Intrinsic factors influencing the maintenance of contractile embryonic heart cells in vitro : II. Biochemical analysis of heart muscle conditioned medium

Seven-day embryonic heart cells are tested for their ability to condition their own medium by comparing cell responses at various inoculum levels. The data show that contractile activity, spreading on glass, and survival increase as inoculum level rises. The data also show that the cell death rate is inversely proportional to the rate at which cell spreading and contractile activity increase.     

Intrinsic and extrinsic factors influencing steroid production in vitro by bovine follicles

Nonatretic bovine follicles were cultured on grids in a conventional static system, in roller tubes or in a continuous flux system. Culture medium from the static and roller tube system was replaced after 24 h of culture, while in the continuous flux system, only a small sample was taken aseptically at that time. Steroid concentrations in these samples were estimated by radioimmunoassay (RIA) and related to intrinsic factors like follicle size, day of cycle and micromorphological appearance at the end of culture and to extrinsic factors like the culture system, O(2)-concentration applied in the gasphase and time of culture. Bovine nonatretic follicles from 2 to 8 mm showed the same amount of estradiol-17beta (E2) production as pmol mg protein(-1) 24 h. Follicles over 8 mm had a significantly higher E2 production. Follicles from the follicular phase of the cycle produced more E2 than follicles from the luteal phase, independent of follicular size and culture system. Degeneration of follicles during culture resulted, independent of the culture system, in a decline of E2-production, and in an increase of P4-production; whereas the T-production initially (primary atresia) rose but subsequently (secondary and tertiary atresia) declined. The difference between the culture systems were reflected by quantitative differences in the production of the steroids measured. The most striking difference between the continuous flux system on one hand and the static and rolling tube system on the other is the predominant E2 production in the former by every follicle. It is thought that this difference might be caused by a better 02 supply in the continuous flux system. This hypothesis is tested in the static culture system. The more 02 the more E2 production. The increase in culture time resulted in an increase of E2 and P4, whereas the testosterone production was not significantly decreased.     

Mycelial morphology: The effect of spore inoculum level

Summary Image analysis has been used to characterise the effect of spore inoculum level on the morphology ofPenicillium chrysogenum grown in batch culture. As inoculum levels rose towards 5×10⁵ spores mL^–1 there was a sharp transition from pelleted to dispersed forms, but above this level there proved to be little additional effect.     

In vitro studies of beating heart cells in culture. XVI. The effect of a heart stable serum fraction on the level of myosin adenosinetriphosphatase

A heat stable serum factor of low molecular weight maintains the myosin ATPase activity of cultured rat heart cells. Its action is directly on the heart cells and it does not act by selection of heart muscle cells. It has no effect on heart muscle creatine phosphokinase or lactic dehydrogenase nor on myosin in skeletal muscle cultures.     

Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells

Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types including spontaneously contracting cardiac myocytes. We have utilized the ES cell differentiation culture system to study the development of the cardiac contractile apparatus in vitro. Difficulties associated with the cellular and developmental heterogeneity of this system have been overcome by establishing attached cultures of differentiating ES cells, and by the micro-dissection of the contracting cardiac myocytes from culture. The time of onset and duration of continuous contractile activity of the individual contracting myocytes was determined by daily visual inspection of the cultures. A functional assay was used to directly measure force production in ES cell-derived cardiac myocyte preparations. The forces produced during spontaneous contractions in the membrane intact preparation, and during activation by Ca2+ subsequent to chemical permeabilization of the surface membranes were determined in the same preparation. Results showed a transition in contractile sensitivity to Ca2+ in ES cell-derived cardiac myocytes during development in vitro. Cardiac preparations isolated from culture following the initiation of spontaneous contractile activity showed marked sensitivity of the contractile apparatus to activation by Ca2+. However, the Ca2+ sensitivity of tension development was significantly decreased in preparations isolated from culture following prolonged continuous contractile activity in vitro. The alteration in Ca2+ sensitivity obtained in vitro paralleled that observed during murine cardiac myocyte development in vivo. This provides functional evidence that ES cell-derived cardiac myocytes recapitulate cardiogenesis in vitro. Alterations in Ca2+ sensitivity could be important in optimizing the cardiac contractile response to variations in the myoplasmic Ca2+ transient during embryogenesis. The potential to stably transfect ES cells with cardiac regulatory genes, together with the availability of a functional assay using control and genetically modified ES cell- derived cardiac myocytes, will permit determination of the functional significance of altered cardiac gene expression during cardiogenesis in vitro.     

Long-term maintenance of in vitro cultured honeybee (Apis mellifera) embryonic cells

Background  

In vitro cultivation of cells allows novel investigation of in vivo- mechanisms and is a helpful tool in developmental biology, biochemistry and functional genomics. Numerous cell lines of insect species, e.g., silkworm and mosquito, have been reported. However, this is not the case for successful long-term cultivation of cells in honeybees.     

