Skip to main content
SpringerLink
Log in

Assessment of the green florescence protein labeling method for tracking implanted mesenchymal stem cells

  • Original Research
  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

Although green fluorescent protein (GFP) labeling is widely accepted as a tracking method, much remains uncertain regarding the retention of injected GFP-labeled cells implanted in ischemic organs. In this study, we evaluate the effectiveness of GFP for identifying and tracking implanted bone marrow- mesenchymal stem cells (BM-MSCs) and the effect of GFP on the paracrine actions of these cells. MSCs isolated from rat femur marrow were transduced with a recombinant adenovirus carrying GFP. After transplantation of the GFP-labeled BM-MSCs into the infarct zone of rat hearts, the survival, distribution, and migration of the labeled cells were analyzed at 3, 7, 14, and 28 days. To evaluate the effect of GFP on the paracrine actions of BM-MSCs, Western blot analysis was performed to detect the expression of vascular endothelial growth factor (VEGF), b fibroblast growth factor (b FGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinases-2 (MMP-2). GFP was successfully expressed by BM-MSCs in vitro. At 14 days after cell transplantation the GFP-positive cells could not be detected via confocal microscopy. By using a GFP antibody, distinct GFP-positive cells could be seen and quantitative analysis showed that the expression volume of GFP was 6.42 ± 0.92 mm3 after 3 days, 1.24 ± 0.76 mm3 after 7 days, 0.33 ± 0.03 mm3 after 14 days, and 0.09 ± 0.05 mm3 after 28 days. GFP labeling did not adversely affect the paracrine actions of BM-MSCs. GFP labeling could be used to track MSC distribution and their fate for at least 28 days after delivery to rat hearts with myocardial infarction, and this stem cell tracking strategy did not adversely affect the paracrine actions of BM-MSCs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

GFP:

Green fluorescent protein

BM-MSCs:

Bone marrow-mesenchymal stem cells

SC:

Stem cell

Ad-GFP:

Adenovirus vector carrying GFP

SD rats:

Sprague–Dawley rats

MI:

Myocardial infarction

MOIs:

Multiplicities of infection

PBS:

Phosphate-buffered saline

LV:

Left ventricle

VEGF:

Vascular endothelial growth factor

FGF:

Fibroblast growth factor

TIMP-1:

Tissue inhibitor of metalloproteinase-1

MMP-2:

Matrix metalloproteinases-2

vWF:

von Willebrand factor

References

  • Amado LC, Saliaris AP, Schuleri KH, St John M, Xie JS, Cattaneo S, Durand DJ, Fitton T, Kuang JQ, Stewart G, Lehrke S, Baumgartner WW, Martin BJ, Heldman AW, Hare JM (2005) Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci 102:11474–11479

    Article  CAS  Google Scholar 

  • Beggs KJ, Lyubimov A, Borneman JN, Bartholomew A, Moseley A, Dodds R, Archambault MP, Smith AK, McIntosh KR (2006) Immunologic consequences of multiple, high-dose administration of allogeneic mesenchymal stem cells to baboons. Cell Transpl 15:711–721

    Article  Google Scholar 

  • Caplan AI, Dennis JE (2006) Mesenchymal stem cells as trophic mediators. J Cell Biochem 98:1076–1084

    Article  CAS  Google Scholar 

  • Conget PA, Minguell JJ (1999) Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 181:67–73

    Article  CAS  Google Scholar 

  • Dai W, Hale SL, Martin BJ, Kuang JQ, Dow JS, Wold LE, Kloner RA (2005) Allogeneic mesenchymal stem cell transplantation in postinfarcted rat myocardium: Short- and long-term effects. Circulation 112:214–223

    Article  Google Scholar 

  • Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy 8:315–317

  • Duan HF, Wu CT, Wu DL, Lu Y, Liu HJ, Ha XQ, Zhang QW, Wang H, Jia XX, Wang LS (2003) Treatment of myocardial ischemia with bone marrow-derived mesenchymal stem cells overexpressing hepatocyte growth factor. Mol Ther 8:467–474

