Identifying distinct oxygen diffusivity through type I pneumocyte-like cell layers using microfluidic device

A number of human cytokeratins are expressed during the development of stratified epithelia from one-layered polar epithelia and continue to be expressed in several adult epithelial tissues. For studies of the regulation of the synthesis of stratification-related cytokeratins in internal tissues, we have prepared cDNA and genomic clones encoding cytokeratin 4, as a representative of the basic (type II) cytokeratin subfamily and cytokeratin 15, as representative of the acidic (type I) subfamily, and determined their nucleotide sequences. The specific expression of mRNAs encoding these two polypeptides in certain stratified tissues and cultured cell lines is demonstrated by Northern blot hybridization. Hybridization in situ with antisense riboprobes and/or synthetic oligonucleotides shows the presence of cytokeratin 15 mRNA in all layers of esophagus, whereas cytokeratin 4 mRNA tends to be suprabasally enriched, although to degrees varying in different regions. We conclude that the expression of the genes encoding these stratification-related cytokeratins starts already in the basal cell layer and does not depend on vertical differentiation and detachment from the basal lamina. Our results also show that simple epithelial and stratification-related cytokeratins can be coexpressed in basal cell layers of certain stratified epithelia such as esophagus. Implications of these findings for epithelial differentiation and the formation of squamous cell carcinomas are discussed.

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PCOLCE2Encodes a Functional Procollagen C-Proteinase Enhancer (PCPE2) That Is a Collagen-binding Protein Differing in Distribution of Expression and Post-translational Modification from the Previously Described PCPE1

Journal of Biological Chemistry ◽

10.1074/jbc.m209891200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 49820-49830 ◽

Cited By ~ 70

Author(s):

Barry M. Steiglitz ◽

Douglas R. Keene ◽

Daniel S. Greenspan

Keyword(s):

Expressed Sequence Tag ◽

Open Angle Glaucoma ◽

Type I ◽

Post Translational Modification ◽

Collagen Binding ◽

Type I Procollagen ◽

Morphogenetic Protein ◽

Fibrillar Collagens ◽

Cell Layers ◽

Novel Protein

The procollagen COOH-terminal proteinase enhancer (PCPE) is a glycoprotein that binds the COOH-terminal propeptide of type I procollagen and potentiates its cleavage by procollagen C-proteinases, such as bone morphogenetic protein-1 (BMP-1). Recently, sequencing of a human expressed sequence tag, which maps near the primary open angle glaucoma region on chromosome 3q21, showed it to encode a novel protein with only 43% identity with PCPE but with a similar domain structure. Here we show this novel protein to be a functional procollagen COOH-terminal proteinase enhancer with activity comparable with that of PCPE and thus propose the designations PCPE2 and PCPE1, respectively. PCPE2 is shown to have a much more limited distribution of expression than does PCPE1, with strong expression primarily in nonossified cartilage in developing tissues and at high levels in the adult heart. PCPE2 is shown to be a glycoprotein that differs markedly in the nature of its glycosylation from that of PCPE1. PCPE2 is also shown to have markedly stronger affinity for heparin than PCPE1, which may account for higher affinities for cell layers. Unexpectedly, both PCPE1 and PCPE2 were found to be collagen-binding proteins, capable of binding at multiple sites on the triple helical portions of fibrillar collagens and also capable of competing for such binding with procollagen C-proteinases. The latter observations may provide insights into the ways PCPEs affect the kinetics of the C-proteinase reaction and into the physical interactions that occur between procollagen C-proteinases and their substrates.

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Measurement of in-plane elasticity of live cell layers using a pressure sensor embedded microfluidic device

Scientific Reports ◽

10.1038/srep36425 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 5

Author(s):

Chien-Han Lin ◽

Chien-Kai Wang ◽

Yu-An Chen ◽

Chien-Chung Peng ◽

Wei-Hao Liao ◽

...

