Alveolar type II cells inhibit fibroblast proliferation: role of IL-1alpha

Alveolar type II (ATII) cells inhibit fibroblast proliferation in coculture by releasing or secreting a factor(s) that stimulates fibroblast production of prostaglandin E2 (PGE2). In the present study, we sought to determine the factors released from ATII cells that stimulate PGE2 production in fibroblasts. Exogenous addition of rat IL-1alpha to cultured lung fibroblasts induced PGE2 secretion in a dose-response manner. When fibroblasts were cocultured with rat ATII cells, IL-1alpha protein was detectable in ATII cells and in the coculture medium between days 8 and 12 of culture, correlating with the highest levels of PGE2. Furthermore, under coculture conditions, IL-1alpha gene expression increased in ATII cells (but not fibroblasts) compared with either cell cultured alone. In both mixed species (human fibroblasts-rat ATII cells) and same species cocultures (rat fibroblasts and ATII cells), PGE2 secretion was inhibited by the presence of IL-1 receptor antagonist (IL-1Ra) or selective neutralizing antibody directed against rat IL-1alpha (but not IL-1beta). Conditioned media from cocultures inhibited fibroblast proliferation, and this effect was abrogated by the addition of IL-1Ra. Addition of keratinocyte growth factor (KGF) resulted in an earlier increase in PGE2 secretion and fibroblast inhibition (day 8 of coculture). This effect was inhibited by indomethacin but was not altered by IL-1Ra. We conclude that in this coculture system, IL-1alpha secretion by ATII cells is one factor that stimulates PGE2 production by lung fibroblasts, thereby inhibiting fibroblast proliferation. In addition, these studies demonstrate that KGF enhances ATII cell PGE2 production through an IL-1alpha-independent pathway.     

Interactions between cardiac cells enhance cardiomyocyte hypertrophy and increase fibroblast proliferation

In cardiac hypertrophy, both excessive enlargement of cardiac myocytes (CMs) and progressive fibrosis are known to occur simultaneously. To investigate the nature of interactions between ventricular CMs and cardiac fibroblasts (CFs) in these conditions, we have established a "dedifferentiated model" of adult murine CMs in coculture with CFs. In such a model, which is recognized to study cardiac cell hypertrophy in vitro, dedifferentiated CMs in culture and in coculture were characterized by immunopositive staining to ANP (atrial natriuretic peptide) and beta-myosin heavy chain (beta-MHC). The results confirm that ANP secretion by CMs was significantly increased during the cultures. The increase size of cultured CMs was significantly higher in CM/CF cocultures than in CM cultures which was also observed when CMs were cultured with fibroblast conditioned medium (FCM). In addition, fibroblast proliferation studies showed that CMs favored fibroblast adhesion and/or growth at the beginning of the coculture and fibroblast proliferation throughout the time course of the coculture. Furthermore, a significant level of interleukin-6 (IL-6) production was detected by ELISA in CM/CF cocultures. A similar higher increase was observed when CMs were cultured in the presence of FCM. These results demonstrate that CFs enhance myocyte hypertrophy and that CMs regulate fibroblast adhesion and/or proliferation, suggesting a paracrine interaction between CMs and CFs which could involve IL-6.     

Stimulation of growth of primary cultured adult rat hepatocytes without growth factors by coculture with nonparenchymal liver cells

DNA synthesis of adult rat parenchymal hepatocytes alone in primary culture can be stimulated only by the addition of humoral growth factors to the culture medium. However, when parenchymal hepatocytes were cocultured with nonparenchymal liver cells from adult rats, their DNA synthesis was markedly stimulated in the absence of added growth factors or calf serum. DNA synthesis of parenchymal hepatocytes was not stimulated by conditioned medium from nonparenchymal liver cells and was greatest when the parenchymal cells were plated on 24-h cultures of nonparenchymal liver cells. A dead feeder layer of nonparenchymal cells was almost as effective as a feeder layer of viable nonparenchymal cells. These results suggest that the stimulation of DNA synthesis in parenchymal hepatocytes was not due to some soluble factors secreted by nonparenchymal liver cells but to an insoluble material(s) produced by the nonparenchymal liver cells. This insoluble material(s) was collagenase- and acid-sensitive, suggesting that it was a protein containing collagen. The effect of nonparenchymal liver cells was specific: coculture with hepatoma cells, liver epithelial cells, or Swiss 3T3 cells did not stimulate DNA synthesis in parenchymal hepatocytes. Added insulin and epidermal growth factor showed additive effects with nonparenchymal cells in the cocultures. These results suggest that DNA synthesis in parenchymal hepatocytes is stimulated not only by various humoral growth factors but also by cell-cell interaction between parenchymal and nonparenchymal hepatocytes, possibly endothelial cells. This cell-cell interaction may be important in repair of liver damage and liver regeneration.     

