Julie H. Chamley

Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and in subculture.

SummaryA method is presented for growing large numbers of pure isolated smooth muscle cells from adult human, monkey, and rabbit blood vessels in primary culture.In the first few days in culture these cells closely resembled those in vivo and could be induced to contract with angiotensin II, noradrenaline and mechanical stimulation. They stained intensely with antibodies against smooth muscle actin and myosin. Fibroblasts and endothelial cells did not stain with these antibodies thereby allowing the purity of each batch of cultures to be monitored. This was consistently found to be better than 99%. The smooth muscle cells modified or “dedifferentiated” after about 9 days in culture to morphologically resemble fibroblasts. At this stage cells could no longer be induced to contract and did not stain with the myosin antibodies. Intense proliferation of these cells soon resulted in a confluent monolayer being formed at which stage some differentiated characteristics returned. The modification or “dedifferentiation” process could be inhibited by the presence of a feeder layer of fibroblasts or endothelial cells, or the addition of cAMP to the culture medium.Smooth muscle cells which had migrated from explants in primary culture, and cells in subculture, had morphological and functional properties of “dedifferentiated” cells at all times.The advantages of differentiated rather than “dedifferentiated” smooth muscle cells in culture for the study of mitogenic agents in atherosclerosis is discussed.

Selective growth of sympathetic nerve fibers to explants of normally densely innervated autonomic effector organs in tissue culture

Abstract Explants of sympathetic ganglia were cultured 1–2 mm from explants of two different autonomic effector organs in modified Rose chambers. The tissues used were taken from mid-term fetal to 20-day-old rats. While there was sympathetic nerve fiber growth to small, highly proliferating clumps of cells from all tissues (“nonspecific” growth), there also appeared to be preferential growth to explants of normally densely innervated atrium and vas deferens in competition with explants of normally sparsely innervated kidney medulla, uterus, and distal portion of ureter (“specific” growth). This effect was more pronounced with atrium than with vas deferens. In addition, the total amount of sympathetic nerve fiber growth was greater in cultures where atrium was present. The nature of the “specific” growth was examined by observing the pattern of developing nerve fiber growth to the various tissues over several days. Nerve fibers grew readily from the side of the sympathetic ganglion explant opposite atrium or vas deferens explants and appeared to be directed toward them through all stages of growth. In contrast, nerve fibers appeared at a later time from the side of the sympathetic ganglion explants opposite kidney, uterus, and ureter explants, and their growth appeared to be random through all stages. It is suggested that the direction and extent of nerve fiber growth may be related to the density of the normal potential sympathetic innervation of the tissues, and that a chemical substance diffusing from the explants is involved in this process. The possibility that nerve growth factor (NGF) is the chemical substance is discussed.

Sympathetic ganglia in culture. I. Neurons.

Summary1. Phase-contrast microscopy, time-lapse cinematography, electron microscopy and fluorescence histochemistry were used to study neurons in cultured sympathetic ganglia of rat, guinea-pig and embryo chick.2. There was considerable variation in the morphology and size of neurons in all three species. Many neurons migrated into the outgrowth; those that migrated freely in rat and guinea-pig differed sufficiently from non-migratory neurons to allow a classification into type I (migratory) and type II (non-migratory). Type II neurons appeared to correspond to those seen in situ, but whether type I neurons represent immature or retarded neurons, or are due to culture conditions, is not clear. In the chick, no classification on this basis was possible; instead both migratory and non-migratory neurons showed a large and similar variation in nuclear size.3. In all three species both migratory and non-migratory neurons remained viable and noradrenaline was demonstrable histochemically for eight weeks (oldest cultures studied). Species differences were found in the extent of outgrowth of nerve fibers and accessory cells.4. Ultrastructural studies showed processes with features characteristic of sympathetic nerves in situ and of regenerating nerves. Profiles with other ultrastructural features were described and their identity discussed.5. The action of Nerve Growth Factor (NGF) at 1 unit/ml on sympathetic nerve fibers showed that guinea-pig was the most susceptible and chick the least. In the rat and guinea-pig, NGF increased levels of noradrenaline. NGF did not alter the appearance or relative numbers of type I and II neurons in rat and guinea-pig, but did affect the size distribution of both chick migratory and non-migratory neurons; some chick neurons were insensitive to NGF.

