‘Transdifferentiation’ of chicken neural retina into lens and pigment epithelium in culture: controlling influences

Development ◽  
1978 ◽  
Vol 48 (1) ◽  
pp. 1-21
Author(s):  
D. J. Pritchard ◽  
R. M. Clayton ◽  
D. I. De Pomerai
Keyword(s):  
Pigment Cell ◽  
Pigment Epithelium ◽  
Neural Retina ◽  
Medium Composition ◽  
Pigment Cells ◽  
Bicarbonate Concentration ◽  
Experimental Conditions ◽  
Culture Growth ◽  
New Hypothesis

The in vitro transdifferentiation of chicken embryo neural retina into pigment epithelium and lens cells was investigated under a variety of experimental conditions. Our findings suggest that some aspects of the phenomena are a function of medium composition and volume, whereas others depend upon conditions which develop during culture growth. Before melanin is visible, potential pigment cells are recognized as foci within epithelialsheets which remain in contact with the dish. The final area occupied by colonies of potential pigment cells is directly proportional to bicarbonate concentration. Low total medium volume also favours formation of potential pigment cells. In contrast the extent of cells other than potential pigment cells is not related to bicarbonate and is favoured when the volume of medium is large. Accumulation of melanin within the potential pigment cell colonies is suppressed when cells are crowded together. Lentoid bodies are formed from cells which are distinct from potential pigment cells and arise in crowded situations, in association with multilayering. Another type of structure superficially resembling a lentoid is derived from cell aggregates formed during the initial establishment of cultures. The survival of these ‘aggregate bodies’ is inversely related to bicarbonate concentration. Crystallin content is unrelated to lentoid numbers. The results provide the basis for a new hypothesis concerning cytodifferentiation in this system.

Transdifferentiation of chicken embryo neural retina into pigment epithelium: indications of its biochemical basis

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 47-62
Author(s):  
D. J. Pritchard
Keyword(s):  
Pigment Cell ◽  
Pigment Epithelium ◽  
Cell Formation ◽  
Tca Cycle ◽  
Cell Types ◽  
Neural Retina ◽  
Pigment Cells ◽  
Biochemical Basis ◽  
Pigment Synthesis

Neural retina from 8- to 9-day embryo chickens was grown in long-term cell culture in an experiment to test the hpothesis that one step during the in vitro transdifferentiation of neural retina into pigment cells occurs in response to stimulation of tricarboxylic acid (TCA) cycle activity. Time-lapse photography showed that pigment-cell formation occurs through the intermediate stages of ‘undistinguished cells’, ‘pavement epithelium’ and ‘potential pigment cells’. Mitosis of undistinguished cells to pavement epithelium was proportional to malonate over most of the tested range of concentrations and was inhibited by succinate, which respectively depress and stimulate the TCA cycle. Conversely mitosis of pavement epithelium to potential pigment cells occurred in proportion to succinate concentration over most of the tested range and was inhibited by malonate, in support of the hypothesis under test. Melanin synthesis begins in a minority of ‘pigment leader cells’ uniquely stimulated by the lowest concentration of malonate, although higher concentrations blocked pigment synthesis in all cell types. The pigment leader cells appear to act as centres of influence upon neighbouring potential pigment cells, which subsequently also beome pigmented. Lactate inhibited most or all of the steps in formation of pigment epithelium. Between three and five mitoses occur in the production of pigment cells, whereas multilayers and lentoid bodies seem to be formed by expansion of undistinguished cells, probably without mitosis. The observations lead to a general theory that metaplastic conversion between cell types in eye tissues may require the physical isolation of overtly differentiated, multipotent cells from ‘leader’ cells which normally hold them in physiological subjugation.

nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate

Development ◽  
1999 ◽  
Vol 126 (17) ◽  
pp. 3757-3767 ◽  
Author(s):  
J.A. Lister ◽  
C.P. Robertson ◽  
T. Lepage ◽  
S.L. Johnson ◽  
D.W. Raible
Keyword(s):  
Transcription Factor ◽  
Retinal Pigment Epithelium ◽  
Neural Crest ◽  
Cell Fate ◽  
Pigment Cell ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Pigment Cells ◽  
Evolutionary Divergence ◽  
Wild Type

