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PLOS ONE  2014 

The piggyBac Transposon-Mediated Expression of SV40 T Antigen Efficiently Immortalizes Mouse Embryonic Fibroblasts (MEFs)

DOI: 10.1371/journal.pone.0097316

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Abstract:

Mouse embryonic fibroblasts (MEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can undergo self-renewal and differentiate into to multiple lineages, including bone, cartilage and adipose. Primary MEFs have limited life span in culture, which thus hampers MEFs’ basic research and translational applications. To overcome this challenge, we investigate if piggyBac transposon-mediated expression of SV40 T antigen can effectively immortalize mouse MEFs and that the immortalized MEFs can maintain long-term cell proliferation without compromising their multipotency. Using the piggyBac vector MPH86 which expresses SV40 T antigen flanked with flippase (FLP) recognition target (FRT) sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized. The immortalized MEFs (piMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by FLP recombinase. The piMEFs express most MEF markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages upon BMP9 stimulation in vitro. Stem cell implantation studies indicate that piMEFs can form bone, cartilage and adipose tissues upon BMP9 stimulation, whereas FLP-mediated removal of SV40 T antigen diminishes the ability of piMEFs to differentiate into these lineages, possibly due to the reduced expansion of progenitor populations. Our results demonstrate that piggyBac transposon-mediated expression of SV40 T can effectively immortalize MEFs and that the reversibly immortalized piMEFs not only maintain long-term cell proliferation but also retain their multipotency. Thus, the high transposition efficiency and the potential footprint-free natures may render piggyBac transposition an effective and safe strategy to immortalize progenitor cells isolated from limited tissue supplies, which is essential for basic and translational studies.

