Loss of responsiveness in senescent human TIG-1 cells to the DNA synthesis-inducing effect of various growth factors

Responses of human diploid cells, TIG-1, were examined with respect to their ability to initiate DNA synthesis under the influence of various growth factors and their combinations. The following agents stimulated DNA synthesis in quiescent TIG-1 cells at 37-49 PDL (population doubling level) (66-79% of lifespan completed): fetal bovine serum; tumor-derived DNA synthesis factors such as those from rat rhodamine fibrosarcoma, human adenoma and from the conditioned medium of cultured human pituitary cells; human and mouse epidermal growth factors; tumor promotors such as 12-O-tetradecanoylphorbol 13-acetate and teleocidin; microtubule-disrupting agents as colchicine, vinblastine, podophyllotoxin and TN-16; melittin; and dexamethasone. Cells at 58-60 PDL (94-97% of lifespan completed) were stimulated to synthesize DNA by fetal bovine serum, tumor-derived DNA synthesis factors and epidermal growth factors, but not by other agents. Finally, in senescent cells at 62 PDL (100% of lifespan completed), any of these growth factors and of their combinations failed to induce DNA synthesis at all. These senescent cells, however, still retained the ability to initiate DNA synthesis following infection with SV40 as reported previously [Exp. Cell Res., 143 (1983) 343-349].     

Failure in S6 protein phosphorylation by serum stimulation of senescent human diploid fibroblasts, TIG-1

When quiescent young or senescent human diploid cells, TIG-1, were metabolically labeled with 32Pi and stimulated with 10% fetal bovine serum, the phosphorylation of ribosomal S6 protein was enhanced in young cells but not in senescent cells while that of some other proteins were increased in both cells. Inability to stimulate the phosphorylation of S6 protein in senescent cells after serum addition may be the primary cause of the failure of enhancement in protein synthesis followed by the block of prereplicative events dependent on protein synthesis and thus of the failure of cells to enter S phase. However, when the cell-free preparation from serum-stimulated senescent cells was incubated with [gamma-32P]ATP, S6-kinase activity was stimulated and S6 in ribosomal fraction was susceptible to phosphorylation as observed in young cells. Differences in S6 phosphorylation of senescent cells between in vivo and in vitro was discussed.     

Paracrine effects of bombesin/gastrin-releasing peptide and other growth factors on pulmonary neuroendocrine cells in vitro

Pulmonary neuroendocrine cells (PNEC) are numerous in the fetus where they have been implicated to have a role in fetal lung development. We assessed the effects of putative growth factors, gastrin releasing peptide (GRP), cholecystokinin (CCK), gastrin (GN), serotonin (5-HT), and epidermal growth factor (EGF), some of which are produced by PNEC, either alone or in combination, on cultured fetal rabbit PNEC from 20, 24, and 28 day fetuses. GRP increased the total protein of the cultures over a 7 day period in an age-dependent manner, with greatest effect in cultures from the 24 day fetus, no effect with the 28 day fetus, and an inhibitory effect on 20 day cultures. This was accompanied by an increase in PNEC, which could be blocked by treatment of the cultures with a monoclonal antibody to GRP (2A11). There was no increase in ³H-thymidine labeling of PNEC in GRP treated cultures but an increase in numbers of cells partially stained for 5-HT, suggesting the induction of a precursor cell. Other growth factors had neither an inhibitory nor a stimulatory effect either alone or in combination with GRP. Preliminary studies with ¹²⁵I-GRP receptor localization suggests that the GRP receptor is mostly expressed on pulmonary fibroblasts, and less on epithelial cells, so that the role for GRP in fetal lung development, at least in the rabbit, is probably indirect, acting via a paracrine mechanism. © 1993 Wiley-Liss, Inc.     

Production and action of insulin-like growth factor I/somatomedin C in primary cultures of fetal lung fibroblasts

Production of insulin-like growth factor I/somatomedin C (IGF-I) by 19-day-gestation fetal rat lung fibroblasts was studied, and a paracrine (local production and action) mitogenic activity of this growth factor was explored. Specific IGF-I mRNAs were demonstrated in these cells, consistent with production of IGF-I. Using an IGF-I monoclonal antibody, IGF-I-like material was isolated from fetal lung fibroblast conditioned medium (FCM) and separated by molecular weight. Several molecular weight species were identified, including an 8,000 to 10,000 molecular weight species, a weight similar to purified IGF-I. IGF-I binding proteins elaborated by these cells were also demonstrated. The possibility of a paracrine mitogenic activity of the fetal lung fibroblast-produced IGF-I in cultures was suggested by demonstrating a reduction in DNA synthesis in cultures incubated with either the IGF-I monoclonal antibody or an IGF-I receptor antibody. These findings indicate that 19-day-gestation fetal rat lung fibroblasts produce IGF-I that acts in a paracrine fashion and suggest that this growth factor is biologically active during fetal lung development.     

