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.

  • Article
  • Published:

Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells

Abstract

We describe here the large-scale ex vivo production of mature human red blood cells (RBCs) from hematopoietic stem cells of diverse origins. By mimicking the marrow microenvironment through the application of cytokines and coculture on stromal cells, we coupled substantial amplification of CD34+ stem cells (up to 1.95 × 106-fold) with 100% terminal differentiation into fully mature, functional RBCs. These cells survived in nonobese diabetic/severe combined immunodeficient mice, as do native RBCs. Our system for producing 'cultured RBCs' lends itself to a fundamental analysis of erythropoiesis and provides a simple in vitro model for studying important human viral or parasitic infections that target erythroid cells. Further development of large-scale production of cultured RBCs will have implications for gene therapy, blood transfusion and tropical medicine.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Large-scale amplification of erythroid cells.
Figure 2: Maturation of reticulocytes into RBCs.
Figure 3: Confocal microscopy images.
Figure 4: Functionality of cultured RBCs.
Figure 5: Flow cytometry of the CFSE-labeled cRBCs in the NOD/SCID mouse model.

Similar content being viewed by others

References

  1. Bessis, M. Erythroblastic island, functional unity of bone marrow. Rev. Hematol. 13, 8–11 (1958).

    CAS  PubMed  Google Scholar 

  2. Lichtman, M.A. The ultrastructure of the hemopoietic environment of the marrow: a review. Exp. Hematol. 9, 391–410 (1981).

    CAS  PubMed  Google Scholar 

  3. Qiu, L.B., Dickson, H., Hajibagheri, N. & Crocker, P.R. Extruded erythroblast nuclei are bound and phagocytosed by a novel macrophage receptor. Blood 85, 1630–1639 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. Gregory, C.J. & Eaves, A.C. Three stages of erythropoietic progenitor cell differentiation distinguished by a number of physical and biologic properties. Blood 51, 527–537 (1978).

    Article  CAS  PubMed  Google Scholar 

  5. Metcalf, D. Hematopoietic regulators: redundancy or subtlety? Blood 82, 3515–3523 (1993).

    Article  CAS  PubMed  Google Scholar 

  6. Freyssinier, J.M. et al. Purification, amplification and characterization of a population of human erythroid progenitors. Br. J. Haematol. 106, 912–922 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Panzenbock, B., Bartunek, P., Mapara, M.Y. & Zenke, M. Growth and differentiation of human stem cell factor/erythropoietin-dependent erythroid progenitor cells in vitro. Blood 92, 3658–3668 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Fibach, E., Manor, D., Oppenheim, A. & Rachmilewitz, E.A. Proliferation and maturation of human erythroid progenitors in liquid culture. Blood 73, 100–103 (1989).

    Article  CAS  PubMed  Google Scholar 

  9. Wada, H. et al. Expression of major blood group antigens on human erythroid cells in a two-phase liquid culture system. Blood 75, 505–511 (1990).

    Article  CAS  PubMed  Google Scholar 

  10. Sui, X. et al. Erythropoietin-independent erythrocyte production: signals through gp130 and c-kit dramatically promote erythropoiesis from human CD34+ cells. J. Exp. Med. 183, 837–845 (1996).

    Article  CAS  PubMed  Google Scholar 

  11. von Lindern, M. et al. The glucocorticoid receptor cooperates with the erythropoietin receptor and c-Kit to enhance and sustain proliferation of erythroid progenitors in vitro. Blood 94, 550–559 (1999).

    Article  CAS  PubMed  Google Scholar 

  12. Malik, P. et al. An in vitro model of human red blood cell production from hematopoietic progenitor cells. Blood 91, 2664–2671 (1998).

    Article  CAS  PubMed  Google Scholar 

  13. Carlile, G.W., Smith, D.H. & Wiedmann, M. Caspase-3 has a nonapoptotic function in erythroid maturation. Blood 103, 4310–4316 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Migliaccio, G. et al. In vitro mass production of human erythroid cells from the blood of normal donors and of thalassemic patients. Blood Cells Mol. Dis. 28, 169–180 (2002).

    Article  PubMed  Google Scholar 

  15. Lyons, B.L. et al. Mechanisms of anemia in SHP-1 protein tyrosine phosphatase-deficient “viable motheaten” mice. Exp. Hematol. 31, 234–243 (2003).

