Recent advances in molecular biology have led to the identification of hematopoietic growth factors that support and influence the proliferation of hematopoietic progenitor cells in vitro and in vivo. Although these factors have been extensively studied, little is known of their role in the regulation of cell-cycling of hematopoietic progenitors, especially in the early stage of hematopoiesis. In the present study, we examined the effects of early acting growth factors on proliferative kinetics of hematopoietic progenitors by monitoring the number of cells in individual developing colonies, using an in vitro clonal assay. Interleukin-11 (IL-11) or steel factor (SF), alone or in combination, shortened the time for the size of IL-3-dependent colonies to double. Consecutive replating experiments provided evidence for direct action of growth factors on the growth rate of hematopoietic progenitor cells. Shortening of the time for the total cell number in the colonies to double was due to a reduction in time for each single cell within the respective colonies to become two daughter cells, and there was no alteration in the incidence of cells with a proliferative capacity. Cell-cycle analysis demonstrated that IL-11 has the potential to induce a shortened time for cell-cycle of hematopoietic progenitor cells without affecting distribution of each fraction of the cell- cycle, whereas SF has the potential to reduce cell-cycle time mainly by decreasing the time required for hematopoietic progenitor cells to go through the G1 phase. These results suggest that growth factors may modulate cell-cycling of hematopoietic progenitor cells.

Although hematopoietic growth factors have been extensively studied as to their roles in recruitment of hematopoietic progenitors from quiescence state to cell division state, little is known of their effects on cell-cycling of progenitors that have already transited from quiescence into active cell-cycling. We examined the effects of the flt3 ligand (FL) on cell-cycling of hematopoietic progenitors in serum- free culture. Results from our serial observations of colony formation and replating experiments suggest that FL enhances the rate of growth of interleukin-3 (IL-3)-dependent colonies by shortening the time for each progenitor in the colonies to divide. Cell-cycle analysis showed that shortening of cell-cycle time induced by FL is mainly because of alteration in the G1 phase that hematopoietic progenitors go through. We next investigated the role of transforming growth factor-beta (TGF- beta) in cell-cycling of progenitors, using TGF-beta protein and TGF- beta antisense oligonucleotides, because mRNA of TGF-beta was detected by reverse transcriptase polymerase reaction in blast cells that we used as a source of progenitors. TGF-beta lengthened the time required for IL-3-dependent progenitors to become two daughter cells, whereas the effects of TGF-beta antisense oligonucleotides were opposite to those of TGF-beta. The addition of TGF-beta neutralizing monoclonal antibodies to the cultures resulted in effects similar to those seen with TGF-beta antisense oligonucleotides. DNA studies indicated that both TBF-beta and TGF-beta antisense oligonucleotides change the length of G1 phase of the cell-cycle. TGF-beta abrogated the effects of FL on the growth rate of hematopoietic progenitors, whereas the combination of FL with TGF-beta antisense oligonucleotides exerted additive effects. These data show that FL has the potential to accelerate cell- cycling of hematopoietic progenitors, which is susceptible to the modulation by TGF-beta.

We examined the role of the ligand for c-mpl. thrombopoietin (TPO). in murine early haemopoiesis. using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) supported colony formation by marrow cells of 5-fluorouracil (5-FU)-treated mice whereas TPO alone yielded no colony. When blast cell colonies grown in the presence of TPO plus SF or TPO plus IL-3 were individually replated in suspension cultures containing serum and several growth factors, various combinations of myeloid lineages were seen, indicating that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of haemopoietic progenitors. We then studied the effects of TPO on proliferative kinetics of cycling progenitors. TPO hastened IL-3-dependent growth of progenitors by shortening the time required for cell cycling. These results suggest that TPO as a single factor, can support the survival of haemopoietic progenitors and TPO synergizes with SF or IL-3 to act on early multipotential haemopoietic progenitors.

We separated haemopoietic progenitors derived from marrow cells of 5-fluorouracil (5-FU)-treated mice into three groups, based on the stages of stem cell development and studied doubling time, using a serum-free clonal culture system. Stage I progenitors were those present in primary marrow cells from 5-FU-treated mice. Stages II and III progenitors were early and late progenies in culture of stage I progenitors respectively. The morphological analysis of colonies derived from stage I, II and III progenitors demonstrated an association of progression of stages with loss of multipotentiality. The doubling time of haemopoietic progenitors was estimated by sequential analysis of colony formation and studies of growth fraction. The time required for haemopoietic progenitors to double shortened as their stage of development progressed. Alteration in one doubling time of haemopoietic progenitors at progressive stages of stem cell development was seen in cultures supported by various combinations of growth factors, including interleukin-3 (IL-3), IL-11. and steel factor (SF), Cell-cycle analysis suggested that reduction of the doubling time of haemopoietic progenitors is probably due to a decrease in the time spent in the G1 phase of the cell cycle. Our results suggest that in early haemopoiesis the doubling time of haemopoietic progenitors may change with stem cell development.

Proto-oncogene c-mpl is structurally homologous with the hematopoietic growth factor receptor superfamily. The ligand for c-mpl was purified and its gene cloned. Extensive functional studies revealed that the ligand for c-mpl plays a crucial role in megakaryocytopoiesis and platelet production, hence, this ligand proved to be the long-sought hematopoietin, thrombopoietin (TPO). We briefly review here the role for TPO in early hematopoiesis, based on our in vitro data obtained using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) but not TPO alone supported the growth of murine primitive hematopoietic progenitors. Studies on lineage expression indicated that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of primitive hematopoietic progenitors. TPO also hastened IL-3-dependent growth of progenitors by shortening the time required for cell-cycling. While size of the colonies did not differ between colonies supported by IL-3 alone and those supported by IL-3 plus TPO, the incidence of megakaryocyte-containing colonies in cultures supported by IL-3 plus TPO was higher than that in cultures supported by IL-3 alone. Taken together, TPO as a single factor can support the survival of hematopoietic progenitors and TPO synergizes with SF or IL-3 to be active on early multipotential hematopoietic progenitors. These findings suggest that the function of TPO initially thought to be restricted to the megakaryocytic lineage proved to be greater in hematopoiesis. Reports from other laboratories regarding the involvement of TPO in early hematopoiesis are also discussed.