Monoclonal antibody to human epidermal growth factor

A monoclonal antibody directed to a species-specific determinant of human epidermal growth factor (h-EGF) was obtained by fusing murine myeloma cells with BALB/c mouse splenocytes sensitized to h-EGF. This antibody, referred to as 863.D4, did not react with either rat or mouse epidermal growth factor or with 11 other polypeptide hormones tested as shown by solid-phase radioimmunoassay (SPRIA), and immunoprecipitation followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scatchard analysis of the antibody binding to purified h-EGF revealed an apparent equilibrium dissociation constant of 1 X 10(-8) M. The antibody blocked both the binding of h-EGF and h-EGF stimulation of 3H-thymidine incorporation into DNA by greater than 90% in confluent cultures of human foreskin fibroblasts.     

An Autocrine Activity Capable of Substituting for Serum in Cell Cultures

Abstract

Provided that high cell densities (above 10⁶/ml) are maintained, a factor-dependent murine hemopoietic progenitor cell line (FDC-PI) will proliferate in serum-free medium. The conditioned medium (CM) from high-density FDC-PI cells permits the serum-free survival of FDC-PI cells even at low density, indicating the existence of a diffusible autocrine factor. The requirement of FDC-PI for a colony-stimulating factor (either IL-3 or GM-CSF) is not abrogated by culturing the cells at high cell density or in the conditioned medium. Furthermore, the CM from FDC-PI enhances the mitogenic stimulation of normal human skin fibroblasts (HSF) by epidermal growth factor (EGF): i.e., the lag period before entry into the cell cycle is shortened by up to 6 hr. The fibroblasts themselves secrete an activity into serum-free medium that appears to be required during mitogenic stimulation by EGF. The HSF-CM also allows FDC-PI cells to survive and proliferate serum-free at low cell densities. Low concentrations of fetal calf serum or human plasma (0.2-2%) have the same effect as FDC-PI-CM and HSF-CM. We have tested many of the known growth factors, and none of them mimicked the autocrine serum replacing activity (ASRA). The activity in human plasma elutes from a gel-filtration column with an apparent molecular weight of 60,000. It appears as if cultured normal cells and cell lines produce molecules capable of complementing the growth factors required for the survival and proliferation of a range of cells in serum-free cultures.     

Cell density governs the ability of human bronchial epithelial cells to recognize serum and transforming growth factor beta-1 as squamous differentiation-inducing agents.

Sparse (75 to 2000 cells/cm2) density cultures of normal human bronchial epithelial cells uniformly undergo terminal squamous differentiation when incubated in medium containing serum (fetal bovine serum [FBS]) or transforming growth factor beta-1 (TGF-beta 1). It was found that the cell density of the culture affects the probability that a cell will respond to these differentiation-inducing agents. Thus whereas irreversible inhibition of DNA synthesis occurs in sparse cell-density cultures within 24 hours after exposure, only a transient (less than 36 hours) depression in DNA synthesis was seen in high (more than 10,000 cells/cm2) density cultures. In addition, although phase microscopic image analysis revealed that virtually all of the cells displayed a squamous morphology within 1 hour after exposure to FBS or TGF-beta 1, observations made 48 to 72 hours later showed the presence of clusters of small prolate spheroid-shaped cells surrounded by many involucrin-positive squamous-appearing cells. Only the small cells were capable of DNA synthesis and cell division as determined by autoradiography and time-lapse photomicrographic images. These replicating cells immediately undergo squamous differentiation if they are subcultured and reinoculated at low cell density and incubated in medium supplemented with FBS or TGF-beta 1. Therefore the probability that a human bronchial epithelial cell will be refractive to FBS- or TGF-beta 1 induced terminal squamous differentiation is solely a function of the cell density of the culture.     

