Accelerated Cell-Cycling of Hematopoietic Progenitor Cells by Growth Factors

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1 Accelerated Cell-Cycling of Hematopoietic Progenitor Cells by Growth Factors By Ryuhei Tanaka, Naoyuki Katayama, Kohshi Ohishi, Nadim Mahmud, Ryugo Itoh, Yuka Tanaka, Yoshihiro Komada, Nobuyuki Minami, Minoru Sakurai, Shigeru Shirakawa, and Hiroshi Shiku Recent advances in molecular biology have led to the identification of hematopoietic growth factors that support and influence the prolieration 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-l1 (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. M AINTENANCE OF hematopoiesis requires continuous proliferation and differentiation of hematopoietic progenitor cells, which are likely to be regulated by a network of hematopoietic growth fa~t0rs.l.~ Some of these factors have been identified, and the related genes were With the availability of recombinant growth factors, in vitro assays led to acquisition of evidence that several hematopoietic growth factors including interleukin- 1 (IL- l), L-4, IL-6, E-7, IL-11, L-12, granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory proteins (MIP), and steel factor (c-kit ligand) (SF) can act in combination with other factors to augment the proliferative responses of hematopoietic progenitors, yet individually have little stimulatory As most of these results are based on the evaluation of colonies containing mature cells with no further potential for proliferation, they do not provide information on proliferative kinetics of individual hematopoietic progenitors in the early stage of growth, and the precise role of hematopoietic growth factors in the regulation of early hematopoiesis is not well understood. One approach to a better understanding of the mechanism of enhancement of the growth of hematopoietic progenitor cells by hematopoietic growth factors at the cellular level is the monitoring of proliferative behavior of hematopoietic progenitors during the early phase of an in vitro assay, under direct observation using an inverted microscope. We cultured hematopoietic progenitors in the presence of hematopoietic growth factors using a clonal culture system and serially observed and recorded the development of individual colonies. A serum-free culture system was used because the serum itself contains variable concentrations of hematopoietic growth factors or other substances, both of which may influence the proliferation of hematopoietic progenitor^.^"^^ Our results provide further insight into the mechanism of action of hematopoietic growth factors in the control of cellcycling of hematopoietic progenitor cells. MATERIALS AND METHODS Cell preparation. Ten- to 15-week-old male BDFl mice were obtained from Japan SLC, Ltd, Hamarnatsu, Japan. 5-Fluorouracil 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-l1 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 reducecell-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 by The American Society of Hematology. (5-FU) (Kyowa Hakko Kogyo CO, Ltd, Tokyo, Japan) at 150 mg/ kg body weight was administered through the tail veins 2 days before the mice were killed. Single-cell suspensions were prepared from pooled femurs and tibia of the mice. The marrow cells were enriched for progenitors by density gradient separation and immunomagnetic selection using a cocktail of monoclonal antibodies with a modification of the technique, as described previou~ly.~~~ Anti-B220 (14.8), CD4 (GK1.5), CD8 ( ), and Gr-l (RB6-8C5) were purchased from Pharmingen (San Diego, CA). Mac-l was purchased from Serotec (Oxford, England). Immunomagnetic beads (Dynabeads M- 450, coated with sheep antirat IgG) were purchased from Dynal AS (Oslo, Norway). Growth factors. Purified recombinant murine IL-3, murine SF, murine granulocyte/macrophage colony-stimulating factor (GM- CSF), human G-CSF, and human erythropoietin (Ep) were provided by Kirin Brewery CO, Ltd. Tokyo, Japan. IL-3, GM-CSF, and G- CSF has a specific activity of 1 X 10 U/mg. Purified recombinant human IL-11 with a specific activity of 1.39 X lo6 U/mg was a gift from Yarnanouchi Pharmaceutical CO, Ltd, Tokyo, Japan. Purified recombinant human macrophage colony-stimulating factor (M-CSF) with a specific activity of 2 X lo8 U/mg was kindly provided by Morinaga Milk Industry CO, Ltd, Zama, Japan. Purified recombinant murine IL-4 was purchased from R & D Systems Inc, Minneapolis, MN. Concentrations of growth factors in this study were as follows: IL-3, 10 ng/ml; L-11, 100 ng/ml; SF, 100 ng/ml; Ep, 2 U/mL; From the Department of Pediatrics, the Second Department of Internal Medicine, and Blood Transfusion Service, Mie University School of Medicine, Mie University Hospital, Mie, Japan; and the Department of Clinical Oncology, Institute of Medical Science, the University of Tokyo, Tokyo, Japan. Submitted July 5, 1994; accepted February 6, Supported by research grants from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare of Japan, Tokyo. Address reprint requests to Naoyuki Katayama, MD, The Second Department of Internal Medicine, Mie University School of Medicine, Edobashi, Tsu, Mie 514, Japan. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. section 1734 solely to indicate this fact by The American Society of Hematology /95/ $3.00/0 Blood, Vol 86, No 1 (July l), 1995: pp

2 74 TANAKA ET AL GM-CSF, IO ng/ml; G-CSF, 10 ng/ml; M-CSF, 10 ng/ml; and IL-4, IO ng/ml. Primary clonal culture. Serum-free methylcellulose culture was performed in 35-mm Falcon suspension culture dishes (Becton Dickinson Labware, Lincoln Park, NJ), using a modification of the technique previously de~cribed.~~.'~ One milliliter of culture contained 500 enriched marrow cells, a-medium (INC. Imine, CA), 1.2% 1,500 cps methylcellulose (Shinetsu Kagaku Kogyo Ltd, Tokyo, Japan), 1% deionized crystallized globulin-free bovine serum albumin (BSA) (Sigma Chemical CO, St Louis, MO), 1 X mom 2- mercaptoethanol (Sigma), 300 pglml fully iron-saturated human transferrin (Sigma), 160 pg/ml soybean lecithin (Sigma), 96 pgl ml cholesterol (Nakarai Tesque Inc, Kyoto, Japan), and designated hematopoietic growth factors. Dishes were incubated at 37 C in a humidified atmosphere flushed with 5% CO2. Secondary clonal culture. On day 5 of incubation of the primary cultures supported by IL-I 1 and SF, blast cell colonies35 consisting of 20 to 40 blast cells were identified, individually lifted from the cultures using a IO pl Eppendorf pipette under direct inverted microscopic visualization, pooled, and washed three times with a- medium. The pooled blast cells at a concentration of 100 cells per dish were plated in secondary cultures containing a-medium, 1.2% 1,500 cps methylcellulose, 1 % deionized crystallized globulin-free BSA, 1 X moll 2-mercaptoethanol, 300 pg/ml fully ironsaturated human transferrin, 160 pg/ml soybean lecithin, 96 pg/ml cholesterol, and designated hematopoietic growth factors. Serial observation of colony formation in,secondary cultures. The cell culture dishes were daily observed on an inverted microscope. When small cell aggregates consisting of 5 to 15 undifferentiated blast cells were identified, the location and number of cells in each colony were recorded. Cell numbers of individual colonies were estimated daily until the number could not be accurately assessed in situ, usually over 200 cells per colony. The time of daily observation was also recorded. Consecutive replating of secondary blast cell colonies. Blast cell colonies consisting of 20 to 40 cells in secondary cultures supported by IL-3 alone were picked up, pooled, washed three times with a- medium, and replated in tertiary cultures containing IL-3 plus SF. Colony formation in both secondary cultures supported by TL-3 alone and tertiary cultures supported by IL-3 plus SF were observed daily. Individual replating of each secondary blast cell colony. The proliferative capacity of individual blast cells in each growing secondary blast cell colony supported by various combinations of hematopoietic growth factors was determined. Each secondary blast cell colony containing 20 to 40 cells was identified and the number of cells in the colony was counted. Subsequently, each colony was individually pickedupwith a IO pl Eppendorf pipette and suspended in 0.1 ml of a-medium in a respective tertiary 35 mm culture dish, and each sample in the separate dish was mixed well by gentle pipetting. To the respective culture dish containing a single transferred secondary blast cell colony, 0.9 ml of serum-free methycellulose medium containing the same combinations of hematopoietic growth factors as those present in the secondary cultures was individually added. Each dish was then agitated gently to thoroughly disperse blast cells derived from a single transferred secondary blast cell colony. Cultures of individual