scholarly journals Detargeting Lentiviral-Mediated CFTR Expression in Airway Basal Cells Using miR-106b

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1169
Author(s):  
Soon H. Choi ◽  
Rosie E. Reeves ◽  
Guillermo S. Romano Ibarra ◽  
Thomas J. Lynch ◽  
Weam S. Shahin ◽  
...  
Keyword(s):  
Lentiviral Vector ◽  
Cell Types ◽  
Genome Integrity ◽  
Target Sequence ◽  
Flock House Virus ◽  
Basal Cells ◽  
Viral Production ◽  
Rnai Suppressor ◽  
Air Liquid Interface ◽  
Columnar Cells

Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However, CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR, suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein, we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production, obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air–liquid interface and organoid cultures, the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium.

Innate immune responses of human tracheal epithelium toPseudomonas aeruginosaflagellin, TNF-α, and IL-1β

2006 ◽  
Vol 290 (3) ◽  
pp. C678-C690 ◽  
Author(s):  
Jill Tseng ◽  
Jiun Do ◽  
Jonathan H. Widdicombe ◽  
Terry E. Machen
Keyword(s):  
Immune Responses ◽  
Tight Junctions ◽  
Inflammatory Responses ◽  
Cell Types ◽  
Innate Immune ◽  
Apical Surface ◽  
Basal Cells ◽  
Innate Immune Responses ◽  
Tnf Α ◽  
Columnar Cells

We measured innate immune responses by primary human tracheal epithelial (HTE) cells grown as confluent, pseudostratified layers during exposure to inflammatory activators on apical vs. basolateral surfaces. Apical Pseudomonas aeruginosa strain PAK (but not flagellin mutant PAK·fliC), flagellin, and flagellin + PAK·fliC activated NF-κB and IL-8 expression and secretion. In contrast, HTE cells were insensitive to LPS compared to flagellin. Flagellin activated NF-κB in columnar but not basal cells. IL-1β + TNF-α elicited responses similar to those of flagellin. Basolateral flagellin or IL-1β + TNF-α caused 1.5- to 4-fold larger responses, consistent with the fact that NF-κB activation occurred in both columnar and basal cells. MyD88 (toll receptor-associated adapter), IL-1 receptor (IL1R)1, and TNF-α receptor (TNFR)1 were expressed in columnar and basal cells. ZO-1 was localized to tight junctions of columnar cells but not to basal cells. We infer the following. 1) Flagellin is necessary and sufficient to trigger inflammatory responses in columnar cells during accumulation of P. aeruginosa in the airway surface liquid (ASL); columnar cells express toll-like receptor 5 and MyD88, often associated with flagellin-activated cell signaling. 2) IL-1β + TNF-α in the ASL also activate columnar cells, and these cells also express IL1R1 and TNFR1. 3) Apical flagellin, IL-1β, and TNF-α do not activate basal cells because tight junctions between columnar cells prevent access from the apical surface to the basal cells. 4) Exposure of basolateral surfaces to inflammatory activators elicits larger responses because both columnar and basal cells are activated, likely because both cell types express receptors for flagellin, IL-1β, and TNF-α.

The structure of the gills of the axolotl, Ambystoma mexicanum

1987 ◽  
Vol 45 ◽  
pp. 824-825
Author(s):  
Mohinder S. Jarial
Keyword(s):  
Leydig Cells ◽  
Secretory Granules ◽  
Light And Electron Microscopy ◽  
Cell Types ◽  
Ambystoma Mexicanum ◽  
Basal Cells ◽  
Granular Cells ◽  
Gill Filaments ◽  
Mitotic Figures ◽  
Apical Membranes

The axolotl is a strictly aquatic salamander in which the larval external gills are retained throughout life. The external gills of the adult axolotl have been studied by light and electron microscopy for ultrastructural evidence of ionic transport. The thin epidermis of the gill filaments and gill stems is composed of 3 cell types: granular cells, the basal cells and a sparce population of intervening Leydig cells. The gill epidermis is devoid of muscles, and no mitotic figures were observed in any of its cells.The granular cells cover the gill surface as a continuous layer (Fig. 1, G) and contain secretory granules of different forms, located apically (Figs.1, 2, SG). Some granules are found intimately associated with the apical membrane while others fuse with it and release their contents onto the external surface (Fig. 3). The apical membranes of the granular cells exhibit microvilli which are covered by a PAS+ fuzzy coat, termed “glycocalyx” (Fig. 2, MV).

scholarly journals An atlas of cell types in the mammalian epididymis and vas deferens

