Neoplastic progression of rat tracheal epithelial cells is associated with a reduction in the number of growth factors required for clonal proliferation in culture

Summary Factors regulating the proliferation of normal, preneoplastic, and neoplastic rat tracheal epithelial (RTE) cells were investigated to identify changes taking place during the progression of RTE cells to neoplasia. Normal RTE cells exhibit clonal proliferation in a serum-free medium containing pituitary extract, serum albumin, cholera toxin, epidermal growth factor, hydrocortisone, and insulin. All combinations of these six factors were examined for their abilities to support clonal proliferation of normal, preneoplastic, and neoplastic RTE cells. In general, preneoplastic RTE cells required fewer factors for proliferation than normal RTE cells, and neoplastic cells required fewer factors than preneoplastic cells. A common pattern of reductions has been identified in the growth factors required for the clonal proliferation of preneoplastic vs. normal RTE cells and for neoplastic vs. preneoplastic and normal RTE cells. Normal RTE cells exhibit clonal proliferation in a serum-free medium supplemented with a minimum of six factors: bovine serum albumin, bovine pituitary extract, cholera toxin, epidermal growth factor, hydrocortisone, and insulin. Preneoplastic RTE cells exhibit clonal proliferation in a serum-free medium supplemented with four factors: bovine serum albumin, bovine pituitary extract, hydrocortisone, and insulin. Finally, neoplastic RTE cells exhibit clonal proliferation in a serum-free medium supplemented with two factors: bovine serum albumin and bovine pituitary extract. These results suggest that the progression of RTE cells to neoplasia is associated with a series of changes in regulatory pathways that control cell proliferation.     

Clonal growth of normal human uroepithelial cells

Summary We report the development of culture conditions which routinely support clonal growth of normal human uroepithelial cells (HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency (CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3×10⁵ lethally irradiated Swiss 3T3 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum. Using these conditions, the average CFE was 16.1±2.5%. A cloning efficiency of 4.9±1.5% was obtained under the same conditions in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM ethanolamine. The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining for human epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of Swiss 3T3 feeder cells resistant to 5 μg/ml 6-thioguanine (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone supports HUC clonal growth with a CFE of 17.9±2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented MCDB 170 medium containing 70 μg/ml bovine pituitary extract. The average cloning efficiency using these conditions was 5.7±1.7%. This work was supported by NIH grant 29525 to C. A. R. L. J. L. is a recipient of National Science Foundation predoctoral fellowship.     

Epidermal growth factor dependence and TGF alpha autocrine growth regulation in primary rat tracheal epithelial cells.

We have examined dependence of primary rat tracheal epithelial (RTE) on exogenous epidermal growth factor (EGF) and determined whether a TGF alpha autocrine pathway is operating in these cells. Primary RTE cells plated in serum free media (SFM) without EGF and bovine pituitary factor (BPE) show little proliferation compared to cultures propagated in media containing EGF/BPE (CSFM). Removal of EGF/BPE shortly after plating, however, results in significant proliferation, although plateau cell densities are reduced and cell morphology is significantly altered compared to cells propagated in CSFM. Addition of EGF and/or BPE to cultures propagated in SFM minus EGF/BPE restores maximum cell density. The concentration of TGF alpha peptide in media conditioned by cells propagated without EGF/BPE is lower than the concentration in the media of CSFM cultures. TGF alpha mRNA and protein levels are also significantly lower in cells late in culture compared to logarithmically growing cells regardless of the presence or absence of EGF/BPE. The proliferation of primary RTE cells propagated without EGF/BPE is inhibited by neutralizing TGF alpha antiserum and by a tyrphostin compound that blocks TGF alpha/EGF receptor tyrosine kinase activity. These results indicate that primary RTE cells utilize TGF alpha as an autocrine growth factor and that the autocrine pathway is regulated as a function of growth state of the cells. However, this pathway does not provide growth autonomy to primary RTE cells, since cultures remain dependent on exogenous EGF/BPE for sustained proliferation.     

Long-term culture of human endothelial cells

Summary Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10³/cm²) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts. This research was supported by grants from the U.S. Public Health Service (AG 01732, HL 16387, and HL 07080), the Cystic Fibrosis Foundation, and the New York and American Heart Associations. Victor B. Hatcher is an Established Fellow of the New York Heart Association and a recipient of the Ann Weinberg Cystic Fibrosis Research Scholarship Award.     

