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:

In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase

Gene Therapy volume 17, pages 238–249 (2010)Cite this article

Abstract

Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug-resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase the engraftment of gene-modified, hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR–GFP-expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rγcnull (NSG) mice after injection of Tyr22-DHFR-hESC-derived cells significantly increases human CD34+ and CD45+ cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR cells in vivo, and provides a novel approach for combined human cell and gene therapy.

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

Similar content being viewed by others

References

  1. Halene S, Kohn DB . Gene therapy using hematopoietic stem cells: Sisyphus approaches the crest. Hum Gene Ther 2000; 11: 1259–1267.

    Article  CAS  Google Scholar 

  2. 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  Google Scholar 

  3. Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003; 348: 255–256.

    Article  Google Scholar 

  4. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302: 415–419.

    Article  CAS  Google Scholar 

  5. Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002; 296: 2410–2413.

    Article  CAS  Google Scholar 

  6. Gerull S, Beard BC, Peterson LJ, Neff T, Kiem HP . In vivo selection and chemoprotection after drug resistance gene therapy in a nonmyeloablative allogeneic transplantation setting in dogs. Hum Gene Ther 2007; 18: 451–456.

    Article  CAS  Google Scholar 

  7. Neff T, Horn PA, Peterson LJ, Thomasson BM, Thompson J, Williams DA et al. Methylguanine methyltransferase-mediated in vivo selection and chemoprotection of allogeneic stem cells in a large-animal model. J Clin Invest 2003; 112: 1581–1588.

    Article  CAS  Google Scholar 

  8. Cornetta K, Croop J, Dropcho E, Abonour R, Kieran MW, Kreissman S et al. A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase. Cancer Gene Ther 2006; 13: 886–895.

    Article  CAS  Google Scholar 

  9. Nash RA, Antin JH, Karanes C, Fay JW, Avalos BR, Yeager AM et al. Phase 3 study comparing methotrexate and tacrolimus with methotrexate and cyclosporine for prophylaxis of acute graft-versus-host disease after marrow transplantation from unrelated donors. Blood 2000; 96: 2062–2068.

    CAS  PubMed  Google Scholar 

  10. Ratanatharathorn V, Nash RA, Przepiorka D, Devine SM, Klein JL, Weisdorf D et al. Phase III study comparing methotrexate and tacrolimus (prograf, FK506) with methotrexate and cyclosporine for graft-versus-host disease prophylaxis after HLA-identical sibling bone marrow transplantation. Blood 1998; 92: 2303–2314.

    CAS  PubMed  Google Scholar 

  11. Bertino JR . Karnofsky memorial lecture. Ode to methotrexate. J Clin Oncol 1993; 11: 5–14.

    Article  CAS  Google Scholar 

  12. Gori JL, Podetz-Pedersen K, Swanson D, Karlen AD, Gunther R, Somia NV et al. Protection of mice from methotrexate toxicity by ex vivo transduction using lentivirus vectors expressing drug-resistant dihydrofolate reductase. J Pharmacol Exp Ther 2007; 322: 989–997.

    Article  CAS  Google Scholar 

  13. Allay JA, Galipeau J, Blakley RL, Sorrentino BP . Retroviral vectors containing a variant dihydrofolate reductase gene for drug protection and in vivo selection of hematopoietic cells. Stem Cells 1998; 16 (Suppl 1): 223–233.

    PubMed  Google Scholar 

  14. Persons DA, Allay JA, Bonifacino A, Lu T, Agricola B, Metzger ME et al. Transient in vivo selection of transduced peripheral blood cells using antifolate drug selection in rhesus macaques that received transplants with hematopoietic stem cells expressing dihydrofolate reductase vectors. Blood 2004; 103: 796–803.

    Article  CAS  Google Scholar 

  15. Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson JA . Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 2001; 98: 10716–10721.

    Article  CAS  Google Scholar 

  16. Tian X, Morris JK, Linehan JL, Kaufman DS . Cytokine requirements differ for stroma and embryoid body-mediated hematopoiesis from human embryonic stem cells. Exp Hematol 2004; 32: 1000–1009.

    Article  CAS  Google Scholar 

  17. Tian X, Kaufman DS . Hematopoietic development of human embryonic stem cells in culture. Methods Mol Biol 2008; 430: 119–133.

    Article  CAS  Google Scholar 

  18. Vodyanik MA, Bork JA, Thomson JA, Slukvin II . Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood 2005; 105: 617–626.

    Article  CAS  Google Scholar 

  19. Qiu C, Olivier EN, Velho M, Bouhassira EE . Globin switches in yolk sac-like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells. Blood 2008; 111: 2400–2408.

    Article  CAS  Google Scholar 

  20. Chadwick K, Wang L, Li L, Menendez P, Murdoch B, Rouleau A et al. Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood 2003; 102: 906–915.

    Article  CAS  Google Scholar 

  21. Woll PS, Martin CH, Miller JS, Kaufman DS . Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity. J Immunol 2005; 175: 5095–5103.