A heterologous 3-D coculture model of breast tumor cells and fibroblasts to study tumor-associated fibroblast differentiation

The objective of our study was to establish spheroid cocultures as a valid 3-D in vitro model mimicking tumor-fibroblast interactions in scirrhous breast tumors. The experimental setup was designed to verify if in cocultures (a) adherence and migration reflect the invasive potential of breast tumor cells, (b) breast tumor cells induce tumor-associated fibroblast differentiation, and (c) tumor-derived fibroblasts better reflect the in vivo situation than normal skin fibroblasts. Only one (SK-BR-3) out of five tumor cell types showed extensive fibroblast infiltration, MCF-7 cells frequently invaded fibroblast spheroids; BT474, T47D, and ZR-75-1 were noninvasive. While tumor cell invasion was independent of fibroblast origin, tumor-associated myofibroblast differentiation defined by alpha-SMA expression was demonstrated for tumor-derived but not normal skin fibroblasts in coculture indicating that (a) tumor cell invasion and myofibroblast differentiation are autonomous processes and (b) cocultures with tumor-derived fibroblasts resemble advanced stages of desmoplastic carcinomas while cocultures with normal skin fibroblasts rather reflect the early tumor development. The latter is also implied by fibroblast-associated alterations in tumor cell morphology and ECM distribution in the system. By using RNA arbitrarily primed PCR and cells isolated from cocultures by fluorescence-activated and magnetic cell separation, peripheral myelin protein PMP22/SR13 has been identified as a novel candidate with potential relevance in the interaction between tumor cell and normal fibroblast since PMP22 mRNA was significantly reduced in normal skin fibroblasts in coculture with BT474 cells.     

Depletion of 43-kD growth-associated protein in primary sensory neurons leads to diminished formation and spreading of growth cones

Epithelial-mesenchymal interactions control epidermal growth and differentiation, but little is known about the mechanisms of this interaction. We have examined the effects of human dermal microvascular endothelial cells (DMEC) and fibroblasts on keratinocytes in conventional (feeder layer) and organotypic cocultures (lifted collagen gels) and demonstrated the induction of paracrine growth factor gene expression. Clonal keratinocyte growth was similarly stimulated in cocultures with irradiated DMEC and fibroblasts as feeder cells. This effect is most probably caused by induction of growth factor expression in cocultured dermal cells. Keratinocytes stimulated mRNA levels for KGF and IL-6 in both mesenchymal cell types and GM-CSF in fibroblasts. The feeder effect could not be replaced by conditioned media or addition of isolated growth factors. In organotypic cocultures with keratinocytes growing on collagen gels (repopulated with dermal cells), a virtually normal epidermis was formed within 7 to 10 d. Keratinocyte proliferation was drastically stimulated by dermal cells (histone 3 mRNA expression and BrdU labeling) which continued to proliferate as well in the gel. Expression of all typical differentiation markers was provoked in the reconstituted epithelium, though with different localization as compared to normal epidermis. Keratins K1 and K10 appeared coexpressed but delayed, reflecting conditions in epidermal hyperplasia. Keratin localization and proliferation were normalized under in vivo conditions, i.e., in surface transplants on nude mice. From these data it is concluded that epidermal homeostasis is in part controlled by complex reciprocally induced paracrine acting factors in concert with cell-cell interactions and extracellular matrix influences.     

Positive regulation of normal and tumoral mammary epithelial cell proliferation by fibroblasts in coculture

Summary In the mammary gland, mesenchymal-epithelial interactions are of paramount importance during normal and tumoral developments. We have studied the paracrine growth regulation of a variety of breast epithelial cells in coculture with normal or pathological breast fibroblasts. Two models of coculture were used in which the two cell types were seeded and grown, either together in microchamber slides or separated by a microporous membrane. Under these two conditions, all fibroblasts were shown to stimulate the proliferation of the hormono-responsive breast carcinoma MCF-7 cell line, suggesting that cell contacts were not indispensable for the paracrine stimulation of MCF-7 cell growth by fibroblasts. Moreover, in the Transwell coculture system, the proliferation of a variety of other breast carcinoma cells (MDA-MB231, T47D, and BT-20) was also stimulated by fibroblasts. However, the amplitude of the proliferative response seemed to be dependent on the carcinoma cell line considered. Moreover, the proliferative response of normal mammary epithelial cells to the presence of fibroblasts was shown to be significantly higher than the tumor cell response. The nature of the tissue of fibroblast origin, normal or pathological, did not influence the growth response of the epithelial cells. In this study, we thus demonstrate that fibroblasts are able to stimulate the proliferation of normal and carcinoma cells through paracrine exchange mechanisms. We also conclude that the target epithelial cell phenotype will essentially determine the extent of the proliferative response.     

