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.

  • Research Article
  • Published:

Efficient retrovirus-mediated transduction of primitive human peripheral blood progenitor cells in stroma-free suspension culture

Gene Therapy volume 8, pages 687–696 (2001)Cite this article

Abstract

Retroviral transduction of hematopoietic cells has resulted in unsatisfactory gene marking in clinical studies. Since cytokine-stimulated stem cells have engrafted poorly in animal models, we investigated phenotypic changes during culture of peripheral blood progenitor cells (PBPC). Human CD34+ HLA-DRlow cells, immunomagnetically separated from PBPC collections, were found to extrude rhodamine-123, which is characteristic for primitive hematopoietic cells. Cells were grown in suspension cultures supplemented with cytokines. While interleukin-3-containing factor combinations promoted cell proliferation they caused loss of rhodamine-123 extrusion and reduced the frequencies of cobblestone area-forming cells (CAFC). Several other cytokines failed to stimulate cell divisions, which are required for retroviral transduction. A combination including Flt-3 ligand (FL), interleukin-6 and stem cell factor (SCF) preserved an immature phenotype for 5 to 6 days and stimulated cell divisions, which was improved upon addition of leukemia inhibitory factor and interleukin-11. Furthermore, the CAFC frequency among cells treated with these cytokines was increased as compared with widely used cocktails containing interleukin-3, interleukin-6 and SCF. Rhodamine-123 appeared to be a particularly sensitive indicator for differentiation of PBPC. For analysis of gene transfer, amphotropic retroviruses conferring an MDR1 cDNA were added repeatedly for 6 days to cytokine-treated PBPC stroma-free cultures. Proviral cDNA was detected by polymerase chain reaction in 68% of cobblestone areas derived from CD34+HLA-DRlow cells that had been exposed to Flt-3 ligand, interleukin-6 and SCF. In summary, conditions were identified that facilitate efficient transduction of early PBPC with amphotropic retroviruses while preserving a primitive phenotype for extended periods.

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
Figure 6
Figure 7
Figure 8

Similar content being viewed by others

References

  1. Hanania EG et al. Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to posttransplant hematopoietic recovery following intensive systemic therapy Proc Natl Acad Sci USA 1996 93: 15346–15351

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hesdorffer C et al. Phase I trial of retroviral-mediated transfer of the human MDR1 gene as marrow chemoprotection in patients undergoing high-dose chemotherapy and autologous stem-cell transplantation J Clin Oncol 1998 16: 165–172

    Article  CAS  PubMed  Google Scholar 

  3. Moscow JA et al. Reconstitution and expansion of hematopoietic cells transduced with retroviruses containing either the multidrug resistance gene (MDR1) or the neomycin resistance gene (neoR) in breast cancer patients treated with paclitaxel, doxorubicin and vinblastine Blood 1999 94: 52–61

    CAS  PubMed  Google Scholar 

  4. Cowan KH et al. Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients Clin Cancer Res 1999 5: 1619–1628

    CAS  PubMed  Google Scholar 

  5. Sutherland HJ et al. Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro Blood 1989 74: 1563–1570

    CAS  PubMed  Google Scholar 

  6. Srour EF et al. Human CD34+ HLA-DR− bone marrow cells contain progenitor cells capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis Blood Cells 1991 17: 287–295

    CAS  PubMed  Google Scholar 

  7. Chaudhary PM, Roninson IB . Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells Cell 1991 66: 85–94

    Article  CAS  PubMed  Google Scholar 

  8. Leemhuis T et al. Isolation of primitive human bone marrow hematopoietic progenitor cells using Hoechst 33342 and Rhodamine 123 Exp Hematol 1996 24: 1215–1224

    CAS  PubMed  Google Scholar 

  9. Bertolini F et al. Multilineage long-term engraftment potential of drug-resistant hematopoietic progenitors Blood 1997 90: 3027–3036

    CAS  PubMed  Google Scholar 

  10. Drach D et al. Subpopulations of normal and peripheral blood and bone marrow cells express a functional multidrug resistant phenotype Blood 1992 80: 2735–2739

    Google Scholar 

  11. Klimecki WT, Futscher BW, Grogan TM, Dalton WS . P-glycoprotein expression and function in circulating blood cells from normal volunteers Blood 1994 83: 2451–2458

    CAS  PubMed  Google Scholar 

  12. Gottesman MM, Pastan I . Biochemistry of multidrug resistance mediated by the multidrug transporter Annu Rev Biochem 1993 62: 385–427

