Morphological and functional differentiation of cryopreserved lactating bovine mammary cells cultured on floating collagen gels

Cryopreserved bovine mammary epithelial cells prepared from lactating mammary tissue synthesize and secrete the milk proteins alpha_s1-casein, lactoferrin (Lf), and alpha-lactalhumin during in vitro culture on collagen gels in serum-free medium. Each milk protein is differently regulated by detachment and thickness of the collagen substratum, fetal calf scrum, and prolactin in the medium. Collagen detachment did not modulate lactoferrin secretion but strongly induced casein secretion, with detachment on day 6 (after formation of cell sheets) inducing casein secretion to 3 μg/ml medium, which was 2–3-fold higher than for cells on collagen detached on day 2 (prior to cell spreading to form sheets), and ten-fold higher than for cells grown on collagen not detached. Alpha-lactalbumin secretion was also induced, but only to low levels, in cells grown on detached but not on attached collagen. Cells grown on thin collagen gels secreted lower levels of lactoferrin and casein compared to cells on thick collagen. Lactoferrin but not casein secretion was increased in cells grown in the presence of fetal calf serum. Casein but not lactoferrin secretion was completely dependent on prolactin. Cells grown serum-free on collagen gels detached on day 6 of culture showed a polarized epithelial cell layer with high differentiation evidenced by the apical microvilli, tight junctions, and fat droplets surrounded by casein-containing secretory vesicles. An underlying layer of myoepithelial-like cells was also evident. These studies show for eryopreserved primary bovine mammary cells prepared from lactating mammary tissue the induction of highly differentiated and polarized cell morphology and ultrastructure with concomitant induction of the secretion of casein, lactoferrin. and alpha-lactalbumin in vitro, and that the non-coordinate regulation of milk protein secretion by substratum, prolactin, and serum likely involves alternate routing and control of secretion pathways for casein and lactoferrin.     

心肌细胞和血管平滑肌细胞收缩调控机制的研究进展

心脏和血管构成体内的心血管系统,两者都具有收缩性。心脏收缩要求在很短的时间内升高室内压,因此要求细胞收缩快速和有力,这就需要细胞的收缩结构和钙调控过程能满足其要求。血管收缩缓慢而持久,其收缩结构及机制也正好与之功能相适应。本文从细胞水平讨论了心脏和血管的收缩结构和收缩机制,以及钙调控机制,并分别对两者之间的异同点作了介绍。     

Morphological and functional differentiation of human thyroid cells in collagen gel culture

In order to study the expression of the morphological and functional characteristics of human thyroid cells, 3-dimensional cultures were carried out in collagen gel. This substrate allows the cells to retain their organization in follicles with a normal polarity. Cellular polarities appeared normal at the time of collagen embedding, but there was a delay of 4-5 days in culture before the maximal TSH stimulation of 125I- uptake and of cAMP accumulation occurred. In normal and adenoma-derived cells, 125I- uptake, which could be increased by TSH, was demonstrated. cAMP accumulated in the culture medium and thyroglobulin was secreted into the follicle lumen. Of the 4 differentiated carcinomas for which the 72-hr uptake of 125I- was measured, only 2 displayed slight 125I- uptake and response to TSH. Thus, human thyroid cells exhibit better morphological and functional differentiation in collagen gel culture than in monolayer culture. Furthermore, in a variety of pathological cases studied, the expression of specific characteristics in culture varied in a fashion similar to differences observed in vivo.     

Culture and neural differentiation of rat bone marrow mesenchymal stem cells in vitro

Adult bone marrow mesenchymal stem cells (MSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but can also differentiate into non-mesenchymal cells, such as neural cells, under appropriate experimental conditions. Until now, many protocols for inducing neuro-differentiation in MSCs in vitro have been reported. But due to the differences in MSCs' isolation and culture conditions, the results of previous studies lacked consistency and comparability. In this study, we induced differentiation into neural phenotype in the same MSCs population by three different treatments: beta-mercaptoethanol, serum-free medium and co-cultivation with fetal mouse brain astrocytes. In all of the three treatments, MSCs could express neural markers such as NeuN or GFAP, associating with remarkable morphological modifications. But these treatments led to neural phenotype in a non-identical manner. In serum-free medium, MSCs mainly differentiated into neuron-like cells, expressing neuronal marker NeuN, and BME can promote this process. Differently, after co-culturing with astrocytes, MSCs leaned to differentiate into GFAP(+) cells. These data confirmed that MSCs can exhibit plastic neuro-differentiational potential in vitro, depending on the protocols of inducement.     