2020 ◽  
Author(s):  
Vera D. Rinaldi ◽  
Elisa Donnard ◽  
Kyle J. Gellatly ◽  
Morten Rasmussen ◽  
Alper Kucukural ◽  
...  
Keyword(s):  
Vas Deferens ◽  
Immune Cell ◽  
Cell Types ◽  
Receptor Expression ◽  
Basal Cells ◽  
Regional Specialization ◽  
Clear Cells ◽  
Principal Cells ◽  
Mammalian Sperm ◽  
Genomic Clusters

ABSTRACTFollowing spermatogenesis in the testis, mammalian sperm continue to mature over the course of approximately 10 days as they transit a long epithelial tube known as the epididymis. The epididymis is comprised of multiple segments/compartments that, in addition to concentrating sperm and preventing their premature activation, play key roles in remodeling the protein, lipid, and RNA composition of maturing sperm. In order to understand the complex roles for the epididymis in reproductive biology, we generated a single cell atlas of gene expression from the murine epididymis and vas deferens. We recovered all the key cell types of the epididymal epithelium, including principal cells, clear cells, and basal cells, along with associated support cells that include fibroblasts, smooth muscle, macrophages and other immune cells. Moreover, our data illuminate extensive regional specialization of principal cell populations across the length of the epididymis, with a substantial fraction of segment-specific genes localized in genomic clusters of functionally-related genes. In addition to the extensive region-specific specialization of principal cells, we find evidence for functionally-specialized subpopulations of stromal cells, and, most notably, two distinct populations of clear cells. Analysis of ligand/receptor expression reveals a network of potential cellular signaling connections, with several predicted interactions between cell types that may play roles in immune cell recruitment and other aspects of epididymal function. Our dataset extends on existing knowledge of epididymal biology, and provides a wealth of information on potential regulatory and signaling factors that bear future investigation.

Regeneration of mammary glands in vivo from isolated mammary ducts

Development ◽  
1986 ◽  
Vol 96 (1) ◽  
pp. 229-243
Author(s):  
E. Jane Ormerod ◽  
Philip S. Rudland
Keyword(s):  
Epithelial Cells ◽  
Milk Fat ◽  
Cell Types ◽  
Mammary Glands ◽  
Myoepithelial Cells ◽  
Basal Cells ◽  
Alveolar Cells ◽  
Terminal End Buds ◽  
Mammary Cell

Rat mammary ducts, free of buds, can alone regenerate complete mammary trees when transplanted into the interscapular fat pads of syngeneic host rats. All the main mammary cell types are identified within such outgrowths. Epithelial cells, which show the presence of milk fat globule membrane antigens and microvilli on their luminal surfaces, line the ducts. Basal cells surrounding the ducts show characteristic features of myoepithelial cells: immunoreactive actin and keratin within the cytoplasm, myofilaments, pinocytotic vesicles and hemidesmosomal attachments to the basement membrane. Cells within the end buds and lateral buds, however, show few if any cytoplasmic myofilaments and are relatively undifferentiated in appearance. Intermediate morphologies between these cells and myoepithelial cells are seen nearer the ducts. In this respect they exactly resemble the cap cells found in terminal end buds (TEBs) of normal mammary glands. Occasional epithelial cells within alveolar buds show the presence of immunoreactive casein, which is a product of secretory alveolar cells in the normal rat mammary gland. Dissected terminal end buds can regenerate similar ductal outgrowths. Thus, ductal tissue alone can generate all the major mammary cell types seen in the normal gland, including the cap cells.

scholarly journals Isolation, Maintenance and Differentiation of Primary Tracheal Basal Cells from Adult Rhesus Macaque

2019 ◽  
Vol 2 (4) ◽  
pp. 79
Author(s):  
Anna E. Engler ◽  
Gustavo Mostoslavsky ◽  
Lisa Miller ◽  
Jason R. Rock
Keyword(s):  
Rhesus Macaque ◽  
Rhesus Macaques ◽  
Enzymatic Digestion ◽  
Basal Cells ◽  
Early Passage ◽  
Long Term Storage ◽  
Air Liquid Interface ◽  
Nonadherent Cells ◽  
Level 2

In this report, we describe methodologies for the isolation and culture of primary rhesus macaque tracheal basal cells, their cryopreservation, long term storage and differentiation. These are comparable to state-of-the-art protocols that have been developed for mouse and human airway basal cells. This method is based on the use of proprietary media, providing an easily reproducible and applicable protocol for usage in biosafety level 2 (BSL2) settings. Tracheas from rhesus macaques were isolated after animal euthanasia and subjected to enzymatic digestion overnight. Cells of the epithelial layer were scraped off of the trachea for cell culture. Twenty-four hours after plating basal cells had attached and nonadherent cells were removed. First passages of basal cells can be frozen for early passage storage in liquid nitrogen or propagated and differentiated on an air–liquid interface and in a tracheosphere assay up to passage seven. This protocol provides a platform for the analysis of basal cells from a close evolutionary relative to humans.

