Lens Phenotype Research Articles

Enhancement of expression of lens phenotype in cultures of pigmented epithelial cells by hyaluronidase in the presence of phenylthiourea

Pigmented epithelial cells isolated from 8–9-day-old chick embryos can transdifferentiate into lens-like cells at the terminal period of the third generation of culture. However, efficiency of this transdifferentiation is usually rather low. Phenylthiourea, a potent inhibitor of melanin synthesis, effectively enhances transdifferentiation of pigmented epithelial cells into lens-like cells in vitro. Lentoid bodies began to appear in the multilayered region of primary cultures of pigmented epithelial cells maintained in medium containing phenylthiourea at concentrations between 0.5 and 1.0 mM. Furthermore, the enhancing effect of phenylthiourea can be amplified with testicular hyaluronidase. Under these conditions, pigmented epithelial cells grow vigorously and lose their differentiative properties, efficiently switching their phenotype into lens-like cells some 20 days after initiation of culture in the presence of both substances. Semiquantitative analysis revealed that testicular hyaluronidase amplified the effect of phenylthiourea more than 100-fold. It has been suggested that phenotypic expression of pigmented epithelial cells during transdifferentiation can be regulated by manipulating the microenvironment in which these cells reside.

Transformation of retinal glia cells into lens phenotype: expression of MP26, a lens plasma membrane antigen.

We describe experiments in which dissociated cells from differentiated, post-mitotic neural retina of late chicken embryos (13 and 16 days) rapidly and consistently transform (transdifferentiate) in vitro into lens-like phenotype and form spherical lentoids. Using immunohistochemical and other tests, we have established that the lentoids arise from the progeny of definitive retinal glia cells (Müller cells). An early event in their transformation is the appearance in the cell surface of MP26, a plasma membrane protein characteristic of lens but not found in the retina. The results support the hypothesis that the phenotype of definitive glia cells in the retina is stabilized by contact-mediated interactions with neurons; disruption of cell contacts and cell separation alter surface properties of the glia cells, decontrol their phenotype, and predispose them to phenotype transformation.

Commitment to transdifferentiation into lens occurs in neural retina cells after brief spreading culture of the dissociated cells

Cells dissociated from neural retina (NR) of 8-day-old chick embryos transdifferentiated into lens and pigment cells under conditions of stationary culture (spreading culture, SpC), whereas such an alteration in the pathways of differentiation did not occur under conditions of aggregate culture (AgC) with constant gyration for 28 days. When NR cells precultivated for longer than 10 days in SpC were transferred to AgC, extensive transdifferentiation into lens (and pigment cells) occurred in aggregates after a total of 28 days' cultivation in vitro. This was confirmed by immunofluorescent observations of histological sections of aggregates as well as by quantitative immunoelectrophoresis using specific antiserum against δ-crystallin. In 10-day SpC, the presence of δ-crystallin was not detected by immunological assay. Our results suggest that NR cells become committed or ‘transdetermined’ into lens direction before detectable expression of the lens phenotype, when cultured in SpC for 10 days.

Lentoidogenesis from neural retina cells in culture is affected by interactive relationships between different cell types

By centrifugation in a Percoll gradient, two cell fractions were separated from cell populations harvested from 8-day cultures of neutral retina cells of 3-5-day-old quail embryos. The heavy (H-) fraction contained mostly N-cells, which are considered to be putative neuronal cells, while the light (L-) fraction contained both E-cells, putative retinal glial cells, and N-cells. Determination of choline acetyltransferase activity in both fractions suggested that this enzyme is predominantly localised in N-cells. After replating the separated L-fraction for further culturing, frequent lentoidogenesis occurred from clusters of N-cells which, though few in number, were included in this fraction. Addition of H-fraction to L-fraction cells caused a significant increase in lentoidogenesis up to a ratio of N- to E-cells of 3:1. However, addition of excess H-fraction cells beyond this ratio inhibited lens differentiation. This difference in the expression of lens phenotypes resulting from the different ratios of H- and L-fraction was confirmed by monitoring the level of delta-crystallin in cultures. These results are discussed in the light of interactive relationships between N- and E-cells in the transdifferentiation of neural cells into lens in cell culture.