References

[1]  Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276: 71–74.
[2]  Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284: 143–147.
[3]  Caplan AI, Bruder, S P (2001) Mesenchymal stem cells: building blocks for molecular medicine in the 21st century. Trends in Mol Med 7: 259–264.
[4]  Tuan RS, Boland G, Tuli R (2003) Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther 5: 32–45.
[5]  Rastegar F, Shenaq D, Huang J, Zhang W, Zhang BQ, et al. (2010) Mesenchymal stem cells: Molecular characteristics and clinical applications. World J Stem Cells 2: 67–80.
[6]  Shenaq DS, Rastegar F, Petkovic D, Zhang BQ, He BC, et al. (2010) Mesenchymal Progenitor Cells and Their Orthopedic Applications: Forging a Path towards Clinical Trials. Stem Cells Int 2010: 519028.
[7]  Hermann A, Gastl R, Liebau S, Popa MO, Fiedler J, et al. (2004) Efficient generation of neural stem cell-like cells from adult human bone marrow stromal cells. J Cell Sci 117: 4411–4422.
[8]  Keilhoff G, Goihl A, Langnase K, Fansa H, Wolf G (2006) Transdifferentiation of mesenchymal stem cells into Schwann cell-like myelinating cells. Eur J Cell Biol 85: 11–24.
[9]  Wislet-Gendebien S, Wautier F, Leprince P, Rogister B (2005) Astrocytic and neuronal fate of mesenchymal stem cells expressing nestin. Brain Res Bull 68: 95–102.
[10]  Makino S, Fukuda K, Miyoshi S, Konishi F, Kodama H, et al. (1999) Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 103: 697–705.
[11]  Noel D, Djouad F, Jorgense C (2002) Regenerative medicine through mesenchymal stem cells for bone and cartilage repair. Curr Opin Investig Drugs 3: 1000–1004.
[12]  Ren G, Zhang L, Zhao X, Xu G, Zhang Y, et al. (2008) Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell 2: 141–150.
[13]  Chan JL, Tang KC, Patel AP, Bonilla LM, Pierobon N, et al. (2006) Antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-gamma. Blood 107: 4817–4824.
[14]  Corcione A, Benvenuto F, Ferretti E, Giunti D, Cappiello V, et al. (2006) Human mesenchymal stem cells modulate B-cell functions. Blood 107: 367–372.
[15]  Djouad F, Charbonnier LM, Bouffi C, Louis-Plence P, Bony C, et al. (2007) Mesenchymal stem cells inhibit the differentiation of dendritic cells through an interleukin-6-dependent mechanism. Stem Cells 25: 2025–2032.
[16]  Aaronson SA, Todaro GJ (1968) Development of 3T3-like lines from Balb-c mouse embryo cultures: transformation susceptibility to SV40. J Cell Physiol 72: 141–148.
[17]  vom Brocke J, Schmeiser HH, Reinbold M, Hollstein M (2006) MEF immortalization to investigate the ins and outs of mutagenesis. Carcinogenesis 27: 2141–2147.
[18]  Borowiec JA, Dean FB, Bullock PA, Hurwitz J (1990) Binding and unwinding–how T antigen engages the SV40 origin of DNA replication. Cell 60: 181–184.
[19]  Prives C (1990) The replication functions of SV40 T antigen are regulated by phosphorylation. Cell 61: 735–738.
[20]  Zhu JY, Abate M, Rice PW, Cole CN (1991) The ability of simian virus 40 large T antigen to immortalize primary mouse embryo fibroblasts cosegregates with its ability to bind to p53. J Virol 65: 6872–6880.
[21]  Westerman KA, Leboulch P (1996) Reversible immortalization of mammalian cells mediated by retroviral transfer and site-specific recombination. Proc Natl Acad Sci U S A 93: 8971–8976.
[22]  Bi Y, Huang J, He Y, Zhu GH, Su Y, et al. (2009) Wnt antagonist SFRP3 inhibits the differentiation of mouse hepatic progenitor cells. J Cell Biochem 108: 295–303.
[23]  Huang E, Bi Y, Jiang W, Luo X, Yang K, et al. (2012) Conditionally Immortalized Mouse Embryonic Fibroblasts Retain Proliferative Activity without Compromising Multipotent Differentiation Potential. PLoS One 7: e32428.
[24]  Huang J, Bi Y, Zhu GH, He Y, Su Y, et al. (2009) Retinoic acid signalling induces the differentiation of mouse fetal liver-derived hepatic progenitor cells. Liver Int 29: 1569–1581.
[25]  Yang K, Chen J, Jiang W, Huang E, Cui J, et al. (2012) Conditional Immortalization Establishes a Repertoire of Mouse Melanocyte Progenitors with Distinct Melanogenic Differentiation Potential. J Invest Dermatol 132: 2479–2483.
[26]  Li M, Chen Y, Bi Y, Jiang W, Luo Q, et al. (2013) Establishment and characterization of the reversibly immortalized mouse fetal heart progenitors. Int J Med Sci 10: 1035–1046.
