Skip to main content
SpringerLink
Log in

Molecular Imaging of Human Embryonic Stem Cells

  • Protocol
Viral Applications of Green Fluorescent Protein

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 515))

Summary

Human embryonic stem cells (hESCs) are a renewable source of differentiated cell types that may be employed in various tissue regeneration strategies. However, clinical implementation of cell transplantation therapy is hindered by legitimate concerns regarding the in vivo teratoma formation of undifferentiated hESCs and host immune reactions to allogeneic cells. Investigating in vivo hESC behavior and the ultimate feasibility of cell transplantation therapy necessitates the development of novel molecular imaging techniques to longitudinally monitor hESC localization, proliferation, and viability in living subjects. An innovative approach to harness the respective strengths of various imaging platforms is the creation and use of a fusion reporter construct composed of red fluorescent protein (RFP), firefly luciferase (fluc), and herpes simplex virus thymidine kinase (HSV-tk). The imaging modalities made available by use of this construct, including optical fluorescence, bioluminescence, and positron emission tomography (PET), may be adapted to investigate a variety of physiological phenomena, including the spatio-temporal kinetics of hESC engraftment and proliferation in living subjects. This chapter describes the applications of reporter gene imaging to accelerate basic science research and clinical studies involving hESCs through (1) isolation of a homogeneous hESC population, (2) noninvasive, longitudinal tracking of the location and proliferation of hESCs administered to a living subject, and (3) ablation of the hESC graft in the event of cellular misbehavior.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Cao, F. et al. Transcriptional and functional profiling of human embryonic stem cell-derived cardiomyocytes. PLoS ONE 3(10), e3474 (2008)

    Article  PubMed  Google Scholar 

  2. Baharvand, H. et al Neural differentiation from human embryonic stem cells in a defined adherent culture condition. Int J Dev Biol 51, 371–8 (2007).

    Article  Google Scholar 

  3. Schulz, T.C. et al Directed neuronal differentiation of human embryonic stem cells. BMC Neurosci 4, 27 (2003).

    Article  PubMed  Google Scholar 

  4. Jiang, J. et al Generation of insulin-producing Islet-like clusters from human embryonic stem cells. Stem Cells 17, 17 (2007).

    Google Scholar 

  5. Swijnenburg, R.J. van der Bogt, K.E.A., Sheikh, A.Y., Cao, F. & Wu, J.C. Clinical hurdles for the transplantation of cardiomyocytes derived from human embryonic stem cells: role of molecular imaging. Curr Opin Biotechnol 18, 38–45 (2007).

    Article  PubMed  CAS  Google Scholar 

  6. Wollert, K.C. et al Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 364, 141–8 (2004).

    Article  PubMed  Google Scholar 

  7. Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A. & Bongso, A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18, 399–404 (2000).

    Article  PubMed  CAS  Google Scholar 

  8. Thomson, J.A. et al Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–7 (1998).

    Article  PubMed  CAS  Google Scholar 

  9. Sidhu, K.S. & Tuch, B.E. Derivation of three clones from human embryonic stem cell lines by FACS sorting and their characterization. Stem Cells Dev 15, 61–9 (2006).

    Article  PubMed  CAS  Google Scholar 

  10. Nicholas, C.R., Gaur, M., Wang, S., Pera, R.A. & Leavitt, A.D. A method for single-cell sorting and expansion of genetically modified human embryonic stem cells. Stem Cells Dev 16, 109–17 (2007).

    Article  PubMed  CAS  Google Scholar 

  11. Franz, W.M., Rothmann, T., Frey, N. & Katus, H.A. Analysis of tissue-specific gene delivery by recombinant adenoviruses containing cardiac-specific promoters. Cardiovasc Res 35, 560–6 (1997).

    Article  PubMed  CAS  Google Scholar 

  12. Huber, I. et al Identification and selection of cardiomyocytes during human embryonic stem cell differentiation. FASEB J 13, 13 (2007).

    Google Scholar 

  13. Yaghoubi, S.S. & Gambhir, S.S. PET imaging of herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk reporter gene expression in mice and humans using [18F]FHBG. Nat Protoc 1, 3069–75 (2006).

