Inhibitors of Protein Biosynthesis Can Stimulate Proliferation of Mouse Hepatocytes in Vitro

Hepatocyte proliferation in the liver regenerating after partial hepatectomy ceases when the organ is restored, and the mechanism of this phenomenon is still unclear. In the experiments on fusing hepatocytes from the regenerated mouse liver (15 days after partial hepatectomy) with NIH 3T3 mouse fibroblasts, we revealed no DNA synthesis in the nuclei of stimulated fibroblasts in heterokaryons (in the presence of hepatocyte nuclei), whereas DNA synthesis in nonfused cells was undisturbed. In this work, our purpose was to find out whether the suppression of DNA synthesis in heterokaryons could be due to the appearance in hepatocytes of some endogenous factors having an inhibitory effect on proliferation. To this end, hepatocytes from the mouse liver regenerated after partial hepatectomy were treated with cycloheximide for 1–4 h and were then fused with stimulated fibroblasts. Such a short-term treatment of hepatocytes with cycloheximide proved to result in the loss of their ability to inhibit DNA synthesis in the nuclei of stimulated or quiescent fibroblasts in heterokaryons, but hepatocytes proper actively proliferated in the medium with a low serum content (0.2%). When the mice with the liver regenerated after partial hepatectomy were treated with a single sublethal dose of cycloheximide (3 mg/kg), their hepatocytes taken two days after this treatment had no inhibitory effect. Puromycin, another inhibitor of protein synthesis, had the same effect on hepatocytes. These results may be interpreted as evidence that the final stage of liver regeneration after damage is controlled by the factors having a negative effect on cell proliferation.     

Hepatocytes in heterokaryons do not retard the nuclei of stimulated fibroblasts entering the S period]

Serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with hepatocytes from intact, regenerating and embryonic mouse livers to elucidate mechanisms of liver cell proliferation, DNA synthesis being investigated in nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes in heterokaryons were found to have no inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period, but on the contrary they were involved in DNA synthesis. In addition, the nuclei in heterokaryons mutually stimulated each other to enter the S-period. In their turn, the resting fibroblasts did not prevent the proliferating hepatocytes from the regenerating and embryonic livers to enter the S-period. Possible reasons of the absence of inhibitory effect of differentiated cells in heterokaryons are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in resting immortalized cells differs from that in differentiated cells where proliferation seems to be stopped without affecting the endogenous inhibitor postulated for the resting and ageing fibroblasts.     

ACTH-like peptides in postimplantation mouse embryos: a possible role in myoblast proliferation and muscle histogenesis.

ACTH and related peptides are mitogens for certain mesodermal cell types such as adrenocortical cells, T-lymphocytes, and skeletal myoblasts. In order to postulate a possible physiological role for these peptides in skeletal muscle histogenesis, it is necessary to establish whether they are present in muscle forming anlagens of postimplantation mouse embryos. By radioimmunoassay and immunofluorescence with antibodies specific for ACTH, we have detected these peptides in many areas of mouse embryos including neural tube, limb buds, eye lens, and myotomal muscles. During fetal development, immunoreactivity decreased in muscle tissue and appeared in visceral ganglia. Furthermore, primary myotubes or C2C12 myotubes, but not muscle or 3T3 fibroblasts, release significant levels of ACTH immunoreactive peptides into the culture medium. Using a microassay for mitogen production, primary myotubes or C2C12 myotubes, but not other mesodermal cells (with the exception of dermal fibroblasts) were shown to release factors into the medium which support myoblast proliferation. Neutralizing antibodies against ACTH inhibit myoblast but not fibroblast proliferation in a dose-dependent fashion. Based on these results, we propose that myotube-derived mitogens (including ACTH-like peptides) promote the proliferation of surrounding myoblast during muscle histogenesis in vivo.     

