Options for tracking GFP-Labeled transplanted myoblasts using in vivo fluorescence imaging: implications for tracking stem cell fate

BMC Biotechnology

Zhong Yang³  Yaming Wang²  Yanan Li³  Qiang Liu²  Qing Zeng¹  Xiaoyin Xu¹ 

[1] Department of Radiology, Functional and Molecular Imaging Center, Brigham & Women’s Hospital, 75 Francis Street SR 153, Boston, MA 02115, USA

[2] Department of Anesthesia, Brigham & Women’s Hospital, Boston, MA 02115, USA

[3] Department of Clinical Hematology, College of Laboratory Medicine, Southwest Hospital, Third Military Medical University, No. 29, GaoTanYan Street, ChongQing 400038, PR China

关键词: Stem Cell;    Fluorescence Imaging;    GFP-Labeled Transplanted Myoblasts;   

Others  :  1085027
DOI  :  10.1186/1472-6750-14-55

 received in 2014-01-25, accepted in 2014-05-29,  发布年份 2014

【 摘 要 】

Background

Green fluorescent protein (GFP) is a useful biomarker, widely used in biomedical research to track stem cells after transplantation and/or to assess therapeutic transgene expression. However, both GFP and therapeutic gene products themselves may be immunogenic to the recipient. The main aim of this study was to use animal models to evaluate potential impact of GFP on the cell engraftment and to optimize tracking strategies prior to transplantation.

Results

By using a fluorescent imaging (FLI) system, we investigated the dynamic cell behavior of GFP-transduced myoblasts in tibialis anterior (TA) muscles of immunocompetent mdx mice and immuno-compromised nude mice over a period of three months. The results suggested an apparent underlying host immunorejection in the mdx mice. Dystrophin immunostaining showed that the engraftment of wild type myoblasts was much more effective than that of the GFP-labeled counterparts in the mdx mice, further confirming an antigen role of GFP in this process. We tracked the GFP-transduced myoblasts in C57BL/6 mice and found GFP to be minimally immunogenic in these animals, as indicated by the GFP signal maintaining a much stronger level than that found in mdx and BALB/c mice at parallel time points. We also compared the in vivo cell behavior differences between myoblasts from virally GFP-transduced and GFP transgenic mice. The latter displayed much better engraftment, as determined both biomaging and histological observations.

Conclusions

Our results not only demonstrated the immunogenicity of GFP in immunocompetent mice, but determined the optimized conditions for GFP-based in vivo stem cells tracking, that can potentially be extrapolated to human biomedical research.