    Article  CAS  Google Scholar 

  • Gnecchi M, He H, Noiseux N, Liang OD, Zhang L, Morello F, Mu H, Melo LG, Pratt RE, Ingwall JS, Dzau VJ (2006) Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J 20:661–669

    Article  CAS  Google Scholar 

  • Guo YH, He JG, Wu JL, Yang L, Zhang DS, Tan XY, Qi RD (2008) Hepatocyte growth factor and granulocyte colony-stimulating factor form a combined neovasculogenic therapy for ischemic cardiomyopathy. Cytotherapy 10:857–867

    Article  Google Scholar 

  • Hamm A, Krott N, Breibach I, Blindt R, Bosserhoff AK (2002) Efficient transfection method for primary cells. Tissue Eng 8:235–245

    Article  CAS  Google Scholar 

  • Hare JM, Traverse JH, Henry TD, Dib N, Strumpf RK, Schulman SP, Gerstenblith G, DeMaria AN, Denktas AE, Gammon RS, Hermiller JB Jr, Reisman MA, Schaer GL, Sherman W (2009) A randomized, double-blind, placebo-controlled, dose-escalation study of intravenous adult human mesenchymal stem cells (prochymal) after acute myocardial infarction. J Am Coll Cardiol 54:2277–2286

    Article  CAS  Google Scholar 

  • Hatzistergos KE, Quevedo H, Oskouei BN, Hu Q, Feigenbaum GS, Margitich IS, Mazhari R, Boyle AJ, Zambrano JP, Rodriguez JE, Dulce R, Pattany PM, Valdes D, Revilla C, Heldman AW, McNiece I, Hare JM (2010) Bone marrow mesenchymal stem cells stimulate cardiac stem cell proliferation and differentiation. Circ Res 107:913–922

    Article  CAS  Google Scholar 

  • Huang J, Zhang Z, Guo J, Ni A, Deb A, Zhang L, Mirotsou M, Pratt RE, Dzau VJ (2010) Genetic modification of mesenchymal stem cells overexpressing CCR1 increases cell viability, migration, engraftment, and capillary density in the injured myocardium. Circ Res 106:1753–1762

    Article  CAS  Google Scholar 

  • Keyser KA, Beagles KE, Kiem HP (2007) Comparison of mesenchymal stem cells from different tissues to suppress T-cell activation. Cell Transpl 16:555–562

    Google Scholar 

  • Kinnaird T, Stabile E, Burnett MS, Lee CW, Barr S, Fuchs S, Epstein SE (2004) Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines promote in vitro in vivo arteriogenesis through paracrine mechanisms. Circ Res 94:678–685

    Article  CAS  Google Scholar 

  • Kruglyakov PV, Sokolova IB, Zin’kova NN, Viide SK, Aleksandrov GV, Petrov NS, Polyntsev DG (2006) In vitro and in vivo differentiation of mesenchymal stem cells in the cardiomyocyte direction. Bull Exp Biol Med 142:503–506

    Article  CAS  Google Scholar 

  • Le Blanc K, Pittenger M (2005) Mesenchymal stem cells: progress toward promise. Cytotherapy 7:36–45

    Article  CAS  Google Scholar 

  • Leiker M, Suzuki G, Iyer VS, Canty JM Jr, Lee T (2008) Assessment of a nuclear affinity labeling method for tracking implanted mesenchymal stem cells. Cell Transpl 17:911–922

    Article  Google Scholar 

  • Lippincott-Schwarhz J, Patterson GH (2003) Development and use of fluorescent protein markers in living cells. Science 300:87–91

    Article  Google Scholar 

  • Lu ZZ, Ni F, Hu ZB, Wang L, Wang H, Zhang QW, Huang WR, Wu CT, Wang LS (2006) Efficient gene transfer into hematopoietic cells by a retargeting adenoviral vector system with a chimeric fiber of adenovirus serotype 5 and 11p. Exp Hematol 34:1171–1182

    Article  CAS  Google Scholar 

  • Mangi AA, Noiseux N, Kong D, He H, Rezvani M, Ingwall JS, Dzau VJ (2003) Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med 9:1195–1201

    Article  CAS  Google Scholar 

  • Meyer GP, Wollert KC, Lotz J, Steffens J, Lippolt P, Fichtner S, Hecker H, Schaefer A, Arseniev L, Hertenstein B, Ganser A, Drexler H (2006) Intracoronary bone marrow cell transfer after myocardial infarction: Eighteen months’ follow-up data from the randomized, controlled BOOST (Bone marrow transfer to enhance ST-elevation infarct regeneration) trial. Circulation 113:1287–1294

    Article  Google Scholar 

  • Ohnishi S, Yanagawa B, Tanaka K, Miyahara Y, Obata H, Kataoka M, Kodama M, Ishibashi-Ueda H, Kangawa K, Kitamura S, Nagaya N (2007) Transplantation of mesenchymal stem cells attenuates myocardial injury and dysfunction in a rat model of acute myocarditis. J Mol Cell Cardiol 42:88–97

    Article  CAS  Google Scholar 

  • Rizzo MA, Davidson MW, Piston DW (2009) Fluorescent protein tracking and detection: fluorescent protein structure and color variants. Cold Spring Harb Protoc 12: pdb.top63

  • Stroh A, Boltze J, Sieland K, Hild K, Gutzeit C, Jung T, Kressel J, Hau S, Reich D, Grune T, Zimmer C (2009) Impact of magnetic labeling on human and mouse stem cells and their long-term magnetic resonance tracking in a rat model of Parkinson disease. Mol Imag 8:166–178

    CAS  Google Scholar 

  • Suzuki G, Lee T, Fallavollita JA, Canty JM Jr (2005) Adenoviral gene transfer of FGF-5 to hibernating myocardium improves function stimulates myocytes to hypertrophy and reenter the cell cycle. Circ Res 96:767–775

    Article  CAS  Google Scholar 

  • Wang X, Hu Q, Mansoor A, Lee J, Wang Z, Lee T, From AH, Zhang J (2006) Bioenergetic and functional consequences of stem cell-based VEGF delivery in pressure-overloaded swine hearts. Am J Physiol Heart Circ Physiol 290: H1393–H1405

    Google Scholar 

  • Wu Y, Chen L, Scott PG, Tredget EE (2007) Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells 25:2648–2659

    Article  CAS  Google Scholar 

  • Xu X, Yang Z, Liu Q, Wang Y (2010) In vivo fluorescence imaging of muscle cell regeneration by transplanted EGFP-labeled myoblasts. Mol Ther 18:835–842

    Article  CAS  Google Scholar 

  • Yan L, Han Y, He Y, Xie H, Liu J, Zhao L, Wang J, Gao L, Fan D (2007) Cell tracing techniques in stem cell transplantation. Stem Cell Rev 3:265–269

    Article  Google Scholar 

Download references

Acknowledgments

This study was supported by the National Natural Science Foundation of China (No. 81000018), the Major Program of the Chinese PLA General Hospital Nursery funds (No. 10KMZ04) and the opening foundation of the State Key Laboratory of Space Medicine Fundamentals and Application, Chinese Astroaut Research and Training Center (No. SMFA11K02).

Author information

Authors and Affiliations

  1. Department of Nanlou Respirtory Disease, Chinese PLA General Hospital, Chinese PLA Postgraduate Medical School, Beijing, 100853, People’s Republic of China

    Yinghua Guo, Longxiang Su, Dong Zhang, Xiaojun Zhang, Guizhi Zhang, Tianzhi Li, Junfeng Wang & Changting Liu

  2. From Shandong Provincial Hospital, Shandong University School of Medicine, Jinan, 250021, China

    Junlou Wu

Authors
  1. Yinghua Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Longxiang Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Junlou Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaojun Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Guizhi Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tianzhi Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Junfeng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Changting Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Changting Liu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Guo, Y., Su, L., Wu, J. et al. Assessment of the green florescence protein labeling method for tracking implanted mesenchymal stem cells. Cytotechnology 64, 391–401 (2012). https://doi.org/10.1007/s10616-011-9417-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10616-011-9417-y

Keywords

Search

Navigation