Protein Substrate Alters Cell Physiology in Primary Culture of Vocal Fold Epithelial Cells

2021 ◽  
pp. 1-14
Author(s):  
Emily E. Kimball ◽  
Lea Sayce ◽  
Xiaochuan C. Xu ◽  
Chase M. Kruszka ◽  
Bernard Rousseau
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Vocal Fold ◽  
Phase Contrast ◽  
Mesenchymal Cell ◽  
Mesenchymal Cells ◽  
Collagen Iv ◽  
Collagen I ◽  
Protein Substrate

The basement membrane interacts directly with the vocal fold epithelium. Signaling between the basement membrane and the epithelium modulates gene regulation, differentiation, and proliferation. The purpose of this study was to identify an appropriate simple single-protein substrate for growth of rabbit vocal fold epithelial cells. Vocal folds from 3 New Zealand white rabbits (<i>Oryctolagus cuniculus</i>) were treated to isolate epithelial cells, and cells were seeded onto cell culture inserts coated with collagen I, collagen IV, laminin, or fibronectin. Transepithelial electrical resistance (TEER) was measured, and phase contrast microscopy, PanCK, CK14, and E-cadherin immunofluorescence were utilized to assess for epithelial cell-type characteristics. Further investigation via immunofluorescence labeling was conducted to assess proliferation (Ki67) and differentiation (Vimentin). There was a significant main effect of substrate on TEER, with collagen IV eliciting the highest, and laminin the lowest resistance. Assessment of relative TEER across cell lines identified a larger range of TEER in collagen I and laminin. Phase contrast imaging identified altered morphology in the laminin condition, but cell layer depth did not appear to be related to TEER, differentiation, or morphology. Ki67 staining additionally showed no significant difference in proliferation. All conditions had confluent epithelial cells and dispersed mesenchymal cells, with increased mesenchymal cell numbers over time; however, a higher proportion of mesenchymal cells was observed in the laminin condition. The results suggest collagen IV is a preferable basement membrane substrate for in vitro vocal fold epithelial primary cell culture, providing consistent TEER and characteristic cell morphology, and that laminin is an unsuitable substrate for vocal fold epithelial cells and may promote mesenchymal cell proliferation.

Role of laminin polymerization at the epithelial mesenchymal interface in bronchial myogenesis

Development ◽  
1998 ◽  
Vol 125 (14) ◽  
pp. 2621-2629 ◽  
Author(s):  
Y. Yang ◽  
K.C. Palmer ◽  
N. Relan ◽  
C. Diglio ◽  
L. Schuger
Keyword(s):  
Smooth Muscle ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Mesenchymal Cell ◽  
Cell Types ◽  
Mesenchymal Cells ◽  
Cell Contact ◽  
Alpha Actin ◽  
Muscle Myosin

Undifferentiated mesenchymal cells were isolated from mouse embryonic lungs and plated at subconfluent and confluent densities. During the first 5 hours in culture, all the cells were negative for smooth muscle markers. After 24 hours in culture, the mesenchymal cells that spread synthesized smooth muscle alpha-actin, muscle myosin, desmin and SM22 in levels comparable to those of mature smooth muscle. The cells that did not spread remained negative for smooth muscle markers. SM differentiation was independent of cell-cell contact or proliferation. In additional studies, undifferentiated lung mesenchymal cells were cocultured with lung embryonic epithelial cells at high density. The epithelial cells aggregated into cysts surrounded by mesenchymal cells and a basement membrane was formed between the two cell types. In these cocultures, the mesenchymal cells in contact with the basement membrane spread and differentiated into smooth muscle. The rest of the mesenchymal cells remained round and negative for smooth muscle markers. Inhibition of laminin polymerization by an antibody to the globular regions of laminin beta1/gamma1 chains blocked basement membrane assembly, mesenchymal cell spreading and smooth muscle differentiation. These studies indicated that lung embryonic mesenchymal cells have the potential to differentiate into smooth muscle and the process is triggered by their spreading along the airway basement membrane.

