Derivation of Mature Erythrocytes from Human Pluripotent Stem Cells by Coculture with Murine Fetal Stromal Cells

  • Bin Mao
  • Xulin Lu
  • Shu Huang
  • Jinfeng Yu
  • Mowen Lai
  • Kohichiro Tsuji
  • Tatsutoshi Nakahata
  • Feng MaEmail author
Part of the SpringerBriefs in Stem Cells book series (BRIEFSSTEM, volume 6)


Transfusion of red blood cells (RBCs) is a requisite cell therapy today, while RBCs supplied by donors cannot match the huge demand of patients. Human pluripotent stem cells (hPSCs) are promising cell sources to obtain RBCs as an alternative transfusion product for clinical application. Several in vitro culture systems have been reported that in which mature erythrocytes can be efficiently generated from hPSCs. However, different efficiency and maturity of hPSC-derived erythrocytes could be obtained when using different culture systems. We still lack a complete understanding of the regulatory pathways controlling human erythrocyte development and maturation, especially the origination of erythrocytes early in the embryo and enucleation at the terminal stage of differentiation. In this chapter, we focus on an efficient method established successfully in our laboratory to derive functionally mature erythrocytes from hPSCs by coculture with mouse fetal stromal cells [aorta–gonad–mesonephros stromal cells (mAGM) and fetal liver stromal cells (mFLSCs), respectively]. The procedures to investigate the characteristics of these hPSC-derived erythrocytes are also introduced, including colony formation assay to detect the hematopoietic potential, flow cytometry assay to detect the phenotypic expression pattern, and immuno-staining assay of the Hb components to evaluate the maturity. At the end of this review, several future prospects are also be addressed in this research fields.


Erythrocytes hPSCs AGM Fetal liver Hematopoiesis 


  1. 1.
    WHO. Blood safety and availability. 2014.Google Scholar
  2. 2.
    Migliaccio AR, Whitsett C, Papayannopoulou T, Sadelain M. The potential of stem cells as an in vitro source of red blood cells for transfusion. Cell Stem Cell. 2012;10:115–9.PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Neildez-Nguyen TM, Wajcman H, Marden MC, Bensidhoum M, Moncollin V, Giarratana MC, Kobari L, Thierry D, Douay L. Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo. Nat Biotechnol. 2002;20:467–72.CrossRefPubMedGoogle Scholar
  4. 4.
    Douay L, Giarratana MC. In vitro generation of mature and functional human red blood cells: a model with multidisciplinary perspectives. Bulletin de l’Academie nationale de medicine. 2005;189:903–13 (discussion 914-905).Google Scholar
  5. 5.
    Douay L, Andreu G. Ex vivo production of human red blood cells from hematopoietic stem cells: what is the future in transfusion? Transfus Med Rev. 2007;21:91–100.CrossRefPubMedGoogle Scholar
  6. 6.
    Giarratana MC, Rouard H, Dumont A, Kiger L, Safeukui I, Le Pennec PY, Francois S, Trugnan G, Peyrard T, Marie T, Jolly S, Hebert N, Mazurier C, Mario N, Harmand L, Lapillonne H, Devaux JY, Douay L. Proof of principle for transfusion of in vitro-generated red blood cells. Blood. 2011;118:5071–9.PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Flores-Guzman P, Fernandez-Sanchez V, Mayani H. Concise review: ex vivo expansion of cord blood-derived hematopoietic stem and progenitor cells: basic principles, experimental approaches, and impact in regenerative medicine. Stem Cells Transl Med. 2013;2:830–8.PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Jing Q, Cai H, Du Z, Ye Z, Tan WS. Effects of agitation speed on the ex vivo expansion of cord blood hematopoietic stem/progenitor cells in stirred suspension culture. Artif Cells Nanomed Biotechnol. 2013;41:98–102.CrossRefPubMedGoogle Scholar
  9. 9.
    Migliaccio AR, Whitsett C, Migliaccio G. Erythroid cells in vitro: from developmental biology to blood transfusion products. Curr Opin Hematol. 2009;16:259–68.CrossRefPubMedGoogle Scholar
  10. 10.
    Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–7.CrossRefPubMedGoogle Scholar
  11. 11.
    Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–72.CrossRefPubMedGoogle Scholar
  12. 12.
