Accelerated cell-cycling of hematopoietic progenitor cells by growth factors

Abstract

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.

A possible change in doubling time of haemopoietic progenitor cells with stem cell development

“…Since the growth fraction at stage III in the presence of IL-3 plus IL-11 plus SF was significantly low, we next estimated the average of the actual time for cell division of each cell in the process of formation of colonies (Table IV). When the time for the number of cells in colonies containing 20-40 cells to double in number is DTp, the time for a single cell in the colonies containing 20-40 cells to become two daughter cells is DTc, and the growth fraction is E, the association among DTp, DTc and E is given by the following equation: 40 20 2 E 2 2 DTp/DTc 20(1 ÿ E), as we previously reported (Tanaka et al, 1995). Therefore DTc can be calculated by the equation: DTc DTp 2 log2/log(1 1/E).…”

“…Previous studies using 3 H-thymidine labelling and CFU-S assay explored the kinetics of growth of haemopoietic progenitors (Testa & Lord, 1973;Schofield et al, 1980). In prior studies we found that the doubling time of haemopoietic progenitors can be modulated by growth factors, determined by monitoring the proliferative kinetics of haemopoietic progenitors during the early phase of an in vitro clonal assay (Tanaka et al, 1995;Ohishi et al, 1996). In the present study we used the same technique to characterize growth of haemopoietic progenitors at different stages of stem cell development and focused on changes in doubling time.…”

“…Singlecell suspensions were prepared from pooled femora and tibiae of normal and 5-FU-treated mice. The marrow cells were enriched for progenitors by density gradient separation and negative immunomagnetic selection, using a cocktail of lineage-specific monoclonal antibodies with a modification of the technique, as described previously (Shih et al, 1992;Katayama et al, 1993a, b;Tanaka et al, 1995). Anti-B220 (14.8), anti-CD4 (GK1.5), anti-CD8 (53-6.72), and anti-Gr-1 (RB6-8C5) were purchased from Pharmingen (San Diego, U.S.A.).…”

“…Clonal culture of enriched marrow cells from normal and 5-FUtreated mice. Serum-free methylcellulose culture was carried out in 35 mm Falcon suspension culture dishes (Becton Dickinson Labware, Lincoln Park, U.S.A.), using a modification of the technique described elsewhere (Iscove et al, 1980;Tanaka et al, 1995). Enriched marrow cells of normal and 5-FU-treated mice were plated at the concentration of 500 cells per plate.…”

“…1% sodium citrate containing 0 . 05% Nonidet P-40 (Sigma) and 10 g/ml of RNase (Sigma) for 20 min in the dark and then analysed on a FACScan cytometer (Becton Dickinson Immunocytometry Systems, San Jose, U.S.A.), using the technique previously described (Tanaka et al, 1995;. For each sample, DNA histograms were analysed, using CellFIT software (Becton Dickinson Immunocytometry Systems) to determine the distribution of cells in G1, S and G2 M phases of the cell cycle.…”