Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Optimal conditions for lentiviral transduction of engrafting human CD34+ cells

Gene Therapy volume 18, pages 1078–1086 (2011)Cite this article

Subjects

Abstract

Cytokines are required for γ-retroviral transduction of human CD34+ cells. However, cytokines may reduce engraftment of CD34+ cells and may not be necessary for their lentiviral transduction. We sought to optimize transduction and engraftment of human CD34+ cells using lentiviral vectors. Single 24 h transduction of human CD34+ cells with human immunodeficiency virus type 1 (HIV1)-based lentiviral vectors in media containing stem cell factor (SCF), FMS-like tyrosine kinase 3 (FLT3) ligand, thrombopoietin (each 100 ng ml−1) and 10% fetal bovine serum was compared with various cytokine conditions during ex vivo culture and assayed using humanized xenograft mice for 6 months after transplantation. Serum-free media improved transduction efficiency of human CD34+ cells. Interleukin-3 (20 ng ml−1) had little effect on transduction efficiency or engraftment. Threefold higher cytokine mixture (each 300 ng ml−1) reduced engraftment of CD34+ cells. SCF alone (100 ng ml−1) proved insufficient for maintaining engraftment ability and reduced transduction efficiency. Short-term prestimulation had little effect on transduction efficiency or engraftment, yet 24 h prestimulation showed higher transduction efficiency, higher gene expression levels and lower engraftment. In summary, 24 h prestimulation followed by single 24-h lentiviral transduction in serum-free media with SCF, FLT3 ligand and thrombopoietin yields high transduction efficiency to engrafting human CD34+ cells, and is applicable in human clinical gene therapy trials.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Hacein-Bey-Abina S, Le Deist F, Carlier F, Bouneaud C, Hue C, De Villartay JP et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 2002; 346: 1185–1193.

    Article  CAS  PubMed  Google Scholar 

  2. Aiuti A, Cattaneo F, Galimberti S, Benninghoff U, Cassani B, Callegaro L et al. Gene therapy for immunodeficiency due to adenosine deaminase deficiency. N Engl J Med 2009; 360: 447–458.

    Article  CAS  PubMed  Google Scholar 

  3. May C, Rivella S, Callegari J, Heller G, Gaensler KM, Luzzatto L et al. Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin. Nature 2000; 406: 82–86.

    Article  CAS  PubMed  Google Scholar 

  4. Imren S, Payen E, Westerman KA, Pawliuk R, Fabry ME, Eaves CJ et al. Permanent and panerythroid correction of murine beta thalassemia by multiple lentiviral integration in hematopoietic stem cells. Proc Natl Acad Sci USA 2002; 99: 14380–14385.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Levasseur DN, Ryan TM, Pawlik KM, Townes TM . Correction of a mouse model of sickle cell disease: lentiviral/antisickling beta-globin gene transduction of unmobilized, purified hematopoietic stem cells. Blood 2003; 102: 4312–4319.

    Article  CAS  PubMed  Google Scholar 

  6. Pawliuk R, Westerman KA, Fabry ME, Payen E, Tighe R, Bouhassira EE et al. Correction of sickle cell disease in transgenic mouse models by gene therapy. Science 2001; 294: 2368–2371.

    Article  CAS  PubMed  Google Scholar 

  7. Persons DA, Allay ER, Sabatino DE, Kelly P, Bodine DM, Nienhuis AW . Functional requirements for phenotypic correction of murine beta-thalassemia: implications for human gene therapy. Blood 2001; 97: 3275–3282.

    Article  CAS  PubMed  Google Scholar 

  8. Rivella S, May C, Chadburn A, Riviere I, Sadelain M . A novel murine model of Cooley anemia and its rescue by lentiviral-mediated human beta-globin gene transfer. Blood 2003; 101: 2932–2939.

    Article  CAS  PubMed  Google Scholar 

  9. Slaets H, Hendriks JJ, Van den Haute C, Coun F, Baekelandt V, Stinissen P et al. CNS-targeted LIF expression improves therapeutic efficacy and limits autoimmune-mediated demyelination in a model of multiple sclerosis. Mol Ther 2010; 18: 684–691.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Cavazzana-Calvo M, Payen E, Negre O, Wang G, Hehir K, Fusil F et al. Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature 2010; 467: 318–322.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Shultz LD, Ishikawa F, Greiner DL . Humanized mice in translational biomedical research. Nat Rev Immunol 2007; 7: 118–130.

    Article  CAS  PubMed  Google Scholar 

  12. Ito M, Hiramatsu H, Kobayashi K, Suzue K, Kawahata M, Hioki K et al. NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. Blood 2002; 100: 3175–3182.

