Characterisation of cholinesterase expression during murine embryonic stem cell differentiation.

It is already established that cholinesterases (ChEs) appear in every embryonic blastema at a very early stage of development, independently from innervation. Embryonic butyrylcholinesterase (BChE) is typically found in cells engaged in proliferation processes, while acetylcholinesterase (AChE) is expressed by cells undergoing morphogenetic processes. In order to better define the regulation of cholinesterases during development, we examined their expressions during in vitro differentiation of two murine embryonic stem cell lines by reverse transcription polymerase chain reaction, histochemistry and enzyme activity measurements. AChE and BChE activity and mRNA were present in the undifferentiated stem cells. To test whether the ChEs expression is regulated during differentiation, we employed the embryoid bodies (EBs) culture method, allowing the cells to differentiate, to then collect them at various stages in culture. Interestingly, phases of differentiation were accompanied by increased AChE transcripts; BChE expression was constant, decreasing at later differentiation stages. Cholinesterase activities showed corresponding patterns, with AChE activity increasing at later stages in culture and BChE slightly decreasing. Histochemistry revealed that AChE and BChE activities were mutually exclusive, being expressed by different cell subpopulations. Thus, we have demonstrated that mouse embryonic stem cells express cholinesterases, the enzymes are functional and their expression is regulated during differentiation. Therefore, it appears that their functions under these conditions are not related to synaptic transmission, but for the developmental processes.

Exposure to diazinon alters in vitro retinogenesis: retinospheroid morphology, development of chicken retinal cell types, and gene expression.

Developing embryos are more vulnerable than adults to acute cholinergic intoxication by anticholinesterases, including organophosphorus pesticides. These agents affect the process of neural development itself, leading to permanent deficits in the architecture of the nervous system. Recent evidence on direct roles of acetylcholinesterase (AChE) on neuronal differentiation provides additional grounds for investigating the developmental toxicity of anticholinesterases. Therefore, the effect of the organophosphate diazinon on the development of chick retinal differentiation was studied by an in vitro reaggregate approach. Reaggregated spheres from dissociated retinal cells of the E6 chick embryo were produced in rotation culture. During the whole culture period of 10 days, experimental cultures were supplemented with different concentrations of the pesticide, from 20 to 120 microM diazinon. The pesticide-treated spheres were reduced in size, and their outer surface was irregular. More importantly, inner structural distortions could be easily traced because the structure of control spheroids can be well characterized by a histotypical arrangement of laminar parts homologous to the normal retina. Acetylcholinesterase activity in diazinon-treated spheres was reduced when compared with controls. As a dramatic effect of exposure to the pesticide, inner plexiform layer (IPL)-like areas in spheroids were not distinguishable anymore. Similarly, photoreceptor rosettes and Müller radial glia were strongly decreased, whereas apoptosis was stimulated. The expression of transcripts for choline-acetyltransferase and muscarinic receptors was affected, revealing an effect of diazinon on the cholinergic system. This further proves the significance of cholinesterases and the cholinergic system for proper nervous system development and shows that further studies of debilitating diazinon actions on development are necessary.

Interaction between organophosphate compounds and cholinergic functions during development.

Organophosphate (OP) compounds exert inhibition on cholinesterase (ChE) activity by irreversibly binding to the catalytic site of the enzymes. For this reason, they are employed as insecticides for agricultural, gardening and indoor pest control. The biological function of the ChE enzymes is well known and has been studied since the beginning of the XXth century; in particular, acetylcholinesterase (AChE, E.C. 3.1.1.7) is an enzyme playing a key role in the modulation of neuromuscular impulse transmission. However, in the past decades, there has been increasing interest concerning its role in regulating non-neuromuscular cell-to-cell interactions mediated by electrical events, such as intracellular ion concentration changes, as the ones occurring during gamete interaction and embryonic development. An understanding of the mechanisms of the cholinergic regulation of these events can help us foresee the possible impact on environmental and human health, including gamete efficiency and possible teratogenic effects on different models, and help elucidate the extent to which OP exposure may affect human health. The chosen organophosphates were the ones mainly used in Europe: diazinon, chlorpyriphos, malathion, and phentoate, all of them belonging to the thionophosphate chemical class. This research has focused on the comparison between the effects of exposure on the developing embryos at different stages, identifying biomarkers and determining potential risk factors for sensitive subpopulations. The effects of OP oxonisation were not taken into account at this level, because embryonic responses were directly correlated to the changes of AChE activity, as determined by histochemical localisation and biochemical measurements. The identified biomarkers of effect for in vitro experiments were: cell proliferation/apoptosis as well as cell differentiation. For in vivo experiments, the endpoints were: developmental speed, size and shape of pre-gastrula embryos; developmental anomalies on neural tube, head, eye, heart. In all these events, we had evidence that the effects are mediated by ion channel activation, through the activation/inactivation of acetylcholine receptors (AChRs).

