Regeneration of a chimeric retina from single cells in vitro: cell-lineage-dependent formation of radial cell columns by segregated chick and quail cells (original) (raw)
Related papers
Neuroscience Letters, 1984
We report here that, in comparison to aggregates from retinal cells alone, addition of pigmented epithelial cells to retinal cells in rotary culture results in a pronounced increase of spatial order. A particularly high level of organization is found in about 15-20°70 of the aggregates. In these 'retinoids' the main layers characteristic of developing in vivo retinae can be distinguished in correct sequential arrangement on the basis of morphological criteria and by using acetylcholinesterase histochemistry [5, 6, 15], peanut agglutinin-lectin binding [11] and Lucifer Yellow staining [7-9]. Dissociated single cells from embryonic chick retinae reaggregate quickly and form relatively organized histotypic structures in a rotary culture system. Reaggregation of dissociated single cells starts with a random alignment of retinal cells, followed by the formation of primary rosettes and by the incorporation of additional cells into the aggregate [1, 4, 10-14]. We studied the internal order of these aggregates upon prolonged incubation of 2-3 weeks. Fujisawa [3] described specific layers which are formed upon aggregation of retinal cells on the chorioallantoic membrane. We were able to demonstrate a similar high degree of laminar organization in a pure in vitro system of aggregates derived from mixed cultures of retinal and pigmentepithelial (PE) cells. Methodologically, the neuroretinae and pigmented epithelia from E6 chicken embryos (White Leghorn) were dissected and washed with Hank's solution. Pigmented epithelia were first incubated in 1 mg/ml collagenase and 300 U/ml hyaluronidase (both from Boehringer) in Eagle's Minimal Essential Medium (Ea-MEM) for 10 min at 37°C, washed once in Hank's solution and treated with 1 mg/ml trypsin (Difco) for 20 min at 37°C. Isolated retinae were digested only by trypsin (10 min, 20°C and 10 min, 37°C). Then the tissues were rinsed in Ea-MEM and mildly dissociated into single cells in the presence of 0.05 mg/ml DNAase. After a 3-fold wash in Ea-MEM the cells were resuspended in aggregation medium (10070 fetal calf serum, 2°70 chicken serum, 1 070 glutamine, 0.1 070 penicillin/streptomycin and 0.02 mg/ml gentamycin in Ea-MEM). A 2 ml suspension of cells consisting either of 2-5 x 106 retina
Proceedings of the Royal Society B: Biological Sciences, 1997
Reaggregation of dispersed retinal cells of the chick embryo leads to histotypic retinospheroids in which the laminar organization remains incomplete: photoreceptors form rosettes which are surrounded by constituents of the other retinal layers. Here, for the ¢rst time, a complete arrangement of layers is achieved in cellular spheres (stratoids), provided that fully dispersed retinal cells are younger than embryonic day E6, and are reaggregated in the presence of a monolayer of retinal pigmented epithelium (RPE). A remarkable mechanism of stratoid formation from 1 to 15 days in vitro is revealed by the establishment of a radial MÏller glia sca¡old and of photoreceptors. During the ¢rst two days of reaggregation on RPE, rosettes are still observed. At this stage immunostaining with vimentin and F11 antibodies for radial MÏller glia reveal a disorganized pattern. Subsequently, radial glia processes organize into long parallel ¢bre bundles which are arranged like spokes to stabilize the surface and centre of the stratoid. The opsin-speci¢c antibody CERN 901 detects photoreceptors as they gradually build up an outer nuclear layer at the surface. These ¢ndings assign to the RPE a decisive role for the genesis and regeneration of a vertebrate retina.
