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 t heir ability to generate colonies containing from 3 to >100 Mk, detect able as glycoprotein IIb/IIIa(+) cells in APAAP-stained whole mount ag arose cultures. Optimal growth conditions were achieved through the us e 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 nu mber of CFC-Mk at 18-21 d was linearly related to the number of cells plated, Variation in the cytokines added showed that thrombopoietin (T PO) or IL-3 alone would support the formation of large numbers of CFC- Mk. However, optimal yields of colonies containing cells of both Mk an d non-Mk lineages required the addition of other growth factors, of wh ich 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 t o this combination reduced the number of large 'pure' Mk colonies seen and in their place a corresponding number of mixed erythroid-Mk colon ies became detectable, Flt-ligand alone was unable to support the grow th of CFC-Mk nor did it enhance their growth when combined with other factors. Plating of FAGS-sorted subpopulations of CD34(+) marrow cells in both serum-fret agarose and methylcellulose assays demonstrated th at most CFC-Mk are generated from CD34(+) cells that are CD45RA(-) and CD71(+), approximately half of which are CD41(+). Thus, CFC-Mk are mo re similar to primitive clonogenic erythroid progenitors than to their granulopoietic counterparts in their expression of CD34, CD45RA and C D71, Taken together, these findings support the concept that some eryt hroid and Mk progenitors may share a common developmental pathway, The availability of sensitive and reproducible procedures for isolating a nd detecting human Mk progenitors should facilitate future investigati ons of their biology and role in a variety of haematological condition s.