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Measurement of Cell-Penetrating Peptide-Mediated Transduction of Adult Hematopoietic Stem Cells

  • Aziza P. Manceur
  • Julie Audet
Part of the Methods in Molecular Biology book series (MIMB, volume 482)

Abstract

The ability of cell-penetrating peptides (CPPs) to cross cell membranes and transport cargo into cells makes them an attractive tool for the molecular engineering of stem cells. Even though the exact mechanism of transduction remains unclear, their potential has been demonstrated for diverse applications, including hematopoietic stem cell expansion and the generation of islets cells from embryonic stem cells. Several parameters can affect the intracellular delivery of CPP-based constructs. Those include the type of cells targeted, the type of CPP used, and the properties of the cargo. For this reason, it is important to have a means to quantitatively assess the transduction efficiency of specific constructs in the cell type of interest in order to select the best vector for a specific application. In this chapter, we describe a method to measure the uptake of HIV transactivator of transcription (TAT) and the homeobox protein Antennapedia (Antp) constructs in primary hematopoietic progenitor cells and hematopoietic cell lines. This method is useful to compare, select, and optimize different strategies to deliver CPP-based constructs into a given cell type.

Key words

Hematopoietic stem cell cell-penetrating peptides protein transduction domains TAT antennapedia intracellular delivery biological transport flow cytometry confocal microscopy 

References

  1. 1.
    Green, I., Christison, R., Voyce, C. J., Bundell, K. R., and Lindsay, M. A. (2003) Protein transduction domains: are they delivering? Trends Pharmacol Sci 24, 213–215.PubMedGoogle Scholar
  2. 2.
    Kobayashi, N., Rivas-Carrillo, J. D., Soto-Gutierrez, A., Fukazawa, T., Chen, Y., Navarro-Alvarez, N., and Tanaka, N. (2005) Gene delivery to embryonic stem cells. Birth Defects Res C Embryo Today 75, 10–18.CrossRefPubMedGoogle Scholar
  3. 3.
    Dietz, G. P., and Bahr, M. (2004) Delivery of bioactive molecules into the cell: the Trojan horse approach. Mol Cell Neurosci 27, 85–131.CrossRefPubMedGoogle Scholar
  4. 4.
    Gupta, B., Levchenko, T. S., and Torchilin, V. P. (2005) Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Adv Drug Deliv Rev 57, 637–651.CrossRefPubMedGoogle Scholar
  5. 5.
    Noguchi, H., and Matsumoto, S. (2006) Protein transduction technology: a novel therapeutic perspective. Acta Med Okayama 60, 1–11.PubMedGoogle Scholar
  6. 6.
    Fischer, R., Fotin-Mleczek, M., Hufnagel, H., and Brock, R. (2005) Break on through to the other side-biophysics and cell biology shed light on cell-penetrating peptides. Chembiochem 6, 2126–2142.CrossRefPubMedGoogle Scholar
  7. 7.
    Richard, J. P., Melikov, K., Vives, E., Ramos, C., Verbeure, B., Gait, M. J., Chernomordik, L. V., and Lebleu, B. (2003) Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J Biol Chem 278, 585–590.CrossRefPubMedGoogle Scholar
  8. 8.
    Thomas, T. E., Miller, C. L., and Eaves, C. J. (1999) Purification of hematopoietic stem cells for further biological study. Methods 17, 202–218.CrossRefPubMedGoogle Scholar
  9. 9.
    Henriques, S. T., Costa, J., and Castanho, M. A. (2005) Re-evaluating the role of strongly charged sequences in amphipathic cell-penetrating peptides: a fluorescence study using Pep-1. FEBS Lett 579, 4498–4502.CrossRefPubMedGoogle Scholar
  10. 10.
    Letoha, T., Gaal, S., Somlai, C., Venkei, Z., Glavinas, H., Kusz, E., Duda, E., Czajlik, A., Petak, F., and Penke, B. (2005) Investigation of penetratin peptides. Part 2. In vitro uptake of penetratin and two of its derivatives. J Pept Sci 11, 805–811.CrossRefPubMedGoogle Scholar
  11. 11.
    Miller, C. L., Audet, J., and Eaves, C. J. (2002) Ex vivo expansion of human and murine hematopoietic stem cells, in Hematopoietic Stem Cell Protocols (Klug, A.A. and Jordan, C.T., eds.), Humana Press, Totowa, New Jersey, pp.189–208.Google Scholar
  12. 12.
    Szeto, H. H., Schiller, P. W., Zhao, K., and Luo, G. (2005) Fluorescent dyes alter intracellular targeting and function of cell-penetrating tetrapeptides. Faseb J 19, 118–120.PubMedGoogle Scholar
  13. 13.
    Van Amersfoort, E. S., and Van Strijp, J. A. (1994) Evaluation of a flow cytometric fluorescence quenching assay of phagocytosis of sensitized sheep erythrocytes by polymorphonuclear leukocytes. Cytometry 17, 294–301.CrossRefPubMedGoogle Scholar
  14. 14.
    Hallbrink, M., Oehlke, J., Papsdorf, G., and Bienert, M. (2004) Uptake of cell-penetrating peptides is dependent on peptide-to-cell ratio rather than on peptide concentration. Biochim Biophys Acta 1667, 222–228.CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Aziza P. Manceur
    • 1
  • Julie Audet
    • 1
  1. 1.Terrence Donnelly Centre for Cellular and Biomolecular ResearchInstitute of Biomaterials and Biomedical Engineering, University of TorontoTorontoCanada

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