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.     

Effects of feeder layers made of human,mouse, hamster,and rat cells on the cloning efficiency of transformed human cells

Summary The effect of feeder layers on cloning efficiency of transformed human cells was investigated. Embryonic human skin or lung fibroblasts; adult human skin fibroblasts; early passage cells from embryos of mouse, rat, and hamster; established mouse cell lines; 3T3 and 10T1/2 were used as feeder layers after they were lethally exposed to Co-60 gamma-rays at 3,000 rad. As test cells to study the effect of feeder layers on cloning efficiency, WI-38 CT-1 cells transformed in vitro by Co-60 gamma-rays and HGC cells cultured from a human gastric cancer were used. The effect of feeder layers on the cloning efficiency of the test cells was dependent on cell density of feeder layer cells, sources of the feeder layer cells, and kinds of test cells. An optimal density of feeder cels produced cloning efficiencies 3 to 15 times higher than in cultures without a feeder layer. Generally, high density of cells in feeder layers decreased the cloning efficiency of the test cells, presumably owing to contact inhibition of growth and depletion of essential nutrients by the feeder layer cells. Regarding the effect of the feeder layers made of human fibroblasts, there were no significant differences in population doubling levels; tissue origins of fibroblasts; or fibroblasts derived from normal individuals, patients with cancer, or with a genetically high familial incidence of cancer, hereditary adenomatosis of the colon and rectum. This study was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan.     

Functional differentiation of alveolar type II epithelial cells in vitro: effects of cell shape, cell-matrix interactions and cell-cell interactions

Alveolar type II epithelial cells rapidly lose characteristics of differentiated function when cultured on plastic dishes. We have attempted to circumvent this problem by culturing type II cells under conditions that might better reproduce their environment in vivo. Cell-matrix interactions were studied by culturing isolated adult rat type II cells on Engelbreth-Holm-Swarm (EHS) tumor basement membrane. Aggregates of type II cells formed on the surface of the matrix during 4 days in culture. Microscopic examination of these aggregates revealed cuboidal cells that retained more characteristics of differentiated type II cells than did cells cultured on plastic. Type II cells cultured on EHS matrix incorporated a higher percentage of acetate into phosphatidylcholine (PC) than did cells on plastic, and a higher percentage of this PC was saturated. Phosphatidylglycerol (PG) synthesis by these cells was no different from that seen in cells on plastic. The effects of cell-cell interactions and cell shape were evaluated by culturing type II cells on feeder layers that in turn were grown on collagen gels. The feeder layer cells included fetal rat lung fibroblasts, adult rat lung fibroblasts, fetal rat skin fibroblasts, bovine aortic endothelial cells, and rat mammary tumor epithelial cells. One-half of the gels remained attached to the culture dish and one-half of the gels were detached after 24 h and allowed to float free in the medium. Type II cells grown in association with any of the attached feeder layers became flattened and lost their differentiated phenotype. These cells incorporated no greater percentage of acetate into PC than did cells on plastic. Saturated PC synthesis was modestly increased. PG synthesis declined in parallel with that seen in cells cultured on plastic. Type II cells cultured on feeder layers that were detached assumed their native cuboidal shape and also exhibited many morphological characteristics of differentiated function. These cells incorporated a significantly greater percentage of acetate into PC compared to cells on either plastic or attached feeder layers. Saturated PC synthesis also increased markedly. These cells, however, incorporated no greater percentage of acetate into PG than did cells on plastic or attached feeder layers. These data suggest an important role for cell shape and cell-matrix interactions and maintenance of type II cell differentiation. The effects of cell-cell interactions, while beneficial, appear to be non-specific.     

