021, 2008.
[11]
F. dos Santos, P. Z. Andrade, G. Eibes, C. L. da Silva, and J. M. S. Cabral, “Ex vivo expansion of human mesenchymal stem cells on microcarriers,” Methods in Molecular Biology, vol. 698, pp. 189–198, 2011.
[12]
C. A. Herberts, M. S. G. Kwa, and H. P. H. Hermsen, “Risk factors in the development of stem cell therapy,” Journal of Translational Medicine, vol. 9, article 29, 2011.
[13]
D. Hanahan and R. A. Weinberg, “The hallmarks of cancer,” Cell, vol. 100, no. 1, pp. 57–70, 2000.
[14]
J. Bartkova, Z. Ho？ej？í, K. Koed et al., “DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis,” Nature, vol. 434, no. 7035, pp. 864–870, 2005.
[15]
D. Rubio, J. Garcia-Castro, M. C. Martín et al., “Spontaneous human adult stem cell transformation,” Cancer Research, vol. 65, no. 8, pp. 3035–3039, 2005.
[16]
Y. Wang, D. I. Huso, J. Harrington et al., “Outgrowth of a transformed cell population derived from normal human BM mesenchymal stem cell culture,” Cytotherapy, vol. 7, no. 6, pp. 509–519, 2005.
[17]
G. V. R？sland, A. Svendsen, A. Torsvik et al., “Long-term cultures of bone marrow-derived human mesenchymal stem cells frequently undergo spontaneous malignant transformation,” Cancer Research, vol. 69, no. 13, pp. 5331–5339, 2009.
[18]
M. Miura, Y. Miura, H. M. Padilla-Nash et al., “Accumulated chromosomal instability in murine bone marrow mesenchymal stem cells leads to malignant transformation,” Stem Cells, vol. 24, no. 4, pp. 1095–1103, 2006.
[19]
S. Aguilar, E. Nye, J. Chan et al., “Murine but not human mesenchymal stem cells generate osteosarcoma-like lesions in the lung,” Stem Cells, vol. 25, no. 6, pp. 1586–1594, 2007.
[20]
M. E. Bernardo, N. Zaffaroni, F. Novara et al., “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, vol. 67, no. 19, pp. 9142–9149, 2007.
[21]
Z. X. Zhang, L. X. Guan, K. Zhang et al., “Cytogenetic analysis of human bone marrow-derived mesenchymal stem cells passaged in vitro,” Cell Biology International, vol. 31, no. 6, pp. 645–648, 2007.
[22]
S. Redaelli, A. Bentivegna, D. Foudah et al., “From cytogenomic to epigenomic profiles: monitoring the biologic behavior of in vitro cultured human bone marrow mesenchymal stem cells,” Stem Cell Research & Therapy, vol. 3, no. 6, article 47, 2012.
[23]
F. Z. Yi, M. Bosch-Marce, H. Okuyama et al., “Spontaneous transformation of cultured mouse bone marrow-derived stromal cells,” Cancer Research, vol. 66, no. 22, pp. 10849–10854, 2006.
[24]
D. Furlani, W. Li, E. Pittermann et al., “A transformed cell population derived from cultured mesenchymal stem cells has no functional effect after transplantation into the injured heart,” Cell Transplantation, vol. 18, no. 3, pp. 319–331, 2009.
[25]
D. Foudah, S. Redaelli, E. Donzelli et al., “Monitoring the genomic stability of in vitro cultured rat bone-marrow-derived mesenchymal stem cells,” Chromosome Research, vol. 17, no. 8, pp. 1025–1039, 2009.
[26]
S. Garcia, M. C. Martín, R. de la Fuente, J. C. Cigudosa, J. Garcia-Castro, and A. Bernad, “Pitfalls in spontaneous in vitro transformation of human mesenchymal stem cells,” Experimental Cell Research, vol. 316, no. 9, pp. 1648–1650, 2010.
[27]
A. Torsvik, G. V. R？sland, A. Svendsen et al., “Spontaneous malignant transformation of human mesenchymal stem cells reflects cross-contamination: putting the research field on track—letter,” Cancer Research, vol. 70, no. 15, pp. 6393–6396, 2010.
[28]
R. De la Fuente, A. Bernad, J. Garcia-Castro, M. C. Martin, and J. C. Cigudosa, “Retraction: spontaneous human adult stem cell transformation,” Cancer Research, vol. 70, no. 16, article 6682, 2010.
[29]
K. Tarte, J. Gaillard, J. J. Lataillade et al., “Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation,” Blood, vol. 115, no. 8, pp. 1549–1553, 2010.
[30]
R. Binato, T. de Souza Fernandez, C. Lazzarotto-Silva et al., “Stability of human mesenchymal stem cells during in vitro culture: considerations for cell therapy,” Cell Proliferation, vol. 46, no. 1, pp. 10–22, 2013.