Cell cycle-dependent multiplication of avian adenoviruses in chicken embryo fibroblasts

Summary Propagation of CELO virus employing confluent monolayers of chicken embryo fibroblasts (CEF) yielded virus titers one to two logs lower than those from confluent chicken kidney (CK) cells. An enhancement of virus production in CEF as measured by plaque formation was obtained by infecting cultures in the growing non confluent state. Measurements of³H-thymidine incorporation revealed a positive correlation between the DNA synthesis of CEF cultures at the time of inoculation and the amount of progeny virus, whereas in the CK-CELO-system no such relation was observed.Requirement of replicative fibroblasts for CELO multiplication was also demonstrated by comparison of virus replication in synchronized stationary and serum stimulated CEF cells. In stationary CEF cells arrested in the G₁ phase of the cell replication cycle by serum deprivation and infected with CELO virus, no cytopathic effect could be observed, and only very low amounts of virus were produced. But 24 hours after release of these cells for growth by serum stimulation a logarithmic rate of virus multiplication and a complete CPE occurred. Infection of synchronized CEF cultures at different stages of the cell cycle revealed that CELO multiplication was correlated with the S phase of the infected cell.In synchronized CELO infected CEF cultures viral DNA synthesis started 12 to 14 hours after growth stimulation when cells were near the end of the S phase. In contrast, no viral DNA synthesis could be measured in growth arrested CELO infected CEF cells, when cellular DNA synthesis was low. Therefore not only production of infectious virus but also viral DNA synthesis is correlated with events during the S phase of the infected CEF cell.With 7 FiguresPresented in part at the Arbeitstagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie, Mainz, September 27–29, 1976.     

An autocrine activity capable of substituting for serum in cell cultures

Provided that high cell densities (above 10(6)/ml) are maintained, a factor-dependent murine hemopoietic progenitor cell line (FDC-P1) will proliferate in serum-free medium. The conditioned medium (CM) from high-density FDC-P1 cells permits the serum-free survival of FDC-P1 cells even at low density, indicating the existence of a diffusible autocrine factor. The requirement of FDC-P1 for a colony-stimulating factor (either IL-3 or GM-CSF) is not abrogated by culturing the cells at high cell density or in the conditioned medium. Furthermore, the CM from FDC-P1 enhances the mitogenic stimulation of normal human skin fibroblasts (HSF) by epidermal growth factor (EGF): i.e., the lag period before entry into the cell cycle is shortened by up to 6 hr. The fibroblasts themselves secrete an activity into serum-free medium that appears to be required during mitogenic stimulation by EGF. The HSF-CM also allows FDC-P1 cells to survive and proliferate serum-free at low cell densities. Low concentrations of fetal calf serum or human plasma (0.2-2%) have the same effect as FDC-P1-CM and HSF-CM. We have tested many of the known growth factors, and none of them mimicked the autocrine serum replacing activity (ASRA). The activity in human plasma elutes from a gel-filtration column with an apparent molecular weight of 60,000. It appears as if cultured normal cells and cell lines produce molecules capable of complementing the growth factors required for the survival and proliferation of a range of cells in serum-free cultures.     

Density-dependent inhibition of cell growth by transforming growth factor-beta 1 in normal human fibroblasts

We report here that transforming growth factor-beta 1 (TGF-beta 1) inhibits platelet-derived growth factor (PDGF)-induced DNA synthesis in normal human fibroblasts in a cell density-dependent manner; no inhibition was seen in sparse cultures, approximately 50% inhibition in confluent cell cultures, and an almost total inhibition in dense cultures. The PDGF-inducible genes c-myc and c-fos were induced also by TGF-beta 1. Simultaneous addition of TGF-beta 1 and PDGF resulted in sustained, rather than transient, expression of c-fos mRNA; c-fos mRNA was detected as long as 24 hr after addition of PDGF and TFG-beta 1. TGF-beta 1 also induced mRNA for the A chain, but not the B chain, of PDGF. Conversely, PDGF induced TGF-beta 1 mRNA in sparse but not in dense cultures. These data indicate the existence of a complex interdependent regulation of PDGF and TGF-beta mRNA expression which is influenced by the cell density.     

Regulation of carcinoembryonic antigen expression in human colon carcinoma cells by the organ microenvironment.