    Migliaccio G, Di Pietro R, di Giacomo V, Di Baldassarre A, Migliaccio AR, Maccioni L, Galanello R, Papayannopoulou T. In vitro mass production of human erythroid cells from the blood of normal donors and of thalassemic patients. Blood Cells Mol Dis. 2002;28:169–80.CrossRefPubMedGoogle Scholar
  13. 13.
    Mali P, Ye Z, Hommond HH, Yu X, Lin J, Chen G, Zou J, Cheng L. Improved efficiency and pace of generating induced pluripotent stem cells from human adult and fetal fibroblasts. Stem Cells. 2008;26:1998–2005.CrossRefPubMedGoogle Scholar
  14. 14.
    Papapetrou EP, Lee G, Malani N, Setty M, Riviere I, Tirunagari LM, Kadota K, Roth SL, Giardina P, Viale A, Leslie C, Bushman FD, Studer L, Sadelain M. Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells. Nat Biotechnol. 2011;29:73–8.PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Sebastiano V, Maeder ML, Angstman JF, Haddad B, Khayter C, Yeo DT, Goodwin MJ, Hawkins JS, Ramirez CL, Batista LF, Artandi SE, Wernig M, Joung JK. In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases. Stem Cells. 2011;29:1717–26.PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Hanna J, Wernig M, Markoulaki S, Sun CW, Meissner A, Cassady JP, Beard C, Brambrink T, Wu LC, Townes TM, Jaenisch R. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science. 2007;318:1920–3.CrossRefPubMedGoogle Scholar
  17. 17.
    Cheng L. Human stem cell models for red blood disease modeling and treatment. In 2014 International symposium on erythrocyte biology, Zhengzhou, China. 2014.Google Scholar
  18. 18.
    Lu SJ, Feng Q, Park JS, Vida L, Lee BS, Strausbauch M, Wettstein PJ, Honig GR, Lanza R. Biologic properties and enucleation of red blood cells from human embryonic stem cells. Blood. 2008;112:4475–84.PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    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.CrossRefPubMedGoogle Scholar
  20. 20.
    Lapillonne H, Kobari L, Mazurier C, Tropel P, Giarratana MC, Zanella-Cleon I, Kiger L, Wattenhofer-Donze M, Puccio H, Hebert N, Francina A, Andreu G, Viville S, Douay L. Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine. Haematologica. 2010;95:1651–9.PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Chang CJ, Mitra K, Koya M, Velho M, Desprat R, Lenz J, Bouhassira EE. Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells. PLoS ONE. 2011;6:e25761.PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Chang KH, Nelson AM, Cao H, Wang L, Nakamoto B, Ware CB, Papayannopoulou T. Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin. Blood. 2006;108:1515–23.PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Kobari L, Yates F, Oudrhiri N, Francina A, Kiger L, Mazurier C, Rouzbeh S, El-Nemer W, Hebert N, Giarratana MC, Francois S, Chapel A, Lapillonne H, Luton D, Bennaceur-Griscelli A, Douay L. Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model. Haematologica. 2012;97:1795–803.PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    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;98:10716–21.PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    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;111:2400–8.PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Ma F, Wang D, Hanada S, Ebihara Y, Kawasaki H, Zaike Y, Heike T, Nakahata T, Tsuji K. Novel method for efficient production of multipotential hematopoietic progenitors from human embryonic stem cells. Int J Hematol. 2007;85:371–9.CrossRefPubMedGoogle Scholar
  27. 27.
    Ma F, Ebihara Y, Umeda K, Sakai H, Hanada S, Zhang H, Zaike Y, Tsuchida E, Nakahata T, Nakauchi H, Tsuji K. Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis. Proc Natl Acad Sci USA. 2008;105:13087–92.PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Dias J, Gumenyuk M, Kang H, Vodyanik M, Yu J, Thomson JA, Slukvin II. Generation of red blood cells from human induced pluripotent stem cells. Stem Cells Dev. 2011;20:1639–47.PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Qiu C, Hanson E, Olivier E, Inada M, Kaufman DS, Gupta S, Bouhassira EE. 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;33:1450–8.CrossRefPubMedGoogle Scholar
  30. 30.