[27]  Wang X, Cui J, Zhang BQ, Zhang H, Bi Y, et al. (2014) Decellularized liver scaffolds effectively support the proliferation and differentiation of mouse fetal hepatic progenitors. J Biomed Mater Res A. 102: 1017–1025.
[28]  Kim A, Pyykko I (2011) Size matters: versatile use of PiggyBac transposons as a genetic manipulation tool. Mol Cell Biochem 354: 301–309.
[29]  Fraser MJ, Smith GE, Summers MD (1983) Acquisition of Host Cell DNA Sequences by Baculoviruses: Relationship Between Host DNA Insertions and FP Mutants of Autographa californica and Galleria mellonella Nuclear Polyhedrosis Viruses. J Virol 47: 287–300.
[30]  Wu SC, Meir YJ, Coates CJ, Handler AM, Pelczar P, et al. (2006) piggyBac is a flexible and highly active transposon as compared to sleeping beauty, Tol2, and Mos1 in mammalian cells. Proc Natl Acad Sci U S A 103: 15008–15013.
[31]  Wilson MH, Coates CJ, George AL Jr (2007) PiggyBac transposon-mediated gene transfer in human cells. Mol Ther 15: 139–145.
[32]  Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, et al. (2003) Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am 85-A: 1544–1552.
[33]  Kang Q, Sun MH, Cheng H, Peng Y, Montag AG, et al. (2004) Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery. Gene Ther 11: 1312–1320.
[34]  Luu HH, Song WX, Luo X, Manning D, Luo J, et al. (2007) Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells. J Orthop Res 25: 665–677.
[35]  Kang Q, Song WX, Luo Q, Tang N, Luo J, et al. (2009) A comprehensive analysis of the dual roles of BMPs in regulating adipogenic and osteogenic differentiation of mesenchymal progenitor cells. Stem Cells Dev 18: 545–559.
[36]  Luther G, Wagner ER, Zhu G, Kang Q, Luo Q, et al. (2011) BMP-9 Induced Osteogenic Differentiation of Mesenchymal Stem Cells: Molecular Mechanism and Therapeutic Potential. Curr Gene Ther 11: 229–240.
[37]  Luo X, Chen J, Song WX, Tang N, Luo J, et al. (2008) Osteogenic BMPs promote tumor growth of human osteosarcomas that harbor differentiation defects. Lab Invest 88: 1264–1277.
[38]  Haydon RC, Zhou L, Feng T, Breyer B, Cheng H, et al. (2002) Nuclear receptor agonists as potential differentiation therapy agents for human osteosarcoma. Clin Cancer Res 8: 1288–1294.
[39]  Peng Y, Kang Q, Cheng H, Li X, Sun MH, et al. (2003) Transcriptional characterization of bone morphogenetic proteins (BMPs)-mediated osteogenic signaling. J Cell Biochem 90: 1149–1165.
[40]  Tang N, Song WX, Luo J, Luo X, Chen J, et al. (2009) BMP9-induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/beta-catenin signaling. J Cell Mol Med 13: 2448–2464.
[41]  Sharff KA, Song WX, Luo X, Tang N, Luo J, et al. (2009) Hey1 Basic Helix-Loop-Helix Protein Plays an Important Role in Mediating BMP9-induced Osteogenic Differentiation of Mesenchymal Progenitor Cells. J Biol Chem 284: 649–659.
[42]  Zhang W, Deng ZL, Chen L, Zuo GW, Luo Q, et al. (2010) Retinoic acids potentiate BMP9-induced osteogenic differentiation of mesenchymal progenitor cells. PLoS One 5: e11917.
[43]  He BC, Chen L, Zuo GW, Zhang W, Bi Y, et al. (2010) Synergistic antitumor effect of the activated PPARgamma and retinoid receptors on human osteosarcoma. Clin Cancer Res 16: 2235–2245.
[44]  He BC, Gao JL, Zhang BQ, Luo Q, Shi Q, et al. (2011) Tetrandrine inhibits Wnt/beta-catenin signaling and suppresses tumor growth of human colorectal cancer. Mol Pharmacol 79: 211–219.
[45]  He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, et al. (1998) A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 95: 2509–2514.
[46]  Luo J, Deng ZL, Luo X, Tang N, Song WX, et al. (2007) A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nat Protoc 2: 1236–1247.
[47]  He TC, Sparks AB, Rago C, Hermeking H, Zawel L, et al. (1998) Identification of c-MYC as a target of the APC pathway [see comments]. Science 281: 1509–1512.
[48]  Hu N, Jiang D, Huang E, Liu X, Li R, et al. (2013) BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells. J Cell Sci 126: 532–541.
[49]  Luther GA, Lamplot J, Chen X, Rames R, Wagner ER, et al. (2013) IGFBP5 Domains Exert Distinct Inhibitory Effects on the Tumorigenicity and Metastasis of Human Osteosarcoma. Cancer Lett 336: 222–230.
[50]  Rastegar F, Gao JL, Shenaq D, Luo Q, Shi Q, et al. (2010) Lysophosphatidic acid acyltransferase beta (LPAATbeta) promotes the tumor growth of human osteosarcoma. PLoS One 5: e14182.
