Erythropoiesis pp 117-132 | Cite as

Functional Analysis of Erythroid Progenitors by Colony-Forming Assays

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1698)

Abstract

The capacity of erythroid-lineage progenitors to form colonies of maturing red blood cells in semisolid media has provided a functional assay for these progenitors and has greatly contributed to our understanding of erythropoiesis. Studies since the 1970s have led to the development of a model of the erythron, whereby the earliest erythroid-committed progenitor, the immature burst-forming unit erythroid (BFU-E), gives rise sequentially to late-stage BFU-E and to colony-forming units erythroid (CFU-E). CFU-E give rise, in turn, to maturing erythroblast precursors that hemoglobinize. It is these terminal cells that comprise the mature colonies of erythroid cells derived from the progenitors cultured in semisolid media. The in vitro generation of erythroid colonies requires cytokine support, most notably erythropoietin (EPO), which is critical for CFU-E survival and for promoting erythroblast maturation.

During mouse embryogenesis, a transient population of primitive erythroid colony-forming progenitors (EryP-CFC) emerges in the yolk sac and gives rise to a wave of maturing primitive erythroblasts in the fetal bloodstream. This wave of EryP-CFC is followed closely by a wave of BFU-E in the yolk sac that enter the bloodstream and seed the fetal liver to generate the first definitive red cells in the fetus. BFU-E in the fetal liver, unlike those in the adult bone marrow, can give rise to colonies in vitro when cultured with EPO alone and also are more sensitive to EPO levels. Here, we describe methods for the in vitro culture of murine embryonic (primitive) and fetal/adult (definitive) erythroid progenitors in semisolid media.

Key words

Erythropoiesis Progenitor Yolk sac Colony-forming cell Erythropoietin 

Notes

Acknowledgments

We thank Gordon Keller and Marion Kennedy for sharing so generously of their knowledge and expertise of tissue culture and hematopoietic progenitor colony assays. Scott Peslak and Paul Kingsley photographed the erythroid colonies. This work has been supported by funds from the National Institutes of Health and from the Strong Children’s Research Center, University of Rochester, Rochester, NY.