    Peschle C, Mavilio F, Care A, Migliaccio G, Migliaccio AR, Salvo G, Samoggia P, Petti S, Guerriero R, Marinucci M, et al. Haemoglobin switching in human embryos: asynchrony of zeta—alpha and epsilon—gamma-globin switches in primitive and definite erythropoietic lineage. Nature. 1985;313:235–8.CrossRefPubMedGoogle Scholar
  31. 31.
    Tavian M, Coulombel L, Luton D, Clemente HS, Dieterlen-Lievre F, Peault B. Aorta-associated CD34+ hematopoietic cells in the early human embryo. Blood. 1996;87:67–72.PubMedGoogle Scholar
  32. 32.
    Xu MJ, Tsuji K, Ueda T, Mukouyama YS, Hara T, Yang FC, Ebihara Y, Matsuoka S, Manabe A, Kikuchi A, Ito M, Miyajima A, Nakahata T. Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines. Blood. 1998;92:2032–40.PubMedGoogle Scholar
  33. 33.
    Ma F, Kambe N, Wang D, Shinoda G, Fujino H, Umeda K, Fujisawa A, Ma L, Suemori H, Nakatsuji N, Miyachi Y, Torii R, Tsuji K, Heike T, Nakahata T. Direct development of functionally mature tryptase/chymase double-positive connective tissue-type mast cells from primate embryonic stem cells. Stem Cells. 2008;26:706–14.CrossRefPubMedGoogle Scholar
  34. 34.
    Ma F, Nashihama YG, Yang W, Yasuhiro E, Tsuji K. Differentiation oh human embryonic and induced pluripotent stem cells into blood cells in coculture with murine stromal cells. In Jin KYAS, editor. Human embryonic and induced pluripotent stem cells: lineage-specific differentiation protocols. Clifton: Humana Press; 2011. pp. 321–335.Google Scholar
  35. 35.
    Keller G, Lacaud G, Robertson S. Development of the hematopoietic system in the mouse. Exp Hematol. 1999;27:777–87.CrossRefPubMedGoogle Scholar
  36. 36.
    Matsuoka S, Tsuji K, Hisakawa H, Xu M, Ebihara Y, Ishii T, Sugiyama D, Manabe A, Tanaka R, Ikeda Y, Asano S, Nakahata T. Generation of definitive hematopoietic stem cells from murine early yolk sac and paraaortic splanchnopleures by aorta-gonad-mesonephros region-derived stromal cells. Blood. 2001;98:6–12.CrossRefPubMedGoogle Scholar
  37. 37.
    Muller AM, Medvinsky A, Strouboulis J, Grosveld F, Dzierzak E. Development of hematopoietic stem cell activity in the mouse embryo. Immunity. 1994;1:291–301.CrossRefPubMedGoogle Scholar
  38. 38.
    Medvinsky A, Rybtsov S, Taoudi S. Embryonic origin of the adult hematopoietic system: advances and questions. Development. 2011;138:1017–31.CrossRefPubMedGoogle Scholar
  39. 39.
    Ledran MH, Krassowska A, Armstrong L, Dimmick I, Renstrom J, Lang R, Yung S, Santibanez-Coref M, Dzierzak E, Stojkovic M, Oostendorp RA, Forrester L, Lako M. Efficient hematopoietic differentiation of human embryonic stem cells on stromal cells derived from hematopoietic niches. Cell Stem Cell. 2008;3:85–98.CrossRefPubMedGoogle Scholar
  40. 40.
    Ma F, Wada M, Yoshino H, Ebihara Y, Ishii T, Manabe A, Tanaka R, Maekawa T, Ito M, Mugishima H, Asano S, Nakahata T, Tsuji K. Development of human lymphohematopoietic stem and progenitor cells defined by expression of CD34 and CD81. Blood. 2001;97:3755–62.CrossRefPubMedGoogle Scholar
  41. 41.
    Takayama N, Nishikii H, Usui J, Tsukui H, Sawaguchi A, Hiroyama T, Eto K, Nakauchi H. Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors. Blood. 2008;111:5298–306.CrossRefPubMedGoogle Scholar
  42. 42.
    Nakahata T, Spicer SS, Cantey JR, Ogawa M. Clonal assay of mouse mast cell colonies in methylcellulose culture. Blood. 1982;60:352–61.PubMedGoogle Scholar
  43. 43.