blast cells derived from each replated secondary blast cell colony were incubated. All colonies, including doublets, formed in each tertiary culture dish were respectively counted, and the cumulative numbers of tertiary colonies derived from each replated secondary blast cell colony were enumerated. We separately calculated the replating efficiency of each secondary blast cell colony, using the respective cell number per secondary blast cell colony and the respective number of tertiary colonies derived from individual blast cells of the replated secondary blast cell colony. Liquid culture. To obtain a large number of blast cells with the potential to yield colony formation, liquid culture was also performed using a 50 ml tissue culture flask (Nunc, Roskilde, Denmark). Enriched marrow cells were cultured with IL-I I plus SF at 2 to 5 X IO4 cells per milliliter in the same culture mixture as for the methylcellulose culture, except for the absence of methylcellulose. On day 5 of incubation, cells were harvested, washed three times with a-medium, and cultured again with IL-3 alone, IL-3 plus IL-I I, or IL-3 plus SF in suspension culture. Three to 6 days later, the cells were collected, washed three times with a-medium, and analyzed for DNA content. Cell-cycle analysis. Cells obtained from liquid cultures were prepared for cell-cycle analysis. The cells were stained with 50 pglml of propidium iodide (PI) (Sigma) in 0.1% sodium citrate containing 0.05% Nonidet P-40 (Sigma) and IO pg/ml of RNase (Boehringer-Mannheim Biochemicals, Indianapolis, IN) for 20 minutes in the dark and then analyzed on the FACScan cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) using a modification ofthe technique previously described." For each sample, DNA histograms were analyzed, using CellFIT software (Becton Dickinson Immunocytometry Systems) to determine the distribution of cells in GI, S, and G2 + M phases of the cell-cycle. Starisrical analysis. Statistical analyses were made using Student's t-test. RESULTS Serial observation of blast cell colony formation. To examine the potential for enhancement of hematopoietic growth factors that directly affect proliferative kinetics of hematopoietic progenitors in the early phase of colony formation, we studied populations highly enriched for progenitors. Accordingly, we used pooled blast cells from blast cell colonies that appear to be completely devoid of accessory cells.3' The combination of SF with IL-6 or IL-11, both of which are known to share a common signal transducing receptor component37 and have similar biologic effects on the early stage of hematopoiesis: supports proliferation of the primitive hematopoietic progenitor^.^,^^.^'.'^ Thus, the combination of IL-11 and SF was chosen for support of primary blast cell colonies. Blast cell colonies supported by IL-11 and SF, each containing 20 to 40 blast cells, were picked up, pooled, washed with a-medium three times, and plated into secondary cultures. As a single factor, IL-3 was most effective in support of formation of secondary colonies, including multilineage colonies in the serum-free culture system, compared with GM-CSF, G-CSF, M-CSF, IL-4, IL- I 1, or SF (data not shown). In these cultures, we examined the effects of early acting growth factors, including IL- 1 l '' and ~~ on the IL-3-dependent proliferation of hematopoietic progenitor cells. In sequential observation of the development of blast cell colonies, newly appearing small blast cell colonies with less than 20 cells were identified and their cell numbers recorded until the colonies reached over 40 cells. The time of daily observation was also recorded. These results are presented in Fig 1. Ten, 13, 1 l, and 10 colonies were observed in cultures containing IL-3 alone, IL-3 plus IL-I 1, IL-3 plus SF, and IL-3 plus IL-11 plus SF, respectively. Because the difference in slopes of the lines, which represent logarithmically the proliferative kinetics, seemed apparent among the combinations of hematopoietic growth factors, we estimated the average number of hours for indi-