鸡胚与胎鼠的成纤维细胞条件培养液促进成肌细胞增殖和融合的比较

用培养过鸡胚(来亨鸡)或胎鼠(ICR小鼠)肌组织的成纤维细胞的条件培养液,定量地研究它们对胎鼠或鸡胚的成肌细胞的增殖和融合的影响。所得结果如下:(1) 胎鼠的成纤维细胞条件培养液促进胎鼠或鸡胚成肌细胞增殖,分别为对照组的2.65倍,(P<0.001)或2.35倍,(P<0.01);(2) 鸡胚的成纤维细胞条件培养液促进鸡胚或胎鼠的成肌细胞增殖,分别为对照组的2.66倍,(P<0.01)或2.17倍,(P<0.01);(3) 胎鼠的成纤维细胞条件培养液增加胎鼠或鸡胚的成肌细胞的融合率,分别为对照组的1.9倍或2.6倍;鸡胚的成纤维细胞条件培养液只增加鸡胚成肌细胞的融合率,为对照组的2.1倍,但对胎鼠成肌细胞的融合无明显的影响。 实验结果提示:成纤维细胞条件培养液促进成肌细胞的增殖,两种动物间无明显的差异,但在融合上却有一定的种属特异性。     

DNA synthesis in heterokaryons obtained by the fusion of proliferating cells of the NIH 3T3 line with cells in transition to the resting state

NIH 3T3 mouse fibroblasts cultured in the medium containing 0.5% serum for 2, 4, 8, 24, 48 and 72 hours were fused to cells stimulated for proliferation by replacing the medium with a fresh one containing 10% serum; DNA synthesis was studied in monokaryons, homo- and heterodikaryons using radioautography and double-labelling technique. Cells that were cultured in the medium with a low serum content for 72 hours exerted their inhibitory effect on the entry of stimulated nuclei into the S period in heterodikaryons, whereas cells deprived of serum for shorter periods failed to exert this effect. It thus appears that in cell fusion studies with NIH 3T3 cells, the effects of endogenous growth inhibitor(s) produced by resting cells may be revealed not earlier than by the 3rd day of serum depletion.     

Transforming viruses directly reduce the cellular growth requirement for a platelet derived growth factor

Early passage mouse embryo fibroblasts, mouse 3T3 cell lines, and early passage diploid human fibroblasts grew to higher cell densities in tissue culture medium supplemented with serum than in medium supplemented with defibrinogenated platelet-poor plasma (PPP). Unlike the mouse cells, the human fibroblasts displayed this differential growth response only in the presence of hypophysiologic concentrations of calcium. The addition of heat-treated extracts of human platelets to PPP-supplemented medium stimulated the replication of both the normal mouse cells and early passage human embryo fibroblasts. Human or mouse fibroblasts transformed by either retroviruses or by SV40, including SV40 infected “serum revertants” and “flat transformants,” grew to equal cell densities in medium supplemented with either serum or PPP. Infection of Balb/c-3T3 cells with SV40 rapidly induced them to grow in PPP-supplemented medium demonstrating that the ability of SV40-transformed cell lines to proliferate in PPP-supplemented medium does not arise from the cell culture selection procedures usually employed to obtain stable virus-transformed cell lines. 3T3 cells infected but not transformed by retroviruses do not replicate in PPP-supplemented medium demonstrating that reduction of the growth requirement for the platelet growth factor(s) by retroviruses is a transformation-specific response. Cell cultures that did not proliferate well in PPP-supplemented medium did not form tumors when inoculated into athymic nude mice. Many, although not all, of the lines which grew well in PPP medium were tumorigenic in nude mice. Together, these findings indicate that: (1) normal fibroblast-like cells display a growth requirement for factor(s) present in serum but not found in PPP; (2) this serum specific growth factor is derived from platelets; (3) a primary response to viral transforming genes is a reduction in the growth requirement for these platelet-derived factors; and (4) cells that have a reduced requirement for the platelet-derived growth factor are often tumorigenic.     

Cytokines in adipose-derived mesenchymal stem cells promote the healing of liver disease

Adipose-derived mesenchymal stem cells(ADSCs) are a treatment cell source for patients with chronic liver injury. ADSCs are characterized by being harvested from the patient's own subcutaneous adipose tissue, a high cell yield(i.e., reduced immune rejection response), accumulation at a disease nidus, suppression of excessive immune response, production of various growth factors and cytokines, angiogenic effects, antiapoptotic effects, and control of immune cells via cellcell interaction. We previously showed that conditioned medium of ADSCs promoted hepatocyte proliferation and improved the liver function in a mouse model of acute liver failure. Furthermore, as found by many other groups, the administration of ADSCs improved liver tissue fibrosis in a mouse model of liver cirrhosis. A comprehensive protein expression analysis by liquid chromatography with tandem mass spectrometry showed that the various cytokines and chemokines produced by ADSCs promote the healing of liver disease. In this review, we examine the ability of expressed protein components of ADSCs to promote healing in cell therapy for liver disease. Previous studies demonstrated that ADSCs are a treatment cell source for patients with chronic liver injury. This review describes the various cytokines and chemokines produced by ADSCs that promote the healing of liver disease.     