The expression of carcinoembryonic antigen (CEA) is thought to be involved in homotypic adhesion and has been associated with the progression of human colon carcinomas (HCC) to the metastatic state. Three cell lines established from surgical specimens of Dukes' stage D (KM20) or Dukes' stage B (KM12C, KM12SM) with high and low preoperative CEA serum levels, respectively, were studied subsequent to growth in culture, in the subcutis (ectopic) or cecal wall (orthotopic) of nude mice. In all cell lines, CEA expression was higher in cecal tumors than in subcutaneous lesions. The degree of differentiation and CEA expression by HCC growing in the cecal wall of nude mice correlated with the pathological diagnosis and preoperative CEA level of the original patients. To better understand the regulation of CEA expression, the HCC cells were grown in culture as sparse and confluent monolayers or as multicell spheroids. The CEA expression level increased in all three cell lines growing as confluent monolayers and was highest in multicell spheroids. Treatment of sparse monolayer cultures of KM12SM cells with mitomycin-C inhibited cell division and was associated with higher production of CEA protein. Moreover, conditioned media from confluent monolayer cultures or from spheroids up-regulated CEA production in sparse monolayer cells. These data show that CEA expression in HCC cells may be regulated by cell density and by factors from the organ environment.     

Growth factor responsiveness declines during adulthood for human skin-derived cells

Keratinocytes and fibroblasts were obtained from upper arm biopsies of young (22-27 years) and old (60-82 years) adult donors. Keratinocytes were grown in serum-free medium containing variable quantities of either epidermal growth factor (EGF) or a bovine hypothalamic extract known to contain keratinocyte growth factor (KGF). Fibroblasts were grown in serum-free medium containing variable quantities of EGF or insulin. Paired keratinocyte cultures were plated in serum-free medium containing 20% newborn keratinocyte-conditioned medium (NM) or 20% control conditioned medium (CM). Newborn foreskin keratinocytes were plated in 20% conditioned media derived from newborn, young adult or old adult keratinocyte cultures. In spite of large inter-donor variability, keratinocyte growth significantly decreased with age (0.05 greater than P greater than 0.01). Cell yield at 7 days showed an 8-fold increase for young adults over the KGF dose range treated, but only a 4-fold increase for old adults. Young adult cells in varying concentrations of EGF achieved 3-fold to 5-fold higher densities than old, although EGF was not stimulatory for either adult age group. Donor age-associated loss of growth factor responsiveness was confirmed with dermal fibroblasts derived from the same biopsies. Newborn but not adult keratinocytes were stimulated by NM, while newborn cells were not stimulated by either young or old adult conditioned media (YM or OM). An epidermal proliferation index, incorporating both donor cell yield and cell yield of newborn cells in donor conditioned medium, was significantly different (P less than 0.01) for newborn vs. young or old adult cells. Our findings confirm that a decreased proliferative capacity is measurable within adulthood, and suggest that this decrease may be due to a reduced ability to synthesize or respond to mitogens, including autocrine factors.     

Alpha 2-macroglobulin and serum preferentially counteract the mitoinhibitory effect of transforming growth factor-beta 2 in rat hepatocytes.

Transforming growth factors-beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) are potent, multifunctional modifiers of cellular proliferation and differentiation in many cell types. To evaluate factors which may alter the activity of the TGF-beta s during hepatocyte proliferation, we examined the influences of bovine serum and purified bovine or human alpha 2-macroglobulin (alpha 2M) on the mitoinhibitory effects of the TGF-beta in primary cultures of rat hepatocytes. The inhibitory activity of TGF-beta was evaluated by autoradiographic labeling index at 48 hours in hepatocyte cultures exposed to [3H]thymidine between hours 24 and 48 in culture. In the absence of serum or alpha 2M, TGF-beta 1 and TGF-beta 2 were equivalently potent in inhibiting S-phase DNA synthesis in hepatocytes cultured with or without epidermal growth factor. However, bovine serum and purified bovine or human alpha 2M consistently counteracted the mitoinhibitory effects of TGF-beta 2. S-phase DNA synthesis increased five- to six-fold when bovine serum (15%) or alpha 2M (200 micrograms/ml) were included with TGF-beta 2 (0.1 ng/ml) and epidermal growth factor (20 ng/ml). The mitoinhibitory effect of TGF-beta 1 was not influenced by the addition of purified bovine alpha 2M. Human alpha 2M or bovine serum counteracted inhibition by TGF-beta 1 to a lesser extent. [125I]TGF-beta 1 and 125I]TGF-beta 2 formed complexes with purified bovine alpha 2M and serum proteins migrating at identical positions to purified alpha 2M during nondenaturing polyacrylamide gel electrophoresis. However, [125I]TGF-beta 1 associated preferentially with the "fast" migrating form of alpha 2M, whereas [125I]TGF-beta 2 associated with both the "slow" and "fast" migrating forms. Inhibitory activity of TGF-beta 1 and TGF-beta 2 coeluted with alpha 2M in the high molecular weight fractions from a Sephacryl S-200 column. These results support the hypothesis that purified alpha 2M and alpha 2M in bovine serum binds both TGF-betas but preferentially counteracts the mitoinhibitory effect of TGF-beta 2 on rat hepatocytes.     