3 CELL-CYCLING OF HEMATOPOIETIC PROGENITORS A C ib Table 1. Population Doubling Time of Blast Cell Colonies Population Doubling Time (h) Colony No. IL-3* 11-3, IL-1lX 11-3, SF* IL-11, IL-3, Mean 2 SD t t 13.9 t 3.0t t 75 SF* Population doubling time represents the time required for secondary blast cell colonies of 20 cells to reach 40 cells. * Factors present in secondary cultures. t Difference from the numbers of hours in the presence of IL-3 alone is significant at P <, t Fig 1. Proliferative kinetics in secondary blast cellcolonies are graphically presented using cell number changes in individual colonies. Cultures were stimulated by IL-3 alone (A), IL-3 plus IL-l1 (B), IL-3 plus SF (C), and 11-3 plus IL-l1 plus SF (D). vidual colonies of 20 cells to reach 40 cells by calculating slopes of the lines present in Fig l. The results are presented in Table 1. The average numbers of hours were , , , and hours in cultures containing IL-3 alone, IL-3 plus IL-11, IL-3 plus SF, and IL-3 plus IL-11 plus SF, respectively. These data suggest that IL-l1 and SF significantly enhanced the rate of growth of the colonies. Consecutive 10.6 replating and analysis of doubling 22.5 time of colonies. To confirm 11.1 the direct action of 19.0 growth factors, including IL and SF, on IL-3 -dependent growth.9 of hematopoietic progenitors, 15.2 consecutive replating 19.3 studies were done with SF. We 11.5 pooled secondary blast cell 21.6 colonies supported by IL-3 alone 12.5 in secondary cultures and 27.1 replated them into tertiary cultures containing IL-3 plus SF, and serially observed growth of the tertiary blast cell colonies. In parallel, we also observed the development of secondary blast cell colonies supported by IL-3 alone. We estimated the time for secondary blast cell colonies, supported by IL-3 alone, and tertiary blast cell colonies, supported by E-3 plus SF, to double in size. Consistent with the results shown in Table 1, the average time for the size of each blast cell colony to double was shortened to 11.3? 2.0 hours, whereas that of secondary blast cell colonies supported by IL-3 alone was 22.7? 3.7 hours (Table 2). Replating efficiency of individual blast cell colonies. Possible explanations for the significant difference of the growth rate among the colonies supported by IL-3 alone and those supported by the combinations of IL-3 with IL-11 and or SF (Table 1) include: (1) an increase in the incidence of cells that are capable of proliferation; (2) shortening of the time required for each single cell in the colony to divide; and (3) a combination of the above factors. To address this issue, we individually examined the replating efficiency of each secondary blast cell colony growing in the presence of IL-3 alone and the combinations of IL-3 with IL- 11 andor Table 2. Population Doubling Time of Blast Cell Colonies in a Consecutive Replating Study Colony No. IL-3, IL-3 Doubling Time (h) Mean 2 SD * -C 2.0* Population doubling time represents the time required for secondary blast cell colonies supported by IL-3 alone and tertiary blast cell colonies supported by IL-3 and SF, with a size of 20 cells, to reach 40 cells. Difference between these numbers is significant at P <.001. SF

4 76 TANAKA ET AL Table 3. Replating Efficiencies of Individual Secondary Blast Cell Colonies Cultured With IL-3 Alone, IL-3 Plus IL-11, IL-3 Plus SF, or IL-3 Plus IL-l1 Plus SF IL-3* IL-3, IL-1lx IL-3. SF* IL-11, IL-3, SF* Cell No. per No. of Cell No. per No. of Cell No. per No. of Cell No. per No. of Secondary Tertiary Replating Secondary Tertiary Replating Secondary Tertiary Replating Secondary Tertiary Replating Colony No. Colony Colonies Efficiency Colony Colonies Efficiency Colony Colonies Efficiency Colony Colonies Efficiency oo oo Mean 5 SD ? 0.12t oo oo ? t oo oo oo t oo oo i t Each secondary blast cell colony consisting of 20 to 40 blast cells was individually picked up and transferred respectively to a separate tertiary culture dish containing the same combination of growth factors as that of secondary culture dishes by which the growth of each secondary blast cell colony was supported. All colonies formed in tertiary cultures were tabulated. Factors present in both secondary and tertiary cultures. t There are no statistical differences among these numbers. SF. Each secondary blast cell colony containing 20 to 40 cells in secondary cultures was individually picked up and respectively replated into a separate tertiary culture dish that contained exactly the same combinations of hematopoietic growth factors as those present in the secondary cultures. The replating efficiency of each secondary blast cell colony was calculated using the respective cell number per secondary blast cell colony and the respective number of tertiary colonies derived from individual