[51]  Si W, Kang Q, Luu HH, Park JK, Luo Q, et al. (2006) CCN1/Cyr61 Is Regulated by the Canonical Wnt Signal and Plays an Important Role in Wnt3A-Induced Osteoblast Differentiation of Mesenchymal Stem Cells. Mol Cell Biol 26: 2955–2964.
[52]  Zhu GH, Huang J, Bi Y, Su Y, Tang Y, et al.. (2009) Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells. Differentiation 78 195–204.
[53]  Huang E, Zhu G, Jiang W, Yang K, Gao Y, et al. (2012) Growth hormone synergizes with BMP9 in osteogenic differentiation by activating the JAK/STAT/IGF1 pathway in murine multilineage cells. J Bone Miner Res 27: 1566–1575.
[54]  Luo Q, Kang Q, Si W, Jiang W, Park JK, et al. (2004) Connective Tissue Growth Factor (CTGF) Is Regulated by Wnt and Bone Morphogenetic Proteins Signaling in Osteoblast Differentiation of Mesenchymal Stem Cells. J Biol Chem 279: 55958–55968.
[55]  Peng Y, Kang Q, Luo Q, Jiang W, Si W, et al. (2004) Inhibitor of DNA binding/differentiation helix-loop-helix proteins mediate bone morphogenetic protein-induced osteoblast differentiation of mesenchymal stem cells. J Biol Chem 279: 32941–32949.
[56]  Chen L, Jiang W, Huang J, He BC, Zuo GW, et al. (2010) Insulin-like growth factor 2 (IGF-2) potentiates BMP-9-induced osteogenic differentiation and bone formation. J Bone Miner Res 25: 2447–2459.
[57]  Luo J, Tang M, Huang J, He BC, Gao JL, et al. (2010) TGFbeta/BMP type I receptors ALK1 and ALK2 are essential for BMP9-induced osteogenic signaling in mesenchymal stem cells. J Biol Chem 285: 29588–29598.
[58]  Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, et al. (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8: 315–317.
[59]  Lamplot JD, Qin J, Nan G, Wang J, Liu X, et al. (2013) BMP9 signaling in stem cell differentiation and osteogenesis. Am J Stem Cells 2: 1–21.
[60]  Phinney DG, Prockop DJ (2007) Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair–current views. Stem Cells 25: 2896–2902.
[61]  Phinney DG, Sensebe L (2013) Mesenchymal stromal cells: misconceptions and evolving concepts. Cytotherapy 15: 140–145.
[62]  Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, et al. (2001) Multilineage cells from human adipose tissue: implications for cell- based therapies. Tissue Eng 7: 211–228.
[63]  Sinanan AC, Hunt NP, Lewis MP (2004) Human adult craniofacial muscle-derived cells: neural-cell adhesion-molecule (NCAM; CD56)-expressing cells appear to contain multipotential stem cells. Biotechnol Appl Biochem 40: 25–34.
[64]  Ringe J, Leinhase I, Stich S, Loch A, Neumann K, et al. (2008) Human mastoid periosteum-derived stem cells: promising candidates for skeletal tissue engineering. J Tissue Eng Regen Med 2: 136–146.
[65]  Liu Z, Martin LJ (2004) Pluripotent fates and tissue regenerative potential of adult olfactory bulb neural stem and progenitor cells. J Neurotrauma 21: 1479–1499.
[66]  In 't Anker PS, Scherjon SA, Kleijburg-van der Keur C, de Groot-Swings GM, Claas FH, et al. (2004) Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 22: 1338–1345.
[67]  Davis AA, Temple S (1994) A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature 372: 263–266.
[68]  da Silva Meirelles L, Chagastelles PC, Nardi NB (2006) Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 119: 2204–2213.
[69]  Coles BL, Angenieux B, Inoue T, Del Rio-Tsonis K, Spence JR, et al. (2004) Facile isolation and the characterization of human retinal stem cells. Proc Natl Acad Sci U S A 101: 15772–15777.
[70]  Bjerknes M, Cheng H (2006) Intestinal epithelial stem cells and progenitors. Methods Enzymol 419: 337–383.
[71]  Amoh Y, Li L, Campillo R, Kawahara K, Katsuoka K, et al. (2005) Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves. Proc Natl Acad Sci U S A 102: 17734–17738.
[72]  Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U, et al. (2005) Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol 33: 1402–1416.
[73]  Di Matteo M, Matrai J, Belay E, Firdissa T, Vandendriessche T, et al. (2012) PiggyBac toolbox. Methods Mol Biol 859: 241–254.
[74]  Li X, Burnight ER, Cooney AL, Malani N, Brady T, et al. (2013) piggyBac transposase tools for genome engineering. Proc Natl Acad Sci U S A 110: E2279–2287.
[75]  Yusa K, Zhou L, Li MA, Bradley A, Craig NL (2011) A hyperactive piggyBac transposase for mammalian applications. Proc Natl Acad Sci U S A 108: 1531–1536.

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