    Article  PubMed  CAS  Google Scholar 

  14. Yaghoubi, S. et al Human pharmacokinetic and dosimetry studies of [(18)F]FHBG: a reporter probe for imaging herpes simplex virus type-1 thymidine kinase reporter gene expression. J Nucl Med 42, 1225–34 (2001).

    PubMed  CAS  Google Scholar 

  15. Yaghoubi, S.S. et al Preclinical safety evaluation of 18F-FHBG: a PET reporter probe for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk’s expression. J Nucl Med 47, 706–15 (2006).

    PubMed  CAS  Google Scholar 

  16. Phelps, M.E. Inaugural article: positron emission tomography provides molecular imaging of biological processes. Proc Natl Acad Sci USA 97, 9226–33 (2000).

    Article  PubMed  CAS  Google Scholar 

  17. Moolten, F.L. Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res 46, 5276–81 (1986).

    PubMed  CAS  Google Scholar 

  18. Moolten, F.L., Wells, J.M., Heyman, R.A. & Evans, R.M. Lymphoma regression induced by ganciclovir in mice bearing a herpes thymidine kinase transgene. Hum Gene Ther 1, 125–34 (1990).

    Article  PubMed  CAS  Google Scholar 

  19. Vile, R.G. & Hart, I.R. Use of tissue-specific expression of the herpes simplex virus thymidine kinase gene to inhibit growth of established murine melanomas following direct intratumoral injection of DNA. Cancer Res 53, 3860–4 (1993).

    PubMed  CAS  Google Scholar 

  20. Schuldiner, M., Itskovitz-Eldor, J. & Benvenisty, N. Selective ablation of human embryonic stem cells expressing a “suicide” gene. Stem Cells 21, 257–65 (2003).

    Article  PubMed  CAS  Google Scholar 

  21. Cao, F. et al Molecular imaging of embryonic stem cell misbehavior and suicide gene ablation. Cloning Stem Cells 9, 107–17 (2007).

    Article  PubMed  CAS  Google Scholar 

  22. Gambhir, S.S. et al A mutant herpes simplex virus type 1 thymidine kinase reporter gene shows improved sensitivity for imaging reporter gene expression with positron emission tomography. Proc Natl Acad Sci USA 97, 2785–90 (2000).

    Article  PubMed  CAS  Google Scholar 

  23. Tisi, L.C. et al Development of a thermostable firefly luciferase. Anal Chim Acta 457, 115–23 (2002).

    Article  CAS  Google Scholar 

  24. Ray, P., Tsien, R. & Gambhir, S.S. Construction and validation of improved triple fusion reporter gene vectors for molecular imaging of living subjects. Cancer Res 67, 3085–93 (2007).

    Article  PubMed  CAS  Google Scholar 

  25. Cao, F. et al In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery. Circulation 113, 1005–1014 (2006).

    Article  PubMed  Google Scholar 

  26. Ray, P., De, A., Min, J.J., Tsien, R.Y. & Gambhir, S.S. Imaging tri-fusion multimodality reporter gene expression in living subjects. Cancer Res 64, 1323–30 (2004).

    Article  PubMed  CAS  Google Scholar 

  27. Wu, J.C. et al Proteomic analysis of reporter genes for molecular imaging of transplanted embryonic stem cells. Proteomics 6, 6234–49 (2006).

    Article  PubMed  CAS  Google Scholar 

  28. Ray, P., Wu, A.M. & Gambhir, S.S. Optical bioluminescence and positron emission tomography imaging of a novel fusion reporter gene in tumor xenografts of living mice. Cancer Res 63, 1160–5 (2003).

    PubMed  CAS  Google Scholar 

  29. Massoud, T.F. & Gambhir, S.S. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev 17, 545–80 (2003).

    Article  PubMed  CAS  Google Scholar 

  30. Eiges, R. et al Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr Biol 11, 514–8 (2001).

    Article  PubMed  CAS  Google Scholar 

  31. Mohr, J.C., de Pablo, J.J. & Palecek, S.P. Electroporation of human embryonic stem cells: Small and macromolecule loading and DNA transfection. Biotechnol Prog 22, 825–34 (2006).

    Article  PubMed  CAS  Google Scholar 

  32. Siemen, H. et al Nucleofection of human embryonic stem cells. Stem Cells Dev 14, 378–83 (2005).

    Article  PubMed  CAS  Google Scholar 

  33. Ma, Y., Ramezani, A., Lewis, R., Hawley, R.G. & Thomson, J.A. High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors. Stem Cells 21, 111–7 (2003).

