Differentiation of Lens-Like Structures from Newt Iris Epithelial Cells In Vitro

Abstract

(10,11,27,28).We have shown that the switch from pigmented retinal cells of 9-day-old check embryos into lens cells occurs in clonal cell culture without interactions with other cell types (14). The question then arises whether this approach can be extended to iris epithelial cells of newts which possess the capacity for Wolffian regeneration in vivo. This communication describes the differentiation of lens-like structures in cultures of dissociated iris epithelial cells of adult newts. MATERIALS AND METHODSTwenty-five to 50 adult newts, Cynops (Triturus) pyrrhogaster were used in each experiment. Isolated whole eyes were sterilized by three 1-min immersions in 70% ethanol. The irisrings (iris pars iridica) were then removed (11) and treated in an enzyme mixture that contained 5 parts of 0.4% (v/v) Diluted Leibovitz medium IL15 (12) was used as the culture medium and was prepared by dissolving 0.894 g of commercial Leibovitz L-15 powder (GIBCO) in 90 ml of triple-distilled water; this was supplemented with 10 ml of fetal calf serum (GIBCO, Control No. A322102), 3200 IU of penicillin, and 4 mg of streptomycin before filtration by Millipore filter.For the immunological identification of lens products in cultured cells, antisera were prepared in rabbits against the 15,000 X g supernatant of adult newt lenses in 20 mM Tris HCl buffer (pH 7.5) and against the a-, f-, and y-crystallin fractions, respectively. The separation of crystallins from the whole lens extract was made by column chromatography through Sephadex G-50 and G-200, and DEAE-Sephadex A-50 according to established methods (13). Sixteen rabbits were injected, one each with 40, 5, 3, and 1 mg of each protein fraction, respectively. Each rabbit received injections of protein with an incomplete Freund's adjuvant at intervals of two weeks. Antisera were usually obtained 10 days-after the second injection. In immunodiffusion tests (15) anti-whole lens extract and anti-a-crystallin cross-reacted faintly with extracts of heterologous organs of newts. This cross-reactivity was removed by absorbing the antisera with saline extracts of liver homogenate of adult newts. Cross-reactions of anti-acrystallin with the heterologous crystallins were also removed by absorption with 0-crystallin solution, whereas anti-acrystallin and anti-y-crystallin did not cross-react with the heterologous test-antigens (Fig. 1)

In vivo study of lens regeneration in Rana cyanophlyctis under influence of vitamin A and ascorbic acid

“…Results of the present study are also being supported by the finding of Eguchi et al (1974). In their finding, they observed that under certain conditions, dedifferentiation of extra ordinary cells enable the acquisition of new and higher developmental potencies.…”

Heterogeneous proliferative potential in regenerative adult newt cardiomyocytes

“…This is manifest in different ways depending on tissue type. Iris pigmented epithelial cells transdifferentiate and proliferate during lens regeneration (Eguchi et al, 1974). Multinucleate newt myotubes and myofibres re-enter the cell cycle and undergo conversion to mononucleate cells during limb and tail regeneration (Echeverri et al, 2001;Kumar et al, 2000;Lo et al, 1993;Velloso et al, 2000).…”

In Vivo and in Vitro Experimental Analysis of Lens Regeneration in Larval Xenopus laevis

“…There is evidence that in different experimental systems the extent of aggregation of cells in culture can influence cell differentiation (for a review, see 10). In tissue culture, newt dorsal iris fails to transdifferentiate into lens unless retina or pituitary is present (32, 12), but under cell culture conditions iris epithelial cells can transdifferentiate into lens fibers (18,13). Moreover, in tissue culture lens fiber formation from hatched chick lens epithelium also does not occur unless the retina or pituitary is present.…”