ORDERLY PROCESS OF SEQUENTIAL CYTOKINE STIMULATION IS REQUIRED FOR ACTIVATION AND MAXIMAL PROLIFERATION OF PRIMITIVE HUMAN BONE-MARROW CD34(+) HEMATOPOIETIC PROGENITOR CELLS RESIDING IN G(0)

Bone marrow (BM) CD34(+) cells residing in the G(0) phase of cell cycl e may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cel ls (HPCs). We designed a double simultaneous labelling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectiv ely, to isolate CD34(+) cells residing in G(0)(G(0)CD34(+)). Using lon g-term BM cultures and limiting dilution analysis, G(0)CD34(+) cells w ere found to be enriched for primitive HPCs. In vitro proliferation of G(0)CD34(+) cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a prim ary stimulation consisting of either stem cell factor (SCF), Flt3-liga nd (FL), interleukin-3 (IL-3), or IL-6 far 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the fou r primary cytokines individually, or in combination. Tracking of proge ny cells was accomplished by staining cells with PKH2 on day 0 and wit h PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferat ion. Phase I contained cytokine nonresponsive cells that failed to pro liferate. Phase II contained cells dividing up to three times within t he first 7 days. Phases III and IV consisted of cells dividing up to f ive divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary sti mulation, G(0)CD34(+) cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with <20 % of cells remaining in phases I and II) if IL-3, but not IL-6, was su bstituted for either cytokine on day 7. G(o)CD34(+) cells incubated wi th IL-3 for 14 days proliferated the most and progressed into phase IV ; however, when SCF was substituted on day 7, cells failed to prolifer ate into phase IV. Most intriguing was a group of cells, many of which were CD34(+), detected in cultures initially stimulated with IL-3, wh ich remained as a distinct population, mostly in G(0)/G(1), unable to progress out of phase II regardless of the nature of the second stimul us received on day 7. A small percentage of these cells expressed cycl in E, suggesting that their proliferation arrest may have been mediate d by a cyclin-related disruption in cell cycle. These results suggest that a programmed response to sequential cytokine stimulation may be p art of a control mechanism required for maintenance of proliferation o f primitive HPCs and that unscheduled stimulation of CD34(+) cells res iding in G(0) may result in disruption of cell-cycle regulation. (C) 1 997 by The American Society of Hematology.