    Article  PubMed  CAS  Google Scholar 

  34. Li, Z. et al. Comparison of reporter gene and iron particle labeling for tracking fate of human embryonic stem cells and differentiated endothelial cells in living subjects. Stem Cells 26(4), 864–73 (2008).

    Article  PubMed  CAS  Google Scholar 

  35. Liew, C.G., Draper, J.S., Walsh, J., Moore, H. & Andrews, P.W. Transient and stable transgene expression in human embryonic stem cells. Stem Cells 25, 1521–8 (2007).

    Article  PubMed  CAS  Google Scholar 

  36. Groth, A.C., Olivares, E.C., Thyagarajan, B. & Calos, M.P. A phage integrase directs efficient site-specific integration in human cells. Proc Natl Acad Sci USA 97, 5995–6000 (2000).

    Article  PubMed  CAS  Google Scholar 

  37. Black, M.E., Newcomb, T.G., Wilson, H.M. & Loeb, L.A. Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy. Proc Natl Acad Sci USA 93, 3525–9 (1996).

    Article  PubMed  CAS  Google Scholar 

  38. Kokoris, M.S., Sabo, P. & Black, M.E. In vitro evaluation of mutant HSV-1 thymidine kinases for suicide gene therapy. Anticancer Res 20, 959–63 (2000).

    PubMed  CAS  Google Scholar 

  39. Campbell, R.E. et al A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99, 7877–82 (2002).

    Article  PubMed  CAS  Google Scholar 

  40. Miyoshi, H., Blomer, U., Takahashi, M., Gage, F.H. & Verma, I.M. Development of a self-inactivating lentivirus vector. J Virol 72, 8150–7 (1998).

    PubMed  CAS  Google Scholar 

  41. De, A., Lewis, X.Z. & Gambhir, S.S. Noninvasive imaging of lentiviral-mediated reporter gene expression in living mice. Mol Ther 7, 681–91 (2003).

    Article  PubMed  CAS  Google Scholar 

  42. Chen, C. & Okayama, H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7, 2745–52 (1987).

    PubMed  CAS  Google Scholar 

  43. Naldini, L., Blomer, U., Gage, F.H., Trono, D. & Verma, I.M. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA 93, 11382–8 (1996).

    Article  PubMed  CAS  Google Scholar 

  44. Wu, J.C. et al Molecular imaging of the kinetics of vascular endothelial growth factor gene expression in ischemic myocardium. Circulation 110, 685–91 (2004).

    Article  PubMed  CAS  Google Scholar 

  45. Xu, C., Inokuma, M.S., Denham, J., Golds, K., Kundu, P., Gold, J.D., Carpenter, M.K. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19, 971–4 (2001).

    Article  PubMed  CAS  Google Scholar 

  46. Eiges, R. Genetic manipulation of human embryonic stem cells by transfection. Methods Mol Biol 331, 221–39 (2006).

    PubMed  Google Scholar 

  47. Wu, J.C., Inubushi, M., Sundaresan, G., Schelbert, H.R. & Gambhir, S.S. Optical imaging of cardiac reporter gene expression in living rats. Circulation 105, 1631–4 (2002).

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Medicine, University of California, San Diego School of Medicine, La Jolla, CA, USA

    Kazim H. Narsinh

  2. Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, CA, USA

    Feng Cao

  3. Molecular Imaging Program at Stanford (MIPS), Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, CA, USA

    Joseph C. Wu

Authors
  1. Kazim H. Narsinh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Feng Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joseph C. Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joseph C. Wu .

Editor information

Editors and Affiliations

  1. Department of Chemistry, 2355 Fairchild Drive, US Airforce Academy, CO, 80840, USA

    Barry W. Hicks

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Narsinh, K.H., Cao, F., Wu, J.C. (2009). Molecular Imaging of Human Embryonic Stem Cells. In: Hicks, B.W. (eds) Viral Applications of Green Fluorescent Protein. Methods in Molecular Biology™, vol 515. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-559-6_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-559-6_2

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-934115-87-9

  • Online ISBN: 978-1-59745-559-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation