Basic Cell Culture Protocols

Volume 946 of the series Methods in Molecular Biology pp 267-283


Colony Forming Cell Assays for Human Hematopoietic Progenitor Cells

  • Bert WognumAffiliated withSTEMCELL Technologies Email author 
  • , Ning YuanAffiliated withSTEMCELL Technologies
  • , Becky LaiAffiliated withSTEMCELL Technologies
  • , Cindy L. MillerAffiliated withSTEMCELL Technologies

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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