Activation and proliferation kinetics of single quiescent cd34+ cells residing in GO

R. Pvatl, J. McMahel, S. Griasbv, E. F. Srour

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Exit of primitive hematopoietic progenitor cells from GO phase of ceil cycle in response to in vitro cytokine stimulation is associated with a decrease of hematopoietic potential. We hypothesized that loss of primitive function could be modulated by the nature of the initial cytokine stimulation. To test this hypothesis, we evaluated sequential activation by single cytokines on the most quiescent bone marrow (BM) CD34+ cells assayable, those within GO phase of cell cycle. To this end, individual CD34+ cells exhibiting low DNA staining with Hoechst 33342 and tow RNA labeling with Pyronin Y (GO CD34+) were sorted in 96well plates and incubated for 7 days (initial stimulation) with either IL3, SCF. or Flt3 ligand (FL), after which cells were enumerated in each well. While 59% and 57% of viable cells divided in response to IL3 or FL, respectively, 82% of cells stimulated with SCF proliferated, indicating a higher susceptibility of GO CD34+ cells to SCF activation. The proliferative ability of cells that had remained quiescent during the first week and those that had given rise to 2,4 or 8 cells were compared by an additional week incubation with the combination of IL3, SCF and FL. The most significant findings were: 1 ) regardless of the cytokine used for the initial stimulation, the number of prior divisions correlated with a lesser degree of subsequent expansion in IL3-SCF-FL combination, from 21.5 ±5.8 fold expansion for 2-cell clones to 5 ±2.6 fold expansion for 8-cell clones; quiescent cells were still able to proliferate to the highest extent of 31.8 ±22.9 fold; 2} among primary clones resulting from two or more divisions, those initially treated with SCF or FL could retain a higher protrlerative potential (11.3 ±5.8 and 12.1 ±9.6 fold respectively) than those first stimulated with IL3 (5.5 ±3.4 fold}. Finally, clones generated from initial stimulation with IL3 or SCF and then exposed for an additional week to IL3-SCF-FL were assayed for colonyforming cells (CFC). Whereas 46% of the celts activated by SCF formed colonies, only 31 % of those stimulated by IL3 could do so. The difference was most striking when considering clones of 4 cells or more such that 11% of IL3- stimulated clones were preCFC versus 43% after SCF activation. These results suggest that SCF can achieve effective stimulation of quiescent HSC with limited loss of primitive hematopoietic function and demonstrate the existence of a hierarchy among CD34+ cells residing in GO based on their susceptibility to cytokine activation and relative degree of mitotic quiescence.

Original languageEnglish (US)
Number of pages1
JournalExperimental Hematology
Issue number9
StatePublished - Dec 1 1996

ASJC Scopus subject areas

  • Molecular Biology
  • Hematology
  • Genetics
  • Cell Biology
  • Cancer Research

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