Human vascular smooth muscle cells in culture: growth characteristics and protein pattern by use of serum-free media supplements

This study demonstrates that cultivation of vascular smooth muscle cells from human artery wall is possible under completely serum-free conditions. The effects of attachment factors on cell spreading and cell proliferation are described in detail as well as routine cultivation methods under serum-free conditions (clone cultures, cell migration, subcultivation by use of an exogenous trypsin inhibitor, cryopreservation and readaptation of cells). After a careful adaptation period, only two (BMS and Ultroser G) of the four commercially available serum-free media supplements tested were used successfully for a routine cultivation of the smooth muscle cells over several passages. With both supplements cell proliferation rates were comparable with those obtained in medium containing 10% fetal calf serum. The addition of platelet-derived growth factor or transferrin to serum-free cultures had no growth-stimulating effect. The addition of endothelial cell growth factor isolated from bovine brain caused a significant increase in proliferative activity of cells cultivated with BMS, but not with Ultroser G. Moreover, we report that under the serum-free culture conditions described here, the gamma-actin content of the cells is largely reduced (51% +/- 13% (means +/- SD) for cells cultivated in Ultroser G, and 12% +/- 4% (means +/- SD) for cells cultivated in BMS) when compared with cells cultivated under serum-containing conditions (gamma-actin content = 100%). The alpha-actin content was observed to be unaltered. Even after a careful readaptation of serum-free cultured cells to serum conditions, the gamma-actin content remained reduced.     

Astrocytes produce and secrete FGF-1, which promotes the production of apoE-HDL in a manner of autocrine action

The astrocytes prepared by 1 week secondary culture after 1 month primary culture of rat brain cells (M/W cells) synthesized and secreted apolipoprotein E (apoE) and cholesterol more than the astrocytes prepared by conventional 1 week primary and 1 week secondary culture (W/W cells) (Ueno, S., J. Ito, Y. Nagayasu, T. Furukawa, and S. Yokoyama. 2002. An acidic fibroblast growth factor-like factor secreted into the brain cell culture medium upregulates apoE synthesis, HDL secretion and cholesterol metabolism in rat astrocytes. Biochim. Biophys. Acta. 1589: 261-272). M/W cells also highly expressed fibroblast growth factor-1 (FGF-1) mRNA. FGF-1 was identified in the cell lysate of both cell types, but M/W cells released more of it into the medium. Immunostaining of FGF-1 and apoE revealed that both localized in the cells that produce glial fibrillary acidic protein. The conditioned media of M/W cells and FGF-1 stimulated W/W cells to release apoE and cholesterol to generate more HDL. Pretreatment with a goat anti-FGF-1 antibody or heparin depleted the stimulatory activity of M/W cell-conditioned medium. The presence of the anti-FGF-1 antibody in the medium suppressed apoE secretion by M/W cells. Differential inhibition of signaling pathways suggested that FGF-1 stimulates apoE synthesis via the phosphoinositide 3-OH kinase for PI3K/Akt pathway. Thus, astrocytes release FGF-1, which promotes apoE-HDL production by an autocrine mechanism. These results are consistent with our in vivo observation that astrocytes produce FGF-1 before the increase of apoE in the postinjury lesion of the mouse brain (Tada, T., J. Ito, M. Asai, and S. Yokoyama. 2004. Fibroblast growth factor 1 is produced prior to apolipoprotein E in the astrocytes after cryo-injury of mouse brain. Neurochem. Int. 45: 23-30).     

Long-Term Culture of Rat Hippocampal Neurons at Low Density in Serum-Free Medium: Combination of the Sandwich Culture Technique with the Three-Dimensional Nanofibrous Hydrogel PuraMatrix

The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10⁴ cells/mL (8.9×10³ cells/cm²) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).     

A Modified In vitro Method to Obtain Pure Astrocyte Cultures Induced from Mouse Hippocampal Neural Stem Cells Using Clonal Expansion

The aim of the present study was to produce astrocyte cultures of high purity from mouse hippocampal neural stem cells and to compare their in vitro properties with those isolated from enriched mixed glial cultures prepared from mouse hippocampus, which are commonly contaminated by microglia. We produced primary cultures of newborn mouse hippocampal neural stem cells, which have the potential to differentiate into astrocytes, neurons, and oligodendrocytes. We produced monoclonal neural stem cell colonies by limiting dilution. We induced astrocyte differentiation by plating the colonies on poly-l-lysine and culturing them in induction medium consisting of minimum essential medium/F12 supplemented with 10% fetal bovine serum and 100 ng/ml ciliary neurotrophic factor. We then further purified the cells by differential adherence and shaking at a constant temperature, followed by a second round of limiting dilution. Immunocytochemistry for glial fibrillary acidic protein showed that our method yielded 99.4 ± 0.5% pure astrocytes, whereas traditionally enriched mixed glial cultures yielded 94.2 ± 2% pure astrocytes. Induced cells resembled primary astrocyte cultures in functional properties such as cell proliferation rates and lack of tumorigenicity and p53, and expression of epidermal growth factor receptor, bystin, and nitric oxygen synthase. Our novel method of culture and purification of neural stem cells can therefore be used routinely for the primary culture of highly purified astrocytes from mouse hippocampus.     

Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium. Serum-free growth of IGF-I-producing CHO cells

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10⁶ cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin. This revised version was published online in July 2006 with corrections to the Cover Date.     

Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.     

Short-term primary culture of mouse interstitial cells: effects of culture conditions of androgen production

This study examined the effects of culture conditions and hormone treatment on androgen production by mouse interstitial cells in short-term primary culture. Testicular interstitial cells (18-25% 3 beta-hydroxysteriod dehydrogenase-positive) were maintained in serum-free hormone supplemented medium. Basal (nonstimulated) androgen production was found to be plating-density dependent. Androgen production per cell increased dramatically in a time- and cell concentration-dependent manner. This effect was reproduced in low density cultures by addition of charcoal-stripped conditioned medium from high density cultures. The cell anchorage factors, fibronectin and poly-l-lysine, similarly enhanced basal androgen production but did not augment responsiveness to luteinizing hormone (LH). Coating of the culture surface with serum inhibited androgen production. Cultured cells remained responsive to LH for 4 to 5 days and both insulin (5 micrograms/ml) and epidermal growth factor (EGF) (3 ng/ml) augmented LH-stimulated androgen production. There was a transient increase in LH sensitivity and maximum LH-stimulated androgen production for 5 to 72 h in culture followed by a decline in androgen production to low levels after 4 to 5 days in culture. This loss of activity was partially prevented by addition of antioxidants to the medium or by reduction of the ambient O2 concentration to 1%.     

Serum-free culture of AtT 20 pituitary cells: A system for neuroendocrine studies under defined conditions

Summary The growth of the mouse pituitary cell line AtT 20 was studied under different in vitro conditions. A completely defined, serum-free culture medium supported the survival of cells for a period of more than 2 mo. The medium, designed SFI, consisted of basal medium supplemented with transferrin, insulin, putrescine, and selenium. For maintenance of cells during long-term culture, no additional compounds were necessary. The time-dependent increases in cell number during culture with fetal bovine serum (FBS) and under serum-free conditions showed similar properties. Analysis of the effects of different substrata on cell growth demonstrated that polylysine supported adhesion and initial growth of cells to a greater extent than untreated plastic or FBS adsorbed to culture dishes. Synthesis and regulation of proopiomelanocortin (POMC)-mRNA, the precursor-mRNA of adrenocorticotropin (ACTH), could be detected by Northern blot analysis under basal conditions and after incubation with steroids and corticotropin-releasing hormone (CRH), indicating the serum-independent expression of important cellular properties.     