Effects of serum and serum-derived factors on growth and differentiation of mouse keratinocytes

Summary Mouse epidermal keratinocytes (MK cells) were grown as replicating subcultures at clonal density, in a serum-free, low calcium basal medium supplemented with seven different growth factors (Bertolero et al., Exp. Cell. Res. 155:64–80, 1984). This serum-free system was used to investigate the activity of cells. bovine serum (FBS) and of serum-derived factors on the growth and differentiation of MK cells. Unfractionated, whole FBS inhibited growth and induced terminal differentiation of normal MK cells. The growth inhibitory activity was considerably reduced by passing whole FBS over a resin (Chelex) to remove Ca²⁺ and other di- and trivalent cations. It is not known whether this treatment removed other factors. Addition of individual serum components either stimulated or inhibited cell-growth and differentiation. Fetuin, a major α-globulin of FBS, and high density lipoprotein strongly inhibited the colony forming efficiency (CFE) of MK cells, whereas bovine serum albumin increased the CFE 4.5-fold and stimulated the growth rate as well. The addition of impure commercial preparations of platelet-derived growth factor inhibited the CFE and induced the morphological features of squamous terminally-differentiating keratinocytes. As reported in other systems, transforming growth factor beta (TGF-β) inhibited the growth of secondary keratinocytes in a dose-dependent manner. Thus, at least three factors present in FBS inhibited growth whereas others were stimulatory. These observations explain the difficulties in obtaining replicating subcultures of mouse keratinocytes in serum-supplemented media and emphasize the importance of a serum-free system for studies on growth control and carcinogenesis in keratinocytes. Editor’s Statement This report contributes significantly to our knowledge of keratinocyte cell biology in two ways. First, a serum-free medium has been developed that can now be used by many investigators to define growth versus differentiation factors for these cells. This is important since several impure or relatively crude preparations of factors are known to influence these cells. Second, the finding that TGF-Beta is an inhibitor of keratinocyte growth opens new avenues to investigate the biochemical events leading to differentiation. David A. Sirbasku     

Growth regulation of primary rat tracheal epithelial cell cultures by endogenous transforming growth factor-βs

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to secrete transforming growth factor-β (TGFβ) and to be growth inhibited by exogenous TGFβ. The purpose of the present studies was to determine whether the endogenous TGFβ(s) were regulating the growth of RTE cell cultures and, if so, which isoforms were involved. Neutralizing antibodies specific to TGFβ1 and TGFβ2 were added to cultures, and their effects on several growth parameters were measured. Addition of antibodies to early cultures (day 1), resulted in 1.8-and 3-fold increases in colony formation and cell number, respectively, above control IgG-treated cultures. Antibody dose-response experiments revealed that TGFβ2 was the predominant isoform inhibiting early RTE cell growth. The antibody treatments resulted in similar stimulation of early growth at low and high seeding densities, suggesting that the endogenous TGFβs were acting locally. Anti-TGFβ1 treatment of cultures at various stages of growth resulted in 1.2–1.7-fold increases in DNA synthesis above controls, whereas anti-TGFβ2 treatment resulted in increased DNA synthesis only in early and late cultures (1.7- and 2.5-fold, respectively), but not during midlogarithmic growth. Continuous treatment with a combination of both antibodies resulted in increased growth and decreased exfoliation in early cultures, but had no effect on the slow down of growth in late cultures. Thus endogenous TGFβs inhibited primarily early growth and contributed to, but did not appear to be responsible for, plateau of growth in late stage cultures. Antibody treatment of secondary cultures resulted in 4–70-fold increases in colony formation, depending on the age of the primary cultures when replated, indicating that endogenous production of both TGFβ1 and TGFβ2 greatly inhibits the subculturability of primary RTE cells. Other experiments suggested that cholera toxin enhances RTE cell growth in part by counteracting the inhibitory effects of endogenous TGFβs. © 1993 Wiley-Liss, Inc.     

Neoplastic potential of rat tracheal epithelial cell lines induced by 1-nitropyrene and dibenzo(a,i)pyrene.

Our previous study showed that both 1-nitropyrene (1-NP) and dibenzo(a,i)pyrene (DBP) induced enhanced growth variants (EGVs) in primary cultures of rat tracheal epithelial (RTE) cells exposed in vivo. Cell lines were established from some of the EGVs. Further studies, using anchorage-independent growth in soft agar and tumorigenicity in athymic nude mice, were performed to determine the neoplastic potential of EGVs induced by 1-NP and DBP. Results show that three of five from DBP- and five of five from 1-NP-induced cell lines displayed anchorage-independent growth. The colony forming efficiency (CFE) from DBP-induced cell lines was 0.067 per thousand and CFE from 1-NP-induced cell lines was 0.151 per thousand. There is a significant difference between the two CFEs (mu = 12.08, P<0. 01). Two of five DBP- and five of five 1-NP-induced cell lines produced squamous cell carcinomas (SCC) in nude mice. The rate of tumorigenicity counted by injected sites was 20% (6/30) for DBP-induced cell lines and 57% (17/30) for 1-NP-induced cell lines. There is a significant difference between the results of tumorigenicity from the cell lines induced by the two different compounds (chi(2)=8.53, P<0.01). Neither of the two cell lines from spontaneously developed foci grew in soft agar or produced SCC in nude mice. It seems that the neoplastic potential of transformed RTE cells induced by 1-NP was higher than that of DBP.     

Changes in responsiveness of rat tracheal epithelial cells to transforming growth factor-β1 with time in culture

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to be highly sensitive to growth inhibition by transforming growth factor-β1 (TGF-β1) when treated within 1–2 days after plating. The purpose of the present studies was to examine the effects of TGFβ1 on the growth of RTE cells as a function of time in culture. We found that the sensitivity of RTE cells to growth inhibition by TGFβ1 decreased dramatically as the cultures aged. The IC₅₀ for inhibition of colony forming efficiency was 0.18 pM when TGFβ1 was added 24 h after cell plating. When TGFβ1 treatment was begun on day 5 of culture, the IC₅₀ was 3–4 pM as measured by inhibition of growth (cell number) and DNA synthesis. However, when TGFβ1 was begun on day 19, the IC₅₀ was 65 pM or > 500 pM, depending on whether inhibition of growth or DNA synthesis, respectively, was measured. TGFβ1 accelerated cell death, as measured by exfoliation of cells, and inhibited cell proliferation. The decrease in responsiveness to TGFβ1 in late cultures was shown to be dependent on culture age as well as on cell density. No evidence was found for inactivation or degradation of the added TGFβ1 by the late stage cultures. Cells subcultured from late stage primary cultures remained less responsive to TGFβ1 than subcultured cells from early cultures. Similar to its effect on proliferation, TGFβ1 down-regulated the expression of two proliferation-related genes, c-myc and transforming growth factor-α, in early but not late RTE cell cultures. On the other hand, fibronectin expression was increased by TGFβ1 by about twofold at both early and late times in culture. This indicates that the changes in TGFβ1 responsiveness with time in culture are selective, apparently affecting primarily proliferation-related events. © 1992 Wiley-Liss, Inc.     

Factors affecting the differentiation of epithelial transport and responsiveness to hormones

Under standard culture conditions, epithelial cells grow with their basal surface attached to the culture dish and their apical surface facing the medium. Morphological and functional markers are located in the appropriate plasma membrane, and transepithelial transport occurs in a variety of cultured epithelia. As a result of the polarity of the cells and the presence of tight junctions between cells, on standard tissue culture dishes there is restricted access of growth medium to the basolateral surface of the epithelium, which is the surface at which nutrient exchange normally occurs. Greater differentiation of epithelial cultures can be achieved by growing primary cultures or continuous cell lines on permeable surfaces such as porous bottom cultures dishes in which the porous bottom is formed by a filter or membrane of collagen, or on floating collagen gels. In many cultures, differentiation varies with the time after the culture was seeded. Certain chemicals that accelerate differentiation in nonepithelial cells also accelerate the differentiation of epithelial cultures. Ultimately, defined media and specific substrates for cell attachment should lead to further differentiation of epithelia in culture.     

Effect of substrata and fibroblast growth factor on the proliferation in vitro of bovine aortic endothelial cells

The hypothesis that, in the case of clonal or low-density cultures, cells which do not readily proliferate are those that do not produce an extracellular matrix (ECM), while those that proliferate actively are cells that have retained their ability to produce it, has been tested using low-density vascular endothelial cell cultures maintained on either plastic or ECM-coated dishes and exposed to various combinations of media and sera. Proliferation of low-density vascular endothelial cell cultures seeded on plastic and exposed to DMEM, RPMI-1640, or medium 199 plus thymidine is a function of the batch of calf serum used to supplement the various media. In all three cases, such cultures proliferated at a slow rate and fibroblast growth factor (FGF) greatly accelerated their proliferation. In contrast, when similar cultures were seeded on ECM-coated dishes, they actively proliferated regardless of the batch of calf serum to which they were exposed. FGF was no longer required in order for cultures to become confluent. In the case of cultures exposed to RPMI-1640 or medium 199 plus thymidine, it was even toxic. When cultures were exposed to either medium 199 or Waymouth medium, cells did not proliferate, regardless of the substrate (either plastic or ECM) upon which they were maintained and of the batch of serum to which they were exposed. Addition of FGF to such media had no effect. It is therefore likely that nutrient limitations in both of these media restrict the ability of low-density vascular endothelial cells to respond to the mitogenic stimuli provided by either serum or FGF. These restrictions cannot be relieved by maintaining cells on ECM-coated dishes, and modifications of the nutrient composition of both media is required in order to allow cells to respond to either FGF or serum when maintained on plastic or to serum alone when maintained on ECM. These results suggest that, when low-density cell cultures are maintained on plastic and exposed to an adequate medium, their proliferation will be a function of both serum and FGF. When maintained on ECM, their proliferation will depend only on serum. It is therefore possible that the inability of serum to stimulate optimal cell proliferation when cells are maintained on plastic results from an inability of the cells to produce an ECM, and that FGF could induce such production.     

Role of TGF alpha and its receptor in proliferation of immortalized rat tracheal epithelial cells: studies with tyrphostin and TGF alpha antisera

We have shown in the present study and in studies reported previously that preneoplastic and neoplastic rat tracheal epithelial (RTE) cell lines express TGF alpha and do so regardless of the mechanism by which they were transformed. In order to determine whether TGF alpha is an autocrine growth regulator of immortalized RTE cells, we have examined the function of TGF alpha/EGF receptors and the growth requirements for TGF alpha in these cells. The level of immunoprecipitated TGF alpha/EGF receptor protein in immortalized RTE cells was similar to or less than levels in primary RTE cells, indicating that chemically induced transformation of RTE cells does not involve overexpression of TGF alpha/EGF receptors. Scatchard analysis of TGF alpha/EGF receptors in the neoplastic EGV5T cell line revealed the presence of high-affinity (Kd = 0.4 nM) and low-affinity (Kd = 9.8 nM) binding sites. A tyrphostin TGF alpha/EGF receptor tyrosine kinase inhibitor decreased in a dose-dependent manner the proliferation as well as EGF-induced autophosphorylation of the TGF alpha/EGF receptor of transformed RTE cells. The inhibitory effect of tyrphostin on proliferation and receptor kinase activity was attenuated in late log and plateau phase cultures. The phosphotyrosine content of several other EGF-dependent and independent phosphoproteins was also decreased by the tyrphostin. Proliferation of transformed RTE cells was also inhibited when TGF alpha antisera was added to the media of growing cells. These data are consistent with the hypothesis that proliferation of transformed RTE cells involves autocrine regulation by TGF alpha and its receptor.     

Effects of laminin, fibronectin and type IV collagen on liver cell cultures

The effects on mouse liver cells of laminin, fibronectin and type IV collagen, all of which are the main matrix of the basement membrane, were studied. Laminin, a glycoprotein isolated from cultures of rat yolk sac carcinoma cells, promoted the attachment of mouse fetal liver cells to laminin-coated dishes, but did not have a strong influence upon the attachment of normal adult liver cells. On the other hand, fibronectin which was purified from mouse plasma promoted the attachment of adult liver cells but not that of fetal liver cells. The number of neonatal liver cells attached to the surfaces coated was intermediate between those of fetal and adult liver cells in each matrix. DNA synthesis and cell proliferation during the culture of full-term fetal liver cells in laminin-coated dishes were higher than those in fibronectin- or type IV collagen-coated dishes. The amount of alpha-fetoprotein secreted in the laminin-coated dishes was more than in other groups. No differences in secretion of albumin into media, however, were observed in either group. These results suggest that laminin may be necessary for cell growth, tissue organization and cell differentiation during the normal development of liver in vivo.     

