Immortalization of human adipose-derived stromal cells: production of cell lines with high growth rate, mesenchymal marker expression and capability to secrete high levels of angiogenic factors
Tóm tắt
Human adipose-derived stromal cells (hASCs), due to their relative feasibility of isolation and ability to secrete large amounts of angiogenic factors, are being evaluated for regenerative medicine. However, their limited culture life span may represent an obstacle for both preclinical investigation and therapeutic use. To overcome this problem, hASCs immortalization was performed in order to obtain cells with in vitro prolonged life span but still maintain their mesenchymal marker expression and ability to secrete angiogenic factors. hASCs were transduced with the human telomerase reverse transcriptase (hTERT) gene alone or in combination with either SV-40 or HPV E6/E7 genes. Mesenchymal marker expression on immortalized hASCs lines was confirmed by flow cytometry (FC), differentiation potential was evaluated by immunocytochemistry and ELISA kits were used for evaluation of angiogenic factors. Green fluorescent protein (GFP) gene transduction was used to obtain fluorescent cells. We found that hTERT alone failed to immortalize hASCs (hASCs-T), while hTERT/SV40 (hASCs-TS) or hTERT/HPV E6/E7 (hASCs-TE) co-transductions successfully immortalized cells. Both hASCs-TS and hASCs-TE were cultured for up to one year with a population doubling level (PDL) up to 100. Comparative studies between parental not transduced (hASCs-M) and immortalized cell lines showed that both hASCs-TS and hASCs-TE maintained a mesenchymal phenotypic profile, whereas differentiation properties were reduced particularly in hASCs-TS. Interestingly, hASCs-TS and hASCs-TE showed a capability to secrete significant amount of HGF and VEGF. Furthermore, hASCs-TS and hASCs-TE did not show tumorigenic properties in vitr o although some chromosomal aberrations were detected. Finally, hASCs-TS and hASCs-TE lines were stably fluorescent upon transduction with the GFP gene. Here we demonstrated, for the first time, that hASCs, upon immortalization, maintain a strong capacity to secrete potent angiogenic molecules. By combining hASCs immortalization and their paracrine characteristics, we have developed a “hybridoma-like model” of hASCs that could have potential applications for discovering and producing molecules to use in regenerative medicine (process scale-up). In addition, due to the versatility of these fluorescent-immortalized cells, they could be employed in in vivo cell-tracking experiments, expanding their potential use in laboratory practice.
Tài liệu tham khảo
Mizuno H, Tobita M, Uysal AC: Concise review: adipose-derived stem cells as a novel tool for future regenerative medicine. Stem Cells. 2012, 30: 804-810. 10.1002/stem.1076.
Gimble JM, Katz AJ, Bunnell BA: Adipose-derived stem cells for regenerative medicine. Circ Res. 2007, 100: 1249-1260. 10.1161/01.RES.0000265074.83288.09.
Kern S, Eichler H, Stoeve J, Kluter H, Bieback K: Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 2006, 24: 1294-1301. 10.1634/stemcells.2005-0342.
Siddappa R, Licht R, van Blitterswijk C, de Boer J: Donor variation and loss of multipotency during in vitro expansion of human mesenchymal stem cells for bone tissue engineering. J Orthop Res. 2007, 25: 1029-1041. 10.1002/jor.20402.
Schipper BM, Marra KG, Zhang W, Donnenberg AD, Rubin JP: Regional anatomic and and age effects on cell function of human adipose-derived stem cells. Ann Plast Surg. 2008, 60: 538-544. 10.1097/SAP.0b013e3181723bbe.
Binato R, de Souza FT, Lazzarotto-Silva C, Du Rocher B, Mencalha A, Pizzatti L, Bouzas LF, Abdelhay E: Stability of human mesenchymal stem cells during in vitro culture: considerations for cell therapy. Cell Prolif. 2013, 46: 10-22. 10.1111/cpr.12002.
Serrano M, Blasco MA: Putting the stress on senescence. Curr Opin Cell Biol. 2001, 13: 748-753. 10.1016/S0955-0674(00)00278-7.
Campisi J: Cellular senescence as a tumor-suppressor mechanism. Trends Cell Biol. 2001, 11: S27-S31. 10.1016/S0962-8924(01)02151-1.