On the multifunctionality of cholinesterases.

Here we present novel information on non-classical functions of cholinesterases and on a cross-talk linking the two enzymes AChE and BChE. The first part of the article is focussed on the regulation of ChEs and the effects acquired when one of the proteins is knocked down (siRNA for BChE, AChE knock-out mouse). In the second part evidence is presented showing that AChE may exert adhesive properties through its binding to laminin, thus being involved in cell-matrix or cell-cell communication.

Cytochrome-c oxidase is one of several genes elevated in marginal retina of the chick embryo.

The retinal ciliary margin is particularly relevant for the correct generation and regeneration of vertebrate retinae, since pluripotent stem cells are located there throughout development, and--at least in some species--even until adult stages. Our aim was to identify factors (genes) which are involved in processes of proliferation and differentiation in the developing chicken retina. Reverse transcription-polymerase chain reaction differential display was used to identify genes that were differentially expressed in chick central and peripheral embryonic retina. Candidate genes analyzed through sequencing and database searches were confirmed by Northern blot analysis and histochemistry. A series of differentially expressed genes were detected, including a neuronal cell adhesion molecule, an esterase, and homeobox gene products. One of the sequenced products was identified as subunit I of cytochrome-c oxidase (COX-1), an enzyme which is central to energy metabolism and particularly relevant for developing nervous systems. Northern blot analysis confirmed its up-regulation in the chick peripheral retina, being maximal at embryonic day 7. In the retinal pigmented epithelium its expression is lower than in the retinal periphery but higher than in central retina. COX histochemistry revealed distinct laminar patterns in central retina, but also an elevated level of activity in the peripheral retina throughout development. These data not only show that the developing ciliary margin of the chick retina has high energy requirements, but also indicate that COX-1 could play essential roles in developing cells and in stem cells of the eye periphery.

Heterologous expression of acetylcholinesterase affects proliferation and glial cytoskeleton of adherent chicken retinal cells.

Besides its function at cholinergic synapses, acetylcholinesterase (AChE) exerts structural functions on neural differentiation, independent of its enzymatic activity. To elucidate such functions, we have previously heterologously expressed AChE in histotypic retinal reaggregates, revealing strong effects on their histogenesis, particularly on Müller glia processes. To further resolve these findings at a less complex cellular level, in this study we transfected adherent retinal cells of the chick embryo after 2 days i.c. with a sense pSVK3-AChErab-cDNA expression vector encoding for the entire rabbit AChE gene by calcium phosphate precipitation. Northern blots using digoxigenin (DIG)-labeled rabbit cDNA revealed a pronounced level of rabbit AChE mRNA in AChE-transfected cells. Western blot analysis established an increase in the endogenous AChE protein in transfected cells. Noticeably, AChE activity was not much affected, indicating a post-translational regulation of overall AChE activity. As a corollary, 5'-bromo-2'-deoxyuridine (BrdU) studies showed a decrease in cell proliferation. Exploring changes of the Müller glia, the cytoskeletal protein vimentin was found to be increased in transfected cells. Vimentin-stained processes are longer, thicker and more orderly arranged. In conclusion, exogenous expression of rabbit AChE in chicken retinal monolayers exerts a structural function on glial cytoskeletal organization, independent of AChE activity.

Anticholinesterase treatment of chicken retinal cells increases acetylcholinesterase protein independently of protein kinase C.