We report the isolation and identification of a new mutation affecting pigment cell fate in the zebrafish neural crest. Homozygous nacre (nac(w2)) mutants lack melanophores throughout development but have increased numbers of iridophores. The non-crest-derived retinal pigment epithelium is normal, suggesting that the mutation does not affect pigment synthesis per se. Expression of early melanoblast markers is absent in nacre mutants and transplant experiments suggested a cell-autonomous function in melanophores. We show that nac(w2) is a mutation in a zebrafish gene encoding a basic helix-loop-helix/leucine zipper transcription factor related to microphthalmia (Mitf), a gene known to be required for development of eye and crest pigment cells in the mouse. Transient expression of the wild-type nacre gene restored melanophore development in nacre(−/−) embryos. Furthermore, misexpression of nacre induced the formation of ectopic melanized cells and caused defects in eye development in wild-type and mutant embryos. These results demonstrate that melanophore development in fish and mammals shares a dependence on the nacre/Mitf transcription factor, but that proper development of the retinal pigment epithelium in the fish is not nacre-dependent, suggesting an evolutionary divergence in the function of this gene.

scholarly journals Neural retina limits the nonviral gene transfer to retinal pigment epithelium in an in vitro bovine eye model

2004 ◽  
Vol 6 (3) ◽  
pp. 72-80 ◽  
Author(s):  
Leena Pitkänen ◽  
Jukka Pelkonen ◽  
Marika Ruponen ◽  
Seppo Rönkkö ◽  
Arto Urtti
Keyword(s):  
Retinal Pigment Epithelium ◽  
Gene Transfer ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Neural Retina ◽  
Nonviral Gene Transfer ◽  
Eye Model

The control of pigment cell pattern formation in the California newt, Taricha torosa

Development ◽  
1986 ◽  
Vol 97 (1) ◽  
pp. 141-168
Author(s):  
R. P. Tucker ◽  
C. A. Erickson
Keyword(s):  
Neural Crest ◽  
Pigment Cell ◽  
Contact Inhibition ◽  
Pigment Cells ◽  
Lateral Plate Mesoderm ◽  
Lateral Plate ◽  
Pronephric Duct ◽  
Taricha Torosa ◽  
Dorsal Stripe

Neural crest-derived pigment cells form species-specific patterns of pigmentation in amphibian embryos. We have characterized the appearance and changes in pigment cell distribution in the embryos of the California newt, Taricha torosa. Black melanophores first appear scattered over the surface of the somites intermingled with yellow xanthophores in stage 34/35 embryos. The melanophores then migrate either dorsally to form a dorsal stripe at the apex of the somites or ventrally along the intersomitic furrows to form a midbody stripe at the somite—lateral plate mesoderm border. Xanthophores remain between the two melanophore stripes and are also found in the dorsal fin and head. The formation of the dorsal stripe coincides with a change in melanophore tissue affinity from the surface of the somites to the subectodermal extracellular matrix (ECM). The latter substratum is the location of the cue used to organize the dorsal stripe. In addition, melanophores become elongate and highly arborized, which would allow them to extend to the region where the dorsal stripe forms. In contrast, xanthophores do not form long processes in vitro. This suggests that the ability of melanophores but not xanthophores to search for a cue at the apex of the somites may account in part for the segregation of these cells types. Melanophores and xanthophores are trapped to form the midbody stripe by the pronephric duct, which is located just beneath the ectoderm at the bases of the intersomitic furrows. Ablation of the duct prevents formation of the midbody stripe, although melanophores and xanthophores still fail to migrate ventrally over the lateral plate mesoderm. Melanophores grafted to the ventral midline fail to leave the confines of the donor tissue. This suggests that a factor in the lateral plate mesoderm in addition to the pronephric duct is inhibiting further ventral migration. There is no gross morphological difference in the organization of the subectodermal ECM dorsal and ventral to the pronephric duct as revealed by alcian blue, ruthenium red and staining with antibodies to fibronectin. We also conclude that the directed dispersal of the neural crest into the space between the somites and ectoderm is due to contact inhibition of cell movement, since T. torosa neural crest cells demonstrate contact inhibition in vitro and there are enough cells in the lateral migratory spaces to make contact events likely during dispersal.

