Primitive hematopoietic progenitor cete (HPC) residing in the GO phase of cell cycle may be the most suited candidates for the examination olcell activation and prolifération. We designed a double simultaneous labeling technique utilizing both DNA and RNA staining with Hoechst 33342 and Pyronin V, respectively, in order to isolate BM CD34+ cells residing in GO (GO CD34+). In vitro proliferation of these cells in response to sequential cytokine stimulation was examined in a two step assay. In the first step, cells were incubated for 7 days (initiât sti mutation) with either SCF, Fits-1 ig and (FL), IL3, or IL6. In the second, each group was washed and split into 4 groups, each of which was cultured again for another week with one of the 4 initial cytokines individually. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7 Overall examination of proliferation patterns of these cells for 2 weeks revealed that cells could progress into four phases ot proliferation. Phase I contained cytokine nonresponsive cells which failed to proliferate. Phase II contained cells dividing up to 3 times within the first 7 days. Phases III and IV consisted of cells dividing up to 5 divisions and between 6 and B divisions, respectively, by the end of the 14 day period Regardless of the cytokine used for initial stimulation, GO CD34+ cells moved only to phase II by day 7 and a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL lor the entire 14 day period did not progress beyond phase lit but proliferated into phase IV (with <20% of cells remaining in phases I and II) if IL3, but not IL6, was substituted for either cytokine on day 7 GO CD34+ ceHs incubated with IL3 for 14 days proliferated the most and progressed into phase IV, however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34+, detected in cultures initially stimulated with IL3, which remained as a distinct population unable to progress out of phase II regardless of the nature of the second stimulus received on day 7 These results suggest that orderly activation of CD34+ cells residing in GO and a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPC.
|Original language||English (US)|
|Number of pages||1|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology
- Cancer Research