    Article  CAS  PubMed  Google Scholar 

  16. Terstappen, L.W. et al. Multidimensional flow cytometric blood cell differentiation without erythrocyte lysis. Blood Cells 17, 585–602; discussion 603–605 (1991).

    CAS  PubMed  Google Scholar 

  17. Loeuillet, C. et al. Distinct hematopoietic support by two human stromal cell lines. Exp. Hematol. 29, 736–745 (2001).

    Article  CAS  PubMed  Google Scholar 

  18. Suzuki, J., Fujita, J., Taniguchi, S., Sugimoto, K. & Mori, K.J. Characterization of murine hemopoietic-supportive (MS-1 and MS-5) and non-supportive (MS-K) cell lines. Leukemia 6, 452–458 (1992).

    CAS  PubMed  Google Scholar 

  19. Miller, A.D. & Chen, F. Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry. J. Virol. 70, 5564–5571 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Jansen, G., Koenderman, L., Rijksen, G., Cats, B.P. & Staal, G.E. Characteristics of hexokinase, pyruvate kinase, and glucose-6-phosphate dehydrogenase during adult and neonatal reticulocyte maturation. Am. J. Hematol. 20, 203–215 (1985).

    Article  CAS  PubMed  Google Scholar 

  21. Cynober, T., Mohandas, N. & Tchernia, G. Red cell abnormalities in hereditary spherocytosis: relevance to diagnosis and understanding of the variable expression of clinical severity. J. Lab. Clin. Med. 128, 259–269 (1996).

    Article  CAS  PubMed  Google Scholar 

  22. Neildez-Nguyen, T.M. et al. Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo. Nat. Biotechnol. 20, 467–472 (2002).

    Article  CAS  PubMed  Google Scholar 

  23. Lemischka, I.R. Microenvironmental regulation of hematopoietic stem cells. Stem Cells (Suppl. 1) 15, 63–68 (1997).

    Article  PubMed  Google Scholar 

  24. Koller, M.R., Oxender, M., Jensen, T.C., Goltry, K.L. & Smith, A.K. Direct contact between CD34+lin– cells and stroma induces a soluble activity that specifically increases primitive hematopoietic cell production. Exp. Hematol. 27, 734–741 (1999).

    Article  CAS  PubMed  Google Scholar 

  25. Friedenstein, A.J. et al. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp. Hematol. 2, 83–92 (1974).

    CAS  PubMed  Google Scholar 

  26. Ogawa, M. Differentiation and proliferation of hematopoietic stem cells. Blood 81, 2844–2853 (1993).

    Article  CAS  PubMed  Google Scholar 

  27. Verfaillie, C.M. Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation. Blood 82, 2045–2053 (1993).

    Article  CAS  PubMed  Google Scholar 

  28. Hanspal, M. & Hanspal, J.S. The association of erythroblasts with macrophages promotes erythroid proliferation and maturation: a 30-kD heparin-binding protein is involved in this contact. Blood 84, 3494–3504 (1994).

    Article  CAS  PubMed  Google Scholar 

  29. Hanspal, M., Smockova, Y. & Uong, Q. Molecular identification and functional characterization of a novel protein that mediates the attachment of erythroblasts to macrophages. Blood 92, 2940–2950 (1998).

    Article  CAS  PubMed  Google Scholar 

  30. Ohneda, O. & Bautch, V.L. Murine endothelial cells support fetal liver erythropoiesis and myelopoiesis via distinct interactions. Br. J. Haematol. 98, 798–808 (1997).

    Article  CAS  PubMed  Google Scholar 

  31. Yanai, N., Sato, Y. & Obinata, M. A new type-II membrane protein in erythropoietic organs enhances erythropoiesis. Leukemia (Suppl. 3) 11, 484–485 (1997).

    PubMed  Google Scholar 

  32. Loeuillet, C., Douay, L., Herve, P. & Chalmers, D.E. Preservation of the myofibroblastic phenotype of human papilloma virus 16 E6/E7 immortalized human bone marrow cells using the lineage limited alpha-smooth muscle actin promoter. Cell Growth Differ. 12, 233–242 (2001).

    CAS  PubMed  Google Scholar 

  33. Bianchi, G. et al. Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Exp. Cell Res. 287, 98–105 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Moore, J.M., Kumar, N., Shultz, L.D. & Rajan, T.V. Maintenance of the human malarial parasite, Plasmodium falciparum, in scid mice and transmission of gametocytes to mosquitoes. J. Exp. Med. 181, 2265–2270 (1995).