scholarly journals Extracellular chloride signals collagen IV network assembly during basement membrane formation

2016 ◽  
Vol 213 (4) ◽  
pp. 479-494 ◽  
Author(s):  
Christopher F. Cummings ◽  
Vadim Pedchenko ◽  
Kyle L. Brown ◽  
Selene Colon ◽  
Mohamed Rafi ◽  
...  
Keyword(s):  
Cell Culture ◽  
Basement Membrane ◽  
Collagen Iv ◽  
Atomic Level ◽  
Basement Membranes ◽  
Tissue Architecture ◽  
Dynamic Interactions ◽  
Conformational Switch ◽  
And Function ◽  
Fundamental Mechanism

Basement membranes are defining features of the cellular microenvironment; however, little is known regarding their assembly outside cells. We report that extracellular Cl− ions signal the assembly of collagen IV networks outside cells by triggering a conformational switch within collagen IV noncollagenous 1 (NC1) domains. Depletion of Cl− in cell culture perturbed collagen IV networks, disrupted matrix architecture, and repositioned basement membrane proteins. Phylogenetic evidence indicates this conformational switch is a fundamental mechanism of collagen IV network assembly throughout Metazoa. Using recombinant triple helical protomers, we prove that NC1 domains direct both protomer and network assembly and show in Drosophila that NC1 architecture is critical for incorporation into basement membranes. These discoveries provide an atomic-level understanding of the dynamic interactions between extracellular Cl− and collagen IV assembly outside cells, a critical step in the assembly and organization of basement membranes that enable tissue architecture and function. Moreover, this provides a mechanistic framework for understanding the molecular pathobiology of NC1 domains.

scholarly journals Single-Cell RNA-seq Identifies Cell Diversity in Embryonic Salivary Glands

2019 ◽  
Vol 99 (1) ◽  
pp. 69-78 ◽  
Author(s):  
R. Sekiguchi ◽  
D. Martin ◽  
K.M. Yamada ◽  
Keyword(s):  
Epithelial Cells ◽  
Parotid Gland ◽  
Single Cell ◽  
Salivary Glands ◽  
Cell Fate ◽  
Mesenchymal Cell ◽  
Muscle Cells ◽  
Mesenchymal Cells ◽  
Organ Specificity ◽  
Bud Initiation

Branching organs, including the salivary and mammary glands, lung, and kidney, arise as epithelial buds that are morphologically very similar. However, the mesenchyme is known to guide epithelial morphogenesis and to help govern cell fate and eventual organ specificity. We performed single-cell transcriptome analyses of 14,441 cells from embryonic day 12 submandibular and parotid salivary glands to characterize their molecular identities during bud initiation. The mesenchymal cells were considerably more heterogeneous by clustering analysis than the epithelial cells. Nonetheless, distinct clusters were evident among even the epithelial cells, where unique molecular markers separated presumptive bud and duct cells. Mesenchymal cells formed separate, well-defined clusters specific to each gland. Neuronal and muscle cells of the 2 glands in particular showed different markers and localization patterns. Several gland-specific genes were characteristic of different rhombomeres. A muscle cluster was prominent in the parotid, which was not myoepithelial or vascular smooth muscle. Instead, the muscle cluster expressed genes that mediate skeletal muscle differentiation and function. Striated muscle was indeed found later in development surrounding the parotid gland. Distinct spatial localization patterns of neuronal and muscle cells in embryonic stages appear to foreshadow later differences in adult organ function. These findings demonstrate that the establishment of transcriptional identities emerges early in development, primarily in the mesenchyme of developing salivary glands. We present the first comprehensive description of molecular signatures that define specific cellular landmarks for the bud initiation stage, when the neural crest–derived ectomesenchyme predominates in the salivary mesenchyme that immediately surrounds the budding epithelium. We also provide the first transcriptome data for the largely understudied embryonic parotid gland as compared with the submandibular gland, focusing on the mesenchymal cell populations.

scholarly journals Epithelial HVEM promotes basement membrane synthesis and intraepithelial T cell survival and migration