blast cells, which composed the replated secondary blast cell colony. Eleven, 12, 10, and 11 secondary blast cell colonies supported by IL-3 alone, IL-3 plus IL-11, IL-3 plus SF, and IL-3 plus IL-11 plus SF, respectively, were individually analyzed. The results are presented in Table 3. The average of replating efficiencies was not statistically different among the various combinations of hematopoietic growth factors, hence, enhancement of growth rates of blast cell colonies in the secondary cultures was not due to an increase in the incidence of proliferating cells within each secondary blast cell colony, but rather, due to shortening of the time required for individual cells in each developing secondary blast cell colony to divide. Assumption of doubling time of blast cells in secondary cultures. Using the parameters in Tables 1 and 3, we assumed the average of actual time required for cell division of each blast cell in the process of development of the colonies. When the time for the number of cells in blast cell colonies containing 20 cells to become 40 is DTp and the time for a single cell in the colonies containing 20 to 40 cells 10.3 to become two 11.9 daughter cells is DTc, 15.6 the relationship between DTp and DTc is given by the following equation: 40 = 20 x E x 2DTp T + 20 (1 - E) where E is the replating efficiency that can be calculated from the replating studies. Therefore, DTc is: DTc = DTp X log 2Aog (1 + 1E). As summarized in Table 4, the estimated time for cell division in blast cells differed depending on the culture conditions. These data suggest that the length of the cell-cycle of IL-3-dependent hematopoietic progenitors can be modulated by IL-11 andor SF. Cell-cycle analysis of blast cells. To examine more precisely the modulation of the length of cell-cycling by IL- 11 or SF, the fractions in each phase of the cell-cycle of blast cells highly enriched for progenitors were measured by staining cellular DNA with PI. For a flow cytometric study, we obtained enough blast cells from liquid cultures, in the same manner as methylcellulose culture, except for the absence of methylcellulose. Cell-cycle analysis was made on blast cells supported primarily by IL-11 plus SF and secondarily by IL-3 alone, IL-3 plus IL-11, or IL-3 plus SF. Enriched marrow cells were plated in suspension culture in the pres- Table 4. Assumption of Doubling Time of Cells in Developing Colonies Doublino Time (h) IL-3 IL-3, IL-l1 IL-3, SF IL-3, IL-11, SF 21.6 The calculated doubling time represents the time required for each single cell in developing secondary blast cell colonies to become N O daughter cells. Time was calculated by the equation given in the Results section.

5 PROGENITORS CELL-CYCLING OF HEMATOPOIETIC Table 5. Cell-Cycle Distributions of Blast Cells Cultured With 11-3 Alone, 11-3 Plus L-11, or 11-3 Plus SF IL-3* IL-3, IL-11" IL-3, SF' Experiment No. G1 S G2 + M G1 S G2 + M G1 S G2 c M Mean 2 SD t t * $ Blast cells cultured with IL-3 alone, IL-3 plus IL-11, or IL-3 plus SF were stained with PI and analyzed by flow cytometry. Cell-cycle profiles (percentages) of the blast cells from four independent experiments are shown with mean 2 SD. Factors present in secondary cultures. t Difference from the numbers in the presence of IL-3 alone is not significant. * Difference from the numbers in the presence of IL-3 alone is significant at P <.001. ence of IL-l1 plus SF. After 5 days of incubation, the cells were harvested, washed three times with a-medium, and replated again in suspension culture supplemented with IL- 3 alone, IL-3 plus IL-11, or IL-3 plus SF. On days 3 to 6 of incubation, cells were collected, washed three times with a-medium, and prepared for analysis of DNA contents. The results are presented in Table 5. Blast cells cultured with IL- 3 plus IL-11 showed a similar distribution of each fraction of the cell-cycle tothatof blast cells cultured with L-3 alone, while the combination of IL-3 with SF had profound effects on cell-cycle distribution, in that there was a decrease in accumulation of cells in G1 phase with a commensurately increased fraction of cells in S phase. In a representative experiment (Fig 2), analysis of blast cells cultured with IL- 3 alone indicated that 43.0% of the cells remained in G1 phase, whereas 48.9% and 8.1% had entered into S and G2 + M phases, respectively (Fig 2A). For blast cells cultured with IL-3 plus IL-11, 47.9% of the cells were