    Article  CAS  PubMed  Google Scholar 

  13. Ishikawa F, Yasukawa M, Lyons B, Yoshida S, Miyamoto T, Yoshimoto G et al. Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice. Blood 2005; 106: 1565–1573.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Watanabe S, Terashima K, Ohta S, Horibata S, Yajima M, Shiozawa Y et al. Hematopoietic stem cell-engrafted NOD/SCID/IL2Rgamma null mice develop human lymphoid systems and induce long-lasting HIV-1 infection with specific humoral immune responses. Blood 2007; 109: 212–218.

    Article  CAS  PubMed  Google Scholar 

  15. Roesler J, Brenner S, Bukovsky AA, Whiting-Theobald N, Dull T, Kelly M et al. Third-generation, self-inactivating gp91(phox) lentivector corrects the oxidase defect in NOD/SCID mouse-repopulating peripheral blood-mobilized CD34+ cells from patients with X-linked chronic granulomatous disease. Blood 2002; 100: 4381–4390.

    Article  CAS  PubMed  Google Scholar 

  16. Piacibello W, Bruno S, Sanavio F, Droetto S, Gunetti M, Ailles L et al. Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multilineage repopulation in NOD/SCID mice. Nonobese diabetic/severe combined immunodeficient. Blood 2002; 100: 4391–4400.

    Article  CAS  PubMed  Google Scholar 

  17. Naumann N, De Ravin SS, Choi U, Moayeri M, Whiting-Theobald N, Linton GF et al. Simian immunodeficiency virus lentivector corrects human X-linked chronic granulomatous disease in the NOD/SCID mouse xenograft. Gene Therapy 2007; 14: 1513–1524.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Miyoshi H, Smith KA, Mosier DE, Verma IM, Torbett BE . Transduction of human CD34+ cells that mediate long-term engraftment of NOD/SCID mice by HIV vectors. Science 1999; 283: 682–686.

    Article  CAS  PubMed  Google Scholar 

  19. Sellers SE, Tisdale JF, Agricola BA, Donahue RE, Dunbar CE . The presence of the carboxy-terminal fragment of fibronectin allows maintenance of non-human primate long-term hematopoietic repopulating cells during extended ex vivo culture and transduction. Exp Hematol 2004; 32: 163–170.

    Article  CAS  PubMed  Google Scholar 

  20. Tisdale JF, Hanazono Y, Sellers SE, Agricola BA, Metzger ME, Donahue RE et al. Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability. Blood 1998; 92: 1131–1141.

    CAS  PubMed  Google Scholar 

  21. Kiem HP, Andrews RG, Morris J, Peterson L, Heyward S, Allen JM et al. Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor. Blood 1998; 92: 1878–1886.

    CAS  PubMed  Google Scholar 

  22. Williams DA, Rios M, Stephens C, Patel VP . Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions. Nature 1991; 352: 438–441.

    Article  CAS  PubMed  Google Scholar 

  23. Dunbar CE . Gene transfer to hematopoietic stem cells: implications for gene therapy of human disease. Annu Rev Med 1996; 47: 11–20.

    Article  CAS  PubMed  Google Scholar 

  24. Bukrinsky MI, Haggerty S, Dempsey MP, Sharova N, Adzhubel A, Spitz L et al. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature 1993; 365: 666–669.

    Article  CAS  PubMed  Google Scholar 

  25. Abonour R, Williams DA, Einhorn L, Hall KM, Chen J, Coffman J et al. Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells. Nat Med 2000; 6: 652–658.

    Article  CAS  PubMed  Google Scholar 

  26. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000; 288: 669–672.

    Article  CAS  PubMed  Google Scholar 

  27. Kluge KA, Bonifacino AC, Sellers S, Agricola BA, Donahue RE, Dunbar CE . Retroviral transduction and engraftment ability of primate hematopoietic progenitor and stem cells transduced under serum-free versus serum-containing conditions. Mol Ther 2002; 5: 316–322.

    Article  CAS  PubMed  Google Scholar 

  28. Millington M, Arndt A, Boyd M, Applegate T, Shen S . Towards a clinically relevant lentiviral transduction protocol for primary human CD34 hematopoietic stem/progenitor cells. PLoS One 2009; 4: e6461.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Selvaggi TA, Walker RE, Fleisher TA . Development of antibodies to fetal calf serum with arthus-like reactions in human immunodeficiency virus-infected patients given syngeneic lymphocyte infusions. Blood 1997; 89: 776–779.

    CAS  PubMed  Google Scholar 

  30. Yonemura Y, Ku H, Hirayama F, Souza LM, Ogawa M . Interleukin 3 or interleukin 1 abrogates the reconstituting ability of hematopoietic stem cells. Proc Natl Acad Sci USA 1996; 93: 4040–4044.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bodine DM, Karlsson S, Nienhuis AW . Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells. Proc Natl Acad Sci USA 1989; 86: 8897–8901.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Piacibello W, Gammaitoni L, Bruno S, Gunetti M, Fagioli F, Cavalloni G et al. Negative influence of IL3 on the expansion of human cord blood in vivo long-term repopulating stem cells. J Hematother Stem Cell Res 2000; 9: 945–956.