Gudjonsson T, Villadsen R, Rǿnnov-Jessen L, Petersen OW: Immortalization protocols used in cell culture models oh human breast morphogenesis. Cell Mol Life Sci. 2004, 61: 2523-2534. 10.1007/s00018-004-4167-z.
Linder S, Marshall H: Immortalization of primary cells by DNA tumor viruses. Exp Cell Res. 1990, 191: 1-7. 10.1016/0014-4827(90)90027-8.
Jha KK, Banga S, Palejwala V, Ozer HL: SV40-mediated immortalization. Exp Cell Res. 1998, 245: 1-7. 10.1006/excr.1998.4272.
Hung SC, Yang DM, Chang CF, Lin RJ, Wang JS, Low-Tone Ho L, Yang WK: Immortalization without neoplastic transformation of human mesenchymal stem cells by transduction with HPV16 E6/E7 genes. Int J Cancer. 2004, 110: 313-319. 10.1002/ijc.20126.
Hahn WC, Meyerson M: Telomerase activation, cellular immortalization and cancer. Ann Med. 2001, 33: 123-129. 10.3109/07853890109002067.
Salmon P, Oberholzer J, Occhiodoro T, Morel P, Lou J, Trono D: Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes. Mol Ther. 2000, 2: 404-414. 10.1006/mthe.2000.0141.
Darimont C, Zbinden I, Avanti O, Leone-Vautravers P, Giusti V, Burckhardt P, Pfeifer AMA, Macé K: Reconstitution of telomerase activity combined with HPV-E7 expression allow human preadipocytes to preserve their differentiation capacity after immortalization. Cell Death Differ. 2003, 10: 1025-1031. 10.1038/sj.cdd.4401273.
Zuk PA, Zhu M, Ashjan P, De Ugarte DA, Haung JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hendrick MH: Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002, 13: 4979-4995.
Böcker W, Yin Z, Drosse I, Haasters F, Rossmann O, Wierer M, Popov C, Locher M, Mutschier W, Docheva D, Schieker M: Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer. J Cell Mol Med. 2008, 12: 1347-1359. 10.1111/j.1582-4934.2008.00299.x.
Zuk PA: Viral transduction of adipose-derived stem cells. Methods Mol Biol. 2011, 702: 345-357. 10.1007/978-1-61737-960-4_25.
Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L: A third-generation lentivirus vector with a conditional packaging system. J Virol. 1998, 72: 8463-8471.
Itahana K, Campisi J, Dimri GP: Methods to detect biomarkers of cellular senescence: the senescence-associated beta-galactosidase activity. Methods Mol Biol. 2007, 371: 21-31. 10.1007/978-1-59745-361-5_3.
Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, Deans RJ, Keating A, Prockop DJ, Horwitz EM: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006, 8: 315-317. 10.1080/14653240600855905.
De Ugarte DA, Alfonso Z, Zuk PA, Elbarbary A, Zhu M, Ashjian P, Benhaim P, Hedrick MH, Fraser JK: Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow. Immunol Lett. 2003, 89: 267-270. 10.1016/S0165-2478(03)00108-1.
Sorrentino A, Ferracin M, Castelli G, Biffoni M, Tomaselli G, Baiocchi M, Fatica A, Negrini M, Peschle C, Valtieri M: Isolation and characterization of CD146+ multipotent mesenchymal stromal cells. Exp Hematol. 2008, 36: 1035-1046. 10.1016/j.exphem.2008.03.004.
Gang EJ, Bosnakovski D, Figueiredo CA, Visser JW, Perlingeiro RCR: SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood. 2007, 109: 1743-1751. 10.1182/blood-2005-11-010504.
Rehman J, Traktuev D, Li J, Merfeld-Clauss S, Temm-Grove CJ, Bovenkerk JE, Pell CL, Johnstone BH, Considine RV, March KL: Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation. 2004, 109: 1292-1298. 10.1161/01.CIR.0000121425.42966.F1.
Li B, Zeng Q, Ang H, Mao X: Effect of cytokines secreted by human adipose stromal cells on endothelial cells. J Huazhong Univ Sci Technol Med Sci. 2006, 26: 396-398. 10.1007/s11596-006-0403-0.
