Pm. Lansdorp et W. Dragowska, MAINTENANCE OF HEMATOPOIESIS IN SERUM-FREE BONE-MARROW CULTURES INVOLVES SEQUENTIAL RECRUITMENT OF QUIESCENT PROGENITORS, Experimental hematology, 21(10), 1993, pp. 1321-1327
We previously described that cells with a CD34(+)CD71(lo) phenotype fr
om adult human bone marrow are maintained at constant numbers in long-
term suspension cultures supplemented with interleukin-6 (IL-6), IL-3,
mast growth factor (MGF) (a c-kit ligand), and erythropoietin (Epo).
In view of the large increase in cell numbers in such cultures (for ex
ample, >10(6)-fold per cell), this was an unexpected finding. The foll
owing models for the observed maintenance of CD34(+)CD71(lo) cells in
our cultures were considered: (1) survival of non-dividing cells; (2)
self-renewal balanced by loss of cells; (3) asymmetrical divisions; an
d (4) combinations of the above. Two experimental strategies were expl
ored to discriminate between these models. In the first, sorted CD34()CD45RA(lo)CD71(lo) cells were labeled with the fluorescent tracking d
ye PKH26, followed by analysis of PKH26 fluorescence of CD34(+)CD71(lo
) and other cells present in the cultures at various times (up to 11 w
eeks). In the second approach, single CD34(+)CD45RA(lo)CD71(lo) cells
were directly sorted into individual wells, and growing cells were the
n analyzed by flow cytometry. Results from these experiments indicated
a considerable variability in (1) the number of surviving input cells
(ranging from 30 to 80%); (2) the proportion of cells that contribute
d significantly to the total cell production measured at day 20 (rangi
ng from 1 to 5%); and (3) the number of CD34(+) cells present in indiv
idual clones. Taken together, the observed maintenance of primitive CD
34(+) cells in our cultures apparently involves a combination of survi
val of CD34(+)CD71(lo) cells with a very low turnover together with a
very limited production of CD34(+) cells. Clonal heterogeneity, differ
ences in cell cycle kinetics between CD34(+) and CD34(-) cells, and ob
servations that the majority of bone marrow-derived CD34(+)CD45RA(lo)C
D71(lo) cells do not show a rapid proliferative response to a mixture
of IL-6, IL-3, MGF, and Epo will have to be taken into account in the
development of experimental strategies aimed at clinically useful expa
nsion of primitive hematopoietic cells ex vivo.