Keyword(s):

Microfluidic Device ◽

Pressure Sensor ◽

Plane Elasticity ◽

Live Cell ◽

Cell Layers

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Co-culture of type I and type II pneumocytes as a model of alveolar epithelium

10.1101/2021.03.08.434368 ◽

2021 ◽

Author(s):

Oliver Brookes ◽

Sonja Boland ◽

René Lai Kuen ◽

Armelle Baeza-Squiban

Keyword(s):

Cell Lines ◽

Barrier Function ◽

Single Cells ◽

Light And Electron Microscopy ◽

Alveolar Epithelium ◽

Type I ◽

Apical Surface ◽

Type Ii ◽

Cell Layers ◽

Distal Lung

AbstractThe epithelial tissues of the distal lung are continuously exposed to inhaled air, and are of research interest in studying respiratory exposure to both hazardous and therapeutic materials. Pharmaco-toxicological research depends on the development of sophisticated models of the alveolar epithelium, which better represent the different cell types present in the native lung and interactions between them.We developed an air-liquid interface (ALI) model of the alveolar epithelium which incorporates cell lines representative of both type I (NCI-H441) and type II (hAELVi) epithelial cells. We compared morphology of single cells and the structure of cell layers of the two lines using light and electron microscopy. Working both in monotypic cultures and cocultures, we measured barrier function by trans-epithelial electrical resistance (TEER), and demonstrated that barrier properties can be maintained for 30 days. We created a mathematical model of TEER development over time based on these data in order to make inferences about the interactions occurring in these culture systems. We assessed expression of a panel of relevant genes that play important roles in barrier function and differentiation.The coculture model was observed to form a stable barrier akin to that seen in hAELVi, while expressing surfactant protein C, and having a profile of expression of claudins and aquaporins appropriate for the distal lung. We described cavities which arise within stratified cell layers in NCI-H441 and cocultured cells, and present evidence that these cavities represent an aberrant apical surface. In summary, our results support the coculture of these two cell lines to produce a model which better represents the breadth of functions seen in native alveolar epithelium.

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Enhancement of outer cell layers of a methanotrophic bacterium using uranyl acetate en bloc

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102456 ◽

1988 ◽

Vol 46 ◽

pp. 74-75

Author(s):

K. Putzer ◽

T.A. Fassel ◽

C.C. Remsen

Keyword(s):

Uranyl Acetate ◽

Outer Cell ◽

Type I ◽

Type Ii ◽

En Bloc ◽

Wall Structures ◽

Intracytoplasmic Membranes ◽

Cell Layers ◽

Ethanol Series ◽

Autotrophic Microorganisms

Obligate methanotrophic bacteria are autotrophic microorganisms capable of oxidizing methane to supply both their carbon and energy requirements. Methanotrophs are placed in one of two groups based on certain biochemical features and the arrangement of intracytoplasmic membranes (ICM). Type I methanotrophs contain ICM arranged as bundles of disc-like vessicles in the interior of the cell, while Type II methanotrophs display paired peripheral ICM. The organism used in this study, Methylosinus trichosporium OB3b, is a Type II methanotroph and has a capsular structure surrounding the cell as well as several complex cell wall structures described as cup-like, filamentous, and spike-shaped. This study examines the external surface of M. trichosporium OB3b following en bloc staining with uranyl acetate.Cells were harvested in mid-log phase, fixed in 0.5% glutaraldehyde and postfixed in 1% OsO4. Cells were enrobed in 4% agar prior to dehydration in a graded ethanol series.

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Collagen expression, ultrastructural assembly, and mineralization in cultures of chicken embryo osteoblasts.

The Journal of Cell Biology ◽

10.1083/jcb.106.3.979 ◽

1988 ◽

Vol 106 (3) ◽

pp. 979-989 ◽

Cited By ~ 125

Author(s):

L C Gerstenfeld ◽

S D Chipman ◽

C M Kelly ◽

K J Hodgens ◽

D D Lee ◽

...

Keyword(s):