    Article  CAS  PubMed  Google Scholar 

  33. Zandstra PW, Conneally E, Petzer AL, Piret JM, Eaves CJ . Cytokine manipulation of primitive human hematopoietic cell self-renewal. Proc Natl Acad Sci USA 1997; 94: 4698–4703.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Dunbar CE, Takatoku M, Donahue RE . The impact of ex vivo cytokine stimulation on engraftment of primitive hematopoietic cells in a non-human primate model. Ann NY Acad Sci 2001; 938: 236–244; discussion 244–245.

    Article  CAS  PubMed  Google Scholar 

  35. Miller DG, Adam MA, Miller AD . Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 1990; 10: 4239–4242.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Karlsson S . Treatment of genetic defects in hematopoietic cell function by gene transfer. Blood 1991; 78: 2481–2492.

    CAS  PubMed  Google Scholar 

  37. Vigna E, Naldini L . Lentiviral vectors: excellent tools for experimental gene transfer and promising candidates for gene therapy. J Gene Med 2000; 2: 308–316.

    Article  CAS  PubMed  Google Scholar 

  38. Geronimi F, Richard E, Redonnet-Vernhet I, Lamrissi-Garcia I, Lalanne M, Ged C et al. Highly efficient lentiviral gene transfer in CD34+ and CD34+/38−/lin− cells from mobilized peripheral blood after cytokine prestimulation. Stem Cells 2003; 21: 472–480.

    Article  CAS  PubMed  Google Scholar 

  39. Higashimoto T, Grassman E, Cancelas JA, Malik P . Small but powerful, predicting the outcome of gene therapy trial using NSG Mice. Mol Ther 2010; 18 (Suppl 1): 203 (abstract 526).

    Google Scholar 

  40. Peters SO, Kittler EL, Ramshaw HS, Quesenberry PJ . Murine marrow cells expanded in culture with IL-3, IL-6, IL-11, and SCF acquire an engraftment defect in normal hosts. Exp Hematol 1995; 23: 461–469.

    CAS  PubMed  Google Scholar 

  41. Herrera C, Sanchez J, Torres A, Bellido C, Rueda A, Alvarez MA . Early-acting cytokine-driven ex vivo expansion of mobilized peripheral blood CD34+ cells generates post-mitotic offspring with preserved engraftment ability in non-obese diabetic/severe combined immunodeficient mice. Br J Haematol 2001; 114: 920–930.

    Article  CAS  PubMed  Google Scholar 

  42. Malim MH . APOBEC proteins and intrinsic resistance to HIV-1 infection. Philos Trans R Soc Lond Ser B 2009; 364: 675–687.

    Article  CAS  Google Scholar 

  43. Luban J . Cyclophilin A, TRIM5, and resistance to human immunodeficiency virus type 1 infection. J Virol 2007; 81: 1054–1061.

    Article  CAS  PubMed  Google Scholar 

  44. Hayakawa J, Joyal EG, Gildner JF, Washington KN, Phang OA, Uchida N et al. 5% dimethyl sulfoxide (DMSO) and pentastarch improves cryopreservation of cord blood cells over 10% DMSO. Transfusion 2010; 50: 2158–2166.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Hanawa H, Kelly PF, Nathwani AC, Persons DA, Vandergriff JA, Hargrove P et al. Comparison of various envelope proteins for their ability to pseudotype lentiviral vectors and transduce primitive hematopoietic cells from human blood. Mol Ther 2002; 5: 242–251.

    Article  CAS  PubMed  Google Scholar 

  46. Uchida N, Hanawa H, Dan K, Inokuchi K, Shimada T . Leukemogenesis of b2a2-type p210 BCR/ABL in a bone marrow transplantation mouse model using a lentiviral vector. J Nippon Med Sch 2009; 76: 134–147.

    Article  CAS  PubMed  Google Scholar 

  47. Uchida N, Washington KN, Hayakawa J, Hsieh MM, Bonifacino AC, Krouse AE et al. Development of an HIV1-based lentiviral vector that allows efficient transduction of both human and rhesus blood cells. J Virol 2009; 83: 9854–9862.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the intramural research program of the National Heart, Lung and Blood Institute (NHLBI) and the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) at the National Institutes of Health (NIH).

Author information

Authors and Affiliations

  1. Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD, USA

    N Uchida, M M Hsieh, J Hayakawa, C Madison, K N Washington & J F Tisdale

Authors
  1. N Uchida
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M M Hsieh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J Hayakawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C Madison
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. K N Washington
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. J F Tisdale
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J F Tisdale.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on Gene Therapy website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Uchida, N., Hsieh, M., Hayakawa, J. et al. Optimal conditions for lentiviral transduction of engrafting human CD34+ cells. Gene Ther 18, 1078–1086 (2011). https://doi.org/10.1038/gt.2011.63

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2011.63

Keywords

This article is cited by

Search

Advanced search

Quick links