Morphological differentiation of endothelial cells co-cultured with astrocytes on type-I or type-IV collagen

Summary In this study bovine aortic endothelial cells were co-cultured with astrocytes from fetal Wistar Kyoto rats. Endothelial cells growing on type-I collagen, development. Although some cells appeared to be mature, horseradish peroxidase penetrated within 1 min of incubation through the intercellular junctions of these endothelial elements maintained on type-I collagen. In contrast, endothelial cells on type-IV collagen, co-cultured with astrocytes, were well developed; their intercellular junctions were well established, and plasmalemmal vesicles reduced in number. As a result, horseradish peroxidase was unable to penetrate through the endothelial cells grown on type-IV collagen and co-cultured with astrocytes because of the reduced extent of the junctional and vesicular transport. These findings reveal that (1) type-IV collagen is essential for the differentiation of endothelial cells, (2) endothelial cell-astrocyte interactions occur during co-culture, and (3) endothelial permeability depends on astrocyte-produced factors, in addition to type-IV collagen.     

Insulin-like growth factor I supports differentiation of cultured osteoblast-like cells

Rat calvaria cells grown in culture for one week had properties of osteoblasts: a high content in alkaline phosphatase and a marked cyclic AMP response to parathyroid hormone (PTH). In short-term experiments, insulin-like growth factor I (IGF I) stimulated the incorporation of [14C] glucose into glycogen. When IGF I was present in the medium during 6 days the cell number increased slightly and there was a substantial, disproportionate rise in alkaline phosphatase activity of the cultures. Thus, IGF I stimulates growth, and in addition, and in contrast to other growth factors, mainly enhances differentiation of osteoblasts.     

Diversity of ultrastructure in different phenotypes of cultured microvessel endothelial cells isolated from bovine corpus luteum

Summary Five different types of cultured microvessel endothelial cells defined by use of light microscopy and scanning electron microscopy in a preceding study were investigated by transmission electron microscopy. Type-1 cells displayed a deep invagination of the cell membrane or a single cilium. Granules of low electron density were abundant. A perinuclear ring of intermediate filaments occurred. Cultures of type-2 cells were subdivided into phenotype A, reminiscent of cell-type 1, and into phenotype B, assumed to be vascular smooth muscle cells. Many highly electron-dense granules appeared in late postconfluent cultures of both phenotypes. Cell-type 3 was conspicuous because of a large intracytoplasmic vacuole. Lysosomes with curvilinear bodies were found in cell-types 3 and 4. Both cell types developed a peripheral regular network of microfilaments. Cell-type 5 showed vesiculation of the rough endoplasmic reticulum, lipid droplets and a peripheral felt-like belt of microfilaments. Tubular forms seen in late postconfluent cultures of cell-types 1 to 3 displayed a core of extracellular matrix. Pseudotubular forms of cell-type 4 contained apoptotic bodies. Thus, as seen at the ultrastructural level, different features are maintained by cultured microvessel endothelial cells, suggesting that they have different inherent properties.     

An ultrastructural study of dentinogenesis and amelogenesis in rat molar tooth germs cultured in vitro

Summary Molar tooth germs from three-day-old rats were cultured successfully for fourteen days, permitting the study of the development in vitro of both extracellular matrix and cellular elements such as odontoblasts and ameloblasts. The ultrastructure of the cultured tooth germs was compared with the ultrastructure of tooth germs in vivo at a comparable developmental stage. Progenitor cells of odontoblasts and ameloblasts were found to differentiate in vitro. Odontoblasts seemed to contain more lysosome-like bodies and fewer secretory granules than in vivo. They formed normally mineralizing dentine or a thick layer of dense, unmineralized predentine with incidentally some amorphous, extracellular material. Enamel was exclusively present opposite well developed dentine. It was often hyperor hypomineralized and enamel rods were not as regularly shaped as in vivo. In places where no enamel formation had taken place, large amounts of amorphous extracellular material were sometimes seen. From these observations it can be concluded that cellular development in cultured tooth germs appeared more or less normal, but extracellular matrix formation and mineralization were sometimes disturbed.     

Cell shape and arrangement of cultured aortic smooth muscle cells grown on collagen gels

Aortic smooth muscle cells (SMC) grown on conventional plastic culture dishes have morphological and functional properties of dedifferentiated cells in sub-culture. We examined the influence of collagen gels on the cell shape and arrangement. The cells grown on collagen gels showed a multilayered growth with formation of nodules. When the edge of the collagen gels was detached from the culture dish, the shape and arrangement of cells on the edge differed from that of the central, still attached region. The cells grown on floating collagen gels exhibited a spindle-like shape and were arranged in concentric circles. These findings suggest that the physical property of the substrate influences the cell shape and arrangement.     

Atrial natriuretic peptide induces breakdown of phosphatidylinositol phosphates in cultured vascular smooth-muscle cells

Discrepancies exist between extent of guanylate cyclase activation by atrial natriuretic peptide (ANP) in cell-free systems and ANP-stimulated levels of cyclic GMP in whole cells, and also between receptor affinity and dose effectiveness of ANP. Therefore, we have investigated whether, in addition to receptor-coupled guanylate cyclase activation, other second-messenger cascade systems may be involved in mediating both an increase in cyclic GMP and the physiological response to ANP. Equilibrium 125I-ANP binding studies on cultured thoracic aorta smooth muscle cells revealed the existence of low-affinity (approximately 10(-8) M, 84.5 fmol/10(5) cells) and high-affinity (approximately 10(-10) M, 12.5 fmol/10(5) cells) binding sites. We confirm that ANP elevates intracellular cyclic GMP (EC50 approximately 10(-8) M) and inhibits agonist-(isoproterenol and forskolin)-induced increases in intracellular cyclic AMP (IC50 approximately 10(-9) M). ANP also stimulated breakdown of phosphatidylinositol phosphates and generation of inositol phosphates with a half-maximally effective concentration of approximately 10(-10) M. The extent of phosphatidylinositol polyphosphate hydrolysis was small (120%) in comparison to that of phosphatidylinositol (Ptd-Ins) (200%). Ptd-Ins hydrolysis was paralleled by the appearance of glycerophosphoinositol, and there was also a close temporal relationship between these processes and the accumulation of intracellular cyclic GMP. Smooth muscle cells released [3H]arachidonic acid label in response to ANP (EC50 approximately 10(-10) M). Taken together, the data suggest that the vasorelaxant hormone ANP has stimulatory effects on phosphoinositol lipid metabolism via both phospholipase C (generation of inositol phosphates) and phospholipase A2 (generation of releasable [3H]arachidonic acid and indirectly glycerophosphoinositol). In contrast, stimulation of phosphatidylinositol phosphate breakdown by the vasoconstrictive hormone angiotensin II is not associated with glycerophosphoinositol formation, and neither cyclic GMP nor cyclic AMP levels were influenced by this hormone.     

Cell culture from lizard skin: a tool for the study of epidermal differentiation

An in vitro system of isolated skin cells has been developed in order to address the understanding on the factors that control the shedding cycle and differentiation of lizard epidermis. The skin from the regenerating lizard tail has been separated in epidermis and dermis, cells have been dissociated, cultivated in vitro, and studied ultrastructurally after 1–30 days of culture condition. Dissociated keratinocytes after 12 days in culture show numerous cell elongations and contain bundles of keratin or sparse keratin filaments. These cells often contain one to three 0.5–3 μm large and dense “keratinaceous bodies”, an organelle representing tonofilament disassembling. Most keratinocytes have sparse tonofilaments in the cytoplasm and form shorter bundles of keratin in the cell periphery. The dissociated dermis mainly consists of mesenchymal cells containing sparse bundles of intermediate filaments. These cells proliferate and form multi-stratified layers and a dermal pellicle in about 2–3 weeks in vitro in our basic medium. Conversely, cultures of keratinocytes do not expand but eventually reduce to few viable cells within 2–3 weeks of in vitro condition. It is suggested that dermal cells sustain themselves through the production of growth factors but that epidermal cells requires specific growth factors still to be identified before setting-up an in vitro system that allows analyzing the control of the shedding cycle in lizards.     

Growth and differentiation in cultured human thyroid cells: Effects of epidermal growth factor and thyrotropin

Summary Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [³H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 μg/ml), transferrin (5 μg/ml), hydrocortisone (10 μg/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10⁻⁹ M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells. This work was supported by grants from the Medical Research Council of Canada; the Department of Medicine, University of Toronto; and the National Cancer Institute of Canada. J. E. E. is a C.H. Best Foundation and Department of Medicine postdoctoral fellow.     

An in vitro model for studies of intercellular communication in cultured rat anterior pituitary cells

The formation of intimate associations among different hormone-secreting cells within the rat adenohypophysis may serve as a possible site for physiologic regulation. In this report we describe a high density plating method which enables us to study cell-to-cell interactions within anterior pituitary cell cultures. Trypsin-dispersed pituitary cell suspensions attach rapidly (within 6 hr) and quantitatively (95-97%) to glass or plastic surfaces when plated in medium containing microM calcium concentrations (pH 7.6-7.8). Freshly plated cell suspensions obtained from female pituitary glands contained subpopulations of mammotrophs 49.3%, somatotrophs 30.3%, gonadotrophs 12.6%, corticotrophs 3.4% and thyrotrophs 1.5%. Epithelial cell colonies were formed during a 3-day culture period as the cells flattened and re-established contacts with neighboring cells. Freeze-fracture electron microscopic analysis of these colonies produced morphological evidence for direct intercellular contacts among the hormone-secreting cells. Large areas of tight junctions and small gap junctions were identified on the membranes of the epithelial cells within these colonies. Cells which contained tight junctions usually contained microvilli and morphological signs of active hormone secretion. Small junctional plaques containing tightly packed intramembrane particles were also occasionally found on the membranes of cells which were actively secreting pituitary hormones. The high density plating procedure which is described in this report provides greater opportunity for cell-cell interaction and thus may prove to be a useful model for evaluating the role of intercellular communication within this tissue.     

Morphological and electrophysiological changes produced by electrical stimulation in cultured neuroblastoma cells.

Electric fields, which were equivalent to those generated by medical devices, were applied to cultured neuroblastoma cells (mouse and human) to test for morphological damage and to establish damage thresholds. Each of two methods of applying fields permitted flow of electrical current and minimized exposure of cells to electrode-breakdown products. One method consisted of a pair of parallel wires in a Petri dish by which current was delivered within a fixed volume of flowing tissue-culture media. With the other method, the cells were held in a confined geometrical chamber and current was applied via agar bridges. Under a given set of stimulation parameters, damage was found to be variable from cell to cell. By changing the strength of the electric field (frequency and duration of stimulation held constant), thresholds of several V/cm were found above which cell damage could be reliably produced. Depending on the intensity of the field, damage took the form of cell lysis or damage to neurites. Intracellular recordings from the mouse neuroblastoma cells revealed a correlation between a decline in resting transmembrane potential and stimulus intensity. Human neuroblastoma cells were less susceptible to damage than were the mouse neuroblastoma cells, given the same strength of applied electric fields.     