A study of the functional capabilities of human neonatal lymphocytes for in vitro specific antibody production

The ability of neonatal B and T cells to participate in the in vitro production of anti-influenza virus antibody was studied. Peripheral blood mononuclear cells from nearly all normal adults produce anti-influenza virus antibody when stimulated in vitro with type A influenza virus. Cord blood mononuclear cells, however, consistently failed to do so. Using Epstein-Barr virus activation or coculture with irradiated adult T cells in the presence of influenza virus to identify precursor B cells for anti-viral antibody production, newborns were found to have a decreased number of influenza-specific B cells as compared with adults. Thus, a paucity of precursor B cells for anti-influenza virus antibody was one factor contributing to the absent in vitro antibody response. Additional studies were undertaken to investigate the capabilities of newborn T cells in the in vitro response to influenza virus. Newborn T cells failed to proliferate when cultured with influenza virus. Irradiated newborn T cells were, however, able to provide help for specific antibody production in influenza virus-stimulated cocultures with allogeneic adult B cells, and newborn T cells proliferated when stimulated with alloantigens; their helper function in allogeneic coculture was, thus, likely mediated by T cells stimulated by alloantigens rather than by influenza virus. In the absence of T cell irradiation, no antibody was produced in cocultures of adult B cells and neonatal T cells, at least in part as the result of a radiosensitive suppressor T cell. Suppression in influenza virus-stimulated allogeneic cocultures was also observed with normal adult T cells and is, therefore, a property of both newborn and adult T cells. Thus, allogeneic help and suppression can both be manifested in exogenous antigen-stimulated allogeneic cocultures. In addition, both of these allogeneic effects can be mediated by neonatal T cells, indicating that these functions are present at the time of birth and do not require previous exogenous antigenic exposure for expression.     

Gr-1+ myeloid cells lacking T cell protein tyrosine phosphatase inhibit lymphocyte proliferation by an IFN-gamma- and nitric oxide-dependent mechanism

The T cell protein tyrosine phosphatase is involved in the immune system regulation, as evidenced by defective function and development of several hemopoietic cell populations in T cell protein tyrosine phosphatase (TC-PTP)-deficient mice. In particular, B and T cell proliferation is greatly inhibited when total splenocytes are stimulated by LPS or anti-CD3 mAb. To define the functional defect of TC-PTP(-/-) lymphocytes, we isolated T and B cells from the spleen of TC-PTP(-/-) mice. We show that the proliferative response of lymphocytes was greatly increased when cultured as a purified population, indicating that an inhibitory population is present in TC-PTP(-/-) spleen. However, TC-PTP(-/-) lymphocytes have a 2- to 3-fold lower proliferation rate compared with TC-PTP(+/+) lymphocytes, suggesting that, as shown previously in embryonic fibroblasts, TC-PTP is involved in the control of cell cycle in lymphocytes. We have characterized phenotypically and functionally the inhibitory population present in the spleen of TC-PTP(-/-) mice. We show that a Gr-1(+)-enriched cell population isolated from TC-PTP(-/-) mice suppresses the CD3-induced proliferation of T cells in coculture in vitro. The specific inhibition of NO synthesis with N(G)-monomethyl-L-arginine.monoacetate restored splenocyte responses, and there is a strict correlation between NO levels and the degree of suppression. Neutralization of IFN-gamma with specific mAb almost completely abolished the inhibitory activity of Gr-1(+) cells and concomitantly high levels of NO secretion. Moreover, inhibition of lymphocyte proliferative responses required cell-cell contact to achieve sufficient levels of NO. These findings demonstrate an important function of TC-PTP in the induction of the NO pathway that mediates inhibition of T cell proliferation.     

Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krüppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.     

Promoting effect of feeder cells in maintenance of adult rat hepatocytes

A procedure is described for maintaining primary cultures of adult rat hepatocytes for prolonged periods of time on layer of irradiated mouse fibroblast cell line (C3H/1OT1/2) and on a secondary lung fibroblasts obtained from Sprague Dawley rats. Morphologically and ultrastructurally the cocultivated hepatocytes retained many characteristics of hepatocytes in vivo. Within 24 hours after seeding, the individual cells were attached on the feeder cell layer and the in vivo polarity of the liver cells reappeared. Electron microscope studies demonstrated the appearance of newly developed bile ducts and junctions between hepatocytes as well as between hepatocytes and feeder cells. Histochemically, these cells were positive for glucose-6-phosphatase and for glycogen. After 14 days in culture the hepatocytes could be reseeded onto fresh C3H1OT1/2 cells. In contrast, hepatocytes maintained on plastic substrate lost their glycogen content and the epithelial character of the liver cells after 5 days in culture, and by day 10 this culture became predominantly fibroblastic. It is suggested that hepatocytes maintained on an irradiated fibroblast feeder layer provide a valuable approach for studying the morphogenesis, cytotoxicity, or the metabolism of different chemicals in vitro.     

In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.     

