scholarly journals Human induced pluripotent stem cell-derived vocal fold mucosa mimics development and responses to smoke exposure

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Vlasta Lungova ◽  
Xia Chen ◽  
Ziyue Wang ◽  
Christina Kendziorski ◽  
Susan L. Thibeault
Keyword(s):  
Epithelial Cells ◽  
Vocal Fold ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
Mucosal Inflammation ◽  
Three Dimensional Models ◽  
Green Fluorescent

Abstract Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells’ limited proliferative capacity and absence of cell lines. Here we report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa. Furthermore, we demonstrate mucosal inflammation upon exposure of these constructs to 5% cigarette smoke extract. Upregulation of pro-inflammatory genes in epithelium and fibroblasts leads to aberrant VF mucosa remodeling. Collectively, our results demonstrate that hiPSC-derived VF mucosa is a versatile tool for future investigation of genetic and molecular mechanisms underlying epithelium-fibroblasts interactions in health and disease.

Developmental derivation of vocal fold mucosa from human induced pluripotent stem cells

2019 ◽  
Author(s):  
Vlasta Lungova ◽  
Susan Thibeault
Keyword(s):  
Epithelial Cells ◽  
Pluripotent Stem Cells ◽  
Vocal Fold ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Stratified Squamous Epithelium ◽  
Three Dimensional Models ◽  
Induced Pluripotent ◽  
Green Fluorescent

Abstract Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells’ limited proliferative capacity and absence of cell lines. We report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa.

scholarly journals Myosin-X functions in polarized epithelial cells

2012 ◽  
Vol 23 (9) ◽  
pp. 1675-1687 ◽  
Author(s):  
Katy C. Liu ◽  
Damon T. Jacobs ◽  
Brian D. Dunn ◽  
Alan S. Fanning ◽  
Richard E. Cheney
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Paracellular Permeability ◽  
Barrier Formation ◽  
Polarized Epithelial Cells ◽  
Green Fluorescent ◽  
Myosin X ◽  
Membrane Cell

Myosin-X (Myo10) is an unconventional myosin that localizes to the tips of filopodia and has critical functions in filopodia. Although Myo10 has been studied primarily in nonpolarized, fibroblast-like cells, Myo10 is expressed in vivo in many epithelia-rich tissues, such as kidney. In this study, we investigate the localization and functions of Myo10 in polarized epithelial cells, using Madin-Darby canine kidney II cells as a model system. Calcium-switch experiments demonstrate that, during junction assembly, green fluorescent protein–Myo10 localizes to lateral membrane cell–cell contacts and to filopodia-like structures imaged by total internal reflection fluorescence on the basal surface. Knockdown of Myo10 leads to delayed recruitment of E-cadherin and ZO-1 to junctions, as well as a delay in tight junction barrier formation, as indicated by a delay in the development of peak transepithelial electrical resistance (TER). Although Myo10 knockdown cells eventually mature into monolayers with normal TER, these monolayers do exhibit increased paracellular permeability to fluorescent dextrans. Importantly, knockdown of Myo10 leads to mitotic spindle misorientation, and in three-dimensional culture, Myo10 knockdown cysts exhibit defects in lumen formation. Together these results reveal that Myo10 functions in polarized epithelial cells in junction formation, regulation of paracellular permeability, and epithelial morphogenesis.

scholarly journals Targeting Mucosal Sites by Polymeric Immunoglobulin Receptor-directed Peptides

2002 ◽  
Vol 196 (4) ◽  
pp. 551-555 ◽  
Author(s):  
Kendra D. White ◽  
J. Donald Capra
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Secretory Component ◽  
In Vivo Studies ◽  
Polymeric Immunoglobulin Receptor ◽  
Immunoglobulin Receptor ◽  
Potential Binding Site ◽  
Green Fluorescent