2020 ◽  
Author(s):  
Daisuke Takahashi ◽  
Qingyang Wang ◽  
Goo-Young Seo ◽  
Jr-wen Shui ◽  
Zbigniew Mikulski ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Basement Membrane ◽  
Collagen Iv ◽  
Membrane Synthesis ◽  
Basement Membrane Synthesis ◽  
And Migration ◽  
Membrane Components ◽  
T Cell Survival ◽  
Lymphocyte Populations

SummaryIntraepithelial T cells (IET) provide continuous surveillance of the intestinal epithelium, but little was known about how epithelial-derived signals regulate the IET population. We show that epithelial expression of the herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily (TNFRSF), maintained the survival of small intestine IET, especially innate-like TCRαβ+ cells lacking CD4 and CD8β. Patrolling movement of all CD8α+ IET also was impaired in the absence of HVEM. HVEM-deficient epithelial cells exhibited downregulation of synthesis of basement membrane components, including collagen IV. Collagen IV supported IET survival in vitro via interactions with β1 integrins expressed by the IET; absence of β1 integrins decreased some IET subsets. Therefore, these data define a circuit whereby epithelial cells regulate intestine resident T lymphocyte populations through basement membrane synthesis.

The cellular origin of fibronectin in the basement membrane zone of developing tooth

Development ◽  
1986 ◽  
Vol 95 (1) ◽  
pp. 73-80
Author(s):  
Kirsti Hurmerinta ◽  
Pentti Kuusela ◽  
Irma Thesleff
Keyword(s):  
Basement Membrane ◽  
Mesenchymal Cell ◽  
Mesenchymal Cells ◽  
Basement Membrane Zone ◽  
Enamel Organ ◽  
Immunofluorescent Localization ◽  
Cellular Origin ◽  
Chicken Tissue ◽  
Cellular Source ◽  
Species Specific

The cellular source of fibronectin in the dental epitheliomesenchymal interface was studied in interspecies combinations of mouse and quail tissue. Species-specific fibronectin antibodies were produced by immunizing rabbits with purified mouse or chicken fibronectin and by absorbing both antisera with purified heterologous fibronectin and insoluble tissue extract. The absorbed antisera to mouse and chicken fibronectin showed fluorescent staining only in mouse and chicken tissue sections, respectively, but not vice versa. When the mouse mesenchymal dental papilla was combined and cultured either with the mouse enamel organ or with the quail pharyngeal epithelium, mesenchymal cell differentiation was initiated and typical alignment of mesenchymal cells along the basement membrane was seen. Examination with transmission electron microscope revealed a typical bilaminar basal lamina with adherent fibrillar matrix on its mesenchymal aspect. Immunofluorescent localization of fibronectin with the mouse-specific fibronectin antiserum showed a brilliant staining in the mesenchymal tissue and in the basement membrane zone. When the chicken-specific fibronectin antiserum was used, no staining was detected in either tissue recombinations. We have suggested earlier that fibronectin in the dental basement membrane plays an important role during the differentiation of mesenchymal cells into odontoblasts. The present study demonstrates that fibronectin in the basement membrane of the developing tooth is produced exclusively by the differentiating mesenchymal cells.

scholarly journals Mesenchyme-mediated effect of dexamethasone on laminin in cocultures of embryonic gut epithelial cells and mesenchyme-derived cells

1992 ◽  
Vol 101 (1) ◽  
pp. 161-171 ◽  
Author(s):  
P. Simo ◽  
P. Simon-Assmann ◽  
C. Arnold ◽  
M. Kedinger
Keyword(s):  
Epithelial Cells ◽  
Mesenchymal Cell ◽  
Mesenchymal Cells ◽  
Skin Fibroblasts ◽  
Striking Difference ◽  
Intestinal Epithelial ◽  
Fetal Skin ◽  
Differentiation Markers ◽  
Enhanced Expression ◽  
Induced Changes