Mitogen limitation and bone morphogenetic protein-4 promote neurogenesis in SFME cells, an EGF-dependent neural stem cell line

Serum-free mouse embryo (SFME) cells are an epidermal growth factor (EGF)-dependent established line derived from brains of 16-d-old Balb/c mouse embryos. SFME cells grow indefinitely in serum-free medium without replicative senescence, chromosomal abnormalities, or malignant transformation. SFME cells express nestin, a neural stem cell marker, under serum-free conditions. Exposure to serum or transforming growth factor β (TGF-β) leads to a marked increase in differentiation toward the astrocytic lineage with expression of glial fibrillary acidic protein and other astrocyte markers. In this study, we show that treatment of SFME cells with bone morphogenetic protein-4 (BMP-4), another member of the TGF-β family, led to differentiation toward a neuronal lineage under conditions of low mitogenic stimulation (0.5 ng/mL) by EGF and fibroblast growth factor. Maximum mitogenic stimulation with 50 ng/mL EGF blocked the BMP-4 effect on neuronal differentiation, but did not block TGF-β-induced expression of markers of the astrocytic lineage. BMP-4 treatment also enhanced the activity of the neuron-specific enolase (NSE) promoter in SFME-NSE-lacZ cells that carry the gene for bacterial β-galactosidase under the control of the NSE promoter. Extended BMP-4 treatment caused SFME cells to express a neuronal phenotype synthesizing gamma-aminobutyric acid. These results indicate that SFME cells have the capacity to generate both neurons and astrocytes in vitro, which resemble the behavior of EGF-dependent multipotential stem cells in the central nervous system, and establish a relationship between effects of BMP-4 and degree of mitogenic stimulation by other peptide growth factors.     

小鼠大脑皮质星形胶质细胞原代培养与鉴定

为探讨简便、高效的大脑皮质星形胶质细胞体外培养方法,本研究取新生24 h内的ICR小鼠大脑皮层,采用物理方法将其分成约1 mm^3,震荡过滤后进行培养。通过拍照的方式记录原代培养1 d、3 d、7 d、14 d、21 d、28 d、35 d和原代培养14 d后再传代培养14 d(记为P2-14 d)细胞形态;通过实时定量PCR和Western blotting比较原代培养1周、2周、3周、4周、5周和原代培养2周后再传代培养2周(即P2-2)的星形胶质细胞内胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)基因和蛋白水平变化。选取GFAP、S100-β和谷氨酸转运蛋白(excitatory amino acid transporter 1,EAAT1)标记星形胶质细胞,微管相关蛋白(microtubuleassociated protein 2,MAP-2)、离子钙接头蛋白-1(ionized calcium-binding adapter molecule 1,Iba-1)和髓鞘相关糖蛋白(myelin associated glycoprotein,MAG)抗体分别标记神经元、小胶质细胞和少突胶质细胞。通过免疫荧光染色鉴定细胞种类及纯度。研究结果显示细胞生长良好,原代培养4周星形胶质细胞内GFAP比2周、3周、5周和传代培养2周的细胞更加稳定。经免疫荧光鉴定,星形胶质细胞纯度在95%以上。本实验采用相对较简单经济的方法培养出高纯度且生理状态相对较稳定的原代星形胶质细胞,该细胞模型不仅可以用于星形胶质细胞生理功能研究,还可以用于中枢神经系统相关疾病的体外研究。     

Responsiveness of mouse calvaria to parathyroid hormone after explant cryopreservation: 45Ca release in vitro

Newborn mouse calvaria prelabeled with 45Ca and cryopreserved at -196 degrees C in serum-free medium containing dimethylsulfoxide were compared to unpreserved explants for response to parathyroid hormone during subsequent culture. After short-term cryopreservation followed by rapid thawing, the viable explants continued to release 45Ca to the culture medium but additions of parathyroid hormone to the medium did not cause increased bone resorption. The data suggest that cryopreservation and thawing impairs mechanisms responsible for parathyroid hormone action on bone cells.     

