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:

Progenitor Cell Expansion

Ex vivo expansion of CD34-positive peripheral blood progenitor cells from patients with non-Hodgkin's lymphoma: no evidence of concomitant expansion of contaminating bcl2/JH-positive lymphoma cells

Bone Marrow Transplantation volume 26, pages 497–503 (2000)Cite this article

Abstract

The aim of the present study was to evaluate the capacity to expand of hematopoietic stem cell (HSC) samples from eight patients with NHL, and to follow in parallel the fate of tumor cells in four of eight samples still containing bcl2/JH+ tumor cells after CD34+ or CD19−/20−/34+ cell selection. The presence of bcl2/JH+ cells was also investigated after expansion in four of eight samples, two of which were bcl2/JH− at harvesting and two which were initially bcl2/JH+ but became bcl2/JH− (below the level of PCR detection) after cell selection, to assess a possible reappearance of occult tumor cells after expansion culture. We used culture conditions that we previously had established to allow high level expansion of normal precursors, progenitors and LTC-ICs. In this study, particular attention was given to the role of Flt3-ligand, known to favor the growth of B cells. The expansion conditions were: 1.5 × 103 cells/ml in serum-free medium containing stem cell factor (SCF), interleukin-3 (IL-3), IL-6, granulocyte-stimulating factor (G-CSF), erythropoietin (Epo) ± Flt3-ligand (Flt3-L) for 10 days. After culture, total cells, CFU-GMs, BFU-Es and LTC-ICs were expanded to a mean of 833-, 6.6-, 4.6-, and 1.8-fold, respectively with the cocktail of cytokines not including Flt3-L. When Flt3-L was added, the mean expansion values were 1095-, 31-, 15- and three-fold, respectively. Residual bcl2/JH+ cells present in four of eight samples before expansion were not detected after expansion. Similarly, no tumor cells reappeared after expansion of the two samples which had become negative after selection, as well as in the two samples which were bcl2/JH− at harvesting. These results suggest first that ex vivo expansion of hematopoietic stem cells in patients with non-Hodgkin's lymphoma is feasible without incurring the parallel risk of amplifying tumor cells; second, that Flt3-L did not stimulate the growth of tumor cells while it clearly favored the growth of normal progenitors. Bone Marrow Transplantation (2000) 26, 497–503.

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

Similar content being viewed by others

References

  1. Chang J, Coutinho L, Morgenstern G et al. Reconstitution of haemopoietic system with autologous marrow taken during relapse and grown in long-term culture Lancet 1986 i: 294–295

    Article  Google Scholar 

  2. Brugger W, Heimfeld S, Berenson RJ et al. Reconstitution of hematopoiesis after high-dose chemotherapy by autologous progenitor cells generated ex vivo New Engl J Med 1995 333: 283–287

    Article  CAS  PubMed  Google Scholar 

  3. Williams SF, Lee WJ, Bender JG et al. Selection and expansion of peripheral blood CD34+ cells in autologous stem cell transplantation for breast cancer Blood 1996 87: 1687–1691

    CAS  PubMed  Google Scholar 

  4. Poloni A, Giarratana MC, Hussein F et al. The ex vivo expansion capacity of normal human bone marrow cells is dependent on experimental conditions: role of the cell concentration, serum and CD34+ cell selection in stroma-free cultures Hematol Cell Ther 1997 39: 49–58

    Article  CAS  PubMed  Google Scholar 

  5. Reiffers J, Caillot C, Dazey B et al. Abrogation of post-myeloablative chemotherapy neutropenia by ex vivo expanded autologous CD34 positive cells Lancet 1999 354: 1092–1093

    Article  CAS  PubMed  Google Scholar 

  6. Bachier CR, Gokmen E, Teale J et al. Ex vivo expansion of bone marrow progenitor cells for hematopoietic reconstitution following high dose chemotherapy for breast cancer Exp Hematol 1999 27: 615–623

    Article  CAS  PubMed  Google Scholar 

  7. Tisdale JF, Hanazono Y, Sellers SE 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 

  8. Lopez M, Lemoine FM, Firat H et al. Bone marrow vs peripheral blood progenitor cells CD34 selection in patients with non-Hodgkin's lymphoma: different levels of tumour cell reduction. Implications for autografting Blood 1997 90: 2830–2838

