Colony Forming Cell Assays for Human Hematopoietic Progenitor Cells

  • Bert Wognum
  • Ning Yuan
  • Becky Lai
  • Cindy L. Miller
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 946)

Abstract

Hematopoietic stem cells (HSCs) present in small numbers in adult bone marrow (BM), peripheral blood (PB) and umbilical cord blood (CB) produce a heterogeneous pool of progenitors that can be detected in vitro using colony forming cell (CFC) assays. Hematopoietic progenitor cells proliferate and differentiate to produce colonies of maturing cells when cultured in a semisolid methylcellulose-based medium that is supplemented with suitable growth factors and other supplements. The colonies are then classified and enumerated in situ by light microscopy or an automated imaging instrument. CFC assays are important tools in basic hematology research but are also used by clinical cell processing laboratories to measure the progenitor cell content of BM, CB and mobilized PB (MPB) preparations used for cell transplantation. Standard CFC assays for human progenitor cells require a culture period of at least 14 days to enable optimal outgrowth and differentiation of the maximum number of CFCs in a cell preparation. In this chapter protocols are described for the detection and enumeration of myeloid multipotential progenitors and committed progenitors of the erythroid, monocyte, and granulocyte lineages in samples from human PB, MPB, BM, and CB. In addition protocols are described for a modified version of the CFC-assay that allows accurate enumeration of total CFC numbers in CB or MPB after a culture period of only 7 days, but without distinction of colony types.

Key words

Hematopoietic progenitors Bone marrow Peripheral blood Umbilical cord blood Colony-forming cell assays CFU-GEMM BFU-E CFU-E CFU-GM 

