Maintenance of hematopoiesis in serum-free bone marrow cultures involves sequential recruitment of quiescent progenitors.
We previously described that cells with a CD34+CD71lo phenotype from 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 example, > 10(6)-fold per cell), this was an unexpected finding. The following models for the observed maintenance of CD34+CD71lo cells in our cultures were considered: (1) survival of non-dividing cells; (2) self-renewal balanced by loss of cells; (3) asymmetrical divisions; and (4) combinations of the above. Two experimental strategies were explored to discriminate between these models. In the first, sorted CD34+CD45RAloCD71lo cells were labeled with the flourescent tracking dye PKH26, followed by analysis of PKH26 fluorescence of CD34+CD71lo and other cells present in the cultures at various times (up to 11 weeks). In the second approach, single CD34+CD45RAloCD71lo cells were directly sorted into individual wells, and growing cells were then 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 contributed significantly to the total cell production measured at day 20 (ranging from 1 to 5%); and (3) the number of CD34+ cells present in individual clones. Taken together, the observed maintenance of primitive CD34+ cells in our cultures apparently involves a combination of survival of CD34+CD71lo cells with a vary low turnover together with a very limited production of CD34+ cells. Clonal heterogeneity, differences in cell cycle kinetics between CD34+ and CD34- cells, and observations that the majority of bone marrow-derived CD34+CD45RAloCD71lo 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 expansion of primitive hematopoietic cells ex vivo.
Lansdorp
P M
PM
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada.
Dragowska
W
W
D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34; D000944:Antigens, Differentiation, B-Lymphocyte / Q000032:analysis; D001769:Blood; D001854:Bone Marrow Cells; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D002999:Clone Cells / Q000166:cytology; D004921:Erythropoietin / Q000494:pharmacology; D005434:Flow Cytometry; D006410:Hematopoiesis; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D007700:Kinetics; D017493:Leukocyte Common Antigens / Q000032:analysis; D010641:Phenotype; D011990:Receptors, Transferrin; D019089:Stem Cell Factor
D015703:Antigens, CD; D018952:Antigens, CD34; D000944:Antigens, Differentiation, B-Lymphocyte; C068126:CD71 antigen; D016298:Hematopoietic Cell Growth Factors; D007377:Interleukin-3; D015850:Interleukin-6; D011990:Receptors, Transferrin; D019089:Stem Cell Factor; D004921:Erythropoietin; D017493:Leukocyte Common Antigens