Distinction between smooth muscle, fibroblasts and endothelial cells in culture by the use of fluoresceinated antibodies against smooth muscle actin

SummaryFITC-labelled antibodies against native actin from chicken gizzard smooth muscle (Gröschel-Stewart et al., 1976) have been used to stain cultures of guinea-pig vas deferens and taenia coli, rabbit thoracic aorta, rat ventricle and chick skeletal muscle. The I-band of myofibrils of cardiac muscle cells and skeletal muscle myotubes stains intensely. In isolated smooth muscle cells, the staining is located exclusively on long, straight, non-interrupted fibrils which almost fill the cell. Smooth muscle cells which have undergone morphological “dedifferentiation” to resemble fibroblasts with both phase-contrast microscopy and electronmicroscopy still stain intensely with the actin antibody. In those muscle cultures which contain some fibroblasts or endothelial cells, the non-muscle cells are not stained with the actin antibody even when the reactions are carried out at 37° C for 1 h or after glycerination. Prefusion skeletal muscle myoblasts also do not stain with this antibody.It is concluded that the actin antibody described in this report is directed against a particular sequence of amino acids in muscle actin which is not homologous with non-muscle actin. The usefulness of this antibody in determining the origin of cells in certain pathological conditions such as atherosclerosis is discussed.

Comparison of the reaction of cultured smooth and cardiac muscle cells and fibroblasts to specific antibodies to myosin

SummaryImmunofluorescent staining with anti-smooth or anti-striated muscle myosin was carried out for 30 minutes at room temperature (18–20° C) on cultures of smooth muscle cells and fibroblasts from guinea-pig vas deferens, taenia coli and ureter, rabbit aorta and chicken gizzard and of cardiac muscle cells and fibroblasts from rat ventricle. With antismooth muscle myosin, smooth muscle cells showed an intense fluorescent staining in fine fibrils with an “interrupted” appearance running parallel to the longitudinal axis of the cell throughout the cytoplasm, and also in coarser, “non-interrupted” fibrils (termed here “attachment fibrils”) concentrated at the surface of the cell adjacent to the glass coverslip. Fibroblasts in the same cultures showed similar, but much weaker, reactions. When anti-striated myosin was added to the smooth muscle cultures, staining of neither cell type was observed. In contrast, cardiac muscle cells in cultures of rat ventricle did not react with anti-smooth muscle myosin, but gave bright fluorescent A-band staining with anti-striated myosin. Fibroblasts in the ventricle cultures were unreactive with anti-striated muscle myosin but gave the characteristic weak reaction with anti-smooth muscle myosin. Thus immunofluorescent staining with anti-smooth muscle myosin is useful for distinguishing between isolated smooth muscle cells and fibroblasts in tissue culture.

Changes in Myosin Distribution in Dedifferentiating and Redifferentiating Smooth-Muscle Cells in Tissue-Culture

SummaryIsolated smooth muscle cells and fibroblasts from the newborn guinea-pig vas deferens were grown in culture. In the first 2 days, all cells characterized as smooth muscle by phase-contrast microscopy reacted intensely with fluoresceinated antibodies against smooth muscle myosin. The fluorescence was in the form of particles (termed here “myosin aggregates”), which were often aligned to give the cell a striated appearance. After 3–5 days, coarse fluorescent fibrils were also visible. These were termed “attachment fibrils” (“A-fibrils”) since they were thought to represent myosin in microfilament bundles. Between 6 and 7 days in culture, the smooth muscle cells began to dedifferentiate morphologically. At this time, the “myosin aggregates” became clumped and less intensely fluorescent. “A-fibrils” also decreased in fluorescence intensity. By 8 days in culture, the dedifferentiated cells had undergone intense proliferation and gave only a minimal reaction with myosin antibodies. However, when a confluent monolayer of cells formed on day 9 or 10, they immediately began to redifferentiate ultrastructurally and to regain immunofluorescence in both “myosin aggregates” and “A-fibrils”. Throughout the entire culture period, cells characterized as fibroblasts by phase contrast microscopy gave only a weak reaction with fluoresceinated antibodies to myosin showing “A-fibrils” but no “myosin aggregates”.