DNA synthesis in the nuclei of resting and proliferating cells of the NIH 3T3 line in monokaryons, homo- and heterodikaryons

Serum-deprived (0.5%) resting NIH 3T3 mouse fibroblasts were fused with stimulated cells taken at 2 hour intervals after changing the medium to the one containing 10% serum, and DNA synthesis was investigated in monokaryons, homodikaryons, and heterodikaryous using radioautography with double-labeling technique. The presence of the resting nucleus in the common cytoplasm has an inhibitory effect on the entry of the stimulated nucleus into the S period in the medium containing either 0.5 or 10% serum, but DNA synthesis continues. After a 24 hour stay in the common cytoplasm with resting nuclei the stimulated nuclei return into the state of rest. When resting cells are stimulated by 10% serum, their inhibitory effect on stimulated nuclei in heterodikaryons still persists for at least 2 hours following stimulation. Preincubation of resting cells with cycloheximide for 4 hours abolishes their ability to suppress DNA synthesis in stimulated nuclei. The data suggest that the resting cells produce an endogenous inhibitor of cell proliferation whose formation depends upon the synthesis of protein(s). When stimulated, cell can proliferate only upon decreasing the level of this inhibitor. The obtained results are consistent with the idea of a negative control of cell proliferation.     

Hepatocytes in heterokaryons can suppress entry into the S-period of fibroblast nuclei from murine NIH 3T3 cells

Mouse liver regeneration after partial hepatectomy can be considered as a spectacular example of controlled tissue increase. In this study serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with primary hepatocytes isolated from normal (intact) and regenerating adult mouse liver at different times after partial hepatectomy (1-15 days) to elucidate mechanisms of liver cell proliferation cessation at the regeneration end. DNA synthesis was investigated in the nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes isolated from regenerating liver within 1-12 days following operation did not retard the entry of stimulated fibroblast nuclei into the S-period. In contrast, hepatocytes isolated within 15 days after hepatectomy were found to have inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period in heterokaryons. Preincubation of these hepatocytes with cyclocheximide for 2-4 h abolished their ability to suppress DNA synthesis in stimulated fibroblast nuclei in heterokaryons. Possible reasons of inhibitory effect of differentiated cells in heterokaryos are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in regenerating hepatocytes seems to be stopped being affected by the intracellular growth inhibitors, whose formation depends on protein synthesis.     

Enhanced expression of mitochondrial superoxide dismutase leads to prolonged in vivo cell cycle progression and up-regulation of mitochondrial thioredoxin

Mn superoxide dismutase (MnSOD) is an important mitochondrial antioxidant enzyme, and elevated MnSOD levels have been shown to reduce tumor growth in part by suppressing cell proliferation. Studies with fibroblasts have shown that increased MnSOD expression prolongs cell cycle transition time in G1/S and favors entrance into the quiescent state. To determine if the same effect occurs during tissue regeneration in vivo, we used a transgenic mouse system with liver-specific MnSOD expression and a partial hepatectomy paradigm to induce synchronized in vivo cell proliferation during liver regeneration. We show in this experimental system that a 2.6-fold increase in MnSOD activity leads to delayed entry into S phase, as measured by reduction in bromodeoxyuridine (BrdU) incorporation and decreased expression of proliferative cell nuclear antigen (PCNA). Thus, compared to control mice with baseline MnSOD levels, transgenic mice with increased MnSOD expression in the liver have 23% fewer BrdU-positive cells and a marked attenuation of PCNA expression. The increase in MnSOD activity also leads to an increase in the mitochondrial form of thioredoxin (thioredoxin 2), but not in several other peroxidases examined, suggesting the importance of thioredoxin 2 in maintaining redox balance in mitochondria with elevated levels of MnSOD.     