Kilroy GE, Foster SJ, Wu X, Ruiz J, Sherwood S, Heifetz A, Ludlow JW, Stricker DM, Potiny S, Green P, Halvorsen YD, Cheatham B, Storms RW, Gimble JM: Cytokine profile of human adipose-derived stem cells: expression of angiogenic, hematopoietic, and pro-inflammatory factors. J Cell Physiol. 2007, 212: 702-709. 10.1002/jcp.21068.
Bussolino F, Di Renzo MF, Ziche M, Bocchietto E, Olivero M, Naldini L, Gaudino G, Tamagnone L, Coffer A, Comoglio PM: Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol. 1992, 119: 629-641. 10.1083/jcb.119.3.629.
Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N: Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989, 246: 1306-1309. 10.1126/science.2479986.
Polzer H, Volkmer E, Saller MM, Prall WC, Haasters F, Drosse I, Anz D, Mutschler W, Schieker M: Long-term detection of fluorescently labeled human mesenchymal stem cell in vitro and in vivo by semi-automated microscopy. Tissue Eng Part C Methods. 2012, 18: 156-165. 10.1089/ten.tec.2011.0275.
Sen A, Lea-Currie YR, Sujkowska D, Franklin DM, Wilkison WO, Halvorsen YD, Gimble JM: Adipogenic potential of human adipose derived stromal cells from multiple donors is heterogeneous. J Cell Biochem. 2001, 81: 312-319. 10.1002/1097-4644(20010501)81:2<312::AID-JCB1046>3.0.CO;2-Q.
Griesche N, Luttmann A, Stammermann T, Geiger H, Baer PC: A simple modification of the separation method reduces heterogeneity of adipose-derived stem cells. Cells Tissues Organs. 2010, 192: 106-115. 10.1159/000289586.
Gruber HE, Somayaji S, Riley F, Hoelscher GL, Norton HJ, Ingram J, Hanley EN: Human adipose-derived mesenchymal stem cells: serial passaging, doubling time and cell senescence. Biotech Histochem. 2012, 87: 303-311. 10.3109/10520295.2011.649785.
Bourin P, Bunnell BA, Casteilla L, Dominici M, Katz AJ, March KL, Redl H, Rubin JP, Yoshimura K, Gimble JF: Stromal cells from the adipose tissue-derived stromal cells vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy. 2013, 15: 641-648. 10.1016/j.jcyt.2013.02.006.
Voglauer R, Grillari J, Fortschegger K, Wieser M, Sterovsky T, Gunsberg P, Katinger H, Pfragner R: Establishment of human fibroma cell lines from MEN1 patient by introduction of either hTERT or SV40 early region. Int J Oncol. 2005, 26: 961-970.
Tsuruga Y, Kiyono T, Matsushita M, Takahashi T, Kasai H, Matsumoto S, Todo S: Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy. Cell Transplant. 2008, 17: 1083-1094. 10.3727/096368908786991542.
Salgado AJ, Reis RL, Sousa NJ, Gimble JM: Adipose tissue derived stem cells secretome: soluble factors and their roles in regenerative medicine. Curr Stem Cell Res Ther. 2010, 5: 103-110. 10.2174/157488810791268564.
Jun ES, Lee TH, Cho HH, Suh SY, Jung JS: Expression of telomerase extends longevity and enhances differentiation in human adipose tissue-derived stromal cells. Cell Physiol Biochem. 2004, 14: 261-268. 10.1159/000080335.
Kang SK, Putnam L, Dufour J, Ylostalo J, Jung JS, Bunnell BA: Expression of telomerase extends the lifespan and enhances osteogenic differentiation of adipose tissue-derived stromal cells. Stem Cells. 2004, 22: 1356-1372. 10.1634/stemcells.2004-0023.
Wolbank S, Stadler G, Peterbauer A, Gillich A, Karbiener M, Streubel B, Wieser M, Katinger H, van Griensven M, Redl H, Gabriel C, Grillari J, Grillari-Voglauer R: Telomerase immortalized human amnion- and adipose-derived mesenchymal stem cells: maintenance of differentiation and immunomodulatory characteristics. Tissue Eng Part A. 2009, 15: 1843-1854. 10.1089/ten.tea.2008.0205.