Growth characteristics of postnatal rat adrenal medulla in culture

SummaryExplants and enzyme-dispersed cells of adrenal medulla from 10–12 day old rats were studied in culture for up to 3 weeks. Adrenomedullary chromaffin cells, nerve cells and satellite cells were clearly discernible.The nerve cells were few in number and did not show catecholaminespecific fluorescence.Chromaffin cells stored catecholamines, as judged by the Falck and Hillarp method, in varying amounts decreasing with age of the cultures and the distance from the explants. Exocytosis profiles observed with the electron microscope suggested that cultured chromaffin cells also released catecholamines. Moreover, the cells formed processes and frequently migrated into the outgrowth. After 6 days in culture, the great majority of chromaffin cells stored noradrenaline as revealed by electron microscopy with few adrenaline-storing cells being visible. Granular vesicles (∼ 80–240 nm in diameter) with cores of different electron densities were occasionally present in the same cell suggesting the occurrence of mixtures of primary and secondary amines. Apart from “chromaffin” granules, small clear and densecored vesicles (∼ 40–60 nm) were found both in the somata and cell processes. Chromaffin cells and their processes were often closely apposed and occasionally formed specialized attachment zones. As a whole, chromaffin cells in culture resembled small granule-containing cells in sympathetic ganglia.0.5 mM dbcAMP prevented dedifferentiation of chromaffin cells as judged by the lack of processes, the size and amount of “chromaffin” granules and the high number of adrenaline-storing cells present after 6 days in culture.NGF caused a striking increase in the number of axons growing out from expiants.

An analysis of the interactions between sympathetic nerve fibers and smooth muscle cells in tissue culture

Abstract The interactions between sympathetic nerve fibers and smooth muscle cells and fibroblasts from the newborn guinea pig vas deferens were studied in tissue culture with phase contrast microscopy, time-lapse microcinematography, catecholamine fluorescence histochemistry and scanning and transmission electron microscopy. The amount of sympathetic nerve fiber growth, its catecholamine fluorescence reaction and the size of the nerve cell bodies and their nuclei all increased in the presence of vas deferens tissue. Specific growth of nerve fibers to large clumps of vas deferens tissue was seen from distances of up to 2 mm. In contrast, no specific growth from a distance occurred to single cells or small groups of cells. However, random contact with a muscle cell often led to close, extensive, and long-lasting associations. Contact with fibroblasts was always transitory. The rate of sympathetic nerve fiber growth over individual muscle cells was faster than over fibroblasts, which, in turn, was faster than over the collagen-coated surface of the coverslip. Palpation of a muscle cell by a nerve fiber growth cone increased the rate of spontaneous contraction of the muscle cell, the extent of the increase being dependent on the number of nerve fibers involved. Multiple innervation of a smooth muscle cell occurred if nerve fibers reached the cell at about the same time, but not if there was a close association already established. These results are discussed in relation to possible interactions of sympathetic nerve fibers with smooth muscle cells in vivo .

Trophic influences of sympathetic nerves and cyclic AMP on differentiation and proliferation of isolated smooth muscle cells in culture

SummarySmooth muscle cells of the newborn guinea-pig vas deferens dispersed into single cells and grown in culture maintain their differentiation for approximately 5 days before undergoing dedifferentiation and mitosis. The presence of sympathetic nerve fibres in contact with the isolated cells delays this process by 3–7 days (Chamley et al., 1974). A similar delay in dedifferentiation of vas deferens smooth muscle cells in tissue culture in the presence of sympathetic ganglion extract is described in the present report, demonstrating that the trophic effect is elicited by a chemical substance. This effect is mimicked by the presence of either a confluent layer of RKA epithelial cells, dibutyryl cyclic AMP or theophylline. A similar, but considerably weaker, effect is also obtained with spinal cord and liver extracts and noradrenaline. Acetylcholine does not show an effect. It is suggested that a trophic substance (probably not noradrenaline) from sympathetic neurons activates the adenyl cyclase system of smooth muscle cells to increase the intracellular level of cyclic AMP which in turn promotes and maintains the differentiation of the cultured smooth muscle cells.

Sympathetic ganglia in culture. II. Accessory cells.

SummaryThe accessory cells of rat, guinea-pig and chick sympathetic ganglia have been studied in tissue culture with phase-contrast microscopy, time-lapse cinematography and fluorescence histochemistry. Small intensely fluorescent (SIF) cells have been described for the first time in culture. Satellite cells, which are closely associated with nerve cell bodies, and interstitial cells, which are dispersed throughout the ganglion, appeared to be identical cell types; they do not correspond to oligodendrocytes of the central nervous system as has been suggested previously. However, a small number of cells closely resembling central oligodendrocytes were present. Astrocyte-like cells have also been seen occasionally, although their identity is not clear. Several other cell types were present and have been identified as Schwann cells, sheath cells, fibroblasts, perineural epithelium, macrophages and endothelial cells.