European Journal of Neuroscience, 2007
For future retinal tissue engineering, it is essential to understand formation of retinal tissue in a 'cell-by-cell' manner, as can be best studied in retinal reaggregates. In avians, complete laminar spheres can be produced, with ganglion cells internally and photoreceptors at the surface; a similar degree of retinal reconstruction has not been achieved for mammals. Here, we have studied self-organizing potencies of retinal cells from neonatal gerbil retinae to form histotypic spheroids up to 15 days in culture (R-spheres). Shortly after reaggregation, a first sign of tissue organization was detected by use of an amacrine cell (AC)-specific calretinin (CR) antibody. These cells sorted out into small clusters and sent unipolar processes towards the centre of each cluster. Thereby, inner cell-free spaces developed into inner plexiform layer (IPL)-like areas with extended parallel CR + fibres. Occasionally, IPL areas merged to combine an 'inner half retina', whereby ganglion cells (GCs) occupied the outer sphere surface. This tendency was much improved in the presence of supernatants from retinal pigmented cells (RPE-spheres), e.g. cell organization and proliferation was much increased, and cell death shortened. As shown by several markers, a perfect outer ring was formed by GCs and displaced ACs, followed by a distinct IPL and 1-2 rows of ACs internally. The inner core of RPE spheres consisted of horizontal and possibly bipolar cells, while immunostaining and RT-PCR analysis proved that photoreceptors were absent. This shows that (1) mammalian retinal histogenesis in reaggregates can be brought to a hitherto unknown high level, (2) retinal tissue self-organizes from the level of the IPL, and (3) RPE factors promote formation of almost complete retinal spheres, however, their polarity was opposite to that found in respective avian spheroids.
Neuroscience Letters, 1986
Key words." chick retina aggregate-lamination pigment epithelium Wc report here a striking age dependency for tissue reconstruction capacity in rotatmg cultures of chick retinal cells, in particular in mixtures of retinal and pigment epithelial cells. The comparison of sections after acetylcholinesterase histochemistry delivered the following results. (1) In aggregates from retinal cells, sorting out is clearly observable, but only a minor age-dependent effect is detectable, e.g. the laminar order of aggregates derived from 6-day-old cells is increased compared to aggregates from other stages. (2) In aggregates from mixtures of retinal and pigment epithelial cells, the degree of laminar order is highest with cells from 5-day-old embryos and decreases significantly at older ages. The highest number of aggregates with the reconstruction of all main retinal layers was obtained by the reaggregation of retinal cells of embryonic day 5 (25%). (3) To achieve laminar order in aggregates, at all ages, the addition of pigment epithelial cells seems to be a prerequisite.
Cell and Tissue Research, 1989
The capacities of retinal and pigmented cells to regenerate histotypic in-vitro-retinae (IVR) in rotary culture were investigated by dividing the eye cups of 6-day-old chicken embryos into a central and a peripheral part; they were cut along the ora serrata, and the retinal and the pigmented constituents of both parts were isolated. The 4 dissociated cell populations were cultured separately and in all double combinations. Two different types of IVR's were generated; one developed from central or peripheral retinal cells, the other required the addition of pigmented cells from the ciliary margin of the eye. The shape of these IVR's was examined using scanning electron microscopy, and they were also characterized histologically. The acetylcholinesterase pattern marked the inner half of the retina; F11-antibody and a peanut agglutinin marker revealed both plexiform layers and a radial fiber system. In both types, organized histotypical areas consisted of complete sets of retinal layers. In the type containing pigmented cells from the eye periphery, the sequence of layers was identical with that of an in-situ-retina (" laminar IVR"). In IVR's derived from retinal cells only, the sequence of layers was reversed (" rosetted IVR").