Modulation of angiotensin-converting enzyme in cultured human vascular endothelial cells

Summary Previous work has suggested that not all immunoreactive angiotensin-converting enzyme (ACE) in tissues or cells is in a biologically active state. We have explored this possibility in cultured human umbilical vein endothelial cells (HUVEC), one of the most widely studied in vitro endothelial cell systems. Our approach included characterization of the effect of increasing passage number on ACE activity and expression of immunoreactive ACE at the single cell level, the subcellular compartmentalization of active ACE, and the effect of phorbol ester (PMA) treatment. We found that both ACE activity and expression of ACE antigen were downregulated by cultivation (30% of ACE-positive cells at seventh passage vs. 90% in primary culture). ACE downregulation is specific (number of CD31-positive cells did not change with cultivation) and correlated with downregulation of factor VIII-antigen. The percentage of ACE-positive cells in permeabilized HUVEC at third passage was almost twice that in nonpermeabilized HUVEC (90% vs. 50%), indicating that HUVEC contain intracellular immunoreactive ACE. ACE activity, however, was similar when measured in intact cells and in cell lysates. Moreover, diazonium salt of sulfanilic acid (DASA), a membrane-impermeable ACE inhibitor, inhibited ACE activity in intact cells and in cell lysates at the same extent, thus implying that intracellular ACE is inactive. PMA (100 nM) treatment increased the percentage of ACE-positive cells at third passage from 57 to 96%. ACE activity was increased 3-fold in cell and 1.5-fold in the culture medium of PMA-treated cells. Analysis of ACE activity in intact monolayers and cell lysates of control and PMA-treated cells revealed that all enzymatically active ACE in PMA-treated cells is localized on the plasma membrane and acts as an ectoenzyme. We conclude that expression of ACE by HUVEC is downregulated by repeated passage in culture but can be restored by PMA treatment. In addition, ACE expression is heterogeneous between neighboring cells, and total immunoreactive ACE protein associated with HUVEC includes an inactive pool of the enzyme.     

The influence of cell shape on the induction of functional differentiation in mouse mammary cells in vitro

Summary To define more clearly the in vitro conditions permissive for hormonal induction of functional differentiation, we cultured dissociated normal mammary cells from prelactating mice in or on a variety of substrates. Cultivation of an enriched epithelial cell population in association with living adult mammary stroma in the presence of lactogenic hormones resulted in both morphological and biochemical differentiation. This differentiation, however, was not enhanced over that seen when the cells were associated with killed stroma, provided that the killed stroma had a flexibility similar to that of the living stroma. Cells cultured in inflexible killed stroma usually did not differentiate. Cells cultured within the flexible environment of a collagen gel, but removed from the gas-medium interface, differentiated in a manner similar to those cultured in flexible stroma. Cells cultured on the surface of an attached collagen gel were squamous, and their basolateral surfaces were sequestered from the medium; they did not differentiate. Cells cultured on floating collagen gels were cuboidal-columnar, with basolateral surfaces exposed to the medium, and showed good functional differentiation. Cells cultured on inflexible floating collagen gels were extremely flattened and had exposed basolateral surfaces, and showed no evidence of functional differentiation. We infer that assumption of cuboidal to columnar shapes similar to those of mammary cells in vivo may be important to the induction of functional differentiation in vitro. The additional requirement of basolateral cell surface exposure also is important. This work was supported by U.S. Public Health Service Grants CA-05045 and CA-09041 from the National Cancer Institute, Bethesda, MD.     

Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells

The effects of synthetic rat adrenomedullin (rAM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, on receptor binding and cAMP generation were studied in cultured rat vascular smooth muscle cells (VSMC). A binding study using [¹²⁵I]rAM revealed the presence of a single class of high-affinity (K_d1.3 × 10⁻⁸ M) binding sites for rAM in VSMC. The apparent K_i of rat calcitonin gene-related peptide (rCGRP) was 3 × 10⁻⁷ M. Affinity labeling of VSMC membranes with [¹²⁵I]rAM revealed two distinct labeled bands with apparent molecular weights of 120 and 70 kDa, both of which were abolished by excess unlabeled rAM or rCGRP. rAM stimulated cAMP formation with an approximate EC₅₀ of 10⁻⁸ M, the effect of which was additive with isoproterenol, but not with rCGRP. The rAM-induced cAMP response was unaffected by propranalol, indomethacin, or quinaerine, but inhibited by a CGRP receptor antagonist, human CGRP[8–37]. These data suggest that VSMC possesses specific AM receptors functionally coupled to adenylate cyclase with which CGRP interacts.     