It has been reported that anticholinesterase exposure, e.g. by environmental toxins or nerve gases, can increase acetylcholinesterase (AChE) protein, possibly as an autoregulatory stress response. We earlier have transfected retinal cells of the chick embryo with a pSVK3-AChE(rab)-cDNA vector to heterologously express rabbit AChE, which concomitantly also increased AChE protein from chick. To analyse further the cell-internal pathways of these different paradigms (anticholinesterase treatment vs. AChE transfection) which both lead to an AChE increase, we here show that AChE overexpression by transfection leads to an increase in protein kinase C (PKC). Most remarkably, when cells independently of, or in addition to their transfection are treated with 10 microM of the AChE inhibitor BW284c51, AChE protein levels are much more dramatically increased up to 20-fold. This treatment, however, does not affect PKC. These data show that (i) retinal cells respond to anticholinesterase insult by a massive increase of AChE protein; (ii) the response to BW284c51 is not PKC-mediated; and (iii) both strategies of AChE increase follow different cell-internal pathways, their effects being additive. The ecological and biomedical implications of these findings are briefly discussed.

Cerebellar glia cells induce a correct laminar organization in chicken retinal reaggregates.

We investigated the functional role of glia cells during retinogenesis using the rotation culture system. Reaggregating cells from the embryonic chick retina have the unique capacity to reassemble into laminated cellular spheres. These spheres are composed of several compartments holding the constituents of many retinal layers in a topologically correct, yet inverse orientation. However, when these spheres are cultured in the presence of conditioned media derived from monolayers of cerebellar glia cells, the reassembling retinal cells behave totally differently. The anlage of the originally reversed lamina polarity is progressively transformed within a week into a sphere with a compound and correctly laminated orientation. Conditioned media from fibroblasts, other glia cells (except Müller cells) or a set of already characterized retinogenetic factors are not able to produce this dramatic transformation. Additionally, we were able to show that only retinal cells are able to respond with a reorganization process. Reaggregating cells from the chick cerebellum also form spheroids; however, neither in the presence of cerebellar glia cell-derived conditioned medium nor their control counterparts are they able to reassemble histotypically. This indicates that cerebellar glia cells produce diffusible factors to which retinal cells can respond and that these factors can act as important determinants for the correct establishment of the retinal polarity. Since all types of laminar disorganization are of great clinical significance, the knowledge of factors which determine and sustain the normal retinal architecture are biomedically highly relevant.

From stem cells towards neural layers: a lesson from re-aggregated embryonic retinal cells.

Cells from dissociated embryonic avian retinae have the capacity to re-aggregate in rotation culture and form cellular spheres reconstituting a complete arrangement of all retinal layers. This exquisite phenomenon is based upon in vitro proliferation of multipotent precursor stem cells and spatial organization of their differentiating descendants. The addition of soluble factors from cultured retinal pigmented epithelial (RPE) or radial glial cells is essential to revert inside-out spheres (rosetted retinal spheres) into correctly laminated outside-out spheres (stratified spheres). Such complete restoration of a laminated brain tissue by cell re-aggregation has been achieved only for the embryonic avian retina, but not the mammalian retina, nor for other brain parts. This review summarises the history of the re-aggregation approach, presents avian retinal re-aggregate models, and analyses roles of the RPE and Müller cells for successful retinal tissue regeneration. It is predicted that these results will become biomedically relevant, as stem cell biology will soon open ways to produce large amounts of human retinal precursors.

Photoreceptor plasticity in reaggregates of embryonic chick retina: rods depend on proximal cones and on tissue organization.

Plasticity of photoreceptors and their integration into epithelial structures homologous to an outer nuclear layer (ONL), was investigated in embryonic chick retinal cell reaggregates by immunohistochemistry using an antibody specific for red plus green cones (RG-cones) and an antibody for rods. If reaggregates are raised in the presence of pigmented epithelium (RPE), completely reconstructed, stratified retinal spheres are produced, where all rods and cones are integrated into an outer laminar ONL, similar to a normal retina. In the absence of RPE, 'rosetted' spheres form which contain internal rosettes homologous to an ONL. Only a minor fraction of cones and rods of 'rosetted' spheres are located within rosettes, while a larger fraction is diffusely displaced in nonorganized areas, thus, not contributing to an ONL-like epithelium. In both types of spheres, the total percentage of RG-cones was similar to the in vivo retina, indicating that expression of cones is autonomous. Following cones, after about one day, rods developed only within already existing RG-cone clusters. Thereby, the ratio of rods to RG-cones increases as the tissue organization decreases: for stratified spheres this ratio is, 0.50 (1 rod/2 cones; similar to mature retina); for rosettes, 0.74 (3 rods/4 cones) and for nonorganized areas, 1.09 (1 rod/1 cone) -- a higher ratio under our conditions has never been detected. Thus, rod expression depends strictly on the presence of nearby cones; their relative numbers are distinctively adjusted according to the cytoarchitecture of the tissue environment. The biomedical implications of these findings are briefly discussed.