    Article  CAS  PubMed  Google Scholar 

  13. Ploemacher RE et al. Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse Blood 1991 78: 2527–2533

    CAS  PubMed  Google Scholar 

  14. van Hennik PB et al. Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long-term cultures and multilineage engraftment of immunodeficient mice Blood 1998 92: 4013–4022

    CAS  PubMed  Google Scholar 

  15. Uchida N et al. The unexpected G0/G1 cell cycle status of mobilized hematopoietic stem cells from peripheral blood Blood 1997 89: 465–472

    CAS  PubMed  Google Scholar 

  16. 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 

  17. Luskey BD, Rosenblatt M, Zsebo K, Williams DA . Stem cell factor, interleukin-3, and interleukin-6 promote retroviral-mediated gene transfer into murine hematopoietic stem cells Blood 1992 80: 396–402

    CAS  PubMed  Google Scholar 

  18. Shah AJ, Smogorzewska EM, Hannum C, Crooks GM . Flt3 ligand induces proliferation of quiescent human bone marrow CD34+ CD38− cells and maintains progenitor cells in vitro Blood 1996 87: 3563–3570

    CAS  PubMed  Google Scholar 

  19. Conneally E, Eaves CJ, Humphries RK . Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential Blood 1998 91: 3487–3493

    CAS  PubMed  Google Scholar 

  20. Piacibello W et al. Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood Blood 1997 89: 2644–2653

    CAS  PubMed  Google Scholar 

  21. van der Loo JC, Ploemacher RE . Marrow- and spleen-seeding efficiencies of all murine hematopoietic stem cell subsets are decreased by preincubation with hematopoietic growth factors Blood 1995 85: 2598–2606

    CAS  PubMed  Google Scholar 

  22. Yonemura Y et al. 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 

  23. Peters SO, Kittler EL, Ramshaw HS, Quesenberry PJ . Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts Blood 1996 87: 30–37

    CAS  PubMed  Google Scholar 

  24. Kittler EL et al. Cytokine-facilitated transduction leads to low-level engraftment in nonablated hosts Blood 1997 90: 865–872

    CAS  PubMed  Google Scholar 

  25. Podda S et al. Transfer and expression of the human multiple drug resistance gene into live mice Proc Natl Acad Sci USA 1992 89: 9676–9680

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Sorrentino BP et al. Selection of drug-resistant bone marrow cells in vivo after retroviral transfer of human MDR1 Science 1992 257: 99–103

    Article  CAS  PubMed  Google Scholar 

  27. Bertolini F et al. Retrovirus-mediated transfer of the human multidrug resistance gene into human haematopoietic progenitor cells Br J Haematol 1994 88: 318–324

    Article  CAS  PubMed  Google Scholar 

  28. Licht T, Aksentijevich I, Gottesman MM, Pastan I . Efficient expression of functional human MDR1 gene in murine bone marrow after retroviral transduction of purified hematopoietic stem cells Blood 1995 86: 111–121

    CAS  PubMed  Google Scholar 

  29. Ward M et al. Transfer and expression of the human multiple drug resistance gene in human CD34+ cells Blood 1994 84: 1408–1414

    CAS  PubMed  Google Scholar 

  30. Metz MZ, Best DM, Kane SE . Harvey murine sarcoma virus/MDR1 retroviral vectors: efficient virus production and foreign gene transduction using MDR1 as a selectable marker Virology 1995 208: 634–643

    Article  CAS  PubMed  Google Scholar 

  31. Bodine DM et al. Efficient retrovirus transduction of mouse pluripotent hematopoietic stem cells mobilized into the peripheral blood by treatment with granulocyte colony-stimulating factor and stem cell factor Blood 1994 84: 1482–1491

    CAS  PubMed  Google Scholar 

  32. Orlic D et al. The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction Proc Natl Acad Sci USA 1996 93: 11097–11102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Asch J et al. Retroviral gene transfer into cord blood stem/progenitor cells using purified vector stocks Am J Hematol 1998 57: 16–23

    Article  CAS  PubMed  Google Scholar 

  34. Marandin A et al. Retrovirus-mediated gene transfer into human CD34+38low primitive cells capable of reconstituting long-term cultures in vitro and nonobese diabetic-severe combined immunodeficiency mice in vivo Hum Gene Ther 1998 9: 1497–1511

    Article  CAS  PubMed  Google Scholar 

  35. Mazurier F et al. Rapid analysis and efficient selection of human transduced primitive hematopoietic cells using the humanized S65T green fluorescent protein Gene Therapy 1998 5: 556–562