Enhanced formation of mouse hybridomas without hat treatment in a serum-free medium

Summary A newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterinthymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.     

Beneficial effects of serum supplementation during in vitro production of porcine embryos on their ability to survive cryopreservation by open pulled straw vitrification

The ability of porcine blastocysts produced in vitro, in the presence or absence of serum, to survive cryopreservation was investigated in this experiment. Porcine oocytes were matured, fertilized and cultured in vitro using serum-free culture systems. Starting at Day 4 of in vitro embryo culture (Day 0 = fertilization), the culture medium was supplemented with 10% fetal bovine serum (FBS). Embryos were cultured under these conditions until Day 6. Embryos cultured with only BSA supplementation served as serum-free controls. Day 6 blastocysts and expanded blastocysts of excellent quality were vitrified using the open pulled straw method. After warming, blastocysts were cultured in the presence of 10% FBS for an additional 18 h to recover. Portions of blastocysts from both groups, without cryopreservation, were also cultured under the same conditions to serve as non-vitrified controls. To further investigate the influence of FBS on the quality of embryos produced, the total cell numbers in Day 6 blastocysts from both groups were compared. In addition, the ratio of viable to total cells in fully recovered blastocysts at each group was examined. Blastocysts produced in the presence of FBS had an increased ability to survive cryopreservation and also had a higher cell number compared to those produced in serum-free systems (P < 0.05). The fully recovered blastocysts had a normal viable to total cell ratio, compared to non-vitrified controls. Overall, this experiment supports the hypothesis that serum supplementation during in vitro production of porcine embryos is beneficial to the ability of a blastocyst to survive cryopreservation.     

Factors in the rat submaxillary gland that stimulate growth of cultured glioma cells: identification and partial characterization

The effect of rat submaxillary extract on the growth of rat C6 glioma cells in serum-free culture has been examined. Extracts (10-15 microgram/ml) of submaxillary glands from both male and female rats markedly enhanced the growth of serum-deprived C6 cells and, in combination with insulin, transferrin, and NIH-LH (a source of fibroblast growth factor), were able to stimulate C6 cell growth to an extent comparable to that achieved with an optimal amount of fetal calf serum. The mitogenic activity of rat submaxillary extracts was found to be heat-labile, acid-stable, and partially inactivated by protease and 2-mercaptoethanol. Under our assay conditions, biologically active preparations of purified mouse submaxillary gland epidermal growth factor (EGF) or nerve growth factor (NGF) were not mitogenic for C6 cells, nor was the mitogenic activity of rat submaxillary extracts inhibited by antiserum to these mouse submaxillary gland growth factors. These results suggest that the active component(s) of rat submaxillary extracts is unrelated to either EGF or NGF. The growth-enhancing effect also appears unrelated to esteropeptidase activity present in these extracts since the mitogenic activity was unaffected by several protease inhibitors. Moreover, two purified mouse submaxillary gland arginylesteropeptidases, EGF-binding protein and gamma-subunit of 7 S NGF, were unable to elicit a comparable growth response even when added to cell culture medium at unreasonably high concentrations. The C6 cell mitogenic activity of crude submaxillary extracts could be separated into two biologically similar components by either gel filtration on Sephadex G-100, preparative isoelectric focusing in a pH gradient of 3-10, or adsorption to DEAE-cellulose followed by elution with a sodium chloride gradient. One of the active components was acidic in nature and had an apparent molecular weight of 40,000, while the other was near neutral in charge and possessed a molecular weight of approximately 20,000. The relationship between these two C6 cell mitogenic components and the rat submaxillary gland component responsible for stimulating Balb/c-3T3 cell growth in serum-free, factor supplemented medium (McClure et al., 1979, J. Cell Biol. 83:96a) is also discussed.     

