Isolation of Embryonic Hematopoietic Niche Cells by Flow Cytometry and Laser Capture Microdissection

  • Daisuke Sugiyama
  • Tatsuya Sasaki
Part of the Methods in Molecular Biology book series (MIMB, volume 1035)


Hematopoietic stem cells (HSCs) can differentiate into several types of hematopoietic cells, such as erythrocytes, megakaryocytes, lymphocytes, neutrophils, or macrophages, and also undergo self-renewal to sustain hematopoiesis throughout an organism’s lifetime. HSCs emerge and expand during mouse embryogenesis. HSC regulation is governed by two types of activity: intrinsic activity programmed primarily by cell autonomous gene expression, and extrinsic factors, which originate from the so-called niche cells surrounding HSCs. Previously, we reported that endothelial niche cells regulate HSC generation at aorta-gonad-mesonephros region and placenta, and that hepatoblastic niche cells regulate HSC differentiation in mouse embryonic liver. In the course of those studies, we employed immunohistochemistry, flow cytometry, and the laser capture microdissection system to assess embryonic regulation of the mouse hematopoietic niche.

Key words

Hematopoietic stem cells Niche cells Embryo Visualization Laser capture microdissection 



We thank The Ministry of Education, Culture, Sports, Science and Technology; The Ministry of Health, Labor and Welfare; and The Japan Society for the Promotion of Science for grant support, and Dr. Elise Lamar for critical reading of the manuscript.


  1. 1.
    Dzierzak E, Medvinsky A, de Bruijn M (1998) Qualitative and quantitative aspects of hematopoietic cell development in the mammalian embryo. Immunol Today 19:228–236PubMedCrossRefGoogle Scholar
  2. 2.
    McGrath KE, Palis J (2005) Hematopoiesis in the yolk sac: more than meets the eye. Exp Hematol 33:1021–1028PubMedCrossRefGoogle Scholar
  3. 3.
    Ferkowicz MJ, Yoder MC (2005) Blood island formation: longstanding observations and modern interpretations. Exp Hematol 33:1041–1047PubMedCrossRefGoogle Scholar
  4. 4.
    Sugiyama D, Tsuji K (2006) Definitive hematopoiesis from endothelial cells in the mouse embryo; a simple guide. Trends Cardiovasc Med 16:45–49PubMedCrossRefGoogle Scholar
  5. 5.
    Dzierzak E, Speck NA (2008) Of lineage and legacy: the development of mammalian hematopoietic stem cells. Nat Immunol 9:129–136PubMedCrossRefGoogle Scholar
  6. 6.
    Palis J, Robertson S, Kennedy M, Wall C, Keller G (1999) Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development 126:5073–5084PubMedGoogle Scholar
  7. 7.
    Medvinsky A, Dzierzak E (1996) Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 86:897–906PubMedCrossRefGoogle Scholar
  8. 8.
    Cumano A, Dieterlen-Lievre F, Godin I (1996) Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura. Cell 86:907–916PubMedCrossRefGoogle Scholar
  9. 9.
    Yoder MC, Hiatt K, Dutt P, Mukherjee P, Bodine DM, Orlic D (1997) Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac. Immunity 7:335–344PubMedCrossRefGoogle Scholar
  10. 10.
    Matsuoka S, Tsuji K, Hisakawa H, Xu M, Ebihara Y, Ishii T, Sugiyama D, Manabe A, Tanaka R, Ikeda Y et al (2001) Generation of definitive hematopoietic stem cells from murine early yolk sac and paraaortic splanchnopleures by aorta-gonad-mesonephros region-derived stromal cells. Blood 98:6–12PubMedCrossRefGoogle Scholar
  11. 11.
    Sugiyama D, Ogawa M, Nakao K, Osumi N, Nishikawa S, Nishikawa S, Arai K, Nakahata T, Tsuji K (2007) B cell potential can be obtained from pre-circulatory yolk sac, but with low frequency. Dev Biol 301:53–61PubMedCrossRefGoogle Scholar
  12. 12.
    Samokhvalov IM, Samokhvalova NI, Nishikawa S (2007) Cell tracing shows the contribution of the yolk sac to adult haematopoiesis. Nature 446:1056–1061PubMedCrossRefGoogle Scholar
  13. 13.
    Mikkola HK, Gekas C, Orkin SH, Dieterlen-Lievre F (2005) Placenta as a site for hematopoietic stem cell development. Exp Hematol 33:1048–1054PubMedCrossRefGoogle Scholar
  14. 14.
    Zeigler BM, Sugiyama D, Chen M, Guo Y, Downs KM, Speck NA (2006) The allantois and chorion, when isolated before circulation or chorio-allantoic fusion, have hematopoietic potential. Development 133:4183–4192PubMedCrossRefGoogle Scholar
  15. 15.
    Sasaki T, Mizuochi C, Horio Y, Nakao K, Akashi K, Sugiyama D (2010) Regulation of hematopoietic cell clusters in the placental niche through SCF/Kit signaling in embryonic mouse. Development 137:3941–3952PubMedCrossRefGoogle Scholar
  16. 16.
    Johnson GR, Moore MA (1975) Role of stem cell migration in initiation of mouse foetal liver haemopoiesis. Nature 258:726–728PubMedCrossRefGoogle Scholar
  17. 17.
    Houssaint E (1981) Differentiation of the mouse hepatic primordium. II. Extrinsic origin of the haemopoietic cell line. Cell Differ 10:243–252PubMedCrossRefGoogle Scholar
  18. 18.
    Ema H, Nakauchi H (2000) Expansion of hematopoietic stem cells in the developing liver of a mouse embryo. Blood 95:2284–2288PubMedGoogle Scholar
  19. 19.
    Sugiyama D, Arai K, Tsuji K (2005) Definitive hematopoiesis from acetyl LDL incorporating endothelial cells in the mouse embryo. Stem Cells Dev 14:687–696PubMedCrossRefGoogle Scholar
  20. 20.
    Zhang CC, Kaba M, Ge G, Xie K, Tong W, Hug C, Lodish HF (2006) Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells. Nat Med 12:240–245PubMedCrossRefGoogle Scholar
  21. 21.
    Sugiyama D, Kulkeaw K, Mizuochi C, Horio Y, Okayama S (2011) Hepatoblasts comprise a niche for fetal liver erythropoiesis through cytokine production. Biochem Biophys Res Commun 410:301–306PubMedCrossRefGoogle Scholar
  22. 22.
    Sugiyama D, Kulkeaw K, Mizuochi C (2013) TGF-beta-1 up-regulates extra-cellular matrix production in mouse hepatoblasts. Mech Dev 130(2–3):195–206PubMedCrossRefGoogle Scholar
  23. 23.
    Mizuochi C, Fraser ST, Biasch K, Horio Y, Kikushige Y, Tani K, Akashi K, Tavian M, Sugiyama D (2012) Intra-aortic clusters undergo endothelial to hematopoietic phenotypic transition during early embryogenesis. PLoS One 7:e35763PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Daisuke Sugiyama
    • 1
  • Tatsuya Sasaki
    • 1
  1. 1.Division of Hematopoietic Stem Cells, Advanced Medical Initiatives, Department of Advanced Medical InitiativesKyushu University Faculty of Medical SciencesFukuokaJapan

Personalised recommendations