scholarly journals The LIM Domain Protein Lmo4 Is Highly Expressed in Proliferating Mouse Epithelial Tissues

2005 ◽  
Vol 53 (4) ◽  
pp. 475-486 ◽  
Author(s):  
Eleanor Y.M. Sum ◽  
Lorraine A. O'Reilly ◽  
Nadeen Jonas ◽  
Geoffrey J. Lindeman ◽  
Jane E. Visvader
Keyword(s):  
Small Intestine ◽  
Mammary Gland ◽  
Cell Types ◽  
Mammary Epithelial ◽  
Specific Cell ◽  
Basal Cells ◽  
Proliferating Cells ◽  
Terminal End Buds ◽  
Mouse Tissues ◽  
Important Regulator

LMO4 belongs to the LIM-only family of zinc finger proteins that have been implicated in oncogenesis. The LMO4 gene is overexpressed in breast cancer and oral cavity carcinomas, and high levels of this protein inhibit mammary epithelial differentiation. Targeted deletion of Lmo4 in mice leads to complex phenotypic abnormalities and perinatal lethality. To further understand the role of LMO4, we have characterized Lmo4 expression in adult mouse tissues by immunohistochemical staining using monoclonal anti-Lmo4 antibodies. Lmo4 was highly expressed within specific cell types in diverse tissues. Expression was prevalent in epithelial-derived tissues, including the mammary gland, tongue, skin, small intestine, lung, and brain. High levels of Lmo4 were frequently observed in proliferating cells, such as the crypt cells of the small intestine and the basal cells of the skin and tongue. Lmo4 was highly expressed in the proliferative cap cell layer of the terminal end buds in the peripubertal mammary gland and in the lobuloalveolar units during pregnancy. The expression profile of Lmo4 suggests that this cofactor is an important regulator of epithelial proliferation and has implications for its role in the pathogenicity of cancer.

scholarly journals Integrin VLA-3: ultrastructural localization at cell-cell contact sites of human cell cultures.

1989 ◽  
Vol 109 (4) ◽  
pp. 1807-1815 ◽  
Author(s):  
R Kaufmann ◽  
D Frösch ◽  
C Westphal ◽  
L Weber ◽  
C E Klein
Keyword(s):  
Human Cell ◽  
Cultured Cells ◽  
Cell Types ◽  
Cell Surface Receptor ◽  
Cell Contact ◽  
Type I ◽  
Intercellular Contact ◽  
Basal Cells ◽  
Contact Sites ◽  
Cell Cell

The integrin VLA-3 is a cell surface receptor, which binds to fibronectin, laminin, collagen type I and VI (Takada, Y., E. A. Wayner, W. G. Carter, and M. E. Hemler. 1988. J. Cell. Biochem. 37:385-393) and is highly expressed in substrate adherent cultures of almost all human cell types. The ligand specificity of VLA-3 and the inhibition of cell adhesion by anti-VLA-3 monoclonal antibodies suggest its involvement in cell-substrate interaction. In normal tissues, VLA-3 is restricted to few cell types, notably the kidney glomeruli and basal cells of the epidermis. In the epidermis, VLA-3 is generally strongly expressed on the entire plasma membrane of basal cells and is not polarized towards the basement membrane (Klein, C. E., C. Cardon-Cardo, R. Soehnchen, R. J. Cote, H. F. Oettgen, M. Eisinger, and L. J. Old. 1987. J. Invest. Dermatol. 89:500-507). Based on this finding we speculated that, in addition to a role of VLA-3 for adhesion of cells to substrate, it could also be relevant for cell-cell interaction. To investigate this, we ultrastructurally localized VLA-3 on the surface of cultured cells by immunoelectron microscopy. In accordance with our concept, we found VLA-3 strongly associated with intercellular contact sites. Interestingly, very little immunoreactivity was detected at the under-surface of cells which had been cultured for 18-32 h. This observation was unexpected but is consistent with previous findings (Kantor, R. R. S., M. J. Mattes, K. D. Lloyd, L. J. Old, and A. P. Albino. 1987. J. Biol. Chem. 262:15158-15165) which suggest that the association of VLA-3 with the basal surface of substrate adherent tumor cells is a late event occurring after days of culture under confluent conditions. However, we cannot formally rule out VLA-3 expression at the undersurface of cells under our experimental conditions, since VLA-3 molecules at this location could be inaccessible for in situ labeling of unfixed cells because of spatial interferences. In conclusion, our results demonstrate the expression of VLA-3 at intercellular contact sites of cultured cells supporting the concept that it may be relevant for intercellular interactions also.