Primary culture of adult mouse lung fibroblasts in serum-free medium: responses to growth factors

We report a completely serum-free system for primary culture of fibroblasts from explants of adult mouse lung tissue which permits bioassays for cytokine activity to be performed using unselected populations of cells at low passage number, without interference by serum binding proteins or interacting growth factors. Cultures were established on collagen-coated surfaces in medium MCDB 201 containing albumin, transferrin, epidermal growth factor, lipids, prostaglandin E1, vitamin E, and reducing agents. The cells were morphologically and ultrastructurally typical of fibroblasts in culture and demonstrated expression of vimentin and induction of expression of desmin in culture. Proliferation of the cells was reproducible between different primary cultures and was growth factor dependent. Both cycling and growth-arrested cells exhibited increased DNA synthesis when stimulated with epidermal growth factor, platelet-derived growth factor, or basic fibroblast growth factor, which functioned as complete mitogens, but did not respond to insulin, tumor necrosis factor or interleukin-1 beta. Maximal induction of DNA synthesis by epidermal growth factor required the continued presence of the mitogen in the culture medium. These results cannot be satisfactorily explained by the competence-progression model of responses to mitogenic stimuli but support and extend the findings of other studies using diploid fibroblasts.     

The Activin A-Dependent Proliferation of PCC3/A/1 Embryonal Carcinoma Cells in Serum-Free Medium

Examination of the growth requirements of murine embryonal carcinoma cells (EC cells) or embryonic stem cells (ES cells) in serum-free medium revealed that PCC3 EC cells required activin A to grow and/or survive in such medium. In the absence of activin A, PCC3 cells began to disintegrate within 3 days under any serum-free conditions examined. P19 and AT805 EC cells grew even in serum-free medium without activin A but their growth rates were slightly facilitated by its addition. F9 EC cells also grew in the medium without activin A and its addition somewhat inhibited their growth rate. Three independently isolated ES cell lines and feeder-dependent PSA-1 EC cells also grew in serum-free medium without activin A if leukemia inhibitory factor (LIF) was supplemented. The addition of activin A had little effect on their growth rates. These findings suggest that PCC3 EC cells are a sort of nutritional mutant requiring activin A, thus making them useful in stidies on the growth regulatory mechanisms of EC/ES cells and/or the action of activin on EC/ES cells.     

Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel

Summary Sustained growth of mouse ventral and anterior prostatic epithelial cells embedded within collagen gel matrix was achieved in a serum-free medium composed of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1∶1 (vol/vol), supplemented with bovine serum albumin fraction V, epidermal growth factor, transferrin, cholera toxin, prolactin, 5α-dihydrotestosterone, cortisol, putrescine, fibroblast growth factor, and a trace element mixture. Three-dimensional growth of prostatic epithelial cells occurred inside the collagen gel matrix. This serum-free medium allowed cell growth greater than sevenfold over 10 d in culture. Tissue recombination and cell culture techniques were integrated to demonstrate that cultured cells retained prostatic characteristics. Following 10 d of culture, epithelial colonies from mouse ventral and anterior prostatic epithelial cell cultures were isolated and combined with rat fetal urogenital sinus mesenchyme and grown for 4 wk under the renal capsule of intact athymic male mice. These tissue recombinants showed distinctive prostatic histologic characteristic (alveoli and ducts lined with cuboidal or columnar epithelium surrounded by stroma). When histologic sections of recombinants were stained with the Hoechst 33258, epithelial cells of mouse origin were distinguishable from stromal cells of rat origin. Aided by grants CA-05388 and CA-09041 from the National Institutes of Health, Bethesda, MD, and by M. A. R. C. fellowship GM08730 to T. T.     

