Genetically Engineered Mesenchymal Stem Cells Stably Expressing Green Fluorescent Protein

Document Type : Original Article

Authors

1 Research Center, Iranian Blood Transfusion Organization, Tehran Iran

2 Biotechnology Department, Tarbiyat Modares University, Tehran, Iran

3 Department of Anatomy, Faculty of Medicine, Medical University of Tabriz, Tabriz, Iran

4 Molecular Biology, Pasteur Institute of Iran, Tehran, Iran

Abstract

Objective(s)
Mesenchymal stem cells (MSCs) are nonhematopoietic stromal cells that are capable of differentiating into and contribute to the regeneration of mesenchymal tissues. Human mesenchymal stem cells (hMSCs) are ideal targets in cell transplantation and tissue engineering. Enhanced green fluorescent protein (EGFP) has been an important reporter gene for gene therapy. The aim of this study was establishment of MSCs expressing GFP.
Materials and Methods
MSCs were isolated and characterized by Immunophenotyping. The pEGFP-N1 plasmid was extracted from previously transformed Escherichia. coli cells and transfected into MSCs using FuGENE HD transfection reagent. Stable cells were established in the presence of geneticin. Expression of GFP was detected by RT- PCR, western blot analysis and immunoflorecent microscope.
Results
MSCs were successfully isolated and characterized. The MSCs transfected with the pEGFP-N1 plasmid expressed GFP both in mRNA and protein levels while cells transfected with empty vector did not.
Conclusion
The results suggested that this engineered cell line will be used in the future studies and can easily be traced in vivo.

