The differential effects of cadmium exposure on the growth and survival of primary and established cells from fish and mammals

The differential cytotoxic effects of cadmium on fish and mammalian epithelial cells in established and primary culture were assessed by looking at the reduction of the colony-forming ability and reduction in the extent of growth. The influence of medium composition on the toxicity of cadmium was also studied using serum-free and serum-containing media. The experiments using immortalized cell lines showed that mammalian cells were more sensitive than fish cells to cadmium. Both cell types were grown at the same serum concentration. However, using the normal primary system, human epithelial tissue explants showed less sensitivity to cadmium than did similar cultures from rainbow trout. It is likely that cellular mechanisms of cadmium resistance in the different cell types are responsible for these effects. As expected, cadmium proved to be more toxic when tested in serum-free medium. With fish skin primary cultures, reduction of cell numbers was observed at concentrations as low as 5 mol/L in serum-free medium compared to 100 mol/L in serum-containing medium. This was found to be due to the reduction in the activity of free cadmium ions, caused by the presence of serum in the medium. It is concluded that serum-free formulations are probably preferable when testing pollutants in vitro. The results highlight the importance of establishing species-specific pollution tests and standardizing variables.In summary, the results show species and culture media differences in cadmium toxicity and suggest that caution is required when extrapolating results for pollutant effects from one in vitro system to another.Abbreviations CE colony-forming efficiency - EPC epithelioma papulosum cyprini - KGM Clonetics Keratinocyte Growth Medium     

Growth of neural cell cultures in a chemically defined,serum-free culture medium

The composition of a serum-free, completely chemically defined culture medium which supports active growth of dissociated neural-cells in culture is described. This serum-free medium can also be used to grow many types of human cell lines without modification. It is the first report which describes the development of a wholly chemically defined, synthetic culture medium for growth of neural cells.     

An efficient method for <Emphasis Type="Italic">in vitro</Emphasis> regeneration of leafy spurge (<Emphasis Type="Italic">Euphorbia esula</Emphasis> L.)

Leafy spurge (Euphorbia esula L.) is a perennial, invasive weed used as a model to study invasive plant behavior, because molecular tools (such as a deep expressed sequence tag database and deoxyribonucleic acid microarrays) have been developed for this species. However, the lack of effective in vitro regeneration and genetic transformation systems has hampered molecular approaches to study leafy spurge. In this study, we describe an efficient in vitro regeneration system. Three highly regenerative lines were selected by screening the in vitro regeneration capabilities of stem explants of 162 seedlings. The effects of various culture conditions on in vitro regeneration were then evaluated based on explant competence to form calluses and shoots. High rates of shoot regeneration can be obtained using a growth medium containing 1× woody plant basal medium and 1× Murashige and Skoog (MS) basal salts, 1× MS vitamins, 1.11 μM 6-benzylaminopurine, 1.97 μM indole-3-butyric acid, and 3% sucrose, pH 5.6–5.8. After 30 d culture, multiple shoots formed either directly from the stem or indirectly from the callus. This method is a requisite for the development of genetic transformation systems for leafy spurge and may be used to develop in vitro regeneration techniques for other species in the Euphorbiaceae.     

Differentiation of rabbit adipocyte precursor cells in a serum-free medium

Summary A serum-free, hormone-supplemented medium containing insulin, transferrin, and triiodothyronine (ITT medium), able to support differentiation of rat adipose precursor cells, has been used to study the regulation of the development of adipocytes in the rabbit. Adipose conversion was assessed by the appearance of glycerol-3-phosphate dehydrogenase activity. Stromal-vascular cells from rabbit perirenal adipose tissue differentiated to a very low extent or not at all in ITT medium. Supplementation of ITT medium with growth hormone or fibroblast growth factor did not increase the proportion of differentiated cells. In contrast, rabbit stromal-vascular cells were able to differentiate in ITT medium supplemented with glucocorticoids (dexamethasone, corticosterone) whereas sex steroids (β-estradiol, testosterone, progesterone) did not affect the differentiation process. In the presence of both dexamethasone and insulin, 20 to 50% of rabbit stromal-vascular cells differentiated into adipocytes within 2 wk of culture. The stimulatory actions of dexamethasone or insulin were dose-dependent. Insulin-like growth Factor-I (IGF-I), did not replace insulin under our culture conditions and had only a slight effect when added along with dexamethasone (100 nM) and insulin (1.7 nM). The results suggest that glucocorticoids, in association with insulin, may play an important role in the development of adipocytes from rabbit precursor cells. This work was supported by grant 4388 from the Institut National de la Recherche Agronomique, France.     

Formation of dermal skeletal and dental tissues in fish: a comparative and evolutionary approach

Osteichthyan and chondrichthyan fish present an astonishing diversity of skeletal and dental tissues that are often difficult to classify into the standard textbook categories of bone, cartilage, dentine and enamel. To address the question of how the tissues of the dermal skeleton evolved from the ancestral situation and gave rise to the diversity actually encountered, we review previous data on the development of a number of dermal skeletal elements (odontodes, teeth and dermal denticles, cranial dermal bones, postcranial dermal plates and scutes, elasmoid and ganoid scales, and fin rays). A comparison of developmental stages at the tissue level usually allows us to identify skeletogenic cell populations as either odontogenic or osteogenic on the basis of the place of formation of their dermal papillae and of the way of deposition of their tissues. Our studies support the evolutionary affinities (1) between odontodes, teeth and denticles, (2) between the ganoid scales of polypterids and the elasmoid scales of teleosts, and (3) to a lesser degree between the different bony elements. There is now ample evidence to ascertain that the tissues of the elasmoid scale are derived from dental and not from bony tissues. This review demonstrates the advantage that can be taken from developmental studies, at the tissue level, to infer evolutionary relationships within the dermal skeleton in chondrichthyans and osteichthyans.     

Culture of cardiac muscle cells in serum-free media

Cardiac muscle cells from neonatal rats have been cultured in completely defined serum-free media. The most successful system consists of precoating culture flasks with fibronectin at a concentration of 5 μg/cm² of surface area and adding fetuin and either dibutyryl cyclic AMP (db-cAMP), cholera toxin, epidermal growth factor or insulin plus dexamethasone to the medium. In order to define a serum- and a hormone or growth factor-free medium, cardiac muscle cells were grown in the presence of fibronectin, fetuin and db-cAMP for 4 days, after which time db-cAMP was omitted from the medium. Under these conditions the cells continue to maintain their differentiated morphology for at least 4 days thereafter. These morphological studies demonstrate that dissociated neonatal cardiac muscle cells are able to grow and differentiate in a chemically defined medium in the absence of animal serum.     

A new method for study of normal lens developmentin vitro using pulsatile delivery of PDGF or EGF in HL-1 serum-free medium

Summary A new culture method was used to study increases in wet and dry weight and soluble protein during normal development of the transparent lens. Seven different media with more than ten different additives were tested for their effects on cultured lens transparency.In vivo, rat lenses increased 53% in soluble protein content between 3 and 5.5 days of age. Only HL-1 serum-free medium containing 15 μg/ml insulin plus 1–2 ng/ml BB platelet-derived growth factor (PDGF), or 5–7 ng/ml epidermal growth factor (EGF) allowed similar growthin vitro, during the same time period. Normal lens grwoth occurred in culture when fresh medium was delivered to lenses as a pulse every 4–6 hours. Lenses decreased in dry weight and soluble protein content, and became opaque when the same medium was delivered continuously. Lenses increased only 26% and 32% in soluble protein content when delivered pulses of HL-1 medium containing BB PDGF or EGF in the absence of insulin. We suggest that pulsatile delivery of medium containing insulin and PDGF or EGF stimulates lens growth during developmentin vitro. This pulsatile organ culture system is presented as a new approach for studying the effects of growth factors on cell proliferation, differentiation, and receptor regulation in a developing tissue. This work was supported by grants from EY-07031 and EY-04542 from the National Eye Institute and a grant from the Oculon Corporation. Editor's statement This report documents an in vitro system that may mimic lens development and response to growth regulators and hormones. The system may be useful for application to other organs and provide a foundation for cell and molecular level analysis.     

The effects of various nutritional supplements on the growth,migration and differentiation ofxenopus laevis neural crest cells in vitro

Summary This study investigates the nutritional requirements ofXenopus laevis neural crest cells and melanophores developing in vitro. A comparison is made between the growth and differentiation of cells in serum-containing medium and a chemically defined, serum-free medium that we have designed. Our chemically defined medium is more efficient than serum-supplemented medium in promoting proliferation of these cells. Several supplements are required to enhance culture development. These include insulin, α-melanocyte stimulating hormone, somatotropin, luteotrophic hormone, linoleic acid, uridine, and putrescine. In addition, collagen and fibronectin provide the most conductive environment tested for cell migration and adhesion. This work was supported by establishment and major equipment grants from the Alberta Heritage Foundation for Medical Research to N. C. M. Nadine C. Milos is a Heritage Medical Research Scholar of the Alberta Heritage Foundation for Medical Research.     

Human chorion cells respond to growth factors but lose steroidogenic capacity in primary monolayer cell culture

Summary This study has defined a method for preparation and monolayer culture of cells fromchorion laeve. Cell number and cell protein content are table over 7 d in culture. The cells will divide in response to epidermal growth factor in the presence of a supplemented, enriched medium and a collagen matrix, but they lose steroidogenic activity over time in culture. This culture system can be used as the starting point for the development of a chemically defined hormone-supplemented, serum-free culture system for studies of chorion cell differentiation and fetal membrane cell interactions. This work was supported by the Medical Research Council of Canada through Grant MT-7867 (to A. K. T.), Grant MA-7731 (to B. F. M.), and a Summer Studentship (to K. A. F.).     

Teeth before jaws? Comparative analysis of the structure and development of the external and internal scales in the extinct jawless vertebrate Loganellia scotica

Traditional hypotheses posit that teeth evolved from dermal scales, through the expansion of odontogenetically competent ectoderm into the mouth of jawless vertebrates. The discovery of tooth‐like scales inside thelodonts, an extinct group of jawless vertebrates, led to the alternative hypothesis that teeth evolved from endodermal derivatives and that there exists a fundamental developmental and phylogenetic distinction between oral/pharyngeal and external odontodes. We set out a test of this latter hypothesis, examining the development of scales of the thelodont Loganellia scotica using synchrotron radiation X‐ray tomographic microscopy (SRXTM). We reveal that the internal scales are organized into fused patches and rows, a key distinction from the discrete dermal scales. The pattern of growth of oral scale patches is polarized, but not along a particular vector, whereas pharyngeal scale rows grew along a vector. Our test of the phylogenetic distribution of oral and pharyngeal scales and teeth in vertebrates indicates that odontodes are first expressed in an external position. Internal scales, where present, are always located near to external orifices; the sequential development of pharyngeal scales in Loganellia is peculiar among thelodonts and other stem gnathostomes. It represents a convergence on, rather than the establishment of, the developmental pattern underpinning tooth replacement in jawed vertebrates. The available evidence suggests that internal odontodes evolved through the expansion of odontogenic competence from external to internal epithelia.     

Production of multiple shoots from thidiazuron-treated mature embryos and leaf-base/apical meristems of barley (Hordeum vulgare)

The in vitro plant regeneration frequencies for immature scutella, leaf-bases/apical meristems (LB/AM) and mature embryos of four commercially important barley genotypes were compared. Production of shoots from mature embryos or calluses of LB/AM incubated on media containing 1.0 or 2.0 mg l^–1 6-benzylaminopurine (BA) were comparable to regeneration frequencies obtained for scutella-derived calluses of the same genotypes. Incubation of excised mature embryos and LB/AM on media containing the plant growth regulator, thidiazuron (TDZ), resulted in an increased shoot production. However, TDZ treatment did not stimulate plant regeneration from calluses derived from scutella or LB/AM. Shoots formed from TDZ-treated mature embryos and LB/AM were induced without a callus interphase and the in vitro culture system gave a three- to eight-fold higher regeneration frequency than recorded for scutella-derived calluses on BA medium. The simplicity and rapid development of shoots using the mature embryo system could potentially be used for the regeneration and genetic transformation of barley over alternative regeneration systems.     

