Quantitation and characterization of human megakaryocyte colony‐forming cells using a standardized serum‐free agarose assay

Human progenitors of the megakaryocyte (Mk) lineage were detected by their ability to generate colonies containing from 3 to >100 Mk, detectable as glycoprotein IIb/IIIa+ cells in APAAP‐stained whole mount agarose cultures. Optimal growth conditions were achieved through the use of a defined serum substitute and a suitable cocktail of recombinant cytokines. Under these culture conditions, the smallest Mk‐containing colonies (CFC‐Mk) were detectable within a week followed by colonies containing larger numbers of Mk over the ensuing 2 weeks. The total number of CFC‐Mk at 18–21 d was linearly related to the number of cells plated. Variation in the cytokines added showed that thrombopoietin (TPO) or IL‐3 alone would support the formation of large numbers of CFC‐Mk. However, optimal yields of colonies containing cells of both Mk and non‐Mk lineages required the addition of other growth factors, of which a combination of IL‐3, IL‐6, GM‐CSF and Steel factor (SF) ± TPO was the best of those tested. The further addition of erythropoietin to this combination reduced the number of large ‘pure’ Mk colonies seen and in their place a corresponding number of mixed erythroid‐Mk colonies became detectable. Flt3‐ligand alone was unable to support the growth of CFC‐Mk nor did it enhance their growth when combined with other factors. Plating of FACS‐sorted subpopulations of CD34+ marrow cells in both serum‐free agarose and methylcellulose assays demonstrated that most CFC‐Mk are generated from CD34+ cells that are CD45RA− and CD71+, approximately half of which are CD41+. Thus, CFC‐Mk are more similar to primitive clonogenic erythroid progenitors than to their granulopoietic counterparts in their expression of CD34, CD45RA and CD71. Taken together, these findings support the concept that some erythroid and Mk progenitors may share a common developmental pathway. The availability of sensitive and reproducible procedures for isolating and detecting human Mk progenitors should facilitate future investigations of their biology and role in a variety of haematological conditions.