Chapman S, Liu X, Meyers C, Schlegel R, McBride AA: Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor. J Clin Invest. 2010, 120: 2619-2626. 10.1172/JCI42297.
Duncan EL, Wadhwa R, Kaul SC: Senescence and immortalization of human cells. Biogerontology. 2000, 1: 103-121. 10.1023/A:1010000132671.
Tátrai P, Szepesi Á, Matula Z, Szigeti A, Buchan G, Mádi A, Uher F, Német K: Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation. Biochem Biophys Res Commun. 2012, 422: 28-35. 10.1016/j.bbrc.2012.04.088.
Gnecchi M, Zhang Z, Ni A, Dzau VJ: Paracrine mechanisms in adult stem cells signaling and therapy. Circ Res. 2008, 103: 1204-1219. 10.1161/CIRCRESAHA.108.176826.
De Siena R, Balducci L, Blasi A, Montanaro MG, Saldarelli M, Saponaro V, Martino C, Logrieco G, Soleti A, Fiobellot S, Madeddu P, Rossi G, Ribatti D, Crovace A, Cristini S, Invernici G, Parati EA, Alessandri G: Omentum-derived stromal cells improve myocardial regeneration in pig post-infarcted heart through a potent paracrine mechanism. Exp Cell Res. 2010, 316: 1804-1815. 10.1016/j.yexcr.2010.02.009.
Madonna R, Taylor DA, Geng YJ, De Caterina R, Shelat H, Perin EC, Willerson JT: Transplantation of mesenchymal cells rejuvenated by the overexpression of telomerase and myocardin promotes revascularization and tissue repair in a murine model of hindlimb ischemia. Circ Res. 2013, 113: 902-914. 10.1161/CIRCRESAHA.113.301690.
Watson SL, Marcal H, Sarris M, Di Girolamo N, Coroneo MT, Wakefield D: The effect of mesenchymal stem cell conditioned media on corneal stromal fibroblast wound healing activities. Br J Ophthalmol. 2010, 94: 1067-1073. 10.1136/bjo.2009.165837.
Cho YJ, Song HS, Bhang S, Lee S, Kang BG, Lee JC, An J, Cha CI, Nam DH, Kim BS, Joo KM: Therapeutic effects of human adipose stem cell-conditioned medium on stroke. J Neurosci Res. 2012, 90: 1794-1802. 10.1002/jnr.23063.
Song SH, Lee MO, Lee JS, Jeong HC, Kim HG, Kim WS, Hur M, Cha HJ: Genetic modification of human adipose-derived stem cells for promoting wound healing. J Dermatol Sci. 2012, 66: 98-107. 10.1016/j.jdermsci.2012.02.010.
Chen TS, Arslan F, Yin Y, Tan SS, Lai RC, Choo AB, Padmanabhan J, Lee CN, de Klein DP, Lim S: Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs. J Transl Med. 2011, 9: 47-10.1186/1479-5876-9-47.
Mátrai J, Chuah MK, VandenDriessche T: Recent advances in lentiviral vector development and applications. Mol Ther. 2010, 18: 1055-
Picanco-Castro V, de Sousa Russo-Carbolante EM, Tadeu CD: Advances in lentiviral vectors: a patent review. Recent Pat DNA Gene Seq. 2012, 6: 82-90. 10.2174/187221512801327433.
Vilalta M, Dégano IR, Bagó J, Gould D, Santos M, García-Arranz M, Ayats R, Fuster C, Chernajovsky Y, García-Olmo D, Rubio N, Blanco J: Biodistribution, long-term survival, and safety of human adipose tissue-derived mesenchymal stem cells transplanted in nude mice by high sensitivity non-invasive bioluminescence imaging. Stem Cells Dev. 2008, 17: 993-1003. 10.1089/scd.2007.0201.
Progatzky F, Dallman MJ, Lo Celso C: From seeing to believing: labelling strategies for in vivo cell-tracking experiments. Interface Focus. 2013, 3: 20130001-10.1098/rsfs.2013.0001.