Effects of serum collected from rats of different ages on in vitro cell proliferation

It has been reported by Carrel and his co-workers that serum from old hens inhibits cell growth in culture. However, as we had previously demonstrated contradictory results using serum from old rabbits, we examined whether serum from old rats would also show strong induction of cell proliferation. Sera from young and adult rats of either sex strongly stimulated the growth of rat fetal skin fibroblasts and human fetal lung fibroblasts (TIG-1). Sera of old female and male rats (24-29 months old) produced much greater fluctuations in growth-stimulatory activity than sera from young animals. Most samples of serum from old rats stimulated the growth of TIG-1 cells, as did fetal bovine serum and samples from younger rats, even when a higher concentration of serum (up to 50%) was used. On the other hand, a small proportion of samples repressed the growth of the cells. A study on the effects of serial mixtures of both different types of serum samples from old rats on cell growth suggested that this minor proportion of serum samples contain a large amount of inhibitory factor(s). The cell growth-stimulatory activity of serum did not correlate with the total protein and albumin concentrations, albumin/globulin ratio, and the levels of lipid peroxide in the sample. These results therefore seemed to imply that serum induced a striking increase in the heterogeneity of cell growth stimulatory activity with age, although most samples of serum from old rats of either sex stimulated cell proliferation as effectively as samples from younger rats. The biological significance of the small proportion of serum samples from old rats which do inhibit cell proliferation was discussed.     

Establishment of fetal bovine intestinal epithelial cell cultures susceptible to bovine rotavirus infection

Mucosal epithelial cells are infected by a wide variety of pathogens and determining their response to infection is critical for understanding disease pathogenesis. A protocol was developed for culturing primary epithelial cells from fetal bovine intestine and the cultured cells were evaluated for susceptibility to an enteric viral infection. Immunohistochemical staining for cytokeratin confirmed that 60-75% of cultured cells were epithelial cells. Furthermore, following infection with bovine rotavirus (BRV) over 80% of cells in the ileal and jejunal cultures contained viral protein at 16 h post-infection. The intestinal epithelial cell cultures also contained fibroblasts so a jejunal fibroblast culture was established and infected with BRV. Viral protein was detected in jejunal fibroblasts but viral-induced cytopathology was delayed in fibroblast cultures when compared to epithelial cell cultures. This study describes an effective protocol for culturing bovine epithelial cells from fetal intestine and confirmed that the epithelial cells were susceptible to BRV infection. Ileal and jejunal cultures displayed limited growth following continuous passage but early passage epithelial cells provide competent target cells for studying host cell responses to an enteric viral pathogen.     

Induction of host DNA synthesis in senescent human diploid fibroblasts by infection with human cytomegalovirus

A human diploid fibroblast strain, TIG -1, ceased to proliferate at about 60-62 population doubling level. The percentage of nuclei incorporating [3H]thymidine during 24-h culture in fresh medium containing 10% fetal bovine serum was less than 2% in the senescent cells used in this study. Infection of these cells with human cytomegalovirus (HCMV), strain AD-169, increased the percentage of [3H]thymidine-labeled cells by about ten-fold. Equilibrium density gradient centrifugation analysis of purified DNA from infected cells showed that cellular DNA synthesis was stimulated preceded by the viral DNA synthesis. Ultraviolet irradiation of HCMV reduced the ability to induce DNA synthesis. Equilibrium density gradient centrifugation analysis of DNA which was labeled with 5-bromodeoxyuridine indicated semiconservative replication rather than repair synthesis. These results suggested that in a considerable fraction of human senescent cells host DNA replication could be reinitiated by infection with HCMV, but not by the addition of fetal bovine serum.     