    Article  CAS  PubMed  Google Scholar 

  35. Morita, E., Nakashima, A., Asao, H., Sato, H. & Sugamura, K. Human parvovirus B19 nonstructural protein (NS1) induces cell cycle arrest at G(1) phase. J. Virol. 77, 2915–2921 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Carotta, S. et al. Directed differentiation and mass cultivation of pure erythroid progenitors from mouse embryonic stem cells. Blood 104, 1873–1880 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. Koller, M.R. et al. Clinical-scale human umbilical cord blood cell expansion in a novel automated perfusion culture system. Bone Marrow Transplant. 21, 653–663 (1998).

    Article  CAS  PubMed  Google Scholar 

  38. Brott, D.A., Maher, R.J., Parrish, C.R., Richardson, R.J. & Smith, A.K. Flow cytometric characterization of perfused human bone marrow cultures: identification of the major cell lineages and correlation with the CFU-GM assay. Cytometry 53A, 22–27 (2003).

    Article  Google Scholar 

  39. Mendonca, R.Z., Arrozio, S.J., Antoniazzi, M.M., Ferreira, J.M. Jr. & Pereira, C.A. Metabolic active-high density VERO cell cultures on microcarriers following apoptosis prevention by galactose/glutamine feeding. J. Biotechnol. 97, 13–22 (2002).

    Article  CAS  PubMed  Google Scholar 

  40. Douay, L. Experimental culture conditions are critical for ex vivo expansion of hematopoietic cells. J. Hematother. Stem Cell Res. 10, 341–346 (2001).

    Article  CAS  PubMed  Google Scholar 

  41. Kobari, L. et al. In vitro and in vivo evidence for the long-term multilineage (myeloid, B, NK, and T) reconstitution capacity of ex vivo expanded human CD34(+) cord blood cells. Exp. Hematol. 28, 1470–1480 (2000).

    Article  CAS  PubMed  Google Scholar 

  42. Giarratana, M.C. et al. Presence of primitive lymphoid progenitors with NK or B potential in ex vivo expanded bone marrow cell cultures. Exp. Hematol. 28, 46–54 (2000).

    Article  CAS  PubMed  Google Scholar 

  43. Prockop, D.J. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276, 71–74 (1997).

    Article  CAS  PubMed  Google Scholar 

  44. Giarratana, M.C. et al. Cell culture bags allow a large extent of ex vivo expansion of LTC-IC and functional mature cells which can subsequently be frozen: interest for a large-scale clinical applications. Bone Marrow Transplant. 22, 707–715 (1998).

    Article  CAS  PubMed  Google Scholar 

  45. Beutler, E. et al. International Committee for Standardization in Haematology: recommended methods for red-cell enzyme analysis. Br. J. Haematol. 35, 331–340 (1977).

    Article  CAS  PubMed  Google Scholar 

  46. Pic, P., Ducrocq, R. & Girot, R. Separation of hemoglobins F, Fac, S, C, A1c and determination of hemoglobin F using high performance liquid chromatography. Ann. Biol. Clin. 52, 129–132 (1994).

    CAS  Google Scholar 

  47. Papassotiriou, I., Ducrocq, R., Prehu, C., Bardakdjian-Michau, J. & Wajcman, H. Gamma chain heterogeneity: determination of Hb F composition by perfusion chromatography. Hemoglobin 22, 469–481 (1998).

    Article  CAS  PubMed  Google Scholar 

  48. Kister, J., Poyart, C. & Edelstein, S.J. Oxygen-organophosphate linkage in hemoglobin A. The double hump effect. Biophys. J. 52, 527–535 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Marden, M.C., Kister, J., Bohn, B. & Poyart, C. T-state hemoglobin with four ligands bound. Biochemistry 27, 1659–1664 (1988).

    Article  CAS  PubMed  Google Scholar 

  50. Lyons, A.B. & Parish, C.R. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171, 131–137 (1994).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank D. Boyeldieu for donation of the-RhD antibody, G. Trugnan for confocal microscopy and N. Mario for enzymatic assays. This work was supported by INSERM, and grants from the Association pour la Recherche en Transfusion (ART), the Association Combattre La Leucémie (CLL) and the Etablissement Français du Sang (EFS) grant no CS/2002/004.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Luc Douay.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Giarratana, MC., Kobari, L., Lapillonne, H. et al. Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nat Biotechnol 23, 69–74 (2005). https://doi.org/10.1038/nbt1047

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1047

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing