Proliferation,differentiation and ciliary beating of human respiratory ciliated cells in primary culture

Summary The growth, differentiation, ciliary beating pattern and frequency of human respiratory ciliated cells in primary culture were studied by scanning and transmission electron microscopy and by videomicroscopy. The epithelial cells were obtained as outgrowth from explants of adult nasal polyps. When the explants were grown on type-I and type-IV collagen substrates in a standard serum-free, hormone-supplemented medium, a high percentage of ciliated cells (range 29±5% to 37±6%) was present within 2 days of culture. After 5 days of culture, the percentage of ciliated cells near the explant was 51±5%. Most of the cultured ciliated cells (85%) were characterized by individual cilia showing a coordinated movement during the beat cycle and a beating frequency (13.3±1.3 Hz) similar to that reported in vivo. In the other 15% of the ciliated cells, the dyskinetic cilia were aggregated into clumps and characterized by a rigid and planar bending movement and a lower (P<0.01) beating frequency (10.7±1.4 Hz). It is suggested that the latter type of cell, already described during fetal development, might be an intermediate type of ciliated cell which appears temporarily during the surface respiratory epithelial differentiation.     

Differentiation of ciliated cells in the terminal bronchioles of neonatal calves

The bronchiolar ciliated cells are exquisitely sensitive to injury caused by infection or irritation of the airways. The mechanism by which bronchiolar ciliated cells are renewed following injury or during the normal course of differentiation is still debated. The present study aimed at recognizing the progenitor cell population for bronchiolar ciliated cells during early neonatal life of calves and to demonstrate the course of events occurs during its differentiation into ciliated cells. Scanning electron microscopy of the terminal bronchiolar epithelium revealed two distinct cell types namely ciliated and non-ciliated cells. Transmission electron microscopy revealed ciliated, non-ciliated (Clara), intermediate and basal cells. At least two categories of intermediate cells could be distinguished: intermediate cells with abundant glycogen and variable numbers of organelles; intermediate cells with little glycogen, large numbers of polyribosomes, and variable numbers of basal bodies. We conclude that: (1) both bronchiolar non-ciliated and basal cells serve as progenitors for the bronchiolar ciliated cells; (2) differentiation of ciliated cell from the non-ciliated one involves a transitional cell in which glycogen is lost, polyribosomes are synthesized before the synthesis of basal bodies and cilia.     

Granular,ciliated cells in the anterior pituitaries of immature rats

Summary In this communication we demonstrate a new type of ciliated cell in the pituitary gland of immature rats. Anterior pituitary glands of rats, 31 days of age, were examined by electron microscopy. Around the agranular cells, which lined small cavities, there were sparsely granulated cells with many cilia (granular, ciliated cells). Their small granules, which were distributed along the cell membrane, had limiting membranes. Their cilia were of 9+2 type with a central pair of micro tubules. It was suggested that the granular, ciliated cells might be an intermediate-type of cell for the different types of pituitary cells, which appear temporarily during pituitary ontogenesis.     

Tracheal epithelium: cell kinetics and differentiation in normal rat tissue

Abstract. The fate of [³H]thymidine ([³H]Tdr) pulse-labelled cells was followed in tracheal epithelium of young male rats. The time course for cell differentiation, and the relation of events to tissue composition were studied. In vivo labelling and light microscope autoradiography of epoxy embedded sections were used. Labelled and total nuclei for each cell type, and combinations of labelled cells which were adjacent to one another, were tallied. Hierarchical analyses of variance were performed on the several data sets. All cell types, except ciliated, were labelled at 1 hr. A few labelled ciliated cells were seen 24 hr post-label. The frequency of labelled intermediate cells peaked at day 2; goblet and ciliated cells at day 3. No significant changes occurred in the labelling index, but at 24 hr the frequency of adjacent labelled cells (ALC) had increased > 5-fold, and changes had occurred in patterns of ALC combinations. The labelled ciliated cells which were seen at 24 hr were adjacent to labelled intermediate cells. No labelled basal-ciliated cell combinations were seen at any time. Data indicated that ciliated cells can develop from S-phase intermediate cells within 24 hr, and neither basal nor superficial goblet cells are progenitors of ciliated cells. It is proposed that both superficial goblet cell and ciliated cell development is preceded by two divisions: a basal cell division followed by an intermediate cell division.     

The development of ciliated and mucus cells from basal cells in hamster tracheal epithelial cell cultures

Hamster tracheal epithelia consist of three cell types: ciliated, mucus and basal cells. Autoradiographic data from several studies suggest that either basal or non-ciliated columnar cells may serve as stem cells for regeneration of lost or damaged ciliated and mucus cells. The objective of the present study was to examine the role of basal cells in the formation of ciliated and mucus cells in hamster tracheal epithelial (HTE) cell cultures via tritiated thymidine ([3H]-TdR) autoradiography. When 3 day cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (5 day total) label was present in the nuclei of basal and columnar epithelial cells suggesting that the labeled columnar cells may be derived from basal cells. However, the morphological reorganization occurring during this 2 day interval may create difficulties in this interpretation. Since these morphological changes are minimal during the 6 day to 8 day in vitro period, 6 day HTE cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (8 day total), and examined to further study the fate of labeled basal cells during this period. Analysis of these 8 day cultures revealed that labeled nuclei were present in both basal cells and adjacent ciliated and mucus cells. These results do not exclude the possibility of non-basal cell origin of ciliated and mucus cells in other systems but suggest that, at least in HTE cultures, undifferentiated basal cells have the ability to develop into ciliated and mucus cells.     

The role of oestrogen in the control of ciliated cells of the human endometrium.

The incidence of ciliated cells in the human endometrium was determined. Conditions associated with an excess of oestrogenic activity were characterized by an increased incidence of ciliated cells, whilst oestrogen deficiency was associated with decreased numbers. When endometrium was cultured, addition of oestradiol-17 beta caused an increase in the ciliated cell population.     

The fine structure of the secretory cells of the uterus (shell gland) of the chicken

The secretory processes in the shell gland of laying chickens were the subject of this study. Three cell types contribute secretory material to the forming egg: ciliated and non-ciliated columnar cells of the uterine surface epithelium, and cells of tubular glands in the mucosa. The ciliated cells as well as the non-ciliated cells have microvilli, which undergo changes in form and extent during the secretory cycle. At the final stages of shell formation they resemble stereocilia. It is postulated that the microvilli of both cells are active in the production of the cuticle of the shell. The ciliated cell which has both cilia and microvilli manufactures secretory granules which arise from the Golgi complex in varying amounts throughout the egg laying cycle. Granule production reaches its greatest intensity during the early stages of shell deposition. The ciliated cell probably supplies proteinaceous material to the matrix of the forming egg shell. The non-ciliated cell has only microvilli. Secretory granules, containing an acid mucopolysaccharide, arise from the Golgi complex. Some granules are extruded into the uterine lumen where they supply the egg shell with organic matrix. Others migrate towards the supranuclear zone. Here a number of them disintegrate. This is accompanied by the formation of a large membraneless space, which is termed “vacuoloid.” Subsequently the vacuoloid regresses and during regression an extensive rough endoplasmic reticulum with numerous polyribosomes of spiral configuration appears. It is suggested that material in the vacuoloid originating from the disintegrating granules is resynthesized and utilized for the formation of secretory product. The uterine tubular gland cells have irregular, frondlike microvilli. During egg shell deposition, these microvilli form large blebs and are probably related to the elaboration of a watery, calcium-containing fluid.     

Ultrastructure of the Penicillate Podia of the Spatangoid Echinoid Echinocardium cordatum (Echinodermata) with Special Emphasis on the Epidermal Sensory-Secretory Complexes

The spatangoid echinoid Echinocardium cordatum possesses specialized penicillate podia that handle sediment particles during burrowing and feeding. Epidermal complexes, which occur on podial surfaces directly contacting the sediment, each comprise four cells: a non-ciliated secretory cell containing granules rich in mucopolysaccharides (NCS cell), a ciliated secretory cell containing granules of unknown composition (CS cell), and two ciliated non-secretory cells (CNS cells). The cilium of the CS cell is subcuticular whereas that of each CNS cell traverses the cuticle. We propose that these four cells constitute a sensory-secretory complex wherein the ciliated cells are sensory cells and the secretory cells function for adhesion and de-adhesion. More exactly, an NCS cell adhesive and a CS cell de-adhesive would be sequential and would be initiated by two successive stimulations transduced by cilia when the podium touches the sediment. Cilia that first contact the sediment are those protruding through the cuticle from the CNS cells. Their stimulation would result in the secretion of an adhesive material by the NCS cells. Subsequently, the subcuticular cilia of CS cells would be stimulated when the podial digitations closely squeeze the substrate, and this would induce the secretion of a de-adhesive. These two antagonistic secretions would allow the podium to pick up and discharge sediment repetitively during burrowing and feeding.     

The fine structure of the muscle system in the female of the orthonectid Intoshia variabili (Orthonectida)

The contractile system of the female Intoshia variabili (Orthonectida) consists of smooth muscles. The attachment of the longitudinal muscle fibres at the anterior and the posterior tips of the body is rather peculiar, accomplished by means of elongated terminal muscle cells piercing through several ciliated cells. In the last ciliated cell, the muscle cell invaginates the ciliated cell basal membrane almost up to the ciliated cell surface. Here, around the protrusion terminus, there is an electron‐dense zone in contact with the cilia rootlets.     

Ciliated cells in vitamin A-deprived cultured hamster tracheal epithelium do divide

Summary The pseudostratified tracheal epithelium, composed of a heterogeneous phenotypically varying cell population, was studied with respect to the in vitro cell proliferative activity of differentiated epithelial cells. Ciliated tracheal epithelial cells so far have been considered to be terminally differentiated, nonproliferating cells. Tracheal organ cultures obtained from vitamin A-deprived Syrian Golden hamsters were cultured in a vitamin A-deficient, serum-free, hormone-supplemented medium. In vitamin A-deprived tracheal epithelium treated with physiologically active all-trans retinol and low cigarette-smoke condensate concentrations it is possible to stimulate the cell proliferation of both basal and columnar cells. Therefore, the probability of finding proliferating columnar cells was increased compared with the in vivo and the vitamin A-deprived situation in which cell proliferative activity is relatively low. In the presence of cigarette-smoke condensate in a noncytotoxic concentration, basal, small mucous granule, ciliated, and indifferent tracheal epithelial cells incorporated [methyl-³H]-thymidine into the DNA during the S phase. The finding that ciliated cells were labeled was supported by serial sections showing the same labeled ciliated cell in two section planes separated by 2 to 3 μm, without labeled epithelial cells next to the ciliated cell. Furthermore, a ciliated tracheal epithelial cell incorporating [methyl-³H]thymidine into DNA was also seen in tracheal cultures of vitamin A-deprived hamsters treated with all-trans retinol in a physiologic concentration. The present study was financially supported by the Scientific Advisory Committee on Smoking and Health (Dutch Cigarette Industry Foundation) and the Ministry of Welfare, Health and Cutural Affairs.     

Fate of ciliated epidermal cells during early development of Xenopus laevis using whole-mount immunostaining with an antibody against chondroitin 6-sulfate proteoglycan and anti-tubulin: transdifferentiation or metaplasia of amphibian epidermis.

Xenopus embryonic epidermis changes its cellular composition during development: the appearance of ciliated epidermal cells before hatching is a remarkable characteristic. In this study, the functional change of ciliated cells to mucus-secreting cells was examined with immunocytochemistry using anti-tubulin and anti-chondroitin 6-sulfate (C6S). Before hatching, most epidermal cells were labeled with anti-C6S in a granular fashion. Immunoelectron microscopy revealed that the anti-C6S-positive structure was the mucus granule. Ciliated epidermal cells lacked anti-C6S staining, but were strongly labeled with anti-tubulin. After hatching, most ciliated cells in the surface of the embryo disappeared. During their disappearance, some ciliated cells exhibited anti-C6S-positive granular labeling. This strongly suggests that the disappearance of ciliated cells is a functional conversion to mucus-secreting cells instead of shedding through cell death.     

Interaction between SPARC and tubulin in <Emphasis Type="Italic">Xenopus</Emphasis>

Secreted protein, acidic, rich in cysteine (SPARC) is an ancient calcium-binding glycoprotein associated with the extracellular matrices of invertebrates and vertebrates. We have previously reported an intracellular association of SPARC with the 9+2 microtubule arrays of cilia on the surface ectoderm of Xenopus embryos. During early development in Xenopus, ciliated cell precursors are associated with the inner sensorial layer of the two-layered embryonic skin. The ciliated cell precursors migrate to the overlying surface ectoderm where they undergo ciliogenesis. Whole-mount immunohistochemical data indicate SPARC is associated with the ciliary tuffts until ciliated cells begin to disappear from the surface ectoderm during late tailbud development. We now report an association between SPARC and tubulin in Xenopus embryonic cell lysates by co-immunoprecipitation. Tubulin is not co-immunoprecipitated by anti-SPARC antibodies that show no cross-reactivity to Xenopus SPARC by whole-mount immunocytochemical analysis. An association of SPARC with tubulin has also been observed in pull-down assays with biotinylated SPARC as bait. These data indicate that SPARC may have intracellular and extracellular functions during development in Xenopus.     

Fate of ciliated epidermal cells during early development of Xenopus laevis using whole-mount immunostaining with an antibody against chondroitin 6-sulfate proteoglycan and anti-tubulin: transdifferentiation or metaplasia of amphibian epidermis

Xenopus embryonic epidermis changes its cellular composition during development: the appearance of ciliated epidermal cells before hatching is a remarkable characteristic. In this study, the functional change of ciliated cells to mucus-secreting cells was examined with immunocytochemistry using anti-tubulin and anti-chondroitin 6-sulfate (C6S). Before hatching, most epidermal cells were labeled with anti-C6S in a granular fashion. Immunoelectron microscopy revealed that the anti-C6S-positive structure was the mucus granule. Ciliated epidermal cells lacked anti-C6S staining, but were strongly labeled with anti-tubulin. After hatching, most ciliated cells in the surface of the embryo disappeared. During their disappearance, some ciliated cells exhibited anti-C6S-positive granular labeling. This strongly suggests that the disappearance of ciliated cells is a functional conversion to mucus-secreting cells instead of shedding through cell death.     

The ultrastructural organization and the alkaline phosphatase activity of the epithelial surface of the bovine fallopian tube

Summary The epithelial surface of the bovine Fallopian tube has been studied with respect to its ultrastructure and alkaline phosphatase activity. The ciliated cells were found to be provided with cilia and microvilli. The cilia were placed in rows and were most frequent in the central area of each cell surface. The basal corpuscles were provided with indistinct rootlets. The microvilli occurred between the cilia, but showed the reverse distribution, being most frequent towards the periphery of the cell surface. As a rule a marked alkaline phosphatase activity could be revealed on the ciliated cell surface, presumably associated with the microvilli.The secretory cells were bulging over the surface of the ciliated cells. Theywere provided with irregular protrusions, which were as a rule long and slender during the follicular phase but shorter and bulkier during the luteal phase. No alkaline phosphatase activity was detected.This investigation was supported by a grant from the foundation Thérèse och Johan Anderssons Minne.     

Histomorphology of cervical and uterine epithelia of crab-eating macaque, Macaca fasciularis.

Cytological characteristics and pattern of distribution of different cell types in the epithelia of cervix and uterus of crab-eating macaque (Macaca fascicularis) in follicular and luteal phases of the menstrual cyclic and amenorrhea were studied. The cervix uteri and uterus exhibit remarkable structural differenes in the ciliated, secretory, and ciliated-secretory cells. Since the number of ciliated-sexretory cells in the uterus is higher than in the cervix. It is believed that they form an additional source for the secretion of uterine fluid during the menstrual cylce. Both ciliated and secretory cells undergo degeneration; extensive cytoplasmic vacuolation associated with pycnosis and disorganization of the nuclei encountered.     

Activation of NOTCH1 or NOTCH3 Signaling Skews Human Airway Basal Cell Differentiation toward a Secretory Pathway

Airway basal cells (BC) function as stem/progenitor cells capable of differentiating into the luminal ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. The objective of this study was to define the role of Notch signaling in regulating human airway BC differentiation into a pseudostratified mucociliated epithelium. Notch inhibition with γ-secretase inhibitors demonstrated Notch activation is essential for BC differentiation into secretory and ciliated cells, but more so for the secretory lineage. Sustained cell autonomous ligand independent Notch activation via lentivirus expression of the intracellular domain of each Notch receptor (NICD1-4) demonstrated that the NOTCH2 and 4 pathways have little effect on BC differentiation into secretory and ciliated cells, while activation of the NOTCH1 or 3 pathways has a major influence, with persistent expression of NICD1 or 3 resulting in a skewing toward secretory cell differentiation with a parallel decrease in ciliated cell differentiation. These observations provide insights into the control of the balance of BC differentiation into the secretory vs ciliated cell lineage, a balance that is critical for maintaining the normal function of the airway epithelium in barrier defense against the inhaled environment.     

Changes in the surface morphology of the rabbit endometrium related to the estrous and progestational stages of the reproductive cycle

Summary Changes occurring on the surface of the uterine luminal epithelium of the rabbit during the estrous and progestational stages of the reproductive cycle were examined by scanning and transmission electron microscopy. The findings demonstrate that the uterine epithelium, or endometrium, contains two cell types: ciliated cells and nonciliated, microvillous cells. In estrous animals, ciliated cells, although not very numerous, were usually observed in small groups. However, at increasing intervals of time following mating, ciliated cells progressively disappeared from the endometrium until approximately eight to ten days post coitum, when they became scare. From several hours to four to five days following mating, extensive changes occurred on the surfaces of microvillous cells. When observed by TEM, these elements contained organelles typical of cells involved in the synthesis and secretion of glycoproteins. Furthermore, microvillous cells during this period displayed numerous apical protrusions of different sizes and shapes and containing material of varying electron density. Parallel SEM examinations of the same material confirmed the presence of these protrusions. Some of the protrusions appeared as spheroidal masses attached to the cytoplasm by means of a cytoplasmic strand. Other surface masses were clearly unattached to the cell surface and were distributed (1) on the surface of microvillous cells, (2) on the cilia of adjacent ciliated cells, and (3) on the surface of spermatozoa.Changes occurring on the luminal surface during the early postcoital period are interpreted as an expression of morphodynamic processes likely involving coupled secretion (exocytosis) and resorption (endocytosis) of luminal material. The observations presented here also demonstrate that between six and ten days post coitum, the rabbit endometrium contained increasing numbers of enlarged, nonciliated cells that probably arose by the fusion of smaller, microvillous elements.The work reported here was supported by C.N.R. contracts No. CT 760128809 and CT 77014239 (to P.M.) and NIH. Grant HD-04274 (to J.V.B.)     

TGFβ1 induces growth arrest and apoptosis but not ciliated cell differentiation in rat tracheal epithelial cell cultures

Summary We are studying the regulation of ciliated cell differentiation using an in vitro model of tracheal regeneration. Previously, we reported that removal of growth stimulating compounds such as epidermal growth factor (EGF) and cholera toxin reduced DNA synthesis and cell number while increasing ciliated cell differentiation (Clark et al., 1995). This result suggested that the induction of growth arrest may stimulate terminal differentiation of airway epithelial cells into ciliated cells. Transforming growth factor βs (TGFβs) inhibit epithelial cell proliferation and have also been shown to stimulate epithelial cell differentiation. In this study, the effect of TGFβ₁ on growth and ciliated cell differentiation of rat tracheal epithelial (RTE) cells was examined. TGFβ₁ inhibited [³H]thymidine incorporation by RTE cells in a dose-dependent manner. A 40% inhibition was observed after a 24-h incubation with 10 pM TGFβ₁. Continuous treatment with TGFβ₁ (1–50 pM) also reduced cell number during the time when ciliogenesis occurs. This reduction resulted in part from a loss of cells through exfoliation, in addition to the inhibition of proliferation. The exfoliated cells exhibited several morphological features characteristic of apoptosis, including shrunken cells, condensed and fragmented nuclei, and intact organelles. In addition, electrophoretic analysis of genomic DNA analysis isolated from exfoliated cells demonstrated the presence of a nucleosomal ladder. However, in contrast to the removal of EGF, treatment with TGFβ₁ for 7 d did not increase ciliated cell differentiation. TGFβ1 is, therefore, capable of inhibiting proliferation and increasing apoptosis in RTE cells without stimulating ciliated cell differentiation.