    Article  CAS  Google Scholar 

  22. Woll PS, Grzywacz B, Tian X, Marcus RK, Knorr DA, Verneris MR et al. Human embryonic stem cells differentiatiate into a homogeneous population of natural killer cells with highly potent in vivo antitumor activity. Blood 2009; 113: 6094–6101.

    Article  CAS  Google Scholar 

  23. Ma F, Ebihara Y, Umeda K, Sakai H, Hanada S, Zhang H et al. Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis. Proc Natl Acad Sci USA 2008; 105: 13087–13092.

    Article  CAS  Google Scholar 

  24. Qiu C, Hanson E, Olivier E, Inada M, Kaufman DS, Gupta S et al. Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver cells recapitulates the globin switch that occurs early in development. Exp Hematol 2005; 33: 1450–1458.

    Article  CAS  Google Scholar 

  25. Lu SJ, Feng Q, Park JS, Vida L, Lee BS, Strausbauch M et al. Biologic properties and enucleation of red blood cells from human embryonic stem cells. Blood 2008; 112: 4475–4484.

    Article  CAS  Google Scholar 

  26. Ledran MH, Krassowska A, Armstrong L, Dimmick I, Renstrom J, Lang R et al. Efficient hematopoietic differentiation of human embryonic stem cells on stromal cells derived from hematopoietic niches. Cell Stem Cell 2008; 3: 85–98.

    Article  CAS  Google Scholar 

  27. Tian X, Woll PS, Morris JK, Linehan JL, Kaufman DS . Hematopoietic engraftment of human embryonic stem cell-derived cells is regulated by recipient innate immunity. Stem Cells 2007; 24: 1370–1380.

    Article  Google Scholar 

  28. Wang L, Menendez P, Shojaei F, Li L, Mazurier F, Dick JE et al. Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression. J Exp Med 2005; 201: 1603–1614.

    Article  CAS  Google Scholar 

  29. Narayan AD, Chase JL, Lewis RL, Tian X, Kaufman DS, Thomson JA et al. Human embryonic stem cell-derived hematopoietic cells are capable of engrafting primary as well as secondary fetal sheep recipients. Blood 2006; 107: 2180–2183.

    Article  CAS  Google Scholar 

  30. Pfeifer A, Ikawa M, Dayn Y, Verma IM . Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc Natl Acad Sci USA 2002; 99: 2140–2145.

    Article  CAS  Google Scholar 

  31. Wilber A, Linehan JL, Tian X, Woll PS, Morris JK, Belur LR et al. Efficient and stable transgene expression in human embryonic stem cells using transposon-mediated gene transfer. Stem Cells 2007; 25: 2919–2927.

    Article  CAS  Google Scholar 

  32. Giudice A, Trounson A . Genetic modification of human embryonic stem cells for derivation of target cells. Cell Stem Cell 2008; 2: 422–433.

    Article  CAS  Google Scholar 

  33. Ben-Dor I, Itsykson P, Goldenberg D, Galun E, Reubinoff BE . Lentiviral vectors harboring a dual-gene system allow high and homogeneous transgene expression in selected polyclonal human embryonic stem cells. Mol Ther 2006; 14: 255–267.

    Article  CAS  Google Scholar 

  34. Xu XQ, Zweigerdt R, Soo SY, Ngoh ZX, Tham SC, Wang ST et al. Highly enriched cardiomyocytes from human embryonic stem cells. Cytotherapy 2008; 10: 376–389.

    Article  Google Scholar 

  35. Gallo P, Grimaldi S, Latronico MV, Bonci D, Pagliuca A, Gallo P et al. A lentiviral vector with a short troponin-I promoter for tracking cardiomyocyte differentiation of human embryonic stem cells. Gene Therapy 2008; 15: 161–170.

    Article  CAS  Google Scholar 

  36. Davis RP, Ng ES, Costa M, Mossman AK, Sourris K, Elefanty AG et al. Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors. Blood 2008; 111: 1876–1884.

    Article  CAS  Google Scholar 

  37. Huber I, Itzhaki I, Caspi O, Arbel G, Tzukerman M, Gepstein A et al. Identification and selection of cardiomyocytes during human embryonic stem cell differentiation. FASEB J 2007; 21: 2551–2563.

    Article  CAS  Google Scholar 

  38. Lavon N, Yanuka O, Benvenisty N . The effect of overexpression of Pdx1 and Foxa2 on the differentiation of human embryonic stem cells into pancreatic cells. Stem Cells 2006; 24: 1923–1930.

    Article  CAS  Google Scholar 

  39. Zwaka TP, Thomson JA . Homologous recombination in human embryonic stem cells. Nat Biotechnol 2003; 21: 319–321.

    Article  CAS  Google Scholar 

  40. Agarwal S, Nikolai B, Yamaguchi T, Lech P, Somia NV . Construction and use of retroviral vectors encoding the toxic gene barnase. Mol Ther 2006; 14: 555–563.

    Article  CAS  Google Scholar 

  41. Hill KL, Kaufman DS . Hematopoietic differentiation of human embryonic stem cells by cocultivation with stromal layers. Curr Prot Stem Cell Biol 2008; Chapter 1: Unit 1F 6.