[31]
N. Ohtani, D. J. Mann, and E. Hara, “Cellular senescence: its role in tumor suppression and aging,” Cancer Science, vol. 100, no. 5, pp. 792–797, 2009.
[32]
C. López-Otín, M. A. Blasco, L. Partridge, M. Serrano, and G. Kroemer, “The hallmarks of ageing,” Cell, vol. 153, no. 6, pp. 1194–1217, 2013.
[33]
S. M. Welford and A. J. Giaccia, “Hypoxia and senescence: the impact of oxygenation on tumor suppression,” Molecular Cancer Research, vol. 9, no. 5, pp. 538–544, 2011.
[34]
F. J. Rodríguez-Jiménez, V. Moreno-Manzano, R. Lucas-Dominguez, and J. M. Sánchez-Puelles, “Hypoxia causes downregulation of mismatch repair system and genomic instability in stem cells,” Stem Cells, vol. 26, no. 8, pp. 2052–2062, 2008.
[35]
W. L. Grayson, F. Zhao, B. Bunnell, and T. Ma, “Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells,” Biochemical and Biophysical Research Communications, vol. 358, no. 3, pp. 948–953, 2007.
[36]
Y. Jin, T. Kato, M. Furu et al., “Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase,” Biochemical and Biophysical Research Communications, vol. 391, no. 3, pp. 1471–1476, 2010.
[37]
C. C. Tsai, Y. J. Chen, T. L. Yew et al., “Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A-p21 by HIF-TWIST,” Blood, vol. 117, no. 2, pp. 459–469, 2011.
[38]
L. Basciano, C. Nemos, B. Foliguet et al., “Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent status,” BMC Cell Biology, vol. 12, article 12, 2011.
[39]
H. Ueyama, T. Horibe, S. Hinotsu et al., “Chromosomal variability of human mesenchymal stem cells cultured under hypoxic conditions,” Journal of Cellular and Molecular Medicine, vol. 16, no. 1, pp. 72–82, 2012.
[40]
S. McAvoy, S. C. Ganapathiraju, A. L. Ducharme-Smith et al., “Non-random inactivation of large common fragile site genes in different cancers,” Cytogenetic and Genome Research, vol. 118, no. 2–4, pp. 260–269, 2007.
[41]
M. F. Arlt, S. G. Durkin, R. L. Ragland, and T. W. Glover, “Common fragile sites as targets for chromosome rearrangements,” DNA Repair, vol. 5, no. 9-10, pp. 1126–1135, 2006.
[42]
K. R. McEwen, H. G. Leitch, R. Amouroux, and P. Hajkova :, “The impact of culture on epigenetic properties of pluripotent stem cells and pre-implantation embryos,” Biochemical Society Transactions, vol. 41, no. 3, pp. 711–719, 2013.
[43]
L. Hayflick, “The limited in vitro lifetime of human diploid cell strains,” Experimental Cell Research, vol. 37, no. 3, pp. 614–636, 1965.
[44]
K. Stenderup, J. Justesen, C. Clausen, and M. Kassem, “Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells,” Bone, vol. 33, no. 6, pp. 919–926, 2003.
[45]
K. Mareschi, I. Ferrero, D. Rustichelli et al., “Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow,” Journal of Cellular Biochemistry, vol. 97, no. 4, pp. 744–754, 2006.
[46]
W. Wagner, P. Horn, M. Castoldi et al., “Replicative senescence of mesenchymal stem cells: a continuous and organized process,” PLoS ONE, vol. 3, no. 5, article e2213, 2008.
[47]
W. Wagner, S. Bork, G. Lepperdinger et al., “How to track cellular aging of mesenchymal stromal cells?” Aging, vol. 2, no. 4, pp. 224–230, 2010.
[48]
W. Wagner, A. D. Ho, and M. Zenke, “Different facets of aging in human mesenchymal stem cells,” Tissue Engineering B, vol. 16, no. 4, pp. 445–453, 2010.
[49]
V. J. Cristofalo, R. G. Allen, R. J. Pignolo, B. G. Martin, and J. C. Beck, “Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 18, pp. 10614–10619, 1998.
[50]
G. P. Dimri, X. Lee, G. Basile et al., “A biomarker that identifies senescent human cells in culture and in aging skin in vivo,” Proceedings of the National Academy of Sciences of the United States of America, vol. 92, no. 20, pp. 9363–9367, 1995.
[51]
J. L. Osterhage and K. L. Friedman, “Chromosome end maintenance by telomerase,” Journal of Biological Chemistry, vol. 284, no. 24, pp. 16061–16065, 2009.