Bronchial epithelial cell growth regulation in fibroblast cocultures: the role of hepatocyte growth factor

Proliferation of bronchial epithelial cells is an important biological process in physiological conditions and various lung diseases. The objective of this study was to determine how bronchial fibroblasts influence bronchial epithelial cell proliferation. The proliferative activity in cocultures was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and direct cells counts. Concentration of cytokines was measured in cell culture supernatants by means of ELISA. In primary cell cocultures, fibroblasts or fibroblast-conditioned medium enhanced 1.85-fold the proliferation of primary bronchial epithelial cells (P < 0.02) compared with bronchial epithelial cells cultured alone. The proliferative activity in cocultures and in fibroblast-conditioned medium was reduced by neutralizing antibody to hepatocyte growth factor (HGF) and HGF receptor c-met. Neutralizing antibodies to FGF-7 and IGF-1 had no effect. Treatment of fibroblast-epithelial cocultures with anti-IL-6 and anti-TNF-alpha neutralizing antibodies and with indomethacin decreased production of HGF. These results indicate that cytokines and PGE(2) may indirectly mediate epithelial cell proliferation via the regulation of HGF in bronchial stromal cells and that HGF plays a crucial role in proinflammatory cytokine-induced proliferation in the experimental system studied.     

Regulation of the growth rate of mouse fibroblasts by IL-3-activated mouse bone marrow-derived mast cells

When mouse bone marrow-derived mast cells (BMMC) are cocultured with a confluent layer of mouse 3T3 fibroblasts in the presence of WEHI-3-conditioned medium, the mast cells undergo a phenotypic change toward that of a connective tissue mast cell, and the fibroblasts increase their synthesis of globopentaosylceramide. We now demonstrate that fibroblasts lose their contact inhibition and multiply such that by the 2nd and the 4th wk of coculture there are, respectively, approximately four-fold and six-fold more fibroblasts than in the cultures that are not exposed to BMMC. This in vitro increase in the number of fibroblasts is dependent on the number of mast cells (over the range of 6 x 10(4) to 1 x 10(6) BMMC/culture) initially seeded with the fibroblasts and on the concentration of WEHI-3-conditioned medium present during the coculture. That the fibroblasts also multiply in BMMC/fibroblast cocultures exposed to synthetic IL-3 or to purified IL-3 indicates that IL-3 is a component in WEHI-3-conditioned medium that induces mast cells to produce the fibroblast growth factor. The number of fibroblasts does not increase if fibroblasts are exposed to lysates of BMMC, or to BMMC-derived conditioned medium, or if the two cell types are separated from one another during the coculture with a 3-microns filter or a 0.4-microns filter. Thus, IL-3-activated BMMC must be in proximity to fibroblasts to induce them to multiply. Because of their increased numbers per culture dish, total fibroblasts that were cocultured with mast cells synthesized approximately two-fold more 35S-labeled proteoglycans, incorporated approximately 3-fold more [3H] proline into collagenase-sensitive proteins, and had substantially more alpha 2(I) collagen mRNA than fibroblasts that were maintained in the absence of mast cells. These is vitro studies reveal a sequence by which IL-3-activated mast cells may play a role in the induction of fibrosis.     

Dynamics of acid phosphatase activity in the liver in the process of involution of cirrhosis

Changes in the total activity of acid phosphatase in the liver as well as changes in the enzyme activity in hepatocytes and connective tissue cells of fibrosis layers were investigated, using quantitative histochemical method, in the process of mouse cirrhosis involution. After discontinuation of CCl4 injection, the animals with cirrhosis were divided into two groups. In the first group the resection of the left lobe of the liver was performed. The animals of the second group were not subject to operation. The results demonstrate that there is a close correlation between lysosomal hydrolase activity of hepatocytes and connective tissue cells of the liver and collagen resorption during cirrhosis involution. The most intensive lysis of collagen takes place within the first three weeks of cirrhosis involution in both experimental groups. Partial resection in cirrhosis has no significant effect on the changes and level of total activity of lysosomal hydrolase enzymes in the liver during cirrhosis involution.     