    Article  CAS  PubMed  Google Scholar 

  36. Fruehauf S et al. Frequency analysis of multidrug resistance-1 gene transfer into human primitive hematopoietic progenitor cells using the cobblestone area-forming cell assay and detection of vector-mediated P-glycoprotein expression by rhodamine-123 Hum Gene Ther 1996 7: 1219–1231

    Article  CAS  PubMed  Google Scholar 

  37. Bertolini F et al. Engineered stromal layers and continuous flow culture enhance multidrug resistance gene transfer in hematopoietic progenitors Cancer Res 1996 56: 2566–2572

    CAS  PubMed  Google Scholar 

  38. Moritz T, Patel VP, Williams DA . Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors J Clin Invest 1994 93: 1451–1457

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hennemann B et al. Optimization of retroviral-mediated gene transfer to human NOD/SCID mouse repopulating cord blood cells through a systematic analysis of protocol variables Exp Hematol 1999 27: 817–825

    Article  CAS  PubMed  Google Scholar 

  40. Del Canizo C et al. Haematopoietic damage persists 1 year after autologous peripheral blood stem cell transplantation Bone Marrow Transplant 1999 23: 901–905

    Article  CAS  PubMed  Google Scholar 

  41. Mickisch GH et al. Chemotherapy and chemosensitization of transgenic mice which express the human multidrug resistance gene in bone marrow: efficacy, potency and toxicity Cancer Res 1991 51: 5417–5424

    CAS  PubMed  Google Scholar 

  42. Hanania EG, Deisseroth AB . Serial transplantation shows that early hematopoietic precursor cells are transduced by MDR-1 retroviral vector in a mouse gene therapy model Cancer Gene Ther 1994 1: 21–25

    CAS  PubMed  Google Scholar 

  43. Sokolic RA et al. A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression Blood 1996 87: 42–50

    CAS  PubMed  Google Scholar 

  44. Aran JM, Licht T, Gottesman MM, Pastan I . Complete restoration of glucocerebrosidase deficiency in Gaucher fibroblasts using a bicistronic MDR retrovirus and a new selection strategy Hum Gene Ther 1996 7: 2165–2175

    Article  CAS  PubMed  Google Scholar 

  45. Licht T et al. Retroviral transfer of human MDR1 gene to hematopoietic cells: effects of drug selection and of transcript splicing on expression of encoded P-glycoprotein Hum Gene Ther 1999 10: 2173–2185

    Article  CAS  PubMed  Google Scholar 

  46. Luens KM et al. Thrombopoietin, kit ligand, and flk2/flt3 ligand together induce increased numbers of primitive hematopoietic progenitors from human CD34+Thy-1+ Lin− cells with preserved ability to engraft SCID-hu bone Blood 1998 91: 1206–1215

    CAS  PubMed  Google Scholar 

  47. Murray L et al. Optimization of retroviral gene transduction of mobilized primitive hematopoietic progenitors by using thrombopoietin, Flt3, and Kit ligands and RetroNectin culture Hum Gene Ther 1999 10: 1743–1752

    Article  CAS  PubMed  Google Scholar 

  48. Piacibello W et al. Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34+ cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells Blood 1999 93: 3736–3749

    CAS  PubMed  Google Scholar 

  49. Schiedlmeier B et al. Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice Blood 2000 95: 1237–1248

    CAS  PubMed  Google Scholar 

  50. Wu T et al. Prolonged high-level detection of retrovirally marked hematopoietic cells in nonhuman primates after transduction of CD34+ progenitors using clinically feasible methods Mol Ther 2000 1: 285–293

    Article  CAS  PubMed  Google Scholar 

  51. Novelli EM et al. Ex vivo culture of cord blood CD34+ cells expands progenitor cell numbers, preserves engraftment capacity in nonobese diabetic/severe combined immunodeficient mice, and enhances retroviral transduction efficiency Hum Gene Ther 1999 10: 2927–2940

    Article  CAS  PubMed  Google Scholar 

  52. Dunbar CE et al. Improved retroviral gene transfer into murine and Rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor Proc Natl Acad Sci USA 1996 93: 11871–11876

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Hennemann B et al. Efficient retrovirus-mediated gene transfer to transplantable human bone marrow cells in the absence of fibronectin Blood 2000 96: 2432–2439