[52]
S. Di Donna, K. Mamchaoui, R. N. Cooper et al., “Telomerase can extend the proliferative capacity of human myoblasts, but does not lead to their immortalization,” Molecular Cancer Research, vol. 1, no. 9, pp. 643–653, 2003.
[53]
K. Masutomi, E. Y. Yu, S. Khurts et al., “Telomerase maintains telomere structure in normal human cells,” Cell, vol. 114, no. 2, pp. 241–253, 2003.
[54]
S. Zimmermann, S. Glaser, R. Ketteler, C. F. Waller, U. Klingmüller, and U. M. Martens, “Effects of telomerase modulation in human hematopoietic progenitor cells,” Stem Cells, vol. 22, no. 5, pp. 741–749, 2004.
[55]
P. J. Rochette and D. E. Brash, “Human telomeres are hypersensitive to UV-induced DNA damage and refractory to repair,” PLoS Genetics, vol. 6, no. 4, Article ID e1000926, 2010.
[56]
T. de Lange, “How telomeres solve the end-protection problem,” Science, vol. 326, no. 5955, pp. 948–952, 2009.
[57]
C. W. Greider and E. H. Blackburn, “The telomere terminal transferase of tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity,” Cell, vol. 51, no. 6, pp. 887–898, 1987.
[58]
E. H. Blackburn, “Switching and signaling at the telomere,” Cell, vol. 106, no. 6, pp. 661–673, 2001.
[59]
S. Zimmermann, M. Voss, S. Kaiser, U. Kapp, C. F. Waller, and U. M. Martens, “Lack of telomerase activity in human mesenchymal stem cells,” Leukemia, vol. 17, no. 6, pp. 1146–1149, 2003.
[60]
M. M. Bonab, K. Alimoghaddam, F. Talebian, S. H. Ghaffari, A. Ghavamzadeh, and B. Nikbin, “Aging of mesenchymal stem cell in vitro,” BMC Cell Biology, vol. 7, article 14, 2006.
[61]
R. Izadpanah, C. Trygg, B. Patel et al., “Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue,” Journal of Cellular Biochemistry, vol. 99, no. 5, pp. 1285–1297, 2006.
[62]
P. M. Lansdorp, “Telomeres, stem cells, and hematology,” Blood, vol. 111, no. 4, pp. 1759–1765, 2008.
[63]
J. W. Shay and S. Bacchetti, “A survey of telomerase activity in human cancer,” European Journal of Cancer Part A, vol. 33, no. 5, pp. 787–791, 1997.
[64]
C. B. Harley, “Telomerase is not an oncogene,” Oncogene, vol. 21, no. 4, pp. 494–502, 2002.
[65]
W. Wagner, “Implications of long-term culture for mesenchymal stem cells: genetic defects or epigenetic regulation?” Stem Cell Research & Therapy, vol. 3, no. 6, article 54, 2012.
[66]
M. A. Baxter, R. F. Wynn, S. N. Jowitt, J. E. Wraith, L. J. Fairbairn, and I. Bellantuono, “Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion,” Stem Cells, vol. 22, no. 5, pp. 675–682, 2004.
[67]
A. Banfi, A. Muraglia, B. Dozin, M. Mastrogiacomo, R. Cancedda, and R. Quarto, “Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy,” Experimental Hematology, vol. 28, no. 6, pp. 707–715, 2000.
[68]
A. Noer, A. C. Boquest, and P. Collas, “Dynamics of adipogenic promoter DNA methylation during clonal culture of human adipose stem cells to senescence,” BMC Cell Biology, vol. 8, article 18, 2007.
[69]
J. L. Simonsen, C. Rosada, N. Serakinci et al., “Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells,” Nature Biotechnology, vol. 20, no. 6, pp. 592–596, 2002.
[70]
D. S. Bischoff, N. S. Makhijani, and D. T. Yamaguchi, “Constitutive expression of human telomerase enhances the proliferation potential of human mesenchymal stem cells,” BioResearch Open Access, vol. 1, no. 6, pp. 273–279, 2012.
[71]
W. Reik, “Stability and flexibility of epigenetic gene regulation in mammalian development,” Nature, vol. 447, no. 7143, pp. 425–432, 2007.
[72]
M. Berdasco and M. Esteller, “Aberrant epigenetic landscape in cancer: how cellular identity goes awry,” Developmental Cell, vol. 19, no. 5, pp. 698–711, 2010.
[73]
M. Berdasco and M. Esteller, “DNA methylation in stem cell renewal and multipotency,” Stem Cell Research and Therapy, vol. 2, no. 5, article 42, 2011.
[74]
Y. H. Loh, Q. Wu, J. L. Chew et al., “The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells,” Nature Genetics, vol. 38, no. 4, pp. 431–440, 2006.