    Nakahata T, Ogawa M. Identification in culture of a class of hemopoietic colony-forming units with extensive capability to self-renew and generate multipotential hemopoietic colonies. Proc Natl Acad Sci USA. 1982;79:3843–7.PubMedCentralCrossRefPubMedGoogle Scholar
  44. 44.
    Nakahata T, Ogawa M. Hemopoietic colony-forming cells in umbilical cord blood with extensive capability to generate mono and multipotential hemopoietic progenitors. J Clin Invest. 1982;70:1324–8.PubMedCentralCrossRefPubMedGoogle Scholar
  45. 45.
    Liu J, Zhang J, Ginzburg Y, Li H, Xue F, De Franceschi L, Chasis JA, Mohandas N, An X. Quantitative analysis of murine terminal erythroid differentiation in vivo: novel method to study normal and disordered erythropoiesis. Blood. 2013;121:e43–9.PubMedCentralCrossRefPubMedGoogle Scholar
  46. 46.
    Stamatoyannopoulos G. Control of globin gene expression during development and erythroid differentiation. Exp Hematol. 2005;33:259–71.PubMedCentralCrossRefPubMedGoogle Scholar
  47. 47.
    Slukvin II. Deciphering the hierarchy of angiohematopoietic progenitors from human pluripotent stem cells. Cell Cycle. 2013;12:720–7.PubMedCentralCrossRefPubMedGoogle Scholar
  48. 48.
    Choi KD, Vodyanik MA, Togarrati PP, Suknuntha K, Kumar A, Samarjeet F, Probasco MD, Tian S, Stewart R, Thomson JA, Slukvin II. Identification of the hemogenic endothelial progenitor and its direct precursor in human pluripotent stem cell differentiation cultures. Cell Rep. 2012;2:553–67.PubMedCentralCrossRefPubMedGoogle Scholar
  49. 49.
    Sturgeon CM, Ditadi A, Awong G, Kennedy M, Keller G. Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat Biotechnol. 2014;32:554–61.PubMedCentralCrossRefPubMedGoogle Scholar
  50. 50.
    Yoder MC. Inducing definitive hematopoiesis in a dish. Nat Biotechnol. 2014;32:539–41.CrossRefPubMedGoogle Scholar
  51. 51.
    Olivier E, Qiu C, Bouhassira EE. Novel, high-yield red blood cell production methods from CD34-positive cells derived from human embryonic stem, yolk sac, fetal liver, cord blood, and peripheral blood. Stem Cells Transl Med. 2012;1:604–14.PubMedCentralCrossRefPubMedGoogle Scholar
  52. 52.
    Perugini M, Varelias A, Sadlon T, D’Andrea RJ. Hematopoietic growth factor mimetics: from concept to clinic. Cytokine Growth Factor Rev. 2009;20:87–94.CrossRefPubMedGoogle Scholar
  53. 53.
    Smith BW, Rozelle SS, Leung A, Ubellacker J, Parks A, Nah SK, French D, Gadue P, Monti S, Chui DH, Steinberg MH, Frelinger AL, Michelson AD, Theberge R, McComb ME, Costello CE, Kotton DN, Mostoslavsky G, Sherr DH, Murphy GJ. The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation. Blood. 2013;122:376–85.PubMedCentralCrossRefPubMedGoogle Scholar
  54. 54.
    Nakamura Y, Hiroyama T, Miharada K, Kurita R. Red blood cell production from immortalized progenitor cell line. Int J Hematol. 2011;93:5–9.CrossRefPubMedGoogle Scholar
  55. 55.
    Kurita R, Suda N, Sudo K, Miharada K, Hiroyama T, Miyoshi H, Tani K, Nakamura Y. Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells. PLoS ONE. 2013;8:e59890.PubMedCentralCrossRefPubMedGoogle Scholar
  56. 56.
    Hirose S, Takayama N, Nakamura S, Nagasawa K, Ochi K, Hirata S, Yamazaki S, Yamaguchi T, Otsu M, Sano S, Takahashi N, Sawaguchi A, Ito M, Kato T, Nakauchi H, Eto K. Immortalization of erythroblasts by c-MYC and BCL-XL enables large-scale erythrocyte production from human pluripotent stem cells. Stem Cell Rep. 2013;1:499–508.CrossRefGoogle Scholar
  57. 57.
