Novel Method for Efficient Production of Multipotential Hematopoietic Progenitors from Human Embryonic Stem Cells

  • Feng Ma
  • Dan Wang
  • Sachiyo Hanada
  • Yasuhiro Ebihara
  • Hirohide Kawasaki
  • Yuji Zaike
  • Toshio Heike
  • Tatsutoshi Nakahata
  • Kohichiro Tsuji


We propose a novel method for the efficient production of hematopoietic progenitors from human embryonic stem cells (hESC) via coculture with murine fetal liver-derived stromal cells, in which embryonic hematopoiesis dramatically expands at midgestation. We generated various hematopoietic progenitors in coculture, and this hematopoietic activity was concentrated in cobblestone-like cells derived from differentiated hESC. The cobblestone-like cells mostly expressed CD34 and retained an endothelial cell potential. They also contained hematopoietic colony-forming cells, especially erythroid and multilineage colony-forming cells at high frequency. The multipotential hematopoietic progenitors abundant among the cobblestone-like cells produced almost all types of mature blood cells, including adult-type β-globin-expressing erythrocytes and tryptase/chymase double-positive mast cells. These progenitors showed neither the immature properties of ESC nor the potential to differentiate into endoderm and ectoderm at a clonal level. The coculture system developed for hESC can provide a novel source of hematopoietic and blood cells for applications in cellular therapy and drug screening.