[75]
R. D. Hawkins, G. C. Hon, L. K. Lee et al., “Distinct epigenomic landscapes of pluripotent and lineage-committed human cells,” Cell Stem Cell, vol. 6, no. 5, pp. 479–491, 2010.
[76]
C. M. Teven, X. Liu, N. Hu et al., “Epigenetic regulation of mesenchymal stem cells: a focus on osteogenic and adipogenic differentiation,” Stem Cells International, vol. 2011, Article ID 201371, 18 pages, 2011.
[77]
S. Barrand and P. Collas, “Chromatin states of core pluripotency-associated genes in pluripotent, multipotent and differentiated cells,” Biochemical and Biophysical Research Communications, vol. 391, no. 1, pp. 762–767, 2010.
[78]
P. Collas, “Programming differentiation potential in mesenchymal stem cells,” Epigenetics, vol. 5, no. 6, pp. 476–482, 2010.
[79]
J. Tan, J. Lu, W. Huang et al., “Genome-wide analysis of histone H3 lysine9 modifications in human mesenchymal stem cell osteogenic differentiation,” PLoS ONE, vol. 4, no. 8, Article ID e6792, 2009.
[80]
Z. Li, C. Liu, Z. Xie et al., “Epigenetic dysregulation in mesenchymal stem cell aging and spontaneous differentiation,” PLoS ONE, vol. 6, no. 6, Article ID e20526, 2011.
[81]
Y. Wang, T. Chen, H. Yan et al., “Role of histone deacetylase inhibitors in the ageing of human umbilical cord mesenchymal stem cells,” Journal of Cellular Biochemistry, vol. 14, no. 10, pp. 2231–2239, 2013.
[82]
W. Wagner, S. Bork, P. Horn et al., “Aging and replicative senescence have related effects on human stem and progenitor cells,” PLoS ONE, vol. 4, no. 6, Article ID e5846, 2009.
[83]
S. Bork, S. Pfister, H. Witt et al., “DNA methylation pattern changes upon long-term culture and aging of human mesenchymal stromal cells,” Aging Cell, vol. 9, no. 1, pp. 54–63, 2010.
[84]
C. M. Koch, S. Joussen, A. Schellenberg, Q. Lin, M. Zenke, and W. Wagner, “Monitoring of cellular senescence by DNA-methylation at specific CpG sites,” Aging Cell, vol. 11, no. 2, pp. 366–369, 2012.
[85]
A. Schellenberg, Q. Lin, H. Schüler et al., “Replicative senescence of mesenchymal stem cells causes DNA-methylation changes which correlate with repressive histone marks,” Aging, vol. 3, no. 9, pp. 873–888, 2011.
[86]
W. Wagner, F. Wein, A. Seckinger et al., “Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood,” Experimental Hematology, vol. 33, no. 11, pp. 1402–1416, 2005.
[87]
D. No？l, D. Caton, S. Roche et al., “Cell specific differences between human adipose-derived and mesenchymal-stromal cells despite similar differentiation potentials,” Experimental Cell Research, vol. 314, no. 7, pp. 1575–1584, 2008.
[88]
A. P. Bracken, D. Kleine-Kohlbrecher, N. Dietrich et al., “The Polycomb group proteins bind throughout the INK4A-ARF locus and are disassociated in senescent cells,” Genes and Development, vol. 21, no. 5, pp. 525–530, 2007.
[89]
M. Smits, J. Nilsson, S. E. Mir et al., “miR-101 is down-regulated in glioblastoma resulting in EZH2-induced proliferation, migration, and angiogenesis,” Oncotarget, vol. 1, no. 8, pp. 710–720, 2010.
[90]
S. Burdach, S. Plehm, R. Unland et al., “Epigenetic maintenance of stemness and malignancy in peripheral neuroectodermal tumors by EZH2,” Cell Cycle, vol. 8, no. 13, pp. 1991–1996, 2009.
[91]
A. E. Teschendorff, U. Menon, A. Gentry-Maharaj et al., “Age-dependent DNA methylation of genes that are suppressed in stem cells is a hallmark of cancer,” Genome Research, vol. 20, no. 4, pp. 440–446, 2010.
[92]
M. R. Choi, Y. H. In, J. Park et al., “Genome-scale DNA methylation pattern profiling of human bone marrow mesenchymal stem cells in long-term culture,” Experimental & Molecular Medicine, vol. 44, no. 8, pp. 503–512, 2012.
[93]
S. Thomas and D. Bonchev, “A survey of current software for network analysis in molecular biology,” Human Genomics, vol. 4, no. 5, pp. 353–360, 2010.
[94]
R. Izadpanah, D. Kaushal, C. Kriedt et al., “Long-term in vitro expansion alters the biology of adult mesenchymal stem cells,” Cancer Research, vol. 68, no. 11, pp. 4229–4238, 2008.