    Hiroyama T, Miharada K, Kurita R, Nakamura Y. Plasticity of cells and ex vivo production of red blood cells. Stem Cells Int. 2011;2011:195780.PubMedCentralCrossRefPubMedGoogle Scholar
  58. 58.
    Hattangadi SM, Wong P, Zhang L, Flygare J, Lodish HF. From stem cell to red cell: regulation of erythropoiesis at multiple levels by multiple proteins, RNAs, and chromatin modifications. Blood. 2011;118:6258–68.PubMedCentralCrossRefPubMedGoogle Scholar
  59. 59.
    Fujimi A, Matsunaga T, Kobune M, Kawano Y, Nagaya T, Tanaka I, Iyama S, Hayashi T, Sato T, Miyanishi K, Sagawa T, Sato Y, Takimoto R, Takayama T, Kato J, Gasa S, Sakai H, Tsuchida E, Ikebuchi K, Hamada H, Niitsu Y. Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages. Int J Hematol. 2008;87:339–50.CrossRefPubMedGoogle Scholar
  60. 60.
    Xu J, Shao Z, Glass K, Bauer DE, Pinello L, Van Handel B, Hou S, Stamatoyannopoulos JA, Mikkola HK, Yuan GC, Orkin SH. Combinatorial assembly of developmental stage-specific enhancers controls gene expression programs during human erythropoiesis. Dev Cell. 2012;23:796–811.PubMedCentralCrossRefPubMedGoogle Scholar
  61. 61.
    Guvendiren M, Burdick JA. Engineering synthetic hydrogel microenvironments to instruct stem cells. Curr Opin Biotechnol. 2013;24:841–6.PubMedCentralCrossRefPubMedGoogle Scholar
  62. 62.
    Yuan Y, Tse KT, Sin FW, Xue B, Fan HH, Xie Y, Xie Y. Ex vivo amplification of human hematopoietic stem and progenitor cells in an alginate three-dimensional culture system. Int J Lab Hematol. 2011;33:516–25.PubMedGoogle Scholar
  63. 63.
    Zhu J, Marchant RE. Design properties of hydrogel tissue-engineering scaffolds. Expert Rev Med Devices. 2011;8:607–26.PubMedCentralCrossRefPubMedGoogle Scholar
  64. 64.
    Jiang J, Papoutsakis ET. Stem-cell niche based comparative analysis of chemical and nano-mechanical material properties impacting ex vivo expansion and differentiation of hematopoietic and mesenchymal stem cells. Adv Healthc Mater. 2013;2:25–42.CrossRefPubMedGoogle Scholar
  65. 65.
    Mohandas N, An X. Malaria and human red blood cells. Med Microbiol Immunol. 2012;201:593–8.PubMedCentralCrossRefPubMedGoogle Scholar
  66. 66.
    Mel HC, Prenant M, Mohandas N. Reticulocyte motility and form: studies on maturation and classification. Blood. 1977;49:1001–9.PubMedGoogle Scholar
  67. 67.
    Migliaccio G, Sanchez M, Masiello F, Tirelli V, Varricchio L, Whitsett C, Migliaccio AR. Humanized culture medium for clinical expansion of human erythroblasts. Cell Transplant. 2010;19:453–69.PubMedCentralCrossRefPubMedGoogle Scholar
  68. 68.
    Hu Z, Van Rooijen N, Yang YG. Macrophages prevent human red blood cell reconstitution in immunodeficient mice. Blood. 2011;118:5938–46.PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© The Author(s) 2015

Authors and Affiliations

  • Bin Mao
    • 1
  • Xulin Lu
    • 1
  • Shu Huang
    • 1
  • Jinfeng Yu
    • 1
  • Mowen Lai
    • 1
  • Kohichiro Tsuji
    • 2
  • Tatsutoshi Nakahata
    • 3
  • Feng Ma
    • 1
    • 2
    • 4
    Email author
  1. 1.Institute of Blood TransfusionChinese Academy of Medical Sciences and Peking Union Medical CollegeChengduChina
  2. 2.Institute of Medical ScienceThe University of TokyoTokyoJapan
  3. 3.Center for IPS Cell Research and Application (CiRA)Kyoto UniversityKyotoJapan
  4. 4.State Key Lab of Experimental HematologyChinese Academy of Medical Sciences and Peking Union Medical CollegeTianjinChina

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