Inhibition of alpha(1-6)-linked fucose decreases inner retinal cells and increases photoreceptors in chicken retinal reaggregates.

We investigated the developmental role of alpha(1-6)-linked fucose, applying Aleuria aurantia lectin to a specific retinal regeneration system. Thereby, dissociated retinal cells of chicken embryos reaggregate, proliferate, and differentiate in vitro into histotypical spheres, so-called retinospheroids. Under the influence of A. aurantia lectin, processes of proliferation, differentiation and histogenesis of retinospheroids were disturbed. Extending these in vitro studies, we here show that A. aurantia lectin treatment decreases cells of the inner half retina and their processes into inner plexiform layer areas, as revealed by quantitative enzyme histochemistry for butyryl- and acetylcholinesterase, and immunohistochemistry using antibodies to acetylcholinesterase, Pax-6, calbindin-D, and F11. Concomitantly, the number of rod and red/green photoreceptors dramatically increases, using the antibodies rho4D2 and CERN901 (both specific for rods) and CERN906 (specific for red/green cones). These findings show that glycoproteins exhibiting fucose in alpha(1-6)-linkage are involved in processes determining retinal cell fate, strongly shifting the relative ratio of cells of the inner towards cells of the outer retina.

Regulation of the rat oligodendroglia cell line OLN-93 by antisense transfection of butyrylcholinesterase.

Butyrylcholinesterase (BChE) is a glial cell marker with unknown function. For neuroepithelial cells, BChE has been shown to regulate cell division and expression of the postmitotic marker acetylcholinesterase (AChE), while similar studies are lacking for glial cells. By transducing an antisense-5'BChE cDNA expression vector via calcium phosphate precipitation, we have analyzed the effect of BChE inhibition on proliferation and differentiation of rat oligodendroglia-derived OLN-93 cells. OLN-93 cells were chosen because they are highly proliferative, while expressing markers of differentiated oligodendrocytes (Richter-Landsberg and Heinrich, 1996). First, we established that OLN-93 cells do express BChE protein, albeit chiefly in an inactive state, and that BChE was decreased by antisense-5'BChE transfection. Cell proliferation was also strongly diminished, protein kinase C (PKCalpha) was upregulated, and expression of cytoskeletal and cell surface proteins was altered. In particular, immunoreactivities of the intermediate filament proteins vimentin and the cell adhesion protein F11 were detected, indicating that BChE-inhibited OLN-93 cells have shifted toward an astrocytic phenotype. These data support a role of the glia marker BChE in CNS glial cell proliferation and differentiation, achieved via a nonenzymatic mechanism. The possible biomedical impact of BChE protein, e.g., on CNS nerve regeneration, is briefly discussed.

Müller glia cells reorganize reaggregating chicken retinal cells into correctly laminated in vitro retinae.

Müller cells, that belong to the family of radial glia cells, have central functions during retinogenesis. They form a stabilizing scaffold, they are candidate targets for the mediation of extraneous retinogenetic factors, and they are an important source for retina-borne retinogenetic factors. Reaggregate cultures allow the analysis of retinogenesis from dispersed cells to fully laminated tissues. Reaggregating cells from the embryonic chick retina reassemble to reversed laminated cellular spheres including constituents of all retinal layers, yet the outer nuclear layer is represented by internal rosettes. Using spheroids, we tested whether Müller cells have a decisive function in establishing retinal polarity and in determining the lamination pattern. To this end, we established confluent monolayers of highly enriched Müller cells derived from E6 or E13 chicken retinas, and then let dispersed E5.5 retinal cells reaggregate either in the absence of these monolayers or on top of them. In the presence of Müller cells, the reversed lamina polarity of rosetted spheroids progressively transformed within a week into correctly laminated retinal spheres, whereas all initial rosettes vanished. Moreover, photoreceptors formed a regular outer nuclear layer, as visualized by the rod-specific CERN901 antibody. In correctly laminated spheroids, staining for vimentin and glutamine synthetase was much more pronounced than in rosetted spheroids; in particular, a well-established inner limiting membrane stood out wherever the retinal lamination was complete. Because these effects can be similarly achieved by supernatants derived from Müller cells, direct cell-cell contacts or cellular replenishment from the monolayer do not account for these effects. We conclude that Müller cells are involved in the establishment of a correct retinal lamination and in the arrangement of the cells in the reaggregate cultures. In particular, rosette formation is counteracted and the formation of an inner limiting membrane is induced. Because rosettes are objects of concern in several ophthalmological defects, these results are highly relevant, both biomedically and also for normal retinogenesis.