scholarly journals Analysis and Optimization of Conditions for the Use of 2′,7′-Dichlorofluorescein Diacetate in Cultured Hepatocytes

Antioxidants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 674
Author(s):  
Megan J. Reiniers ◽  
Lianne R. de Haan ◽  
Laurens F. Reeskamp ◽  
Mans Broekgaarden ◽  
Rowan F. van Golen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Data Interpretation ◽  
Medium Composition ◽  
Cultured Hepatocytes ◽  
Experimental Conditions ◽  
Membrane Transport Proteins ◽  
Dichlorofluorescein Diacetate ◽  
Cell Type Specific ◽  
The Stability

Numerous liver pathologies encompass oxidative stress as molecular basis of disease. The use of 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH2-DA) as fluorogenic redox probe is problematic in liver cell lines because of membrane transport proteins that interfere with probe kinetics, among other reasons. The properties of DCFH2-DA were analyzed in hepatocytes (HepG2, HepaRG) to characterize methodological issues that could hamper data interpretation and falsely skew conclusions. Experiments were focused on probe stability in relevant media, cellular probe uptake/retention/excretion, and basal oxidant formation and metabolism. DCFH2-DA was used under optimized experimental conditions to intravitally visualize and quantify oxidative stress in real-time in HepG2 cells subjected to anoxia/reoxygenation. The most important findings were that: (1) the non-fluorescent DCFH2-DA and the fluorescent DCF are rapidly taken up by hepatocytes, (2) DCF is poorly retained in hepatocytes, and (3) DCFH2 oxidation kinetics are cell type-specific. Furthermore, (4) DCF fluorescence intensity was pH-dependent at pH < 7 and (5) the stability of DCFH2-DA in cell culture medium relied on medium composition. The use of DCFH2-DA to measure oxidative stress in cultured hepatocytes comes with methodological and technical challenges, which were characterized and solved. Optimized in vitro and intravital imaging protocols were formulated to help researchers conduct proper experiments and draw robust conclusions.

In vitro transdifferentiation of embryonic rat retinal pigment epithelium to neural retina

1995 ◽  
Vol 677 (2) ◽  
pp. 300-310 ◽  
Author(s):  
Shulei Zhao ◽  
Steven C. Thornquist ◽  
Colin J. Barnstable
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Neural Retina

Uveal Melanocytes, Ocular Pigment Epithelium, and Müller Cells in Culture: In Vitro Toxicology

2002 ◽  
Vol 21 (6) ◽  
pp. 465-472 ◽  
Author(s):  
Dan-Ning Hu ◽  
Howard E. Savage ◽  
Joan E. Roberts
Keyword(s):  
Visual Information ◽  
Cultured Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Antioxidant Properties ◽  
Müller Cells ◽  
Neural Retina ◽  
Model Systems ◽  
Muller Cells

Uveal melanocytes and the ocular pigment epithelium are located in the middle and inner layers of the eye. Müller cells (a type of glial cell) are located in the neural retina. Melanocytes, retinal pigment epithelium (RPE), and Müller cells do not participate directly in the detection or transfer of visual information, but they have various functions that support the neural retina and are essential for the maintenance of vision. Methods for the isolation and cultivation of melanocytes, RPE, and Müller cells have been established by us and other investigators. These cultured cells can be used as in vitro model systems for studying the toxicology of visible light, ultraviolet (UV) radiation, drugs, and other potentially toxic agents. Toxic effects on these cells may give rise to altered retinal function and result in impaired vision. Both melanocytes and pigment epithelium contain melanin, which has the ability to bind organic amines and metal ions. This results in the accumulation of these substances in the eye. Melanin may protect cells from chemical stress by binding toxic chemicals; but in chronic exposure, increased and lengthy binding may cause damage to these cells. Two different types of melanin are found in the eye: eumelanin and pheomelanin, which may have photoprotective and phototoxic effects, respectively. Pigment epithelium contains mainly eumelanin, whereas melanocytes contain both eumelanin and pheomelanin. Melanin is an antioxidant and with age, the antioxidant properties may diminish to the point that it may even become a prooxidant. There are also other functions of pigment epithelium and uveal melanocytes not related to melanin and there are also several functions of Müller cells that play a role in the toxicological aspects of the eye. Cultured uveal melanocytes, pigment epithelial cells, and Müller cells can be used to study the toxicology of these cells in vitro.