Adventitious shoot regeneration from leaf explants and stem nodes of Lilium

A method for the regeneration of lily plantlets (Lilium spp.) through different morphogenic pathways is described. Plant regeneration was obtained from in vitro cultured leaves of four lily hybrids, cultured on Murashige and Skoog's basal medium supplemented with cytokinins (TDZ and BA) and auxins (NAA and IBA) at different concentrations. Direct shoot regeneration occurred with all tested media for the Asiatic lilies `Elite' and `Pollyanna' and also for the Oriental hybrid `Star Gazer'. Callus developed on TDZ-enriched medium from leaf segments of L. longiflorum cv. `Snow Queen' regenerated by direct organogenesis. This occurred on a medium with auxin/ cytokinin balance which was lower than other genotypes. There were fewer problems of sterilization with leaves from sprouted bulbs than in vitro scale culture. This suggests that the leaf-segments obtained in this way could be an alternative to the scales as a source of material for propagation. A protocol for micropropagation based on bulblets from in vitro shoot-tip-derived stem nodes was also used. The development of pseudo-bulbets is particularly advantageous since it allows for structures characterised by absent or low dormancy. Regenerated shoots have been rooted and successfully acclimatized to greenhouse conditions where they flowered after the second year giving plants with true-to-type shape and colour.     

Serum-free organ culture of suckling rat jejunum: Effect of regulatory hormones

Summary To facilitate the study of regulators of differentiation and proliferation of small intestinal epithelium in the suckling rat we have developed a serum-free organ culture system and used it to examine epithelial responsiveness to various regulatory hormones. These hormones included the insulin-like growth factors (IGFs) whose action can be blocked by binding proteins in serum. Jejunal explants from 5-day-old suckling rats maintained better brush border enzyme activity and better histology when cultured under hyperbaric conditions for 24 h in serum-free Dulbecco’s modified Eagle’s medium/F12 medium than in RPMI 1640 plus 10% fetal bovine serum. Tissue responsiveness to various regulatory hormones was then tested in the serum-free medium. Insulin had no significant effect on morphology, proliferation rate, or enzyme activity in 5-day explants after 24 h in culture. However, insulin did increase lactase activity and induce the early appearance of sucrase in 10- and 12-day explants after 48 h culture. Dexamethasone increased specific activities of alkaline phosphatase (30%,P<0.001) and lactase (15%,P<0.001), and reduced shedding of alkaline phosphatase into the medium (P<0.001), in explants of 5-day-old rats cultured over 24 h. Dexamethasone combined with insulin had no obvious effect on the rate of protein or DNA synthesis but did increase villus height (P=0.04) and crypt depth (P=0.001) and acted synergistically to further increase lactase activity above levels obtained by either alone. IGF-I and IGF-II, des-(1–3)IGF-I, fibroblast growth factor (FGF), and growth hormone (GH) had no effect on morphology or biochemical activity of explants after 24 or 48 h culture. In conclusion, histology, enzyme activity, protein, and DNA synthesis of suckling rat jejunal explants were equivalent or better in serum-free than in serum-containing organ culture systems. Furthermore, biological responsiveness was demonstrated by dexamethasone and insulin altering the explants morphologically or biochemically. None of the IGFs or GH had any biological effects, raising doubts about their direct biological action on the developing intestinal epithelium.     

Scales and Tooth Whorls of Ancient Fishes Challenge Distinction between External and Oral ‘Teeth’

The debate about the origin of the vertebrate dentition has been given fresh fuel by new fossil discoveries and developmental studies of extant animals. Odontodes (teeth or tooth-like structures) can be found in two distinct regions, the ‘internal’ oropharyngeal cavity and the ‘external’ skin. A recent hypothesis argues that regularly patterned odontodes is a specific oropharyngeal feature, whereas odontodes in the external skeleton lack this organization. However, this argument relies on the skeletal system of modern chondrichthyans (sharks and their relatives), which differ from other gnathostome (jawed vertebrate) groups in not having dermal bones associated with the odontodes. Their external skeleton is also composed of monoodontode ''placoid scales'', whereas the scales of most early fossil gnathostomes are polyodontode, i.e. constructed from several odontodes on a shared bony base. Propagation phase contrast X-ray Synchrotron microtomography (PPC-SRµCT) is used to study the polyodontode scales of the early bony fish Andreolepis hedei. The odontodes constructing a single scale are reconstructed in 3D, and a linear and regular growth mechanism similar to that in a gnathostome dentition is confirmed, together with a second, gap-filling growth mechanism. Acanthodian tooth whorls are described, which show that ossification of the whorl base preceded and probably patterned the development of the dental lamina, in contrast to the condition in sharks where the dental lamina develops early and patterns the dentition.The new findings reveal, for the first time, how polyodontode scales grow in 3D in an extinct bony fish. They show that dentition-like odontode patterning occurs on scales and that the primary patterning unit of a tooth whorl may be the bony base rather than the odontodes it carries. These results contradict the hypothesis that oropharyngeal and external odontode skeletons are fundamentally separate and suggest that the importance of dermal bone interactions to odontode patterning has been underestimated.     