Fucose in alpha(1-6)-linkage regulates proliferation and histogenesis in reaggregated retinal spheroids of the chick embryo.

We have used the lectin from Aleuria aurantia (AAL) which is highly specific for alpha(1-6)-linked fucose, to examine its effect on chicken retinogenesis in a reaggregation culture system. When dispersed cells of the embryonic chick retina are reaggregated to form histotypic retinospheroids, AAL elicits strong inhibition of spheroid growth. The action of AAL is specific, since its effect is dose-dependent, saturable, and inhibited by an excess of fucose. Fucosidase treatment entirely abolishes reaggregation. In contrast, Anguilla anguilla agglutinin (AAA) binding to fucose in alpha(1-2)-linkage does not show any effects. Incubation with CAB4-a specific monoclonal antibody for fucose in alpha(1-6)-linkage-reduces spheroid size and shape. AAL does not much affect primary aggregation, but rather subsequent processes of cell proliferation and histogenesis. In particular, AAL inhibits uptake of bromo-desoxyuridine (BrdU), most efficiently so during days in vitro 2 (div2) and div3. As a consequence, the histological differentiation is entirely disturbed, as evidenced by vimentin immunostaining; particularly, rosettes are not forming and the radial glia scaffold is disorganized. We conclude that glycoproteins exhibiting fucose in alpha(1-6)-linkage may play major roles in early processes of retinal tissue formation.

Butyrylcholinesterase is complexed with transferrin in chicken serum.

The function of the enzyme butyrylcholinesterase (BChE) both in serum and in brain is unclear. In serum, BChE has been found complexed with several biomedically relevant proteins, with which it could function in concert. Here, the existence of a similar complex formed between BChE and sero-transferrin from adult chicken serum was elucidated. In order to identify both proteins unequivocally, we improved methods to highly purify the 81-kDa BChE and the coisolated 75-kDa transferrin, which then allowed us to tryptically digest and sequence the resulting peptides. The sequences as revealed for BChE peptides were highly identical to mammalian BChEs. A tight complex formation between the two proteins could be established (a) since transferrin is coisolated along with BChE over three steps including procainamide affinity chromatography, while transferrin alone is not bound to this affinity column, and (b) since imunoprecipitation experiments of whole serum with a transferrin-specific antiserum allows us to detect BChE in the precipitate with the BChE-specific monoclonal antibody 7D11. The possible biomedical implications of a complex between transferrin and BChE which here has been shown to exist in chicken serum are briefly discussed.

Butyrylcholinesterase antisense transfection increases apoptosis in differentiating retinal reaggregates of the chick embryo.

To investigate the roles of the enzymes butyryl- and acetylcholinesterase (BChE and AChE) in retinal proliferation and differentiation, we use reaggregated spheres from retinal cells of the 6-day-old chick embryo, forming cellular and fibrous areas homologous to all layers of a normal retina. Recently, we could suppress BChE expression by transfecting these so-called retinospheroids during their proliferation period with a pSVK3 expression vector containing a 5' fragment of the rabbit BChE gene in antisense orientation. Along with morphological changes, proliferation was significantly decreased. Here, we have studied the effect of antisense BChE suppression during the differentiation period of retinospheroids. As BChE is suppressed, the differentiation of AChE-positive cells is increased, whereas the immunoreactivities for red and green cone-specific opsins are strongly reduced. Concomitantly, the rate of apoptosis as determined by propidium iodide uptake, by increased CPP 32-like caspase expression, and by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA fragmentation assays is roughly doubled, predominantly at the expense of degenerating photoreceptor precursors. This is further strong evidence that the proliferation marker BChE regulates an intricate balance between cell proliferation, cell differentiation, and programmed cell death in this in vitro retinal system.

Inductive effects of the retinal pigmented epithelium (RPE) on histogenesis of the avian retina as revealed by retinospheroid technology.