References

  1. 1.
    Sun J, Ramos A, Chapman B et al (2014) Clonal dynamics of native hematopoiesis. Nature 514:322–327CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Busch K, Klapproth K, Barile M et al (2015) Fundamental properties of unperturbed haematopoiesis from stem cells in vivo. Nature 518:542–546CrossRefPubMedGoogle Scholar
  3. 3.
    Pluznik DH, Sachs L (1965) The cloning of normal “mast” cells in tissue culture. J Cell Comp Physiol 66:319–324CrossRefGoogle Scholar
  4. 4.
    Bradley TR, Metcalf D (1966) The growth of mouse bone marrow cells in vitro. Aust J Exp Biol Med Sci 44:287–300CrossRefPubMedGoogle Scholar
  5. 5.
    Stephenson JR, Axelrad A, McLeod D, Shreeve M (1971) Induction of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro. Proc Natl Acad Sci U S A 68:1542–1546CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Heath DS, Axelrod AA, McLeod DL, Shreeve MM (1976) Separation of the erythropoietin-responsive BFU-E and CFU-E in mouse bone marrow by unit gravity separation. Blood 47:777–792PubMedGoogle Scholar
  7. 7.
    Iscove NN, Sieber F (1975) Erythroid progenitors in mouse bone marrow detected by macroscopic colony formation in culture. Exp Hematol 3:32–43PubMedGoogle Scholar
  8. 8.
    Koury MJ, Bondurant MC (1990) Erythropoietin retards DNA breakdown and prevents programmed death in erythroid progenitor cells. Science 248:378–381CrossRefPubMedGoogle Scholar
  9. 9.
    Wojchowski DM, Sathyanarayana P, Dev A (2010) Erythropoietin receptor response circuits. Curr Opin Hematol 17:169–176Google Scholar
  10. 10.
    Koury MJ (2016) Tracking erythroid progenitor cells in times of need and times of plenty. Exp Hematol 44:653–663CrossRefPubMedGoogle Scholar
  11. 11.
    Cooper MC, Levy J, Cantor LN, Marks PA, Rifkind AR (1974) The effect of erythropoietin on colonial growth of erythroid precursor cells in vitro. Proc Natl Acad Sci U S A 71:1677–1680CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Gregory CJ, Eaves AC (1977) Human marrow cells capable of erythropoietic differentiation in vitro: definition of three erythroid colony responses. Blood 49:855–864PubMedGoogle Scholar
  13. 13.
    Eliason JF, Van Zant G, Goldwasser E (1979) The relationship of hemoglobin synthesis to erythroid colony and burst formation. Blood 53:935–945PubMedGoogle Scholar
  14. 14.
    Gregory CJ (1976) Erythropoietin sensitivity as a differentiation marker in the hemopoietic system: studies of three erythropoietic colony responses in culture. J Cell Physiol 89:289–301CrossRefPubMedGoogle Scholar
  15. 15.
    Axelrad AA, McLeod DL, Shreeve MM, Heath DS (1974) Properties of cells that produce erythrocytic colonies in vitro. In: Robinson WA (ed) Proceedings of the Second international workshop on hemopoiesis in culture, DHEW publication no NIH 74-205. US Government Printing Office, Washington, DC, pp p226–p234Google Scholar
  16. 16.
    Sonoda Y, Yang YC, Wong GG et al (1988) Erythroid burst-promoting activity of purified recombinant human GM-CSF and interleukin-3: studies with anti-GM-CSF and anti-IL-3 sera and studies in serum-free cultures. Blood 72:1381–1386PubMedGoogle Scholar
  17. 17.
    Mitjavila MT, Natazawa M, Brignaschi P et al (1989) Effects of five recombinant hematopoietic growth factors on enriched human erythroid progenitors in serum-replaced cultures. J Cell Physiol 138:617–623CrossRefPubMedGoogle Scholar
  18. 18.
    Sonoda Y, Sakabe H, Ohmisono Y et al (1994) Synergistic actions of stem cell factor and other burst-promoting activities on proliferation of CD34+ highly purified blood progenitors expressing HLA-DR or different levels of c-kit protein. Blood 84:4099–4106PubMedGoogle Scholar
  19. 19.
    Gregory CJ, McCollough EA, Till TA (1973) Erythropoietic progenitors capable of colony formation in culture: state of differentiation. J Cell Physiol 81:411–420CrossRefPubMedGoogle Scholar
  20. 20.
    Peslak SA, Wenger J, Bemis JC et al (2012) EPO-mediated expansion of late-stage erythroid progenitors in the bone marrow initiates recovery from sublethal radiation stress. Blood 120:2501–2511CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Clarke BJ, Housman D (1977) Characterization of an erythroid precursor cell of high proliferative capacity in normal human peripheral blood. Proc Natl Acad Sci U S A 74:1105–1109Google Scholar
  22. 22.
    Hara H, Ogawa M (1977) Erythropoietic precursors in murine blood. Exp Hematol 5:161–165PubMedGoogle Scholar
  23. 23.
    Wong PMC, Chung SW, Reicheld SM, Chui DH (1986a) Hemoglobin switching during murine embryonic development: evidence for two populations of embryonic erythropoietic progenitor cells. Blood 67:716–721PubMedGoogle Scholar
  24. 24.
    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
  25. 25.
    Wong PMC, Chung SW, Chui DHK, Eaves CJ (1986b) Properties of the earliest clonogenic hemopoietic precursors to appear in the developing murine yolk sac. Proc Natl Acad Sci U S A 83:3851–3854CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Lux C, Yoshimoto M, McGrath KE et al (2008) All primitive and definitive hematopoietic progenitor cells emerging prior to E10 in the mouse embryo are products of the yolk sac. Blood 111:3435–3438CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    McGrath KE, Frame JM, Fromm GJ, Koniski AD, Kingsley PD, Little J, Bulger M, Palis J (2011) A transient definitive erythroid lineage with unique regulation of the beta-globin locus in the mammalian embryo. Blood 117:4600–4608CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Zimmermann F, Rich IN (1996) The sensitivity of in vitro erythropoietic progenitor cells to different erythropoietin reagents during development and the role of cell death in culture. Exp Hematol 24:330–339PubMedGoogle Scholar
  29. 29.
    Emerson SG, Thomas S, Ferrara JL, Greenstein JL (1989) Developmental regulation of erythropoiesis by hematopoietic growth factors: analysis on populations of BFU-E from bone marrow, peripheral blood, and fetal liver. Blood 74:49–55PubMedGoogle Scholar
  30. 30.
    Valtieri M, Gabbianelli M, Pelosi E et al (1989) Erythropoietin alone induces erythroid burst formation by human embryonic but not adult BFU-E in unicellular serum-free culture. Blood 74:460–470PubMedGoogle Scholar
  31. 31.
    Perry JM, Harandi OF, Paulson RF (2007) BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors. Blood 15:4494–4502CrossRefGoogle Scholar
  32. 32.
    Alter BP (1979) Fetal erythropoiesis in stress hematopoiesis. Exp Hematol 7(Suppl 5):200–209PubMedGoogle Scholar
  33. 33.
    Porayette P, Paulson RF (2008) BMP4/Smad5 dependent stress erythropoiesis is required for the expansion of erythroid progenitors during fetal development. Dev Biol 317:24–35CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Hogan BLM, Beddington RSP, Constantini F, Lacy E (1994) Manipulating the mouse embryo: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, PlainviewGoogle Scholar
  35. 35.
    Hara H, Ogawa M (1978) Murine hematopoietic colonies in culture containing normoblasts, macrophages, and megakaryocytes. Am J Hematol 4:23–34CrossRefPubMedGoogle Scholar
  36. 36.
    Downs KM, Davies T (1993) Staging of gastrulating mouse embryos by morphological landmarks in the dissecting microscope. Development 118:1255–1266PubMedGoogle Scholar
  37. 37.
    Kaufman MH (1992) The atlas of mouse development. Academic, New YorkGoogle Scholar

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© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Pediatrics, Center for Pediatric Biomedical ResearchUniversity of Rochester Medical CenterRochesterUSA

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