Key words

Hematopoietic progenitors Human embryonic stem cells Fetal liver Stromal cells 


  1. 1.
    Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;198:154–156.CrossRefGoogle Scholar
  2. 2.
    Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA. 1981;78:7634–7638.PubMedCrossRefGoogle Scholar
  3. 3.
    Nakano T, Kodama H, Honjo T. In vitro development of primitive and definitive erythrocytes from different precursors. Science. 1996;272:722–724.PubMedCrossRefGoogle Scholar
  4. 4.
    Kennedy M, Firpo M, Choi K, et al. A common precursor for primitive erythropoiesis and definitive haematopoiesis. Nature. 1997;386:488–493.PubMedCrossRefGoogle Scholar
  5. 5.
    Thomson JA, Ittsskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–1147.PubMedCrossRefGoogle Scholar
  6. 6.
    Reubinoff BE, Pera MF, Fong C-Y, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol. 2000;18:399–404.PubMedCrossRefGoogle Scholar
  7. 7.
    Kyba M. Genesis of hematopoietic stem cells in vitro and in vivo: new insights into developmental maturation. Int J Hematol. 2005;81:275–280.PubMedCrossRefGoogle Scholar
  8. 8.
    Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson A. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci USA. 2001;96:10716–10721.CrossRefGoogle Scholar
  9. 9.
    Lu SJ, Li F, Vida L, Honig GR. CD34+CD38- hematopoietic precursors derived from human embryonic stem cells exhibit an embryonic gene expression pattern. Blood. 2004;103:4134–4141.PubMedCrossRefGoogle Scholar
  10. 10.
    Vodyanik MA, Bork JA, Thomson JA, Slukvin II. Human embryonic stem cell-derived CD34+ cells: efficient production in the co-culture with OP9 stromal cells and analysis of lymphohe-matopoietic potential. Blood. 2005;105:617–626.PubMedCrossRefGoogle Scholar
  11. 11.
    Wang LS, Li L, Shojaei F, et al. Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties. Immunity. 2004;21:31–41.PubMedCrossRefGoogle Scholar
  12. 12.
    Wang LS, Menendez P, Shojaei F, et al. Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression. J Exp Med. 2005;201:1603–1614.PubMedCrossRefGoogle Scholar
  13. 13.
    Chadwick K, Wang L, Li L, et al. Cytokines and BMP-4 promote hematopoietic differentiation of human embryonic stem cells. Blood. 2003;102:906–915.PubMedCrossRefGoogle Scholar
  14. 14.
    Zambidis ET, Peault B, Park TS, Bunz F, Civin CI. Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development. Blood. 2005;106:860–870.PubMedCrossRefGoogle Scholar
  15. 15.
    Moore M, Metcalf D. Ontogeny of the hematopoietic system: yolk sac origin of in vivo colony-forming cells in the developing mouse embryo. Br J Haematol. 1970;18:279–296.PubMedCrossRefGoogle Scholar
  16. 16.
    Medvinski AL, Samoylina NL, Muller AM, Dzierzak EA. An early pre-liver intraembryonic source of CFU-S in the developing mouse. Nature. 1993;364:64–67.CrossRefGoogle Scholar
  17. 17.
    Godin IE, Garcia-Porrero JA, Coutinho A, Dieterlen-Lievre F, Marcos MA. Para-aortic splanchnopleura from early mouse embryos contains B1a cell progenitors. Nature. 1993;364:67–70.PubMedCrossRefGoogle Scholar
  18. 18.
    Sugiyama D, Arai K, Tsuji K. Definitive hematopoiesis from acetyl LDL incorporating endothelial cells in the mouse embryo. Stem Cells Dev. 2005;14:687–696.PubMedCrossRefGoogle Scholar
  19. 19.
    Kinoshita T, Sekiguchi T, Xu M-J, et al. Hepatic differentiation induced by oncostatin M attenuates fetal liver hematopoiesis. Proc Natl Acad Sci USA. 1999;96:7265–7270.PubMedCrossRefGoogle Scholar
  20. 20.
    Ma F, Wada M, Ebihara Y, et al. Development of human lympho-hematopoietic stem and progenitor cells defined by expression of CD34 and CD81. Blood. 2001;97:3755–3762.PubMedCrossRefGoogle Scholar
  21. 21.
    Mukouyama Y, Hara T, Xu M-J, et al. In vitro expansion of murine multipotential hematopoietic progenitors from the embryonic aorta-gonad-mesonephros region. Immunity. 1998;8:105–114.PubMedCrossRefGoogle Scholar
  22. 22.
    Xu M-J, Tsuji K, Ueda T, et al. Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines. Blood. 1998;92:2032–2040.PubMedGoogle Scholar
  23. 23.
    Sui X, Tsuji K, Tajima S, et al. Erythropoietin-independent erythrocyte production: signals through gp130 and c-Kit dramatically promote erythropoiesis from human CD34+ cells. J Exp Med. 1996;183:837–845.PubMedCrossRefGoogle Scholar
  24. 24.
    Ueda Y, Tsuji K, Yoshino H, et al. Expansion of human NOD/ SCID-repopulating cells by a combination of stem cell factor, Flk2/Flt3 ligand, thrombopoietin and a complex of interleukin-6 and soluble interleukin-6 receptor. J Clin Invest. 2000;105:1013–1021.PubMedCrossRefGoogle Scholar
  25. 25.
    Tajima S, Tsuji K, Ebihara Y, et al. Analysis of IL-6 receptor and gp130 expressions and proliferative capability of human CD34+ cells. J Exp Med. 1996;184:1357–1364.PubMedCrossRefGoogle Scholar
  26. 26.
    Kempuraj D, Saito H, Kaneko A, et al. Characterization of mast cell-committed progenitors present in human umbilical cord blood. Blood. 1999;93:3338–3346.PubMedGoogle Scholar

Copyright information

© The Japanese Society of Hematology 2007

Authors and Affiliations

  • Feng Ma
    • 1
  • Dan Wang
    • 1
  • Sachiyo Hanada
    • 1
  • Yasuhiro Ebihara
    • 1
  • Hirohide Kawasaki
    • 1
  • Yuji Zaike
    • 2
  • Toshio Heike
    • 3
  • Tatsutoshi Nakahata
    • 3
  • Kohichiro Tsuji
    • 1
  1. 1.Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical ScienceUniversity of TokyoTokyoJapan
  2. 2.Department of Laboratory Medicine, Research Hospital, Institute of Medical ScienceUniversity of TokyoTokyoJapan
  3. 3.Department of Pediatrics, Graduate School of MedicineKyoto UniversityKyotoJapan

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