During eye formation, inductive phenomena occurring between retinal pigmented epithelium (RPE) and retina are not well understood. After briefly summarizing the normal development of retina and RPE, we present three-dimensional in vitro models of the chick embryonic retina which allows elucidation of RPE-retina interactions. In such retinospheroids, a complete arrangement of layers is achieved, provided that dispersed retinal cells are: (1) young enough; and (2) reaggregated on a monolayer of RPE. Thereby, the RPE extends cell proliferation, while differentiation is much delayed. These findings assign to the RPE a decisive role for the genesis and regeneration of a vertebrate retina.

Müller glia cells and their possible roles during retina differentiation in vivo and in vitro.

Müller cells are astrocyte-like radial glia cells which are formed exclusively in the retina. Here we present evidence that Müller cells are crucially involved in the development of the retina's architecture and circuitry. There is increasing evidence that Müller cells are present from the very early beginning of retinogenesis. We postulate the "gradual maturation hypothesis of Müller cells". According to this hypothesis, Müller cells are continuously generated by a gradual transition of neuroepithelial stem cells into mature Müller cells. This process may be partly reversible. Müller cells, or their immature precursors, are able to subserve different functions. They are primary candidates for stabilizing the complex retinal architecture and for providing an orientation scaffold. Thereby, they introduce a reference system for the migration and correct allocation of neurons. Moreover, they may provide spatial information and microenvironmental cues for differentiating neurons, and may also be important for the segregation of cell and fibre layers. Additionally, they seem to be involved in the guidance of axonal fibres both in radial and in lateral directions, as they are involved in the support and stabilization of synapses.

Aryl acylamidase activity exhibited by butyrylcholinesterase is higher in chick than in horse, but much lower than in fetal calf serum.

Several side activities have been attributed to butyrylcholinesterase (BChE), including aryl acylamidase (AAA) activity, which is an amidase-like activity with unknown physiological function splitting the artificial substrate o-nitroacetanilide. For avians, extensive developmental data have pointed to neurogenetic functions of BChE, however, a possible AAA activity of BChE has not been studied. In this study, we first compare the relative levels of AAA exhibited by BChE in whole sera from chick, fetal calves (FCS) and horse. Remarkably, FCS exhibits a 400-fold higher ratio of AAA/BChE than horse and 80-fold higher than chick serum. We then show that an immunoisolated preparation of BChE from chicken serum presents significant activity for AAA. Both in sera and with the purified enzyme, the AAA activity is fully inhibited by anticholinesterase drugs, showing that AAA activity is exclusively conveyed by the BChE molecule. Noticeably, AAA inhibition even occurs at lower drug concentrations than that of BChE activity itself. Moreover, AAA is sensitive to serotonin. These data indicate that (1) AAA is a general feature of serum BChE of vertebrates including avians, (2) AAA is effectively inhibited by cholinergic and serotonergic agents, and (3) AAA may have a developmental role, since it is much pronounced in a serum from fetal animals. Functionally, deamination of neuropeptides, a link between cholinergic and serotonergic neurotransmitter systems, and roles in lipoprotein metabolism could be relevant.

Pigmented epithelium sustains cell proliferation and decreases expression of opsins and acetylcholinesterase in reaggregated chicken retinospheroids.

We investigated the effect of the retinal pigmented epithelium on cell proliferation and differentiation in rosetted retinospheroids, which are retina-like spheres reaggregated in the complete absence of retinal pigmented epithelium from dissociated retinal cells of 6-day-old chick embryos in a rotation culture system. In spheroids raised in the absence of retinal pigmented epithelium (controls), acetylcholinesterase was expressed in cells of an inner nuclear-like layer and their neuropil matrices. Moreover, the ratio between rods and cones was found to be approximately normal throughout the spheroid. When spheroids were cultured in the presence of retinal pigmented epithelium monolayers, cell proliferation in spheroids as determined by BrdU labelling was significantly increased and extended for 1 week, while acetylcholinesterase protein levels and specific activities in homogenates were decreased to approximately 30%. At the same time, opsin immunoreactivity was completely suppressed within the spheroid and appeared slowly in cells around its periphery; i.e. the proportion of rhodopsin-positive cells decreased from 14 to 3%. This study reveals that the retinal pigmented epithelium in vitro sustains cell proliferation but inhibits the differentiation of acetylcholinesterase-positive cells and of photoreceptors.