Enhanced growth and quality of St. John's wort (Hypericum perforatum l.) under photoautotrophic in vitro conditions

Summary Photomixotrophic (Pm) micropropagation systems (ones that use a sugar-containing medium) have been used by many rescarchers for transplant production of St. John's wort. However, these methods have not yet been adopted for commercial applications, probably due to the low percentage of regeneration in vitro, and a low growth rate after transplanting ex vitro. In contrast, it is well known that the use of a photoautotrophic (Pa) micropropagation system (one that uses sugar-free medium) can promote the growth and improve the quality of plantlets in vitro, and enhance the growth during acclimatization for many plant species. In the current study, leafy nodal cuttings were cultured under Pa conditions and the growth and quality were compared with those cultured under Pm conditions. After 21d of culture, Pa conditions enhanced the growth and quality of St. John's wort plantlets in vitro, and these plantlets showed faster growth after transplantaing ex vitro compared with those cultured under Pm conditions.     

Factors influencing shoot regeneration from cotyledons of tetraploid <Emphasis Type="Italic">Isatis indigotica</Emphasis> fort

Summary An efficient in vitro plant regeneration system from cotyledons was established in tetraploid Isatis indigotica Fort. Factors influencing shoot regeneration from cotyledons, including culture medium type, combinations of plant growth regulators, and sucrose concentrations in the medium, as well as illumination were investigated. Murashige and Skoog's (MS) medium was found to be best for promoting shoot regeneration, followed by Gamborg's B₅ and White's medium. The highest shoot regeneration frequency was achieved from cotyledons cultured on MS medium supplemented with 2.0 mgl⁻¹ (8.9 μM) 6-benzyladenine and 1.0 mgl⁻¹ (5.4 μM) α-naphthaleneacetic acid (NAA), with 97.9% regeneration, associated with a high number of multiple shoots developed per explant (8.6 shoots per explant). A sucrose concentration of 3% present in the medium and light conditions were beneficial for shoot regeneration. The shoots developed were rooted in a half-strength MS medium supplemented with 1.0 mgl⁻¹ (5.4 μM) NAA and successfully transplanted in soil in pots with over 85% survival. The establishment of an efficient plant regeneration procedure from cotyledons provides a basis for the rapid in vitro multiplication of tetraploid Isatis indigotica Fort., one of the most extensively used medicinal plants in China currently under great shortage.     

Characteristics of strawberry plants propagated by in vitro bioreactor culture and ex vitro propagation method

Reproducible protocol for regeneration of complete plantlets from ‘Bounty’ strawberry (Fragaria ananassa Duch.), using a combination of gelled medium and bioreactor system, has been standardized. Sepals, leaf discs, and petiole halves produced multiple buds and shoots when cultured on semi solid‐gelled medium containing 4 μM thidiazuron (TDZ) for 4 wk followed by transferring in liquid medium containing 2 μM TDZ in a bioreactor system and cultured for another 4 wk. TDZ induced shoot proliferation at 0.1 μM in the bioreactor system but inhibited shoot elongation. TDZ‐induced shoots were elongated and rooted in vitro on gelled medium containing 2 μM zeatin. Such bioreactor‐derived tissue culture (BC) plantlets obtained from sepal explants were grown ex vitro and compared with those propagated by tissue culture on gelled medium (GC) and by conventional runner cuttings (RC), for growth, morphology, anthocyanin content, and antioxidant activity after three growth seasons. The BC and GC plants produced more crowns, runners, leaves, and berries than the RC plants although berry weight per plant did not differ significantly. BC and GC plants produced berries with more anthocyanin contents and antioxidant activities than those produced by the RC plants. However, intersimple sequence repeat (ISSR) marker assay produced a homogenous amplification profile in the tissue culture and donor control plants confirming the clonal fidelity of micropropagated plants. In vitro culture on TDZ and zeatin‐containing nutrient media apparently induced the juvenile branching characteristics that favored enhanced vegetative growth with more crown, runners, leaf, and berry production.     

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.