Ex vivo large-scale generation of human red blood cells from cord blood CD34 + cells by co-culturing with macrophages Authors
First Online: 29 March 2008 Received: 31 October 2007 Revised: 10 January 2008 Accepted: 29 January 2008 DOI:
Cite this article as: Fujimi, A., Matsunaga, T., Kobune, M. et al. Int J Hematol (2008) 87: 339. doi:10.1007/s12185-008-0062-y
We generated red blood cells (RBC) from cord blood (CB) CD34
+ cells using a four-phase culture system. We first cultured CB CD34 + cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34 + cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34 + cells from a different donor using the same co-culture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 × 10 12 RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 × 10 13 RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.
Red blood cell
+ cell Stromal cell
Cooke BM, Mohandas N, Coppel RL. Malaria, the red blood cell membrane. Semi Hematol. 2004;41:173–88.
Penzhorn BL, Schoeman T, Jacobson LS. Feline babesiosis in South Africa: a review. Ann NY Acad Sci. 2004;1026:183–6.
Giarratana MC, Kobari L, Lapillonne H, et al. Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nat Biotechnol. 2005;23:69–74.
Kawano Y, Kobune M, Yamaguchi M, et al. Ex vivo expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subunit (hTERT)-transfected human stromal cells. Blood. 2003;101:532–40.
Matsunaga T, Tanaka I, Kobune M, et al. Ex vivo large-scale generation of human platelets from cord blood CD34
cells. Stem Cells. 2006;24:2877–87.
Miharada K, Hiroyama T, Sudo K, Nagasawa T, Nakamura Y. Efficient enucleation of erythroblasts differentiated in vitro from hematopoietic stem and progenitor cells. Nat Biotechnol. 2006;24:1255–6.
Kobune M, Ito Y, Kawano Y, Sasaki K, Uchida H, Nakamura K. Indian hedgehog gene transfer augments hematopoietic support of human stromal cells including NOD/SCID-β2m
repopulating cells. Blood. 2004;104:1002–9.
Amsellem S, Pflumio F, Bardinet D, Izac B, Charneau P, Romeo PH. Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein. Nat Med. 2003;9:1423–7.
Iavarone A, King ER, Dai XM, Leone G, Stanley ER, Lasorella A. Retinoblastoma promotes definitive erythropoiesis by repressing Id2 in fetal liver macrophages. Nature. 2004;432:1040–5.
Kawane K, Fukuyama H, Kondoh G, et al. Requirement of DNase II for definitive erythropoiesis in the mouse fetal liver. Science. 2001;292:1546–9.
Hanspal M, Hanspal JS. The association of erythroblasts with macrophages promotes erythroid proliferation and maturation: a 30-kD heparin-binding protein is involved in this contact. Blood. 1994;84:3494–504.
Hanspal M, Smockova Y, Uong Q. Molecular identification and functional characterization of a novel protein that mediates the attachment of erythroblasts to macrophages. Blood. 1998;92:2940–50.
Soni S, Bala S, Gwynn B, Sahr KE, Peters LL, Hanspal M. Absence of erythroblast macrophage protein (Emp) leads to failure of erythroblast nuclear extrusion. J Biol Chem. 2006;281:20181–9.
Hashimoto S, Yamada M, Motoyoshi K, et al. Enhancement of macrophage colony-stimulating factor-induced growth and differentiation of human monocytes by interleukin-10. Blood. 1997;89:315–21.
Pic P, Ducrocq R, Girot R. Separation des hemoglobines F, Fac, S, C, A1c et dosage de I’hemoglobine F par chromatographie liquide haute performance. Ann Bio Clin. 1994;52:129–32.
Worthington RE, Bossie-Codreanu J, Zant G. Quantitation of erythroid differentiation in vitro using a sensitive colorimetric assay for hemoglobin. Exp Hematol. 1995;15:85–92.
Van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Meth. 1994;174:83–93.
Hentzen ER, Neelamengham S, Kansas GS, et al. Sequential binding of CD11a/CD18 and CD11b/CD18 defines neutrophil capture and stable adhesion to intercellular adhesion molecule-1. Blood. 2000;95:911–20.
Lewis DE, Schober W, Murrell S, et al. Rare event selection of fetal nucleated erythrocytes in maternal blood by flow cytometry. Cytometry. 1996;23:218–27.
Kolbus A, Blazquez-Domingo M, Carotta S, et al. Cooperative signaling between cytokine receptors and the glucocorticoid receptor in the expansion of erythroid progenitors: molecular analysis by expression profiling. Blood. 2003;102:3136–46.
Neildez-Nguyen TM, Wajcman H, Marden MC, et al. Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo. Nat Med. 2002;20:467–72.
Stowell CP, Levin J, Spiess BD, Winslow RM. Progress in the development of RBC substitutes. Transfusion. 2001;41:287–99.
Winslow RM. Current status of blood substitute research: towards a new paradigm. J Intern Med. 2003;253:508–17.
Issaad C, Croisille L, Katz A, Vainchenker W, Coulombel L. A murine stromal cell line allows the proliferation of very primitive human CD34
progenitor cells in long-term cultures and semisolid assays. Blood. 1993;81:2916–24.
Conneally E, Cashman J, Petzer A, Eaves C. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc Natl Acad Sci USA. 1997;94:9836–41.
McNiece I, Kubegov D, Kerzic P, Shpall EJ, Gross S. Increased expansion and differentiation of cord blood products using a two-step expansion culture. Exp Hematol. 2000;28:1181–6.
Ueda T, Tsuji K, Yoshino H, et al. Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. J Clin Invest. 2000;105:1013–21.
Oda A, Sawada K, Druker B, et al. Erythropoietin induces tyrosine phosphorylation of Jak2, STAT5A, and STAT5B in primary cultured human erythroid precursors. Blood. 1998;92:443–51.
Sadahira Y, Yoshino T, Monobe Y. Very late activation antigen 4-Vascular cell adhesion molecule 1 interaction is involved in the formation of erythroblastic island. J Exp Med. 1995;181:411–5.
Lee G, Spring FA, Parsons SF, et al. Novel secreted isoform of adhesion molecule ICAM-4: potential regulator of membrane-associated ICAM-4 interactions. Blood. 2003;101:1790–979.
Lee G, Lo A, Short SA, et al. Targeted gene deletion demonstrates that the cell adhesion molecule ICAM-4 is critical for erythroblastic island formation. Blood. 2006;108:2064–71.
Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S. Identification of a factor that links apoptotic cells to phagocytes. Nature. 2002;417:182–6.
Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RA, Henson PM. A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature. 2000;405:85–90.
PubMed CrossRef Copyright information
© The Japanese Society of Hematology 2008