scholarly journals Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat

1997 ◽  
Vol 273 (5) ◽  
pp. C1549-C1561 ◽  
Author(s):  
Søren Nielsen ◽  
Landon S. King ◽  
Birgitte Mønster Christensen ◽  
Peter Agre
Keyword(s):  
Plasma Membranes ◽  
Fluid Transport ◽  
Water Movement ◽  
Upper Airway ◽  
Apical Plasma Membrane ◽  
Type I ◽  
Basal Cells ◽  
Basolateral Membranes ◽  
Surface Liquid ◽  
Columnar Cells

The molecular pathways for fluid transport in pulmonary, oral, and nasal tissues are still unresolved. Here we use immunocytochemistry and immunoelectron microscopy to define the sites of expression of four aquaporins in the respiratory tract and glandular epithelia, where they reside in distinct, nonoverlapping sites. Aquaporin-1 (AQP1) is present in apical and basolateral membranes of bronchial, tracheal, and nasopharyngeal vascular endothelium and fibroblasts. AQP5 is localized to the apical plasma membrane of type I pneumocytes and the apical plasma membranes of secretory epithelium in upper airway and salivary glands. In contrast, AQP3 is present in basal cells of tracheal and nasopharyngeal epithelium and is abundant in basolateral membranes of surface epithelial cells of nasal conchus. AQP4 resides in basolateral membranes of columnar cells of bronchial, tracheal, and nasopharyngeal epithelium; in nasal conchus AQP4 is restricted to basolateral membranes of a subset of intra- and subepithelial glands. These sites of expression suggest that transalveolar water movement, modulation of airway surface liquid, air humidification, and generation of nasopharyngeal secretions involve a coordinated network of aquaporin water channels.

scholarly journals Cell–cell Interaction Underlies Formation of Fluid in the Male Reproductive Tract of the Rat

2005 ◽  
Vol 125 (5) ◽  
pp. 443-454 ◽  
Author(s):  
King-ho Cheung ◽  
George P.H. Leung ◽  
Matthew C.T. Leung ◽  
Winnie W.C. Shum ◽  
Wen-liang Zhou ◽  
...  
Keyword(s):  
Reproductive Tract ◽  
Cell Types ◽  
Cell Interaction ◽  
Fluorescence Measurement ◽  
Basal Cells ◽  
Anion Secretion ◽  
Cell Monolayers ◽  
Principal Cells ◽  
Whole Cell Patch Clamp ◽  
Confluent Cell

The epithelia lining the epididymides of many species consists of several cell types. We have provided evidence that the basal cells are essential to the integrated functions of the epithelium. Basal cells, but not principal cells, and other cells in the epididymis express TRPC3 and COX-1. We have isolated basal cells from intact rat epididymis using antibody-coated Dynabeads and subjected them to whole-cell patch-clamp measurement of nonselective cation channel activity, a feature of TRPC3 protein, and Fluo-3 fluorescence measurement of intracellular Ca2+ concentration. The results show that a nonselective cation current blockable by La3+ (0.1 mM), Gd3+ (0.1 mM), or SKF96365 (20 μM) could be activated by lysylbradykinin (200 nM). In cells loaded with Fluo-3, addition of lysylbradykinin (100 nM) caused a sustained increase of intracellular Ca2+. This effect was blocked by Gd3+ (0.1 mM) or SKF96365 (20 μM) and was not observed in Fluo-3–loaded principal cells. Stimulation of basal cell/principal cell cocultures with lysylbradykinin (200 nM) evoked in principal cells a current with CFTR-Cl− channel characteristics. Isolated principal cells in the absence of basal cells did not respond to lysylbradykinin but responded to PGE2 (100 nM) with activation of a CFTR-like current. Basal cells, but not principal cells, released prostaglandin E2 when stimulated with lysylbradykinin (100 nM). The release was blocked by SKF96365 (20 μM) and BAPTA-AM (0.05 or 0.1 mM). Confluent cell monolayers harvested from a mixture of disaggregated principal cells and basal cells responded to lysylbradykinin (100 nM) and PGE2 (500 nM) with an increase in electrogenic anion secretion. The former response was dependent on prostaglandin synthesis as piroxicam blocked the response. However, cell cultures obtained from principal cells alone responded to PGE2 but not to bradykinin. These results support the notion that basal cells regulate principal cells through a Ca2+ and COX signaling pathway.

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