    CAS  PubMed  Google Scholar 

  9. Di Nicola M, Siena S, Corradini P et al. Elimination of bcl-2-IgH-positive follicular lymphoma cells from blood transplants with high recovery of hematopoietic progenitors by the Miltenyi CD34+ cell sorting system Bone Marrow Transplant 1996 18: 1117–1121

    CAS  PubMed  Google Scholar 

  10. Gorin NC, Lopez M, Laporte JP et al. Preparation and successful engraftment of purified CD34+ bone marrow progenitor cells in patients with non-Hodgkin's lymphoma Blood 1995 85: 1647–1654

    CAS  PubMed  Google Scholar 

  11. Paulus U, Schmitz N, Viehmann K et al. Combined positive/negative selection for highly effective purging of PBPC grafts: towards clinical application in patients with B-CLL Bone Marrow Transplant 1997 20: 415–420

    Article  CAS  PubMed  Google Scholar 

  12. Cleary ML, Smith SD, Sklar J . Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation Cell 1986 47: 19–28

    Article  CAS  PubMed  Google Scholar 

  13. Philip T, Armitage JO, Spitzer G et al. High-dose therapy and autologous bone marrow transplantation after failure of conventional chemotherapy in adults with intermediate grade or high-grade non-Hodgkin's lymphoma New Engl J Med 1987 3: 1493–1498

    Article  Google Scholar 

  14. Fouillard L, Laporte JP, Labopin M et al. Autologous stem-cell transplantation for non-Hodgkin's lymphomas: the role of graft purging and radiotherapy post-transplantation: results of a retrospective analysis on 120 patients autografted in a single institution J Clin Oncol 1998 16: 2803–2816

    Article  CAS  PubMed  Google Scholar 

  15. Brenner MK, Rill DR, Moen RC et al. Gene-marking to trace origin of relapse after autologous bone-marrow transplantation Lancet 1993 341: 85–86

    Article  CAS  PubMed  Google Scholar 

  16. Deisseroth AB, Zu Z, Claxton D et al. Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML Blood 1994 83: 3068–3076

    CAS  PubMed  Google Scholar 

  17. Rill DR, Santana VM, Roberts WM et al. Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells Blood 1994 84: 380–383

    CAS  PubMed  Google Scholar 

  18. Gribben JG, Freedman AS, Neuberg D et al. Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B cell lymphoma New Engl J Med 1991 325: 1525–1533

    Article  CAS  PubMed  Google Scholar 

  19. Ross AA, Cooper BW, Lazarus HM et al. Detection and viability of tumour cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques Blood 1993 82: 2605–2610

    CAS  PubMed  Google Scholar 

  20. Lemoli R, Fortuna A, Motta MR et al. Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumour cells Blood 1996 87: 1625–1634

    CAS  PubMed  Google Scholar 

  21. Brugger W, Bross KJ, Glatt M et al. Mobilization of tumour cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors Blood 1994 83: 636–640

    CAS  PubMed  Google Scholar 

  22. Kobari L, Giarratana MC, Poloni A et al. Flt3 ligand, MGDF, Epo and G-CSF enhance ex vivo expansion of hematopoietic cell compartments in the presence of SCF, IL-3 and IL-6 Bone Marrow Transplant 1998 21: 759–767

    Article  CAS  PubMed  Google Scholar 

  23. Hirayama F, Lyman SD, Clark SC, Ogawa M . The flt3 ligand supports proliferation of lymphohematopoietic progenitors and early B lymphoid progenitors Blood 1995 85: 1762–1768

    CAS  PubMed  Google Scholar 

  24. Hunte BE, Hudak S, Campbell D et al. Flk2/flt3 ligand is a potent cofactor for the growth of primitive B cell progenitors J Immunol 1996 156: 489–496

    CAS  PubMed  Google Scholar 

  25. Eaves CJ, Eaves AC . Erythropoiesis. In: Greder DW, Takaku F (eds) Hemopoietic Stem Cells Marcel Dekker: New York 1985 p19

    Google Scholar 

  26. Douay L, Giarratana MC, Labopin M et al. Characterisation of late and early hematopoietic progenitor/stem cell sensitivity to mafosfamide Bone Marrow Transplant 1995 15: 769–775

    CAS  PubMed  Google Scholar 

  27. Sutherland HJ, Lansdorp PM, Henkelman DH et al. Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers Proc Natl Acad Sci USA 1990 87: 3584–3588