Growth and differentiation of alveolar bone cells in tissue-engineered constructs and monolayer cultures

The ability to enhance bone regeneration by implanting autologous osteoblasts in combination with an appropriate scaffold would be of great clinical interest. The aim of our study was to compare the growth and differentiation of alveolar bone cells in tissue-engineered constructs and in monolayer cultures, as the basis for developing procedures for routine preparation of bone-like tissue constructs. Alveolar bone tissue was obtained from four human donors and explant cultures of the cells were established. Expanded cells were seeded on macroporous hydroxyapatite granules, and cultured in medium supplemented with osteogenic differentiation factors for up to 3 weeks. Control monolayer cultures were established in parallel, and cultured in media with or without osteogenic supplements. Cell proliferation, alkaline phosphatase (AP) activity and gene expression of AP, osteopontin and osteocalcin were determined under different culture conditions at weekly intervals. Cells in tissue constructs exhibited growth patterns similar to those in control monolayer cultures: enhanced proliferation was noted during the first 2 weeks of cultivation, followed by a decrease in cell numbers. AP activity at 3 weeks was higher in all cultures in osteogenic medium than in control medium. Gene expression levels were stable in monolayer cultures in both types of media whereas, in tissue constructs, they exhibited patterns of osteogenic differentiation. Light and scanning electron microscopy examination of the cell-seeded constructs showed uniform cell distribution, as well as cell attachment and growth into the interior region of the hydroxyapatite granules. Our results show that bone-like constructs with viable cells exhibiting differentiated phenotype can be prepared by cultivation of alveolar-bone cells on the tested hydroxyapatite granules.     

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.     

Growth control in miniclones of human glial cells

An improved method for the production of haptotactic palladium islands is described. Tissue culture dishes were coated with a thin layer of agarose, which was air-dried. Palladium was evaporated, using electron-microscope grids for masking. When seeded on such dishes, glial cell attachment and spreading was entirely confined to the palladium-coated areas. The method allowed the analysis of the clonal growth of several hundreds of glial cells seeded on 7 200 and 12 500 μm² squares. It was found that the mass population consisted of cells with widely differing potentials for clonal growth. A fraction of non-dividing cells increased with increasing passage level. Eventually, after more than a week of incubation, proliferation ceased on the squares although a considerable part of the periphery of the marginal cells was free of contacts with other cells. The finding is compatible with the idea that restriction of cell spreading, and not cell contact, may cause density-dependent inhibition of proliferation.     

Factors controlling embryonic heart cell proliferation in serum-free synthetic media

Summary Embryonic chick cardiac cell cultures, plated on collagen-coated dishes, containing serum-free synthetic media proliferate actively. The basic medium contained Ham's F12 nutrient mixture, fetuin, ascorbic acid, and bovine serum albumin. This medium was supplemented with various combinations of factors; endothelial cell growth supplement (ECGS), epidermal growth factor (EGF), insulin (I), transferrin (T), selenium (S), hydrocortisone, and thyroxine or supplemented alone. Basic medium supplemented with ECGS alone contributes to the highest final cell density among all other factors used in various combinations or alone. The final cell density of the control culture with 2% fetal bovine serum was higher than those of all experimental cultures and an additional control culture grown in the basic medium. Combinations of factors without ECGS do not promote significant cell proliferation. Thyroxine is required to induce optimal differentiation and contractility of cardiac myocytes in vitro. Fibronectin and laminin did not show any more influence than collagen did on the growth and maintenance of cardiac myocytes in serum-free media. The proportion of cardiac muscle cells in ECGS-containing media was higher than those in other experimental media and control media with the exception of ECGS and ITS-containing medium that showed lower proportion of cardiac myocytes than that of serum-containing medium on Days 3 and 5. The profiles of incorporation of [³H]thymidine into DNA of heart cells in experimental and control cultures showed a peak in incorporation values within the first week of culture and subsequently declined. Autoradiography studies revealed that cardiac myocytes in culture supplemented with ECGS alone attained a peak in labeling index on Day 1 with approximately 62% labeled cells. Subsequently, the labeling indices declined. Cardiac myocytes grown in media without ECGS showed significantly lower labeling indices than those in ECGS-containing media. This study has demonstrated the influence of ECGS, EGF and ITS in promoting the growth of cardiac myocytes and also in contributing to the maintenance of contractile cardiac myocytes in serum-free, long-term culture. The influence of ECGS on heart cell proliferation is considered to be superior to that of EGF and ITS. This study was supported in part by a grant HL-25482 from the National Heart Lung and Blood Institute and a grant from the American Heart Association of Michigan.     