References

  1. 1.
    Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC, Eaves CJ (1990) Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc Natl Acad Sci USA 87:8736–8740PubMedCrossRefGoogle Scholar
  2. 2.
    Conneally E, Cashman J, Petzer A, Eaves C (1997) 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 94:9836–9841PubMedCrossRefGoogle Scholar
  3. 3.
    Szilvassy SJ, Nicolini FE, Eaves CJ, Miller CL (2002) Quantitation of murine and human hematopoietic stem cells by limiting dilution analysis in competitively repopulated hosts. In: Klug CA, Jordan CT (eds) Hematopoietic stem cell protocols. Humana, Totowa, NJGoogle Scholar
  4. 4.
    Sutherland HJ, Lansdorp PM, Henkelman DH, Eaves AC, Eaves CJ (1990) Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc Natl Acad Sci USA 87:3584–3588PubMedCrossRefGoogle Scholar
  5. 5.
    Miller CL, Eaves CJ (2002) Long-term culture-initiating cell assays for human and murine cells. In: Klug CA, Jordan CT (eds) Hematopoietic stem cell protocols. Humana, Totowa, NJGoogle Scholar
  6. 6.
    Ploemacher RE, van der Sluijs JP, van Beurden CA, Baert MR, Chan PL (1991) Use of a limiting-dilution type long-term cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse. Blood 78:2527–2533PubMedGoogle Scholar
  7. 7.
    de Haan G, Ploemacher R (2002) The cobble-area-forming cell assay. In: Klug CA, Jordan CT (eds) Hematopoietic stem cell protocols. Humana, Totowa, NJGoogle Scholar
  8. 8.
    Nakahata T, Ogawa M (1982) 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 79:3843–3847PubMedCrossRefGoogle Scholar
  9. 9.
    Brandt JE, Baird N, Lu L, Srour E, Hoffman R (1988) Characterization of a human hematopoietic progenitor cell capable of forming blast cell containing colonies in vitro. J Clin Invest 82:1017–1027PubMedCrossRefGoogle Scholar
  10. 10.
    McNiece IK, Stewart FM, Deacon DM, Temeles DS, Zsebo KM, Clark SC, Quesenberry PJ (1989) Detection of a human CFC with a high proliferative potential. Blood 74:609–612PubMedGoogle Scholar
  11. 11.
    Fauser AA, Messner HA (1979) Identification of megakaryocytes, macrophages, and eosinophils in colonies of human bone marrow containing neutrophilic granulocytes and erythroblasts. Blood 53:1023–1027PubMedGoogle Scholar
  12. 12.
    Ichikawa Y, Pluznik DH, Sachs L (1966) In vitro control of the development of macrophage and granulocyte colonies. Proc Natl Acad Sci USA 56:488–495PubMedCrossRefGoogle Scholar
  13. 13.
    Stephenson JR, Axelrad AA, McLeod DL, Shreeve MM (1971) Induction of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro. Proc Natl Acad Sci USA 68:1542–1546PubMedCrossRefGoogle Scholar
  14. 14.
    Eaves CJ, Eaves AC (1978) Erythropoietin (Ep) dose-response curves for three classes of erythroid progenitors in normal human marrow and in patients with polycythemia vera. Blood 52:1196–1210PubMedGoogle Scholar
  15. 15.
    Nakeff A, Daniels-McQueen S (1976) In vitro colony assay for a new class of megakaryocyte precursor: colony-forming unit megakaryocyte (CFU-M). Proc Soc Exp Biol Med 151:587–590PubMedGoogle Scholar
  16. 16.
    Dobo I, Allegraud A, Navenot JM, Boasson M, Bidet JM, Praloran VJ (1995) Collagen matrix: an attractive alternative to agar and methylcellulose for the culture of hematopoietic progenitors in autologous transplantation products. J Hematother 4:281–287PubMedCrossRefGoogle Scholar
  17. 17.
    Hogge D, Fanning S, Bockhold K, Petzer A, Lambie K, Lansdorp P, Eaves A, Eaves C (1997) Quantitation and characterization of human megakaryocyte colony-forming cells using a standardized serum-free agarose assay. Br J Haematol 96:790–800PubMedCrossRefGoogle Scholar
  18. 18.
    Paige CJ, Kincade PW, Shinefeld LA, Sato VL (1981) Precursors of murine B lymphocytes. Physical and functional characterization and distinctions from myeloid stem cells. J Exp Med 153:154–165PubMedCrossRefGoogle Scholar
  19. 19.
    Zijlmans JM, Visser JW, Laterveer L, Kleiverda K, Heemskerk DP, Kluin PM, Willemze R, Fibbe WE (1998) The early phase of engraftment after murine blood cell transplantation is mediated by hematopoietic stem cells. Proc Natl Acad Sci USA 95:725–729PubMedCrossRefGoogle Scholar
  20. 20.
    Migliaccio AR, Adamson JW, Stevens CE, Dobrila NL, Carrier CM, Rubinstein P (2000) Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: graft progenitor cells content is a better predictor than nucleated cell quantity. Blood 96:2717–2722PubMedGoogle Scholar
  21. 21.
    Hogge DE, Lambie K, Sutherland HJ, Benny WB, Dalal B, Currie C, Barnett MJ, Eaves AC, Eaves CJ (2000) Quantitation of primitive and lineage-committed progenitors in mobilized peripheral blood for prediction of platelet recovery post autologous transplant. Bone Marrow Transplant 25:589–598PubMedCrossRefGoogle Scholar
  22. 22.
    Iori AP, Cerretti R, De Felice L, Screnci M, Mengarelli A, Romano A, Caniglia M, Cerilli L, Gentile G, Moleti ML, Giona F, Agostini F, Pasqua I, Perrone MP, Pinto MR, Grapulin L, Testi AM, Martino P, De Rossi G, Mandelli F, Arcese W (2004) Pre-transplant prognostic factors for patients with high-risk leukemia undergoing an unrelated cord blood tranplantation. Bone Marrow Transplant 33:1097–1105PubMedCrossRefGoogle Scholar
  23. 23.
    Yang H, Acker JP, Cabuhat M, Letcher B, Larratt L, McGann LE (2005) Association of post-thaw viable CD34+ cells and CFU-GM with time to hematopoietic engraftment. Bone Marrow Transplant 35:881–887PubMedCrossRefGoogle Scholar
  24. 24.
    Yoo KH, Lee SH, Kim HJ, Sung KW, Jung HL, Cho EJ, Park HK, Kim HA, Koo HH (2007) The impact of post-thaw colony-forming units-granulocyte/macrophage on engraftment following unrelated cord blood transplantation in pediatric recipients. Bone Marrow Transplant 39:515–521PubMedCrossRefGoogle Scholar
  25. 25.
    Prasad VK, Mendizabal A, Parikh SH, Szabolcs P, Driscoll TA, Page K, Lakshminarayanan S, Allison J, Wood S, Semmel D, Escolar ML, Martin PL, Carter S, Kurtzberg J (2008) Unrelated donor umbilical cord blood transplantation for inherited metabolic disorders in 159 pediatric patients from a single center: influence of cellular composition of the graft on transplantation outcomes. Blood 112:2979–2989PubMedCrossRefGoogle Scholar
  26. 26.
    Ogawa M (1993) Differentiation and proliferation of hematopoietic stem cells. Blood 81:2844–2853PubMedGoogle Scholar
  27. 27.
    Krystal G, Alai M, Cutler RL, Dickeson H, Mui AL, Wognum AW (1991) Hematopoietic growth factor receptors. Hematol Pathol 5:141–162PubMedGoogle Scholar
  28. 28.
    Kaushansky K, Drachman JG (2002) The molecular and cellular biology of thrombopoietin: the primary regulator of platelet production. Oncogene 21:3359–3367PubMedCrossRefGoogle Scholar
  29. 29.
    Metcalf D (2008) Hematopoietic cytokines. Blood 111:485–491PubMedCrossRefGoogle Scholar
  30. 30.
    Pereira C, Clarke E, Damen J (2007) Hematopoietic colony-forming cell assays. In: Vemuri MC (ed) Methods in molecular biology 407, stem cell assays. Humana, Totowa, NJGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Bert Wognum
    • 1
  • Ning Yuan
    • 1
  • Becky Lai
    • 1
  • Cindy L. Miller
    • 1
  1. 1.STEMCELL TechnologiesVancouverCanada

Personalised recommendations