新生大鼠海马神经干细胞的分离培养及分化研究

研究新生大鼠海马区脑组织中神经干细胞体外培养方法,为治疗神经系统疾病寻找合适的细胞来源。取新生SD大鼠的海马区脑组织,采用accutase结合机械分离法获取神经干细胞,在含有B－27、碱性成纤维生长因子和表皮生长因子的DMEM／F12无血清培养液中培养;Accutase酶消化后传代培养,取第3代细胞行抗巢蛋白免疫荧光染色鉴定并以含10％胎牛血清培养液诱导分化,神经元特异烯醇化酶和胶质纤维酸性蛋白免疫荧光染色检测NSCs向神经元及胶质细胞分化的能力。分离的新生大鼠海马区脑组织中细胞,在无血清培养液中形成大量的神经球,部分神经球出现融合及贴壁分化现象,细胞呈典型NSCs 形态。经巢蛋白染色鉴定,大部分为阳性细胞。神经细胞球经含有胎牛血清培养液培养后,可分化为神经元特异烯醇化酶和胶质纤维酸性蛋白表达阳性的细胞。从新生大鼠海马组织分离培养的NSCs具有自我更新和增殖能力,在含胎牛血清培养液中具有向神经元和神经胶质细胞分化的潜能。     

Production of a monoclonal antibody that recognizes bovine stem cell factor (SCF) and its use in the detection and quantitation of native soluble bovine SCF in fetal bovine serum.

Stem cell factor (SCF) is a pluritropic hematopoietic cytokine that acts predominantly on the proliferation and differentiation of hematopoietic progenitor cells. SCF has long been thought to be present in fetal bovine serum (FBS) as an endogenous factor that stimulates the growth of hematopoietic progenitor cells in FBS-supplemented cultures. To detect and quantitate bovine SCF in FBS, we produced a monoclonal antibody (mAb) by immunizing mice with recombinant soluble bovine SCF protein, which was expressed in insect cells by using a baculovirus system. Using the mAb, we purified native soluble bovine SCF from FBS by immunoaffinity chromatography. Western blot analysis revealed that the purified SCF protein had a molecular weight of 33 kDa. In addition, an enzyme-linked immunosorbent assay (ELISA) incorporating the mAb revealed that the levels of native soluble SCF in commercially available FBS were likely to be <100 pg/ml. These results suggest that the concentration of native soluble bovine SCF present in FBS may be insufficient to promote additive biologic effects on the growth of hematopoietic progenitor cells in FBS-supplemented cultures.     

Human alveolar type II cells: stimulation of DNA synthesis by insulin and endothelial cell growth supplement

Proliferation of type II cells is important for the recovery of the alveolar epithelium after acute lung injury. However, the factors that regulate the proliferation of human type II cells are unknown. Human alveolar type II cells were isolated from resected lung by dissociation with porcine pancreatic elastase and crystalline trypsin and purified by density-gradient centrifugation and serial differential adherence. The purity of the type II cells in the final adherent preparation was 84.4 +/- 1.1% type II cells by alkaline phosphatase and 87.7 +/- 2.8% by cytokeratin (n = 7). The medium MCDB-151 with 0.4% fetal bovine serum (FBS) was used to demonstrate the stimulatory effect of individual growth factors. Under these conditions, thymidine incorporation was stimulated by insulin, epidermal growth factor, endothelial cell growth supplement (ECGS), and acidic and basic fibroblast growth factors. Cholera toxin did not stimulate thymidine incorporation. The most effective stimulation was by the combination of insulin and ECGS. The incorporation of bromodeoxyuridine was used to identify the proportion of cells that were active in DNA synthesis. Insulin and ECGS increased the percentage of cells that incorporated bromodeoxyuridine from 8.5 +/- 1.3% to 21.3 +/- 2.4% (n = 6). Mitotic figures were seen in smears prepared from cultures incubated with insulin and ECGS. This observation was confirmed by electron microscopy, which demonstrated type II cells in metaphase. Increasing the concentration of FBS or human serum in the culture medium to 10% decreased the stimulatory effect of insulin and ECGS.(ABSTRACT TRUNCATED AT 250 WORDS)