    CAS  PubMed  Google Scholar 

  54. Wognum AW et al. Stimulation of mouse bone marrow cells with kit ligand, FLT3 ligand, and thrombopoietin leads to efficient retrovirus-mediated gene transfer to stem cells, whereas interleukin 3 and interleukin 11 reduce transduction of short- and long-term repopulating cells Hum Gene Ther 2000 11: 2129–2141

    Article  CAS  PubMed  Google Scholar 

  55. Dao MA et al. FLT3 ligand preserves the ability of human CD34+ progenitors to sustain long-term hematopoiesis in immune-deficient mice after ex vivo retroviral-mediated transduction Blood 1997 89: 446–456

    CAS  PubMed  Google Scholar 

  56. Rosenzweig M et al. Efficient and durable gene marking of hematopoietic progenitor cells in nonhuman primates after nonablative conditioning Blood 1999 94: 2271–2286

    CAS  PubMed  Google Scholar 

  57. Barquinero J et al. Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice Blood 2000 95: 3085–3093

    CAS  PubMed  Google Scholar 

  58. Uchida N et al. Primitive human hematopoietic cells displaying differential efflux of the rhodamine-123 dye have distinct biological activities Blood 1996 88: 1297–1305

    CAS  PubMed  Google Scholar 

  59. Drach J et al. Expression of MDR1 by normal bone marrow cells and its implication for leukemic hematopoiesis Leuk Lymphoma 1995 16: 419–424

    Article  CAS  PubMed  Google Scholar 

  60. Udomsakdi C, Eaves C, Sutherland H, Lansdorp P . Separation of functionally distinct subpopulations of primitive human hematopoietic cells using rhodamine-123 Exp Hematol 1991 19: 338–342

    CAS  PubMed  Google Scholar 

  61. Pontvert-Delucq S et al. Characterization and functional analysis of adult human bone marrow cell subsets in relation to B-lymphoid development Blood 1993 82: 417–429

    CAS  PubMed  Google Scholar 

  62. Pastan I et al. A retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized expression of P-glycoprotein in MDCK cells Proc Natl Acad Sci USA 1988 85: 4486–4490

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Germann UA, Chin K-V, Pastan I, Gottesmann MM . Retroviral transfer of a chimeric multidrug resistance-adenosine deaminase gene FASEB J 1990 4: 1501–1507

    Article  CAS  PubMed  Google Scholar 

  64. Schrezenmeier H et al. Quantitative analysis of cobblestone area-forming cells in bone marrow of patients with aplastic anemia by limiting dilution assay Blood 1996 88: 4474–4480

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The retroviral MDR1 producer cell line was a generous gift from Dr MM Gottesman. Apheresis cells were kindly provided by Dr F Bertolini and Dr M Wiesneth. We wish to thank Dr MM Gottesman and Professor Dr C Peschel for critical reading of the manuscript, and Dr I Pastan and Dr F Bertolini for valuable suggestions and discussions. This work was in part supported by grants from Associazione Italiana Contro le Leucemie (AIL), Italy (to FB and PB), and from Bundesministerium für Forschung and Technologie (Verbund Klinische Pharmakologie Berlin-Brandenburg), Germany (to TL).

Author information

Author notes

  1. F Berger

    Present address: Paul-Ehrlich-Institut, Langen, Germany

  2. D Soligo and K Schwarz: These authors contributed equally to the work

Authors and Affiliations

  1. Fondazione Matarelli, Ospedale Fatebenefratelli e Oftalmico, Milan, Italy

    F Berger & P Bossolasco

  2. Bone Marrow Transplantation Unit, IRCCS Ospedale Maggiore, Milan, Italy

    D Soligo & G Lambertenghi Deliliers

  3. Technische Universität München, Klinikum rechts der Isar, III Medizinische Klinik, Munich, Germany

    K Schwarz & T Licht

  4. Universitätsklinikum Benjamin Franklin, Freie Universität, Berlin, Germany

    H Schrezenmeier

  5. Universitätsklinikum Ulm, Transfusionsmedizin, Ulm, Germany

    B Kubanek

  6. Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, MD, USA

    T Licht

Authors
  1. F Berger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D Soligo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K Schwarz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P Bossolasco
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. H Schrezenmeier
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. B Kubanek
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. G Lambertenghi Deliliers
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. T Licht
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Berger, F., Soligo, D., Schwarz, K. et al. Efficient retrovirus-mediated transduction of primitive human peripheral blood progenitor cells in stroma-free suspension culture. Gene Ther 8, 687–696 (2001). https://doi.org/10.1038/sj.gt.3301455

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3301455

Keywords

Search

Advanced search

Quick links