  42. Woll PS, Morris JK, Painschab MS, Marcus RK, Kohn AD, Biechele TL et al. Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells. Blood 2008; 111: 122–131.

    Article  CAS  Google Scholar 

  43. Zambidis ET, Park TS, Yu W, Tam A, Levine M, Yuan X et al. Expression of ACE (CD143) identifies and regulates primitive hemangioblasts derived from human pluripotent stem cells. Blood 2008; 112: 3601–3614.

    Article  CAS  Google Scholar 

  44. Warlick CA, Sweeney CL, McIvor RS . Maintenance of differential methotrexate toxicity between cells expressing drug-resistant and wild-type dihydrofolate reductase activities in the presence of nucleosides through nucleoside transport inhibition. Biochem Pharmacol 2000; 59: 141–151.

    Article  CAS  Google Scholar 

  45. Allay JA, Persons DA, Galipeau J, Riberdy JM, Ashmun RA, Blakley RL et al. In vivo selection of retrovirally transduced hematopoietic stem cells. Nat Med 1998; 4: 1136–1143.

    Article  CAS  Google Scholar 

  46. Allay JA, Spencer HT, Wilkinson SL, Belt JA, Blakley RL, Sorrentino BP . Sensitization of hematopoietic stem and progenitor cells to trimetrexate using nucleoside transport inhibitors. Blood 1997; 90: 3546–3554.

    CAS  PubMed  Google Scholar 

  47. Neff T, Beard BC, Peterson LJ, Anandakumar P, Thompson J, Kiem HP . Polyclonal chemoprotection against temozolomide in a large-animal model of drug resistance gene therapy. Blood 2005; 105: 997–1002.

    Article  CAS  Google Scholar 

  48. Kreklau EL, Pollok KE, Bailey BJ, Liu N, Hartwell JR, Williams DA . Hematopoietic expression of O6-methylguanine DNA methly-transferase-P140K allows intenstive treatment of human glioma xenografts with combination O6-benzylguanine and 1,3-bis-(2-chloroethyl)-1-nitrosourea. Mol Cancer Ther 2003; 2: 1321–1329.

    CAS  Google Scholar 

  49. Budak-Alpdogan T, Alpdogan O, Banerjee D, Wang E, Moore MA, Bertino JR . Methotrexate and cytarabine inhibit progression of human lymphoma in NOD/SCID mice carrying a mutant dihydrofolate reductase and cytidine deaminase fusion gene. Mol Ther 2004; 10: 574–584.

    Article  CAS  Google Scholar 

  50. Pollok KE, Hartwell JR, Braber A, Cooper RJ, Jansen M, Ragg S et al. In vivo selection of human hematopoietic cells in a xenograft model using combined pharmacologic and genetic manipulations. Hum Gene Ther 2003; 14: 1703–1714.

    Article  CAS  Google Scholar 

  51. Cai S, Hartwell JR, Cooper RJ, Juliar BE, Kreklau E, Abonour R et al. In vivo effects of myeloablative alkylator therapy on survival and differentiation of MGMTP140K-transduced human G-CSF-mobilized peripheral blood cells. Mol Ther 2006; 13: 1016–1026.

    Article  CAS  Google Scholar 

  52. Ng ES, Davis RP, Azzola L, Stanley EG, Elefanty AG . Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood 2005; 106: 1601–1603.

    Article  CAS  Google Scholar 

  53. Tiscornia G, Singer O, Verma IM . Production and purification of lentiviral vectors. Nat Prot 2006; 1: 241–245.

    Article  CAS  Google Scholar 

  54. Carr F, Medina WD, Dube S, Bertino JR . Genetic transformation of murine bone marrow cells to methotrexate resistance. Blood 1983; 62: 180–185.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by National Institutes of Health Grants CA060803 (RSM), HL77923 (DSK) and CA34196 (LDS). We thank Andrea Mohs, Melinda Hexum, Katie Hill, Julie Morris, Jeremy Allred and Jon Linehan for assistance with the studies.

Author information

Authors and Affiliations

  1. Department of Genetics, Gene Therapy Program, Cell Biology and Development, Institute of Human Genetics, University of Minnesota, Minneapolis, MN, USA

    J L Gori, D Swanson & R S McIvor

  2. Stem Cell Institute and Department of Medicine, University of Minnesota, Minneapolis, MN, USA

    X Tian & D S Kaufman

  3. Research Animal Resources, University of Minnesota, Minneapolis, MN, USA

    R Gunther

  4. The Jackson Laboratory, Bar Harbor, ME, USA

    L D Shultz

Authors
  1. J L Gori
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. X Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. D Swanson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R Gunther
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. L D Shultz
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. R S McIvor
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. D S Kaufman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D S Kaufman.

Additional information

Presented in oral abstract form at the 11th Annual Meeting of the American Society of Gene Therapy, Boston, MA, May 31–June 1, 2008.

Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gori, J., Tian, X., Swanson, D. et al. In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase. Gene Ther 17, 238–249 (2010). https://doi.org/10.1038/gt.2009.131

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2009.131

Keywords

Search

Advanced search

Quick links