D. Hogge et al., QUANTITATION AND CHARACTERIZATION OF HUMAN MEGAKARYOCYTE COLONY-FORMING CELLS USING A STANDARDIZED SERUM-FREE AGAROSE ASSAY, British Journal of Haematology, 96(4), 1997, pp. 790-800
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.