Derivation and maintenance of human embryonic stem cells from poor-quality in vitro fertilization embryos

Human embryonic stem (hES) cells are self-renewing, pluripotent cells that are valuable research tools and hold promise for use in regenerative medicine. Most hES cell lines are derived from cryopreserved human embryos that were created during in vitro fertilization (IVF) and are in excess of clinical need. Embryos that are discarded during the IVF procedure because of poor morphology and a low likelihood for generating viable pregnancies or surviving the cryopreservation process are also a viable source of hES cells. In this protocol, we describe how to derive novel hES cells from discarded poor-quality embryos and how to maintain the hES cell lines.     

The effect of oxidized isoprenaline on the chick embryonic heart

Intra-amnial administration of isoprenaline (IPRO) to chick embryos induces a number of myocardial lesions. The purpose of the present study was to investigate whether similar changes may also be induced after injection of spontaneously oxidized isoprenaline and commercially obtained adrenochrome. Cardiotoxicity of these substances has been demonstrated in adult animals. IPRO, oxidized IPRO, or adrenochrome were administered intra-amnially to 10-day-old chick embryos at doses of 0.1, 1.0, 10.0, and 100.0 mg X kg-1. Parallel experimental groups received propranolol at a dose of 1 mg X kg-1, 15 s before injection of IPRO or oxidized IPRO. The cAMP level in the heart was determined by radioimmunoassay 2 and 30 min after administration of IPRO, oxidized IPRO, or adrenochrome at a single dose of 10.0 mg X kg-1. It has been found that in embryos the effect of IPRO and oxidized IPRO is dose dependent. The rise in mortality and development of cardiomegaly together with increased hydration and disturbances of the development of coronary vascularization were highly significant starting from the dose of 10 mg X kg-1. Furthermore, both drugs significantly increased cAMP levels in the embryonic heart. On the other hand, the administration of adrenochrome was without any effect. The changes induced by IPRO were prevented by the administration of the beta-blocking agent propranolol; the lesions induced by spontaneously oxidized IPRO were, however, prevented only partially.     

The effect of graded hypoxia on the embryonic chick heart

Embryonic ventricular function in the chick was measured in response to graded levels of hypoxia. Myocardial contractility, as measured by cinephotoanalysis and expressed as shortening fraction, was significantly depressed after 1 hour of moderate hypoxia (6% O2) and after 5 hours of milder (16% O2 and 11% O2) levels of hypoxia (P less than .05). Microscopy confirmed associated myocyte damage with cell death noted after 5 hours of moderate hypoxic stress. Heart rate change was not related to the severity of hypoxia. The greatest level of tachycardia was noted with conditions of mildest hypoxia (16% O2). The data confirm that cardiac contractility, as measured by shortening fraction, is depressed on exposure to hypoxia, with impairment of function related to the severity of the hypoxic conditions.     

Pluripotent differentiation in vitro of murine Es-D3 embryonic stem cells

Although the ES-D3 murine embryonic stem cell line was one of the first derived, little information exists on the in vitro differentiation potential of these cells. We have used immunocytochemical and flow cytometric methods to monitor ES-D3 embryoid body differentiation in vitro during a 21-d period. Spontaneous differentiation of embryoid body cells was induced by leukemia inhibitory factor withdrawal in the absence of feeder cells. The pluripotent stem cell markers Oct-3/4, SSEA-1, and EMA-1 were found to persist for at least 7 d, whereas the primitive endoderm marker cytokeratin endo-A was expressed at increasing levels from day 6. The localization of these antigens within the embryoid bodies suggested that embryonic ectoderm- and primitive endoderm-derived tissues were segregated. Localized expression of class III beta-tubulin and sarcomeric myosin also was detected, indicating that representatives of all three embryonic germ layers were present after induction of differentiation in vitro.     

Modulation of the epidermal growth factor receptor of mouse embryonic palatal mesenchyme cells in vitro by growth factors.

A single class of high-affinity receptors for EGF were detected on mouse embryonic palatal mesenchyme (MEPM) cells cultured in vitro. The degree of confluence of the cultured cells did not affect the number or affinity of the binding sites. Culture of MEPM cells in the presence of bFGF, IGF-II or TGF-beta 1 induced changes in 125I-EGF binding. TGF-beta 1 caused a marked reduction in binding to 40% of control levels. This reduction was achieved after 2 h and persisted for 24 h after addition of the growth factor. IGF-II induced a similar reduction but this effect was transitory; after a 12 h pretreatment with IGF-II, binding was restored to control levels. The effects of bFGF were biphasic. Initially, a short pre-treatment period (3-5 h) with bFGF caused a small reduction in 125I-EGF binding; longer periods of pre-incubation (24 h) resulted in a large increase in receptor number. Pre-incubation in medium containing both bFGF and TGF-beta 1 resulted in a decrease in EGF binding. Thus, TGF-beta 1 negated the large increase in receptor number induced by bFGF alone. Changes in receptor number were usually, but not always, directly related to changes in the biological activity of EGF, as assessed by a thymidine incorporation assay. This study highlights the possible interactive role of growth factors known to be present in the developing palate.     