Polymeric immunoglobulins provide first line humoral defense at mucosal surfaces to which they are specifically transported by the polymeric immunoglobulin receptor (pIgR) on mucosal and glandular epithelial cells. Previous studies from our laboratory suggested that amino acids 402–410 of the Cα3 domain of dimeric IgA (dIgA) represented a potential binding site for the pIgR. Here by binding human secretory component to overlapping decapeptides of Cα3, we confirm these residues and also uncover an additional site. Furthermore, we show that the Cα3 motif appears to be sufficient to direct transport of green fluorescent protein through the pIgR-specific cellular transcytosis system. An alternative approach identified phage peptides, selected from a library by the in vitro Madin Darby Canine Kidney transcytosis assay, for pIgR-mediated transport through epithelial cells. Some transcytosis-selected peptides map to the same 402–410 pIgR-binding Cα3 site. Further in vivo studies document that at least one of these peptides is transported in a rat model measuring hepatic bile transport. In addition to identifying small peptides that are both bound and transported by the pIgR, this study provides evidence that the pIgR-mediated mucosal secretion system may represent a means of targeting small molecule therapeutics and genes to mucosal epithelial cells.

scholarly journals Three-Dimensional in Vivo Imaging of Green Fluorescent Protein-Expressing T Cells in Mice with Noncontact Fluorescence Molecular Tomography

2007 ◽  
Vol 6 (2) ◽  
pp. 7290.2007.00007 ◽  
Author(s):  
Anikitos Garofalakis ◽  
Giannis Zacharakis ◽  
Heiko Meyer ◽  
Eleftherios N. Economou ◽  
Clio Mamalaki ◽  
...  
Keyword(s):  
T Cells ◽  
Green Fluorescent Protein ◽  
In Vivo Imaging ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Fluorescence Molecular Tomography ◽  
Green Fluorescent

Neural Development by Transplanted Human Embryonal Carcinoma Stem Cells Expressing Green Fluorescent Protein

2005 ◽  
Vol 14 (6) ◽  
pp. 339-351 ◽  
Author(s):  
R. Stewart ◽  
M. Lako ◽  
G. M. Horrocks ◽  
S. A. Przyborski
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Neural Development ◽  
Molecular Mechanisms ◽  
Embryonal Carcinoma ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Green Fluorescent

For many years, researchers have investigated the fate and potential of neuroectodermal cells during the development of the central nervous system. Although several key factors that regulate neural differentiation have been identified, much remains unknown about the molecular mechanisms that control the fate and specification of neural subtypes, especially in humans. Human embryonal carcinoma (EC) stem cells are valuable research tools for the study of neural development; however, existing in vitro experiments are limited to inducing the differentiation of EC cells into only a handful of cell types. In this study, we developed and characterized a novel EC cell line (termed TERA2.cl.SP12-GFP) that carries the reporter molecule, green fluorescent protein (GFP). We demonstrate that TERA2.cl.SP12-GFP stem cells and their differentiated neural derivatives constitutively express GFP in cells grown both in vitro and in vivo. Cellular differentiation does not appear to be affected by insertion of the transgene. We propose that TERA2.cl.SP12-GFP cells provide a valuable research tool to track the fate of cells subsequent to transplantation into alternative environments and that this approach may be particularly useful to investigate the differentiation of human neural tissues in response to local environmental signals.

scholarly journals Collagen-Like Glycoprotein BclS Is Involved in the Formation of Filamentous Structures of the Lysinibacillus sphaericus Exosporium

2014 ◽  
Vol 80 (21) ◽  
pp. 6656-6663 ◽  
Author(s):  
Ni Zhao ◽  
Yong Ge ◽  
Tingyu Shi ◽  
Xiaomin Hu ◽  
Zhiming Yuan
Keyword(s):  
Fluorescent Protein ◽  
Three Dimensional ◽  
Basal Layer ◽  
Lysinibacillus Sphaericus ◽  
Content Type ◽  
Section Electron ◽  
Wet Heat ◽  
Green Fluorescent ◽  
High Level