DNA synthesis in the nuclei of stimulated NIH 3T3 cells in heterodikaryons obtained by the fusion of these cells with resting cells treated with cycloheximide

Serum-deprived (0.2%) resting NIH 3T3 mouse fibroblasts preincubated with cycloheximide (7.5 micrograms/ml) were fused with stimulated cells taken 10 hours after changing the medium to the one containing 10% serum, and DNA synthesis was investigated in nuclei of heterodikaryons, homodikaryons, and monokaryons, using radioautography with double-labeling technique. Preincubation of resting cells with the inhibitor of protein synthesis cycloheximide for 4, 3, 2, but not for 1 or 0.5 hours abolishes their ability to suppress DNA synthesis in stimulated nuclei in heterodikaryons. Three hours after the removal of cycloheximide from the media, the resting cells acquire once again the inhibitory effect towards the entry of stimulated nuclei into the S-period. The data suggest that the resting cells may produce a labile endogenous inhibitor of cell proliferation, and support the idea on the active metabolic processes occurring in the resting cells.     

Pygmy mouse fibroblasts in tissue culture: evaluation of multiplication stimulating activity (MSA) receptors and growth responses to MSA

The pygmy mouse has been proposed as a model for growth hormone resistance; it has normal serum somatomedin levels and does not respond to growth hormone treatment. In order to determine if the growth impairment is caused by a defect in somatomedin binding or in postreceptor action of somatomedin we compared fibroblasts derived from pygmy mice with those from normal appearing littermates. Using multiplication-stimulating activity (MSA) as a model somatomedin we found a normal Ka of binding to the cell surface MSA receptor but a significantly increased number of MSA receptors on the fibroblasts derived from pygmy mice. Studies of thymidine incorporation into DNA failed to demonstrate a difference between pygmy and normal fibroblasts in their responses to MSA alone, but there was a significantly greater thymidine incorporation into the DNA of normal fibroblasts when both competence factor (platelet-derived growth factor) and progression factors (somatomedins and growth hormone deficient platelet-poor plasma) were present in the test medium. On the other hand, cell proliferation studies did not demonstrate a consistent difference in the growth rate of normal versus pygmy fibroblasts. The data support the conclusion that the imparied growth of the pygmy mouse in vivo may be caused by factors which lie outside of the growth hormone-somatomedin pathway.     

Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines

Experiments were conducted to determine the effects of feeder layers composed of different cell types on the efficiency of isolation and the behavior of porcine embryo-derived cell lines. Inner cell masses (ICM) isolated from 7- to 8-d-old embryos were plated on feeder layers composed of Buffalo rat liver cells (BRL), a continuous cell line of murine embryonic fibroblasts (STO), STO combined with BRL at a 9:1 and 1:1 ratio, STO with BRL-conditioned medium (STO + CM), porcine embryonic fibroblasts (PEF), PEF combined with BRL at a 9:1 and 1:1 ratio, porcine uterine epithelial cells (PUE), murine embryonic fibroblasts (MEF), or an epithelial-like porcine embryo-derived cell line (PH3A). It was found that embryo-derived cell lines could be isolated only from the STO and the STO with BRL-conditioned medium treatments. The isolated cell lines were of epithelial-like and embryonic stem cell-like (ES-like) morphology. The feeders tested had an effect on the behavior of plated ICM. Some feeders, represented by PUE, BRL, STO:BRL (1:1), PEF:BRL (1:1), and PH3A, did not promote attachment of the ICM to the feeder layer; others, represented by STO and MEF, allowed attachment, differentiation and proliferation. On PEF feeders the ICM spread onto the feeder layer after attachment without apparent signs of proliferation or differentiation. None of the feeders tested increased the efficiency of isolation or the growth characteristics of embryo-derived (both ES-like and epithelial-like) cell lines over that of STO feeders.     

Connective tissue mast cells in contact with fibroblasts express IL-3 mRNA. Analysis of single cells by polymerase chain reaction.

