7689481

pubmed:Citation

10

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.

eng

IM

MEDLINE

0301-472X

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NLM

1321-7

pubmed-meshheading:7689481-Antigens, CD, pubmed-meshheading:7689481-Antigens, CD34, pubmed-meshheading:7689481-Antigens, CD45, pubmed-meshheading:7689481-Antigens, Differentiation, B-Lymphocyte, pubmed-meshheading:7689481-Blood, pubmed-meshheading:7689481-Bone Marrow Cells, pubmed-meshheading:7689481-Cell Division, pubmed-meshheading:7689481-Cell Survival, pubmed-meshheading:7689481-Cells, Cultured, pubmed-meshheading:7689481-Clone Cells, pubmed-meshheading:7689481-Erythropoietin, pubmed-meshheading:7689481-Flow Cytometry, pubmed-meshheading:7689481-Hematopoiesis, pubmed-meshheading:7689481-Hematopoietic Cell Growth Factors, pubmed-meshheading:7689481-Hematopoietic Stem Cells, pubmed-meshheading:7689481-Humans, pubmed-meshheading:7689481-Interleukin-3, pubmed-meshheading:7689481-Interleukin-6, pubmed-meshheading:7689481-Kinetics, pubmed-meshheading:7689481-Phenotype, pubmed-meshheading:7689481-Receptors, Transferrin, pubmed-meshheading:7689481-Stem Cell Factor

Maintenance of hematopoiesis in serum-free bone marrow cultures involves sequential recruitment of quiescent progenitors.

Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't