Developmental Brain Research, 1990
The differentiation of presumptive neural retina following its isolation from rat embryos and growth in explant and monolayer culture has been studied to obtain information regarding the extent to which factors extrinsic and intrinsic to the retina participate in determining molecular and cytological differentiation. Explanted retinal epithelium retained the capacity for mitosis, as shown by [3H]thymidine incorporation, and from the undifferentiated neuroepithelium, retinal cell-types emerged and acquired a laminar organization resembling that in vivo. Characterization of rod photoreceptor cells at both the light and electron microscopic level showed that these cells exhibit differentiated structural features including inner segments, connecting cilia and membranous expansions suggestive of forming outer segments. Immunofluorescent labeling with an antibody to a synaptic vesicle protein, and electron microscopic identification of synaptic elements showed formation of synapses by the photoreceptor cells within the explant. Neurites extending from the explants exhibited growth on laminin, fibronectin and collagen substrates. Since the neurites immunolabeled with antibodies to the 140 kDa subunit of neurofilament and with antibodies to Thy-1, they could be identified as axons of ganglion cells. Antibodies to a variety of cell-type specific antigens showed that the cells expressed molecules associated with the fully differentiated cell. Furthermore, since our approach has been to explant embryonic retina at an age when the antigens are not yet expressed in vivo, the appearance of the antigens in culture represented de novo expression. In contrast, neural retinal cells in dissociated cultures did not exhibit de novo expression of differentiated molecular properties. Collectively, these results indicate that the isolated neural retina is capable of cell birth, commitment and differentiation when the cells are maintained together in explant culture, whereas the environment of monolayer cultures is apparently unable to promote the generation of mature neuronal phenotypes.
Experimental Eye Research, 1988
Embryonic retinae from 541-day-old chicks (E5-E6) were cut into stripes either in close contact with (RPE stripes) or in absence of the neighboring retinal pigmented epithelium (R stripes). The stripes were explanted and cultivated in vitro for up to 6 days, during which time they show the following differences in their characteristics of growth and differentiation. Compared with R stripes, RPE stripes morphologically showed a significant increase in size during the first 2 days in culture. Using E5 tissue, this is also demonstrated by a higher rate of cell proliferation (as measured by uptake of radioactive thymidine as well as by DNA contents). In contrast, R stripes after two days in culture show a much stronger neurite growth. After longer periods of culturing (5-6 days) we can show by cholinesterase histochemistry (ACHE and BChE) and by PNA-lectin binding that the RPE stripes have started to form all major layers of the in vivo retina, whereas R stripes remain unstratified and start to degenerate earlier. We conclude that the pigment epithelium might exert a specific stimulus on growth and tissue differentiation of the neural retina not only during in vitro, but possibly also during in vivo development. The in vitro methods introduced here could become useful model systems to further investigate the significance of the RPE for developmental, regenerative and even adult processes of the neural retina. Their future applicability in ophthalmologic research is briefly discussed.
Differentiation and morphogenesis in pellet cultures of developing rat retinal cells
The Journal of Comparative Neurology, 1997
We previously developed a reaggregate cell culture system (pellet cultures) in which retinal neuroepithelial cells proliferate and give rise to rod photoreceptor cells (rods) in vitro (Watanabe and Raff, 1990, Neuron 4:461-467). In the present study, we analyzed cell differentiation and morphogenesis in pellet cultures by using both cell-type-specific markers with immunofluorescence and electron microscopy. We demonstrated that, in addition to rods, the other major retinal cell types, including amacrine cells, bipolar cells, Mü ller cells, and ganglion cells were all present in the pellets, where most were able to develop from dividing precursor cells in vitro. The different cell types in the pellets became organized into two distinct structures: dark rosettes and pale rosettes. The cellular composition of these structures indicated that the dark rosettes correspond to the outer nuclear layer and the pale rosettes to the inner nuclear layer of the normal retina. Ultrastructural studies have indicated that the thin layer of neuronal processes surrounding the dark rosettes correspond to the outer plexiform layer, and the central region of the pale rosettes correspond to the inner plexiform layer of the normal retina. Other features of normal retinal development also occurred in the pellets, including programmed cell death and the formation of inner and outer rod cell segments and synapses. Thus, pellet cultures provide a convenient way to study different aspects of retinal development where one can control the size and the cellular composition of the initial reaggregate.