Mast cell-fibroblast interactions have been extensively investigated in the last few years. Fibroblasts support the in vitro survival but not proliferation of mouse connective-tissue type mast cells. However, the factor(s) that allow their survival on fibroblast monolayers has not been identified. We have investigated the presence of mRNA for IL-3 and granulocyte-macrophage-CSF in single mouse mast cells, before and after co-culture with 3T3 fibroblasts, using the polymerase chain reaction technique. The system was calibrated first by using in vitro generated population of mouse bone-marrow derived mast cells (BMMC). Significant differences in the amplification of IL-3 cDNA were observed in each of the BMMC cells examined, whereas the amplification of cDNA for the alpha-subunit of the Fc epsilon RI were similar. Inasmuch as murine cultured IL-3-dependent mast cells differentiate into connective tissue-like mast cells when co-cultured with 3T3 fibroblasts without any exogenous supply of growth factors, it was of interest to determine whether these connective tissue-like mast cells produce IL-3 message. Separation of the differentiated BMMC from the fibroblast monolayer, by either trypsinization or by single cell manipulation revealed the synthesis of a detectable amount of IL-3 mRNA in these mast cells. Whether this IL-3 mRNA was induced by fibroblasts was further investigated using connective tissue mast cells freshly purified from the mouse peritoneal cavity. Only about 20% of these connective tissue mast cells produced detectable amount of granulocyte-macrophage-CSF mRNA whereas in less than 10% of the cells IL-3 mRNA was detected. However, when these connective tissue mast cells were co-cultured with 3T3 fibroblasts for 18 hours and then separated, IL-3 mRNA were detected in most of the cells whereas no such mRNA was detected in tissue mast cells incubated for 18 h with medium derived from 3T3 fibroblasts. Therefore we conclude that fibroblasts induce the accumulation of IL-3 mRNA in connective tissue mast cells. The production of IL-3 may play a role in the survival of this type of mast cells on the fibroblast monolayer.     

Effects of different forms of chitosan on intercellular junctions of mouse fibroblasts in vitro

Chitosan is a linear polysaccharide that has many biomedical applications. We compared the effects of chitosan, in both solution and membranous form, on intercellular adhesion of Swiss 3T3 mouse fibroblasts. Cells were grown as spheroidal cell cultures. Some control cell spheroids were cultured without chitosan and two experimental groups were cultured with chitosan. Chitosan in solution was used for one experimental group and chitosan in membranous form was used for the other. For each group, intercellular adhesion was investigated on days 5 and 10 of culture. Transmission electron microscopy revealed well-defined cellular projections that were more prominent in cells exposed to either membranous or solution forms of chitosan than to the chitosan-free control. Immunocytochemical staining of ICAM-1 and e-cadherin was used to determine the development of intercellular junctions. Compared to the weakly stained control, strong reactions were observed in both chitosan exposed groups at both 5 and 10 days. Cells were treated with 5-bromo-2-deoxyuridine (BrdU) and incubated with anti-BrdU primary antibody to assess proliferation. Both the solution and membranous forms of chitosan increased proliferation at both 5 and 10 days. Cellular viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The MTT assay indicated high cell viability; maximum viability was obtained with the solution form of chitosan at day 5. Chitosan exposure increased the number of intercellular junctions and showed a significant proliferative effect on 3T3 mouse fibroblasts.     

Activin-A causes Hepatic stellate cell activation via the induction of TNFα and TGFβ in Kupffer cells

Background & aims

TGFβ superfamily member Activin-A is a multifunctional hormone/cytokine expressed in multiple tissues and cells, where it regulates cellular differentiation, proliferation, inflammation and tissue architecture. High activin-A levels have been reported in alcoholic cirrhosis and non-alcoholic steatohepatitis (NASH). Our aim was to identify the cell types involved in the fibrotic processes induced by activin-A in liver and verify the liver diseases that this molecule can be found increased.

The influence of a granulocytic inhibitor(s) on hematopoiesis in an in vivo culture system.

The in vivo diffusion chamber (DC) technique for mouse marrow culture was used to determine the effect of a granulocyte inhibitor on the proliferation of the pluripotent stem cell(CFU-s) and the granulocyte progenitor cell (CFU-c). Granulocyte conditioned medium was injected intraperitoneally into mice bearing DCs during the initial 48 hr of culture. The early injections of inhibitor resulted in a significantly reduced number of granulocytic progeny formed within the DCs while there was no growth inhibition of mouse fibroblasts cultured under identical conditions. The reduced cell production was due in part to a significant reduction in the self-renewal rate of the CFU-c while no apparent direct effect was observed upon the growth of the CFU-s within the same cultures. These data suggest that the granulocytic inhibitor(s) acted to reduce the proliferation within the CFU-c population and thereby diminished the amplification potential inherent in the initial cell inoculum.