Differentiation of isolated murine embryonic palatal epithelium in culture: exogenous transforming growth factor alpha modulates matrix biosynthesis in defined experimental conditions

Summary A novel culture technique, which supports the growth and differentiation of mouse embryonic palatal epithelial cells in the absence of either an extracellular matrix substratum or feeder layers, has been developed. Using this technique we have investigated the effects of exogenous transforming growth factor alpha (TGFα) and serum on extracellular matrix biosynthesis by primary cultures of mouse embryonic epithelial sheets under defined experimental conditions. In all culture treatments (chemically defined medium with and without TGFα or serum) the palatal epithelial sheets differentiated into three regionally distinct cell phenotypes after 36 h. Nasal and oral cells differentiated into pseudostratified, cilliated columnar, and stratified squamous keratinizing epithelium, respectively. In addition, basal medial edge epithelial (MEE) cells at the oral/nasal regional interface assumed an elongated cobblestoned phenotype. In serum-free medium, collagen types IV and V, laminin, fibronectin, and heparan sulphate proteoglycan were detected immunocytochemically throughout the entire epithelial sheet. Tenascin and collagen IX were present almost exclusively in MEE cells. Types I, II, and III collagen were completely absent. Addition of TGFα or serum universally increased the intensity of staining, most notably that for tenascin and collagen IX in MEE cells. These results indicate that mouse embryonic palatal epithelial sheets can be maintained under defined culture conditions during which they exhibit patterns of differentiation similar to those observed in vivo. TGFα, known to localize to the MEE in vivo, can modulate palatal extracellular matrix biosynthesis, particularly by the MEE, suggesting a regulatory role for this factor. The culture system is suitable for further investigating the effects of exogenous factors on mouse embryonic palatal epithelial cell bioactivity and differentiation.     

Regulation of growth inhibitory activity in transformed mouse embryo fibroblasts

Treatment of the transformed mouse embryo fibroblast cell line AKR-MCA with 1% N,N-dimethylformamide (DMF) resulted in the restoration of a nontransformed phenotype in these cells. In order to determine if an increase in growth inhibitory peptides might be responsible for these changes in growth properties of the DMF-treated AKR-MCA cells we examined the serum-free conditioned medium for its ability to inhibit the anchorage-independent growth of a human colon carcinoma cell line. The extracellular levels of inhibitory activity were two-fold higher in conditioned medium derived from AKR-MCA cells than in AKR-MCA cells grown in 1% DMF (AKR-MCA/DMF). Fractionation of the crude conditioned medium indicated the presence of an Mr 20,000 inhibitory fraction in AKR-MCA/DMF conditioned medium which was reduced in AKR-MCA cells. This Mr 20,000 inhibitory activity was acid and heat stable and sensitive to dithiothreitol and trypsin. In addition to inhibiting the growth of a human colon carcinoma cell line this protein induced colony formation in AKR-2B cells and competed for binding to the transforming growth factor beta (TGF-beta) receptor. Therefore, this Mr 20,000 inhibitory polypeptide induced by DMF is probably TGF-beta. TGF-beta was also shown to inhibit the growth of AKR-MCA cells in monolayer culture.     

Serum-free,chemically defined medium to evaluate the direct effects of growth factors and inhibitors on proliferation and function of neonatal rat cardiac muscle cells in culture

Summary Neonatal rat cardiac myocytes were isolated and cultured to evaluate the effects of growth factors and inhibitors on proliferation, survival, and functions in a serum-free medium. Insulin and transferrin in MCDB 107 nutrient medium elicited DNA and protein synthesis in cells on a fibronectin-coated culture surface in serum-free medium. Insulin was most effective on both DNA and protein synthesis in serum-free culture conditions. The serum-free, hormone-supplemented medium eliminated the contamination of noncardiac myocytes and supported the long-term survival (over 18 d) of cardiac myocytes. Dexamethasone was required to induce optimal contractility with or without insulin and transferrin. Serum contained both negative and positive effectors of DNA and protein synthesis of the cardiac myocytes. Concentrations of serum (above 5%) inhibited DNA and protein synthesis. Low density lipoprotein (LDL) accounted in part for the inhibitory activity. The serum-free culture system provides a useful model to elucidate the role of hormones, growth factors, and drugs in heart cell regeneration and function.