【 授权许可】

   
2014 Yang et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Banerjee C: Stem cells therapies in basic science and translational medicine: current status and treatment monitoring strategies. Curr Pharm Biotechnol 2011, 12(4):469-487.
• [2]Janssens S: Stem cells in the treatment of heart disease. Annu Rev Med 2010, 61:287-300.
• [3]Skuk D, Tremblay JP: Intramuscular cell transplantation as a potential treatment of myopathies: clinical and preclinical relevant data. Expert Opin Biol Ther 2011, 11(3):359-374.
• [4]Bhakoo K: In vivo stem cell tracking in neurodegenerative therapies. Expert Opin Biol Ther 2011, 11(7):911-920.
• [5]de Almeida PE, van Rappard JR, Wu JC: In vivo bioluminescence for tracking cell fate and function. Am J Physiol Heart Circ Physiol 2011, 301(3):H663-H671.
• [6]Sheikh AY, Lin SA, Cao F, Cao Y, van der Bogt KE, Chu P, Chang CP, Contag CH, Robbins RC, Wu JC: Molecular imaging of bone marrow mononuclear cell homing and engraftment in ischemic myocardium. Stem Cells 2007, 25(10):2677-2684.
• [7]Waerzeggers Y, Monfared P, Viel T, Winkeler A, Voges J, Jacobs AH: Methods to monitor gene therapy with molecular imaging. Methods 2009, 48(2):146-160.
• [8]Brazelton TR, Blau HM: Optimizing techniques for tracking transplanted stem cells in vivo. Stem cells 2005, 23(9):1251-1265.
• [9]Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA: Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev 2010, 90(3):1103-1163.
• [10]Ma D, Zhang J, Lin HF, Italiano J, Handin RI: The identification and characterization of zebrafish hematopoietic stem cells. Blood 2011, 118(2):289-297.
• [11]Sun N, Lee A, Wu JC: Long term non-invasive imaging of embryonic stem cells using reporter genes. Nat Protocol 2009, 4(8):1192-1201.
• [12]Beagles KE, Peterson L, Zhang X, Morris J, Kiem HP: Cyclosporine inhibits the development of green fluorescent protein (GFP)-specific immune responses after transplantation of GFP-expressing hematopoietic repopulating cells in dogs. Hum Gene Ther 2005, 16(6):725-733.
• [13]Eixarch H, Gomez A, Kadar E, George M, Martinez N, Espejo C, Petriz J, Gimeno R, Barquinero J: Transgene expression levels determine the immunogenicity of transduced hematopoietic grafts in partially myeloablated mice. Mol Ther 2009, 17(11):1904-1909.
• [14]Kung SK, An DS, Bonifacino A, Metzger ME, Ringpis GE, Mao SH, Chen IS, Donahue RE: Induction of transgene-specific immunological tolerance in myeloablated nonhuman primates using lentivirally transduced CD34+ progenitor cells. Mol Ther 2003, 8(6):981-991.
• [15]Stripecke R, Carmen Villacres M, Skelton D, Satake N, Halene S, Kohn D: Immune response to green fluorescent protein: implications for gene therapy. Gene Ther 1999, 6(7):1305-1312.
• [16]Gu E, Chen WY, Gu J, Burridge P, Wu JC: Molecular imaging of stem cells: tracking survival, biodistribution, tumorigenicity, and immunogenicity. Theranostics 2012, 2(4):335-345.
• [17]Rodriguez-Porcel M, Wu JC, Gambhir SS: StemBook [Internet]. Cambridge (MA): Harvard Stem Cell Institute; 2008-2009.
• [18]Bujold M, Caron N, Camiran G, Mukherjee S, Allen PD, Tremblay JP, Wang Y: Autotransplantation in mdx mice of mdx myoblasts genetically corrected by an HSV-1 amplicon vector. Cell Transplant 2002, 11(8):759-767.
• [19]Kinoshita I, Vilquin JT, Guerette B, Asselin I, Roy R, Tremblay JP: Very efficient myoblast allotransplantation in mice under FK506 immunosuppression. Muscle Nerve 1994, 17(12):1407-1415.
• [20]Swijnenburg RJ, Schrepfer S, Govaert JA, Cao F, Ransohoff K, Sheikh AY, Haddad M, Connolly AJ, Davis MM, Robbins RC, Wu JC: Immunosuppressive therapy mitigates immunological rejection of human embryonic stem cell xenografts. Proc Natl Acad Sci U S A 2008, 105(35):12991-12996.
• [21]Denaro M, Oldmixon B, Patience C, Andersson G, Down J: EGFP-transduced EL-4 cells from tumors in C57BL/6 mice. Gene Ther 2001, 8(23):1814-1815.
• [22]Skelton D, Satake N, Kohn DB: The enhanced green fluorescent protein (eGFP) is minimally immunogenic in C57BL/6 mice. Gene Ther 2001, 8(23):1813-1814.
• [23]Bubnic SJ, Nagy A, Keating A: Donor hematopoietic cells from transgenic mice that express GFP are immunogenic in immunocompetent recipients. Hematology 2005, 10(4):289-295.
• [24]Hakamata Y, Murakami T, Kobayashi E: "Firefly rats" as an organ/cellular source for long-term in vivo bioluminescent imaging. Transplantation 2006, 81(8):1179-1184.
• [25]Persons DA, Allay JA, Riberdy JM, Wersto RP, Donahue RE, Sorrentino BP, Nienhuis AW: Use of the green fluorescent protein as a marker to identify and track genetically modified hematopoietic cells. Nat Med 1998, 4(10):1201-1205.
• [26]Partridge TA, Morgan JE, Coulton GR, Hoffman EP, Kunkel LM: Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts. Nature 1989, 337(6203):176-179.
• [27]Tedesco FS, Dellavalle A, Diaz-Manera J, Messina G, Cossu G: Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells. J Clin Invest 2010, 120(1):11-19.
• [28]Grounds MD, Radley HG, Lynch GS, Nagaraju K, De Luca A: Towards developing standard operating procedures for pre-clinical testing in the mdx mouse model of Duchenne muscular dystrophy. Neurobiol Dis 2008, 31:1-19.
• [29]Rando TA, Blau HM: Methods for myoblast transplantation. Meth Cell Biol 1997, 52:261-272.
• [30]Kircher MF, Gambhir SS, Grimm J: Noninvasive cell-tracking methods. Nat Rev Clin Oncol 2011, 8(11):677-688.
• [31]Tangney M, Francis KP: In vivo optical imaging in gene & cell therapy. Curr Gene Ther 2012, 12(1):2-11.
• [32]Xu X, Yang Z, Liu Q, Wang Y: In vivo fluorescence imaging of muscle cell regeneration by transplanted EGFP-labeled myoblasts. Mol Ther 2010, 18(4):835-842.
• [33]Bessis N, GarciaCozar FJ, Boissier MC: Immune responses to gene therapy vectors: influence on vector function and effector mechanisms. Gene Ther 2004, 11(Suppl 1):S10-S17.
• [34]Zhang Z, Kuscu C, Ghazizadeh S: Transgene-specific host responses in cutaneous gene therapy: the role of cells expressing the transgene. Gene Ther 2009, 16(9):1138-1145.
• [35]Lu Z, Ghazizadeh S: Host immune responses in ex vivo approaches to cutaneous gene therapy targeted to keratinocytes. Exp Dermatol 2005, 14(10):727-735.
• [36]Yeung JC, Wagnetz D, Cypel M, Rubacha M, Koike T, Chun YM, Hu J, Waddell TK, Hwang DM, Liu M, Keshavjee S: Ex vivo adenoviral vector gene delivery results in decreased vector-associated inflammation pre- and post-lung transplantation in the pig. Mol Ther 2012, 20(6):1204-1211.