scholarly journals In Vitro Hypoxia Responsiveness Of Fdg And Faza Retention: Influence Of Shaking Versus Stagnant Conditions, Glass Versus Polystyrene Substrata And Cell Number Down-Scaling

2020 ◽  
Author(s):  
Morten Busk ◽  
Michael R Horsman ◽  
Jens Overgaard ◽  
Steen Jakobsen
Keyword(s):  
Single Photon ◽  
Photon Emission ◽  
Cell Number ◽  
Medium Composition ◽  
Emission Computed Tomography ◽  
Experimental Conditions ◽  
Positron Emission ◽  
Orbital Shaking ◽  
Petri Dishes

Abstract Background. In vitro experiments using radiolabeled molecules is fundamental for Positron emission tomography (PET) or single photon emission computed tomography (SPECT) tracer development and various metabolic assays, but no consensus on appropriate incubation conditions exists. Specifically, the use of shaking versus non-shaking conditions, cell number to medium volume and the choice of cell plating material may unintentionally influence cellular oxygenation and medium composition. This is problematic when testing the oxygen-dependence of tracers including 18F-fluoro-2-deoxyglucose (FDG) and hypoxia-selective 2-nitroimidazoles (e.g., 18F-fluoroazomycin-arabinoside, FAZA) or when doing prolonged experiments. The purpose of this study was to assess the influence of various experimental conditions on tracer retention. Methods. Tumor cells were seeded in a) Glass or standard Polystyrene Petri dishes or as b) discrete droplets in polystyrene Petri dishes or on 9 mm glass coverslips positioned in glass Petri dishes. When confluent, cells were pre-equilibrated for 2h to 21%, 0.5% or 0% O2 and FDG or FAZA was added, followed by cell harvest and analysis of radioactivity 1h (FDG) or 3h (FAZA) after. Experiments were conducted with/without orbital shaking. Results. The influence of hypoxia on tracer retention varied widely among cell lines, but shaking-induced convection did not influence uptake. In contrast, hypoxia-driven FAZA, and to some extent FDG, retention was much lower in cells grown on polyethylene than glass. Scaling-down the number of cells did not compromise accuracy. Conclusions. Most experiments can be performed appropriately in the absence of shaking and with downscaling of cell number but the use of conventional plasticware is highly problematic for studies on tracers and drugs that are metabolized and retained or activated at low O2 levels.

Long Term Cultures of Neural Retina and Pigment Epithelium from Newborn Rabbits

1983 ◽  
Vol 38 (1-2) ◽  
pp. 141-145 ◽  
Author(s):  
Tetsuro Tsukamoto ◽  
Hanns Ludwig
Keyword(s):  
Pigment Epithelium ◽  
Amacrine Cells ◽  
Neural Retina ◽  
In Vitro Cultures ◽  
Neural Cells ◽  
Receptor Cells ◽  
Retina Pigment Epithelium ◽  
Frag Ments

In vitro cultures of neural retina, obtained after dispersion and trypsinization of tissue frag ments, were composed of 3 morphologically distinct types o f neural cells, as demonstrated by sil­ver impregnation. They resem bled ganglion or receptor cells, horizontal or amacrine cells, and bi­polar cells of the intact retina. Pigment epithelium was cultured without trypsinization. Both kinds o f techniques may prove helpful for long term experiments in neurobiology and neurovirology.

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