Previous studies have shown that glucocorticoids accelerate intestinal maturation and that this process is mediated by the mesenchymal cells. The possible involvement of laminin (LN), a basement membrane component, in this mesenchymal mediation has been analyzed. For this purpose, the influence of dexamethasone (DX) on the synthesis of LN, its chain composition and its cellular distribution has been examined biochemically and immunocytochemically in two different mesenchyme-derived cell populations, fetal intestinal mesenchymal cells and fetal skin fibroblasts, as well as in cocultures of intestinal endodermal cells seeded on top of confluent fetal skin fibroblasts. Neither the amount of metabolically labeled LN purified by affinity chromatography (expressed per mg cell proteins), nor the A versus B chain ratio monitored after separation on gel electrophoresis and immunoblotting, showed significant differences after 5 days of DX treatment. However, glucocorticoids induced a shift from secreted to cell-associated LN molecules paralleling a striking difference in the immunostaining pattern of intracellular and surface LN in the mesenchyme-derived cell monocultures; the granular intracytoplasmic LN staining in the control cultures was replaced by a fibrillar organization of LN molecules concomitantly with an increased accumulation at the cell surface. In 2-day DX-treated cocultures, there was an acceleration of LN deposition at the epithelial-fibroblastic interface, which accompanied the enhanced expression of epithelial cell differentiation markers (brush border digestive enzymes). These DX-induced changes can be blocked by the addition of anti-LN antibodies in the culture medium. These findings further support the concept that glucocorticoid action on intestinal epithelial cells involves alterations in the extracellular microenvironment, assessed here for LN molecules, occurring at the level of the mesenchymal cell compartment. These changes may contribute to an accelerated organization of LN at the epithelial-mesenchymal interface and subsequently to epithelial differentiation.

P–369 Three-dimensional co-culture of human endometrial cells with aneuploid and euploid embryos

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
S Amiri ◽  
F Amjadi ◽  
M Ashrafi ◽  
R Aflatoonian ◽  
A Akbar. Sene ◽  
...  
Keyword(s):  
Cell Culture ◽  
Epithelial Cells ◽  
Pregnancy Rate ◽  
Stromal Cells ◽  
Phase Contrast ◽  
Three Dimensional ◽  
3D Culture ◽  
Cell Type ◽  
Endometrial Cells ◽  
Culture Systems

Abstract Study question Is implantation different in euploid and aneuploid embryos? Summary answer By simulating the human endometrium using a three-dimensional scaffold, aneuploid embryos were unable to attach to the endometrial cells, while euploid embryos attached. What is known already Although embryo selection for transfer is usually based on morphology, 70% of embryos with high morphological quality have chromosomal abnormalities. The results of implantation and pregnancy rate assessments following Preimplantation Genetic Screening (PGS) are controversial. There is still no in vitro study to compare the implantation of human euploid and aneuploid embryos. Study design, size, duration After informed consent, 10 endometrial biopsies were taken from fertile women. For scaffolding, the stromal cells were resided within the matrix, after 24 hours, the epithelial cells were seeded on the scaffold. Cell culture continued for 5 days to reach the appropriate confluence. The embryos were also examined by performing PGS following CGH Array. 10 euploid and 10 aneuploid blastocysts were selected and co-cultured for 72 hours with the 3D structure of human endometrial cells. Participants/materials, setting, methods Endometrial cells were isolated and expanded in 2D cultures to achieve enough cells. The fibrin-agarose scaffold was made and stromal and epithelial cells were cultured into and on the scaffold, respectively. Then, cell proliferation was assessed by MTT assay. The simulated endometrial construct was confirmed by H&E and IHC. Partial hatching of blastocysts was performed using a laser system. The blastocyst’s attachment to the endometrial-like structure was examined under a phase-contrast microscope and SEM. Main results and the role of chance The MTT OD of scaffolds increased during 5 days of cell culture (P &lt; 0.05). The histological evaluation of the co-culture systems was done under light microscopy by H&E staining. On the top of the 3D culture system, epithelial cells shaped a constricted cell monolayer. Stromal cells combined with the fibrin-agarose scaffold got lengthened and expanded, displaying that the 3D culture systems supplied a suitable environment for the growth of endometrial cells. In the 3D culture, the origins and locations of epithelial and stromal cells were defined by cytokeratin and vimentin immunostaining, respectively. IHC for cytokeratin was only positive for epithelial cells in the surface epithelium. IHC for the vimentin was positive for the stromal cells located in the 3D matrix. These results showed that fibrin-agarose scaffold could simulate the human endometrial structure. Using SEM and phase-contrast microscopy, it was found that only euploid embryos were able to attach to the endometrial construct while aneuploid embryos weren’t. Limitations, reasons for caution Since the co-culture does not contain a unique cell type, and the MTT OD standard curve against cell number is specified for cell type, the number of growing cells in the co-culture cannot be calculated; therefore it is reported as OD. Wider implications of the findings: Our findings determined that PGS allows us to transfer top-quality embryos with higher implantation potential. It improves implantation and pregnancy rate during ART cycles, especially in patients with recurrent implantation failure. Trial registration number Not applicable