in G1 phase, 47.6% in S phase, and 4.5% in G2 + M phase (Fig 2B). The data obtained with blast cells cultured with IL-3 plus SF show that the cells in G1, S, and G2 + M phases accounted for 32.3%, 60.3%, and 7.4%, respectively (Fig 2C). DISCUSSION It appears to be a general rule that, while an appropriate rate of hematopoietic cell production is achieved in the steady state, a high production rate is required for a larger demand. It is likely that hematopoietic growth factors with the potential for enhancement of the growth of hematopoietic progenitor cells accelerate the rate of hematopoietic cell production by induction of an increase in size and incidence of colonies: synergy and recruitment.40 Our present study focused on the mechanism of synergy of colony formation. One approach to investigate synergy of the growth of hematopoietic progenitor cells is to count and record periodically the number of cells and to assume the rate of growth. Our sequential observation of developing blast cell colonies from pooled blast cells showed that, although the time for the total cell number of individual blast cell colonies to double varied among the colonies, IL-11 and SF significantly shortened the average length of time required for the number of cells in IL-3-dependent blast cell colonies to double. Consecutive replating studies provided evidence for the direct action of growth factors resulting in accelerated growth of hematopoietic progenitor cells. On the other hand, these - v) S c 0 0 Z G1, 47.9% S, 47.6% G2/M, 4.5% C GI, 32.3% S, 60.3% G2/M, 7.4% DNA Content Fig 2. DNA histograms of blast cells cultured with IL-3 alone (A), IL-3 plus (B), and IL-3 plus SF (C). Cells were stained with PI and their DNA contents were analyzed on flow cytometry. The percentage of cells in G1 phase, S phase, or G2 + M phase is given inside each figure. X-axis, fluorescence in arbiirary units; Y-axis, cell number in arbitrary units. Data are from a representative experiment (n = 4).

6 78 TANAKA ET AL results raise the question of mechanisms of actions of IL- 11 and SF, which contributed to a substantial change in the time required for the number of cells in colonies to double. To address the question, we did replating studies of individual blast cell colonies containing 20 to 40 cells. As there was no difference in replating efficiencies among growth factor combinations, shortening of the time for the total cell number L, Dzialo R, Fitz L, Ferenz C, Hewick RM, Kellenher K, Hermann SH, Clark SC, Azzoni L, Chan SH, Trinchieri G, Perussia B: Cloning of each blast cell colony to double was due to a shortening of cdna for natural killer cell stimulatory factor, a heterodimeric of time for a single cell within the colonies to divide to give cytokine with multiple biologic effects on T and natural killer cells. rise to two daughter cells and was not due to alteration of J Immunol 146:3074, 1991 the incidence of dividing cells in the developing colonies. 9. Gubler U, Chua AO, Schoenhaut DS, Dwyer CM, McComas These findings suggest that the cell-cycle of hematopoietic W, Motyka R, Nabavi N, Wolitzky AG, Quinn PM, Familletti PC, progenitor cells is susceptible to types of growth factors Gately MK: Coexpression of two distinct genes is required to generpresent in the milieu. Alternatively, it is suggested that hema- ate secreted bioactive cytotoxic lymphocyte maturation factor. Proc topoietic progenitor cells have an unique biologic potential Natl Acad Sci USA 88:4143, 1991 to modulate the length of their cell-cycling, in response to 10. Jubinsky PT, Stanley ER: Purification of hematopoietin l : A multilineage hemopoietic growth factor. Proc Natl Acad Sci USA their environment. It is generally accepted that the difference between cells that divide rapidly and those that divide slowly results mainly from variation in the length of time they spend in the G1 phase of the ~ell-cycle.~' In contrast to this premise, we observed that IL-11 induced a decrease in the time for hematopoietic progenitor cells to go through the cell-cycle without a change in the overall distribution of each phase of the cell-cycle. On the other hand, the combination of IL- 3 with SF resulted in a lower estimate of GI phase fraction of blast cells, compared with the case of 1L-3 alone, thereby implying that a reduced time in cell-cycle by SF is largely due to a shortening of G1 phase. 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