Effect of chromosome instability on the maintenance and differentiation of human embryonic stem cells in vitro and in vivo

The therapeutic potential of human embryonic stem cells (hESCs) has long been appreciated, and the recent FDA approval of hESC derivatives for cell-based therapy encourages the clinical application of hESCs. Here, using CHA3-hESCs with normal and abnormal karyotypes, we report the importance of maintaining normal chromosomes during in vitro culture and the differentiation of hESCs for minimization of posttransplantation complications. We found that undifferentiated CHA3-hESCs with trisomy chromosome 12 undergo abnormal cell division with multiple spindles in comparison to the bipolar cell division of the karyotypically normal CHA3-hESCs. Transplanted karyotypically abnormal CHA3-hESC derivatives formed a tumor-like tissue 6weeks after transplantation in two out of seven mice tested. Our results demonstrate that the preservation of normal chromosomes is indispensable for maintaining the true properties of hESCs in vitro and abolishing adverse effects posttransplantation. Thus, the development of optimized techniques for stabilizing the chromosome state during in vitro hESC culture is a prerequisite for the therapeutic application of hESCs.     

Neuregulin stimulates DNA synthesis in embryonic chick heart cells.

Neuregulins are a family of growth factors that have been shown to promote the growth or differentiation of various cell types. Recently, targeted mutations of the genes for neuregulins or their putative receptors by homologous recombination resulted in embryonic lethality characterized by cardiac malformation. Here we investigate a role for neuregulin in the growth of cultured chick heart cells. Neuregulin induced the tyrosine phosphorylation of a 185-kDa protein in cultured heart cells, and it also stimulated an increase in [(3)H]thymidine incorporation and BrDU labeling in the cell cultures. Immunocytochemistry revealed that the increased DNA synthesis was primarily in mesenchymal cells and not detected in myocytes or endocardial cells. These data suggest that neuregulin may function as a paracrine signal in mesenchymal-endothelial interactions during cardiac development.     

Stronglyoides ransomi: factors influencing the in vitro development of the free-living generation.

Among the progeny of parasitic females of Strongyloides ransomi, ransomi, males did not appear in significant numbers until the 7th week of infection in cases of simple infection, and until the 3rd week of infection in cases of multiple infection. The appearance of males was attributed to the effect of host immunity, the physiological ageing of the parasitic females, or both. Type of culture substrate and other cultural conditions did not influence the percent of larvae developing into males. Sex of larvae was determined prior to hatching, probably during oogenesis or embryogenesis. Culture conditions influenced the direction of development of female larvae. An initial pH below 5.9 or above 7.2 favored differentiation of larvae into infective larvae, whereas, intermediate initial pH levels favored development of free-living females. Baby pig substrate, autoclaved substrate, and substrate washed free of soluble chemicals (adverse cultural conditions) promoted differentiation toward infective larvae. Adult pig substrate, nonautoclaved substrate and unwashed substrate promoted differentiation toward free-living females. In general, adverse conditions inside the host and favor an indirect life cycle.     

Growth and DNA synthesis in embryonic chick heart cells, in vivo and in vitro

On the basis of cell shape, refractility under phase optics, spontaneous beating and nucleolar number, two cell types have been distinguished in embryonic heart cell cultures: M cells (myoblastlike) and F cells (fibroblast-like). However, by different criteria, more than two cell types have been found in the heart in vivo. In the present study, heart cell types are redefined by properties which can be used for identifying cells both in vitro and in vivo. Trypsin-dissociated cells from 7-day chick hearts were cultured for 24 h. PAS staining indicated that all M (thick, refractile) cells contain glycogen (gly⁺), while most F (spread) cells do not (gly⁻). Under the electronmicroscope, only gly⁺ cells contain myofibrils; most of these cells beat spontaneously in culture. Gly⁻ cells do not beat. Gly⁺ cells with myofibrils as well as gly⁻ cells are found in the inoculum and in the heart in vivo. The ultrastructure of gly⁺ cells in vitro and of muscle cells in the myocardium in vivo is similar. Therefore, it is concluded that gly⁺ cells in culture are derived from the myocardium (muscle) of the heart. Gly⁻ cells are apparently derived from the epicardium, the endocardium and the endothelial lining of blood vessels.