Extracellular Matrix ◽

Protein Synthesis ◽

Collagen Synthesis ◽

Collagen Fibrils ◽

Collagen Molecule ◽

Type I ◽

Mrna Levels ◽

Total Collagen ◽

Cell Layers ◽

Kinetics Of

A newly defined chick calvariae osteoblast culture system that undergoes a temporal sequence of differentiation of the osteoblast phenotype with subsequent mineralization (Gerstenfeld, L. C., S. Chipman, J. Glowacki, and J. B. Lian. 1987. Dev. Biol. 122:49-60) has been examined for the regulation of collagen synthesis, ultrastructural organization of collagen fibrils, and extracellular matrix mineralization. Collagen gene expression, protein synthesis, processing, and accumulation were studied in this system over a 30-d period. Steady state mRNA levels for pro alpha 1(I) and pro alpha 2 collagen and total collagen synthesis increased 1.2- and 1.8-fold, respectively, between days 3 and 12. Thereafter, total collagen synthesis decreased 10-fold while mRNA levels decreased 2.5-fold. In contrast to the decreasing protein synthesis after day 12, total accumulated collagen in the cell layers increased sixfold from day 12 to 30. Examination of the kinetics of procollagen processing demonstrated that there was a sixfold increase in the rate of procollagen conversion to alpha chains from days 3 to 30 and the newly synthesized collagen was more efficiently incorporated into the extracellular matrix at later culture times. The macrostructural assembly of collagen and its relationship to culture mineralization were also examined. High voltage electron microscopy demonstrated that culture cell layers were three to four cells thick. Each cell layer was associated with a layer of well developed collagen fibrils orthogonally arranged with respect to adjacent layers. Fibrils had distinct 64-70-nm periodicity typical of type I collagen. Electron opaque areas found principally associated with the deepest layers of the fibrils consisted of calcium and phosphorus determined by electron probe microanalysis and were identified by electron diffraction as a very poorly crystalline hydroxyapatite mineral phase. These data demonstrate for the first time that cultured osteoblasts are capable of assembling their collagen fibrils into a bone-specific macrostructure which mineralizes in a manner similar to that characterized in vivo. Further, this matrix maturation may influence the processing kinetics of the collagen molecule.

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ADAMTS-1 is involved in normal follicular development, ovulatory process and organization of the medullary vascular network in the ovary

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01735 ◽

2005 ◽

Vol 35 (2) ◽

pp. 343-355 ◽

Cited By ~ 64

Author(s):

M Shozu ◽

N Minami ◽

H Yokoyama ◽

M Inoue ◽

H Kurihara ◽

...

Keyword(s):

Granulosa Cell ◽

Ovarian Function ◽

Vascular System ◽

Follicular Development ◽

Vascular Network ◽

Type I ◽

Cell Layers ◽

Serum Gonadotropin ◽

Ovulatory Process

To clarify the role of disintegrin-like and metalloproteinase with thrombospondin type I motifs-1 (ADAMTS-1) in ovarian function, we examined abnormalities in ovulatory processes, folliculogenesis and the vascular system of ADAMTS-1 null ovaries. First, when immature female mice were treated with pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG), the number of ovulated oocytes was markedly decreased in ADAMTS-1 null mice in comparision to ADAMTS-1 (+/−) controls. The proportion of anovulated follicles to total mature follicles was significantly higher in ADAMTS-1 null females when compared with controls. The numbers of growing follicles at each stage were counted. The number of follicles at type 5b (late preantral) and later stages was markedly reduced in ADAMTS-1 null mice, irrespective of gonadotropin treatment (no gonadotropins, PMSG alone or PMSG/hCG). These data demonstrate that impairment of ovarian function to ovulate oocytes in ADAMTS-1 null mice occurs at two different levels: in the development of growing follicles and ovulatory processes. Furthermore, ADAMTS-1 null ovaries included a number of unusual atretic follicles that showed no sign of oocyte degeneration but lost the surrounding granulosa cell layers and were considered to be derived from type 4 or 5a follicles. These results suggest that ADAMTS-1 is important for follicular development beyond the type 4 and/or 5a and for maintaining normal granulosa cell layers in follicles. Finally, the number of large blood vessels in the medullar zone was significantly decreased in ADAMTS-1 null mice ovaries, suggesting that ADAMTS-1 is also involved in the organization of the medullary vascular network.

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The anatomy of two functional types of mechanoreceptive 'free' nerve-ending in the head skin of Xenopus embryos

Proceedings of the Royal Society of London Series B Biological Sciences ◽

10.1098/rspb.1983.0026 ◽

1983 ◽

Vol 218 (1210) ◽

pp. 61-76 ◽

Cited By ~ 17

Keyword(s):

Nerve Ending ◽

Type I ◽

Type Ii ◽

Free Nerve Ending ◽

Skin Cells ◽

Skin Cell ◽

Functional Types ◽

Superficial Skin ◽

Cell Layers ◽

Movement Detectors

The structure of the two functional types of ‘free’ nerve-ending in the head skin of late Xenopus embryos has been examined by horseradish peroxidase staining through their cells in the trigeminal ganglion and by electron microscopy. Type I neurites are identified as the ‘movement’ detectors by their purely homolateral innervation. They have many fine branches between the superficial skin cells, bearing numerous large varicosities. Type II neurites cross the midline to innervate both sides of the head as do the ‘rapid transient’ detectors found by physiology. They have a few fairly straight branches between the skin cell layers with few elongated varicosities.