scholarly journals Requirement of basement membrane for the suppression of programmed cell death in mammary epithelium

1996 ◽  
Vol 109 (3) ◽  
pp. 631-642 ◽  
Author(s):  
S. Pullan ◽  
J. Wilson ◽  
A. Metcalfe ◽  
G.M. Edwards ◽  
N. Goberdhan ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Single Cells ◽  
Control Point ◽  
Collagen I ◽  
Cell Phenotype ◽  
Cell Contact ◽  
Isolated Cells

Apoptosis is an active mechanism of cell death required for normal tissue homeostasis. Cells require survival signals to avoid the engagement of apoptosis. In the mammary gland, secretory epithelial cells are removed by apoptosis during involution. This cell loss coincides with matrix metalloproteinase activation and basement membrane degradation. In this paper we describe studies that confer a new role for basement membrane in the regulation of cell phenotype. We demonstrate that the first passage epithelial cells isolated from pregnant mouse mammary gland die by apoptosis in culture, but that cell death is suppressed by basement membrane. The correct type of extracellular matrix was required, since only a basement membrane, not plastic or a collagen I matrix, lowered the rate of apoptosis. Attachment to a matrix per se was not sufficient for survival, since apoptotic cells were observed when still attached to a collagen I substratum. Experiments with individually isolated cells confirmed the requirement of basement membrane for survival, and demonstrated that survival is enhanced by cell-cell contact. A function-blocking anti-beta1 integrin antibody doubled the rate of apoptosis in single cells cultured with basement membrane, indicating that integrin-mediated signals contributed to survival. We examined the cell death-associated genes bcl-2 and bax in mammary epithelia, and found that although the expression of Bcl-2 did not correlate with cell survival, increased levels of Bax were associated with apoptosis. We propose that basement membrane provides a survival stimulus for epithelial cells in vivo, and that loss of interaction with this type of matrix acts as a control point for cell deletions that occur at specific times during development, such as in mammary gland involution.

Cardiac myocytes cultured on a basement membrane extract of the ehs tumor

1986 ◽  
Vol 44 ◽  
pp. 270-271
Author(s):  
L. Terracio ◽  
A. Dewey ◽  
K. Rubin ◽  
T.K. Borg
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Cardiac Myocytes ◽  
Normal Tissue ◽  
Cell Spreading ◽  
Collagen Iv ◽  
Basement Membrane Extract ◽  
Membrane Extract ◽  
Growth Development ◽  
Multicellular Organisms

The recognition and interaction of cells with the extracellular matrix (ECM) effects the normal physiology as well as the pathology of all multicellular organisms. These interactions have been shown to influence the growth, development, and maintenance of normal tissue function. In previous studies, we have shown that neonatal cardiac myocytes specifically interacts with a variety of ECM components including fibronectin, laminin, and collagens I, III and IV. Culturing neonatal myocytes on laminin and collagen IV induces an increased rate of both cell spreading and sarcomerogenesis.

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