Survival of 3T3-L1 cells induced by fibroblast growth factor depends on cell density and adhesion to the substratum

The effects of cell contacts and the attachment of cells to the substratum on growth-factor-induced survival of 3T3-L1 cells were investigated to clarify their involvement in the maintenance of cell viability. When 3T3-L1 cells in low-density cultures or in high-density cultures were harvested with EDTA solution and cultured in the absence of calf serum, almost all cells from the low-density cultures lost viability 24 h later. However, about 15% of the cells harvested from high-density cultures survived for 24 h in the absence of calf serum. Addition of calf serum also enhanced the survival of cells from high-density cultures to a much greater extent than that of cells from low-density cultures. Addition of fibroblast growth factor enhanced the survival of cells, especially in the case of cells from high-density cultures. However, epidermal growth factor and platelet-derived growth factor failed to enhance survival. Coating of cultures dishes with vitronectin slightly enhanced cell survival. Addition of fibroblast growth factor markedly enhanced the survival of cells on the dishes coated with vitronectin or with fibronectin, but not on the dishes coated with heat-denatured bovine serum albumin. These results suggest that fibroblast growth factor promotes survival of 3T3-L1 cells, depending on cell to-cell contacts during prior culture and on the adhesion of cells to the substratum.     

RPE-derived factors modulate photoreceptor differentiation: A possible role in the retinal stem cell niche

A photoreceptor cell line, designated 661W, was tested for its response to growth factors secreted by retinal pigment epithelial cells including basic fibroblast growth factor, epidermal growth factor, and nerve growth factor. Early passaged 661W cells expressed high levels of retinal progenitor markers such as nestin and Pax6, but not opsin or glial fibrillary acidic protein. 661W cells grown in FGF-2 or EGF exhibited a multiple-process morphology with small phase-bright nuclei similar to neurons, whereas cells cultured in nerve growth factor (NGF) or retinal pigment epithelium (RPE)-conditioned medium (RPE-CM) displayed rounded profiles lacking processes. 661W cells grown in FGF-2 were slightly elevated, but not significantly above, control cultures; but cells treated with RPE-CM or NGF were fewer, ∼63% and 49% of control, respectively. NGF immunodepletion of RPE-CM strongly suppressed the inhibitory activity of RPE-CM on cell proliferation. Cells treated with FGF-2, but not NGF, upregulated their expression of opsin. All treatment conditions resulted in almost 100% viability based on calcium AM staining. Cells grown on extracellular matrix proteins laminin, fibronectin, and/or collagen resembled those grown on untreated dishes. This study showed that early passaged 661W cells displayed characteristics of retinal progenitor cells. The 661W cells proliferated and appeared to mature morphologically expressing rod photoreceptor phenotype in response to FGF-2. In contrast, NGF and RPE-CM inhibited proliferation and morphological differentiation of 661W cells, possibly inducing cell cycle arrest. These findings are consistent with reports that the RPE modulates photoreceptor differentiation and retinal progenitor cells via secreted factors and may play a role in the regulation of the retinal stem cell niche.     

Long-term culture of human endothelial cells

Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10(3)/cm2) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts.     

HEMOPOIETIC STEM CELL PROLIFERATION AND MIGRATION FOLLOWING BORDETELLA PERTUSSIS VACCINE

The ability of a single injection of killed, intact bacteria to effect an increase in the proliferative rate of hemopoietic stem cells was studied. The total numbers of colony forming units in bone marrow, spleen and peripheral blood as well as the proportion of CFU in cycle was assessed. Splenic CFU were observed to rise exponentially due initially to in situ proliferation and later to proliferation in bone marrow with migration via the blood to the spleen. The results are discussed in the light of current concepts of stem cell regulation.