大鼠输精管平滑肌细胞的体外培养及形态学特征(英文）

建立大鼠输精管平滑肌细胞的培养方法。取大鼠输精管,剥离外膜和内膜,用组织块法进行体外培养。用抗α-SMA(anti α-smooth muscle actin)免疫组化染色的方法鉴定培养的细胞。结果显示,在倒置显微镜下观察细胞形态多样,表现为长梭形或星形,细胞伸出突起互相接触,彼此融合,部分区域细胞多层重叠,部分区域细胞单层高低起伏,呈"峰-谷"状生长。免疫组化染色鉴定呈阳性反应,用该方法所分离、培养的输精管平滑肌细胞纯度达99%以上。应用组织块法培养大鼠输精管平滑肌细胞,操作简单,结果稳定。     

Visualisation of specific binding sites for atrial natriuretic peptide on non-neuronal cells of cultured rat sympathetic ganglia

Summary The distribution of atrial natriuretic peptide binding sites on cells in dissociated culture preparations of neonatal rat superior cervical ganglia and in explant cultures of rat thoracic sympathetic chain ganglia has been studied. The autoradiographic visualisation of atrial natriuretic peptide binding sites has been combined with the use of specific immunocytochemical markers for glial cells (antiserum to S-100 protein), fibroblasts (antiserum to fibronectin) and neurones (antiserum to protein gene product 9.5) in order to achieve unambiguous identification of the cell types in culture. Specific binding sites for rat¹²⁵I-atrial natriuretic peptide_(1–28) were observed over subpopulations of fibronectin-like-immunoreactive fibroblasts and S-100-like-immunoreactive glia in the dissociated superior cervical ganglion cultures. However, only a subpopulation of fibronectin-like-immunoreactive fibroblasts possessed atrial natriuretic peptide binding sites in the explant culture preparations. No atrial natriuretic peptide-like-immunoreactive cells were present in either culture. The distribution of autoradiographic grains over individual cell surfaces in culture was uniform, but there were distinct differences in the density of labelling of single cells of the same type. This apparent variation in the number of binding sites on glial cells and fibroblasts in culture did not seem to be related to the morphology of the cells or the surrounding cell types. No sympathetic neurones were labelled with autoradiographic grains in either the dissociated or explant culture preparations. However, the presence of atrial natriuretic peptide binding sites on non-neuronal cells of sympathetic ganglia in culture may be linked to the relationship between atrial natriuretic peptide and the sympathetic nervous system.     

An hypothesis for the interpretation of the contractile response of vascular smooth muscle at the cellular level

The long preservation and recovery of functional (contractile) properties in cultured aortic smooth muscle cells, even after replating or deep-frozen storage and the measurement of their responses are now technically settled issues. We could thus study extensively the responses of single cultured cells from rat thoracic aorta. Responses were elicited by the addition of KCl 40 mmol/L without or with a calcium blocker PN 200-100 (10^–6 mol/L); angiostein II (10^–11–10^–6 mol/L) without or with antagonist (losartan 10^–5 mol/L); or serotonin (10^–9–10^–4 mol/L) without or with antagonist (naftidrofuryl 10^–5 mol/L). Results thus obtained enabled us to propose a new hypothesis for the interpretation of the contractile responses of an elastic vascular smooth muscle. The different maximal effects of different agonists result mainly from the different proportions of cells they can mobilize; the agonist concentration-contraction relationship is mainly due to the increase of the proportion of cells involved up to a maximal value typical of the agonist used. An antagonist primarily reduce the proportion of cells an agonist can mobilize. Some of the consequences of this hypothesis are briefly outlined.