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Co-culture of type I and type II pneumocytes as a model of alveolar epithelium

PLoS ONE ◽

10.1371/journal.pone.0248798 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0248798

Author(s):

Oliver Brookes ◽

Sonja Boland ◽

René Lai Kuen ◽

Dorian Miremont ◽

Jamileh Movassat ◽

...

Keyword(s):

Cell Lines ◽

Barrier Function ◽

Single Cells ◽

Light And Electron Microscopy ◽

Alveolar Epithelium ◽

Type I ◽

Apical Surface ◽

Type Ii ◽

Cell Layers ◽

Distal Lung

The epithelial tissues of the distal lung are continuously exposed to inhaled air, and are of research interest in studying respiratory exposure to both hazardous and therapeutic materials. Pharmaco-toxicological research depends on the development of sophisticated models of the alveolar epithelium, which better represent the different cell types present in the native lung and interactions between them. We developed an air-liquid interface (ALI) model of the alveolar epithelium which incorporates cell lines which bear features of type I (hAELVi) and type II (NCI-H441) epithelial cells. We compared morphology of single cells and the structure of cell layers of the two lines using light and electron microscopy. Working both in monotypic cultures and cocultures, we measured barrier function by trans-epithelial electrical resistance (TEER), and demonstrated that barrier properties can be maintained for 30 days. We created a mathematical model of TEER development over time based on these data in order to make inferences about the interactions occurring in these culture systems. We assessed expression of a panel of relevant genes that play important roles in barrier function and differentiation. The coculture model was observed to form a stable barrier akin to that seen in hAELVi, while expressing surfactant protein C, and having a profile of expression of claudins and aquaporins appropriate for the distal lung. We described cavities which arise within stratified cell layers in NCI-H441 and cocultured cells, and present evidence that these cavities represent an aberrant apical surface. In summary, our results support the coculture of these two cell lines to produce a model which better represents the breadth of functions seen in native alveolar epithelium.

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Enamel protein and collagen production by cells subcultured from porcine tooth bud explants

Biochemistry and Cell Biology ◽

10.1139/o87-092 ◽

1987 ◽

Vol 65 (8) ◽

pp. 698-709 ◽

Cited By ~ 18

Author(s):

Hardy Limeback

Keyword(s):

Type I Collagen ◽

Protein Biosynthesis ◽

Morphological Differentiation ◽

Immunocytochemical Staining ◽

Type I ◽

Type Iv ◽

F Cells ◽

Cell Layers ◽

Enamel Protein

Fibroblast (F) and epithelial (E) cells were obtained as primary outgrowths from explants of fetal porcine maxillary molars and subcultured up to four passages in monolayers enriched with either cell type. Histology of a tooth bud after 1 day in culture showed intact odontogenic E cell layers which were the probable source of the E cell outgrowths. After 2 months in culture, the fourth passage E cells demonstrated morphological differentiation by an alteration in cell packing and the formation of domes and nodules, when E and F cells were cocultured. Occasionally the nodules grew to considerable size, indicating the potential of these cells to aggregate and reorganize into odontogenic tissues even on culture dishes. The cells were characterized in monolayer culture by immunocytochemical staining. Laminin and type IV collagen staining was distributed diffusely throughout the culture, whereas type I collagen and osteonectin staining was predominantly localized in the F cells. Radio-labelled proteins from both E and F cell media produced similar collagen patterns (95% type I, 4% type V, 1% other), except that the F cells appeared to produce active collagenase. In addition, the E cells produced two radiolabelled proteins (relative masses of 50 000 and 53 000) that reacted with an affinity-purified antibody directed against porcine amelogenin. These experiments show that cells subcultured from tooth buds and grown in monolayer cultures can be used to study tooth organogenesis in vitro, as well as enamel protein biosynthesis.

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