MAINTENANCE OF HEMATOPOIESIS IN SERUM-FREE BONE-MARROW CULTURES INVOLVES SEQUENTIAL RECRUITMENT OF QUIESCENT PROGENITORS

Citation
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
Citations number
22
Categorie Soggetti
Medicine, Research & Experimental",Hematology
Journal title
ISSN journal
0301472X
Volume
21
Issue
10
Year of publication
1993
Pages
1321 - 1327
Database
ISI
SICI code
0301-472X(1993)21:10<1321:MOHISB>2.0.ZU;2-X
Abstract
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.