Fibroblast feeder layers inhibit differentiation of retinoic acid-treated embryonal carcinoma cells by increasing the probability of stem cell renewal

The appearance of differentiated cells in embryonal carcinoma (EC) cultures can be inhibited by culturing the cells on fibroblast feeder layers. To determine whether or not feeder layers act by increasing the probability of stem cell renewal, growth and differentiation were monitored in cultures of F9 (subclone OTF9 -63) EC cells exposed to retinoic acid (RA) in either the presence or absence of feeder layers. By measuring the fraction of laminin-positive TROMA 1-positive or alkaline phosphatase-negative cells, it was determined that the frequency of differentiated cells in RA-treated F9 cultures was reduced by 70-80% when cells were cultured on fibroblast feeder layers instead of gelatin-coated dishes. Experiments in which EC cells were cultured in close proximity to a feeder layer demonstrated that cell-cell contact was required for maximal inhibition of differentiation. The probability of stem cell renewal was determined by measuring the number of colony-forming cells in RA-treated cultures as a function of time. Analysis of the data demonstrated that the probabilities of stem cell renewal were 0.5 and 0.25 during the first and second 48 h periods, respectively, following addition of RA for cells cultured without feeder layers. Cultures maintained on feeder layers exhibited a stem cell renewal probability of 0.72. Thus, feeder layers reduce the frequency of differentiated cells in RA-treated cultures by increasing the probability of stem cell renewal. Determining the mechanism by which feeder layers counteract the effect of a chemically defined differentiation inducer should help to uncover the processes that regulate the probability of stem cell renewal.     

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.     

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis

BACKGROUND: To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. METHODS: We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. RESULTS: Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. CONCLUSIONS: The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies.     

FGF4-dependent stem cells derived from rat blastocysts differentiate along the trophoblast lineage

Differentiated trophoblast cell lineages arise from trophoblast stem (TS) cells. To date such a stem cell population has only been established in the mouse. The objective of this investigation was to establish TS cell populations from rat blastocysts. Blastocysts were cultured individually on a feeder layer of rat embryonic fibroblasts (REFs) in fibroblast growth factor-4 (FGF4) and heparin supplemented culture medium. Once cell colonies were established REF feeder layers could be replaced with REF conditioned medium. The blastocyst-derived cell lines, in either proliferative or differentiated states, did not express genes indicative of ICM-derived tissues. In the proliferative state the cells expressed established stem cell-associated markers of TS cells. Cells ceased proliferation and differentiated when FGF4, heparin, and REF conditioned medium were removed. Differentiation was characterized by a decline of stem cell-associated marker gene expression, the appearance of large polyploid cells (trophoblast giant cells), and the expression of trophoblast differentiation-associated genes. Collectively, the data indicate that the rat blastocyst-derived cell lines not only possess many features characteristic of mouse TS cells but also possess some distinct properties. These rat TS cell lines represent valuable new in vitro models for analyses of mechanisms controlling TS cell renewal and differentiation.     

Structural adaptation in adult rabbit ventricular myocytes

The mechanism of induction of cardiomyocyte (CM) dedifferentiation, as seen in chronic hibernating myocardium, is largely unknown. Recently, a cellular model was proposed consisting of long-term cocultures of adult rabbit CMs and cardiac fibroblasts in which typical structural characteristics of hibernation-like dedifferentiation could be induced. Only CMs in close contact with fibroblasts underwent these changes. In this study, we further investigated the characteristics of the fibroblast-CM interaction to seek for triggers and phenomena involved in CM dedifferentiation. Adult rabbit CMs were cocultured with cardiac or 3T3 fibroblasts. Heterocellular interactions and the structural adaptation of the CMs were quantified and studied with vital microscopy and electron microscopy. Immunocytochemical analysis of several adhesion molecules, i.e., N-cadherin, vinculin, β1-integrin, and desmoplakin, were examined. Upon contact with CMs, fibroblasts attached firmly and pulled the former cells, resulting in anisotropic stretch. Quantification of the attachment sites revealed a predominant binding of the fibroblast to the distal ends of the CM in d 1 cocultures and a shift towards the lateral sides of the CMs on d 2 of coculture, suggesting a redistribution of CM membrane proteins. Immunocytochemical analysis of cell adhesion proteins showed that these were upregulated at the heterocellular contact sites. Addition of autologous and nonautologous fibroblasts to the CM culture similarly induced a progressive and accelerated structural adaptation of the CM. Dynamic passive stretch invoked by the fibroblasts and/or intercellular communication involving cell adhesion molecule expression at the interaction sites may play an important role in the induction of hibernation-like dedifferentiation of the cocultured adult rabbit CMs. These authors contributed equally to this study.