ABSTRACTLysinibacillus sphaericusproduces mosquitocidal binary toxins (Bin toxins) deposited within a balloon-like exosporium during sporulation. UnlikeBacillus cereusgroup strains, the exosporium ofL. sphaericusis usually devoid of the hair-like nap, an external filamentous structure formed by a collagen-like protein, BclA. In this study, a new collagen-like exosporium protein encoded by Bsph_0411 (BclS) fromL. sphaericusC3-41 was characterized. Thin-section electron microscopy revealed that deletion ofbclSresulted in the loss of the filamentous structures that attach to the exosporium basal layer and spread through the interspace of spores.In vivovisualization of BclS-green fluorescent protein (GFP)/mCherry fusion proteins revealed a dynamic pattern of fluorescence that encased the spore from the mother cell-distal (MCD) pole of the forespore, and the BclS-GFP fusions were found to be located in the interspace of the spore, as confirmed by three-dimensional (3D) superresolution fluorescence microscopy. Further studies demonstrated that thebclSmutant spores were more sensitive to wet-heat treatment and germinated at a lower rate than wild-type spores and that these phenotypes were significantly restored in thebclS-complemented strain. These results suggested novel roles of collagen-like protein in exosporium assembly and spore germination, providing a hint for a further understanding of the genetic basis of the high level of persistence of Bin toxins in nature.

scholarly journals Use of green fluorescent protein in visualisation of pneumococcal invasion of broncho-epithelial cells in vivo

2001 ◽  
Vol 194 (1) ◽  
pp. 105-110 ◽  
Author(s):  
Aras Kadioglu ◽  
Jason A. Sharpe ◽  
Irene Lazou ◽  
Catharina Svanborg ◽  
Colin Ockleford ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals In vitro cell and tissue models for studying host–microbe interactions: a review

2013 ◽  
Vol 109 (S2) ◽  
pp. S27-S34 ◽  
Author(s):  
Miriam Bermudez-Brito ◽  
Julio Plaza-Díaz ◽  
Luis Fontana ◽  
Sergio Muñoz-Quezada ◽  
Angel Gil
Keyword(s):  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Experimental Models ◽  
Dynamic Interactions ◽  
Basolateral Side ◽  
Microbe Interactions ◽  
Host Microbe Interactions

Ideally, cell models should resemble the in vivo conditions; however, in most in vitro experimental models, epithelial cells are cultivated as monolayers, in which the establishment of functional epithelial features is not achieved. To overcome this problem, co-culture experiments with probiotics, dendritic cells and intestinal epithelial cells and three-dimensional models attempt to reconcile the complex and dynamic interactions that exist in vivo between the intestinal epithelium and bacteria on the luminal side and between the epithelium and the underlying immune system on the basolateral side. Additional models include tissue explants, bioreactors and organoids. The present review details the in vitro models used to study host–microbe interactions and explores the new tools that may help in understanding the molecular mechanisms of these interactions.

scholarly journals Influences on neural lineage and mode of division in the zebrafish retina in vivo

2005 ◽  
Vol 171 (6) ◽  
pp. 991-999 ◽  
Author(s):  
Lucia Poggi ◽  
Marta Vitorino ◽  
Ichiro Masai ◽  
William A. Harris
Keyword(s):  
Cell Fate ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Time Lapse ◽  
Retinal Ganglion ◽  
Fixed Tissue ◽  
Environmental Signals ◽  
Neural Lineage ◽  
Green Fluorescent

Cell determination in the retina has been under intense investigation since the discovery that retinal progenitors generate clones of apparently random composition (Price, J., D. Turner, and C. Cepko. 1987. Proc. Natl. Acad. Sci. USA. 84:156–160; Holt, C.E., T.W. Bertsch, H.M. Ellis, and W.A. Harris. 1988. Neuron. 1:15–26; Wetts, R., and S.E. Fraser. 1988. Science. 239:1142–1145). Examination of fixed tissue, however, sheds little light on lineage patterns or on the relationship between the orientation of division and cell fate. In this study, three-dimensional time-lapse analyses were used to trace lineages of retinal progenitors expressing green fluorescent protein under the control of the ath5 promoter. Surprisingly, these cells divide just once along the circumferential axis to produce two postmitotic daughters, one of which becomes a retinal ganglion cell (RGC). Interestingly, when these same progenitors are transplanted into a mutant environment lacking RGCs, they often divide along the central-peripheral axis and produce two RGCs. This study provides the first insight into reproducible lineage patterns of retinal progenitors in vivo and the first evidence that environmental signals influence the orientation of cell division and the lineage of neural progenitors.

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