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Straetmans N, Herman PH, Van Bockstaele D et al. Haematopoietic defect and decreased expansion potential of bone marow autografts from patients with acute myeloid leukemia in first remission Br J Haematol 1998 101: 571–581

    Article  CAS  PubMed  Google Scholar 

  29. Douay L, Kobari L, Pflumio F et al. Conservation of the potential of primitive hematopoietic cells obtained by amplification of CD34+ cells from cord blood Blood 1999 94: 606a

    Google Scholar 

  30. Gribben JG, Freedman AS, Woo SD et al. All advanced stage non-Hodgkin's lymphomas with a polymerase chain reaction amplifiable breakpoint of bcl-2 have residual cells containing the bcl-2 rearrangement at evaluation and after treatment Blood 1991 78: 3275–3280

    CAS  PubMed  Google Scholar 

  31. Léonard BM, Hétu F, Busque L et al. Lymphoma cell burden in progenitor cell grafts measured by competitive polymerase chain reaction: less than one log difference between bone marrow and peripheral blood sources Blood 1998 91: 331–339

    PubMed  Google Scholar 

  32. Berenson R, Andrew R, Bensinger W et al. Selection of CD34+ marrow cells for autologous marrow transplantation Hematol Oncol Ann 1994 2: 78

    Google Scholar 

  33. Macintyre EA, Belanger C, Debert C et al. Detection of clonal CD34+ 19+ progenitors in bone marrow of bcl2-IgH-positive follicular lymphoma patients Blood 1995 86: 4691–4698

    CAS  PubMed  Google Scholar 

  34. Voso MT, Hohaus S, Moos M, Haas R . Lack of t(14;18) polymerase chain reaction-positive cells in highly purified CD34+ cells and their CD19 subsets in patients with follicular lymphoma Blood 1997 89: 3763–3768

    CAS  PubMed  Google Scholar 

  35. Widmer L, Pichert G, Jost LM, Stahel RF . Fate of contaminating t(14;18)+ lymphoma cells during ex vivo expansion of CD34-selected hematopoietic progenitor cells Blood 1996 88: 3166–3175

    CAS  PubMed  Google Scholar 

  36. Vogel W, Behringer D, Scheding S et al. Ex vivo expansion of CD34+ peripheral blood progenitor cells: implications for expansion of contaminating epithelial tumour cells Blood 1996 88: 2707–2713

    CAS  PubMed  Google Scholar 

  37. Lundell BI, Vredenburgh JJ, DeSombre K, Smith AK . Ex vivo expansion of bone marrow from breast cancer patients: reduction in tumor content through passive purging Bone Marrow Transplant 1998 22: 153–159

    Article  CAS  PubMed  Google Scholar 

  38. Van Riet I, Juge-Morineau N, Schots R et al. Persistence of residual tumour cells after cytokine-mediated ex vivo expansion of mobilized CD34+ blood cells in multiple myeloma Br J Haematol 1997 96: 403–411

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant from the Ligue Nationale Contre le Cancer.

Author information

Authors and Affiliations

  1. Inserm U76, Paris, France

    Ming Yao & M Lopez

  2. Département d'Hématologie et Laboratoire de Thérapie Cellulaire et de Radioprotection Accidentelle, Faculté de Médecine St-Antoine, Paris, France

    Ming Yao, L Fouillard, S Bouchet, NC Gorin & M Lopez

  3. CERVI-CNRS-ESA 7087, Groupe Hospitalier Pitié-Salpétrière, Paris, France

    FM Lemoine

  4. Service d'Hématologie Biologique, Hôpital Trousseau, Paris, France

    H Firat & L Douay

  5. ETS AP-HP, Site St Antoine, Paris, France

    G Andreu

  6. Inserm U417, Hôpital Saint-Antoine, Paris, France

    L Douay

Authors
  1. Ming Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. L Fouillard
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. FM Lemoine
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. S Bouchet
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. H Firat
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. G Andreu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. NC Gorin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. L Douay
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. M Lopez
    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

Yao, M., Fouillard, L., Lemoine, F. et al. Ex vivo expansion of CD34-positive peripheral blood progenitor cells from patients with non-Hodgkin's lymphoma: no evidence of concomitant expansion of contaminating bcl2/JH-positive lymphoma cells. Bone Marrow Transplant 26, 497–503 (2000). https://doi.org/10.1038/sj.bmt.1702539

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.bmt.1702539

Keywords

This article is cited by

Search

Advanced search

Quick links