Keywords

References

1.Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, et al. Bone marrow as a potential source of hepatic oval cells. Science 1999; 284:1168-1170.
2.Theise ND, Nimmakayalu M, Gardner R, Illei PB, Morgan G, Teperman L, et al. Liver from bone marrow in humans. Hepatology 2000; 32:11-16.
3.Alison MR, Poulsom R, Jeffery R, Dhillon AP, Quaglia A, Jacob J, et al. Hepatocytes from non-hepatic adult stem cells. Nature 2000; 406:257-261.
4.Li Y, Chen J, Wang L, Lu M, Chopp M. Treatment of stroke in rat with intracarotid administration of marrow stromal cells. Neurology 2001; 56:1666-1672.
5.Ferrari G , Cusella D, Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998; 279:1528-1530.
6.Korbling M, Katz RL, Khanna A, Ruifrok AC, Rondon G, Albitar M, et al. Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells. N Engl J Med 2002; 346:738-746.
7.Dalakas E, Newsome PN, Harrison DJ, Plevris JN. Hematopoietic stem cell trafficking in liver injury. FASEB J 2005; 19:1225-1231.
8.Shimomura O, Johnson FH, Saiga Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 1962; 59: 223-239.
9.Spees JL, Olson SD, Ylostalo J, Lynch PJ, Smith J, Perry A, et al. Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma. Proc Natl Acad Sci USA 2003; 100:2397-2402.
10.Tai G, Christodoulou I, Bishop AE, Polak JM. Use of green fluorescent fusion protein to track activation of the transcription factor osterix during early osteoblast differentiation. Biochem Biophys Res Commun 2005; 333:1116-1122.
11.Xia Z, Ye H, Locklin RM, Ferguson DJ, Cui Z, Triffitt JT. Efficient characterisation of human cell-bioceramic interactions in vitro and in vivo by using enhanced GFP-labelled mesenchymal stem cells. Biomaterials 2005; 26:5790-5800.
12.Hayakawa J, Migita M, Ueda T, Shimada T, Fukunaga Y. Generation of a chimeric mouse reconstituted with green fluorescent protein-positive bone marrow cells: a useful model for studying the behavior of bone marrow cells in regeneration in vivo. Int J Hematol 2003; 77:456-462.
13.Lee K, Majumdar MK, Buyaner D, Hendricks JK, Pittenger MF, Mosca JD. Human mesenchymal stem cells maintain transgene expression during expansion and differentiation. Molecular therapy 2001; 3: 857-866. Mol Biother 2001; 3:857¬866.
14.Shibata MA, Miwa Y, Morimoto J, Otsuki Y. Easy stable transfection of a human cancer cell line by electrogene transfer with an Epstein-Barr virus-based plasmid vector. Med Mol Morphol 2007; 40:103-107. 
15.Shizhong Z, Zhihao Z, Xiaodan J, Ruxiang X, Wangming Z, Yuan Zh, et al. The therapeutic effects of tyrosine hydroxylase genetransfected hematopoetic stem cells in a rat model of parkinson’s disease. Cell Mol Neurobiol 2008; 28:529-543.
16.Kopen GC, Preckop DJ, Phinney DG. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA 1999; 96: 10711-10716.
17.Chen J, Li Y, Chopp M. Intracerebral transplantation of bone marrow with BDNF after MCAo in rat. Neurepharmacology 2000; 39:711-718.
18.Canola K, Angenieux B, Tekaya M, Quiambao A, Naash MI, Munier F L, et al. Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate. Invest Ophthalmol Vis Sci 2007; 48: 446-454.
19.Boomsma RA, Swaminathan PD, Geenen DL. Intravenously injected mesenchymal stem cells home to viable myocardium after coronary occlusion and preserve systolic function without altering infarct size. In J Cardiol 2007; 122:17-28.
20.Brzoska E , Grabowska I, Hoser G, Streminska W, Wasilewska D, Machaj EK, et al. Participation of stem cells from human cord blood in skeletal muscle regeneration of SCID mice. Exp Hematol 2006; 34:1262-1270.
21.Chu K, Park KI, Lee ST, Jung KH, Ko SY, Kang L, et al. Combined treatment of vascular endothelial growth factor and human neural stem cells in experimental focal cerebral ischemia. Neurosci Res 2005; 53: 384-390.
22.Modo M, Cash D, Mellodew K, Williams SC, Fraser SE, Meade TJ, et al. Tracking transplanted stem cell migration using bifunctional, contrast agent-enhanced, magnetic resonance imaging. Neuroimage 2002; 17:803-811.
23.Amsalem Y, Mardor Y, Feinberg M S, Landa N, Miller L, Daniels D, et al. Iron-oxide labeling and outcome of transplanted mesenchymal stem cells in the infarcted myocardium. Circulation 2007; 116:138-145.
24.Tran N, Franken PR, Maskali F, Nloga J, Maureira P, Poussier S, et al. Intramyocardial Implantation of bone marrow-derived stem cells enhances perfusion in chronic myocardial infarction: Dependency on initial perfusion depth and follow-up assessed by gated pinhole SPECT. J Nucl Med 2007; 48:405-412.
25.Caveliers V, De Keulenaer G, Everaert H, Van Riet I, Van Camp G, Verheye S, et al. In vivo visualization of 111 in labeled CD 13 3+ peripheral blood stem cells after intracoronary administration in patients with chronic ischemic heart disease. Q J Nucl Med Mol Imaging 2007; 51:61-66.
26.Liu HS, Jan MS, Chou CK, Chen PH, Ke NJ. Is green fluorescent protein toxic to the living cells? Biochem Biophys Res Commun. 1999; 3:712-717.
27.Yoo TH, Link AJ,Tirrell DA. Evolution of a fluorinated green fluorescent protein. Proc Nat Acad Sci USA 2007; 104:13887-13890.
28.Beaudry P, Hida Y, Udagawa T, Alwayn IP, Greene AK, Arsenault D, et al. Evolution of a fluorinated green fluorescent protein. J Pediatr Surg 2007; 42:1190-1198.
29.Zhang S, Zou Z, Jiang X, Xu R, Zhang W, Zhou Y, et al. The therapeutic effects of tyrosine hydroxylase gene transfected hematopoetic stem cells in a rat model of Parkinson’s disease. Cell Mol Neurobiol 2008; 4: 529-43.
30.Dai W, Field L J, Rubart M, Reuter S, Hale SL, Zweigerdt R, et al. Survival and maturation of human embryonic stem cell-derived cardiomyocytes in rat hearts. J Mol Cell Cardiol 2007; 3:504-516.
31.Fujita J, Mori M, Kawada H, Ieda Y, Tsuma M, Matsuzaki Y, et al. Administration of granulocyte colony- stimulating factor after myocardial infarction enhances the recruitment of hematopoietic stem cell-derived myofibroblasts and contributes to cardiac repair. Stem Cells 2007; 11: 2750-2759.
32.Li Y, Atmaca-Sonmez P, Schanie CL, Ildstad ST, Kaplan H J, Enzmann V. Endogenous bone marrow derived cells express retinal pigment epithelium cell markers and migrate to focal areas of RPE damage. Invest Ophthalmol Vis Sci 2007; 48: 4321-4327.
33.Falanga V, Iwamoto S, Chartier M, Yufit T, Butmarc J, Kouttab N, et al. Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng 2007; 13:1299-1312.

Files

Share

How to cite

Statistics