Neural Retina Differentiation Research Articles

Direct cell fate conversion of human somatic stem cells into cone and rod photoreceptor-like cells by inhibition of microRNA-203.

Stem cell-based photoreceptor differentiation strategies have been the recent focus of therapies for retinal degenerative diseases. Previous studies utilized embryonic stem (ES) cells and neural retina differentiation cocktails, including DKK1 and Noggin. Here, we show a novel microRNA-mediated strategy of retina differentiation from somatic stem cells, which are potential allogeneic cell sources. Human amniotic epithelial stem cells (AESCs) and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) treated with a retina differentiation cocktail induced gene expressions of retina development-relevant genes. Furthermore, microRNA-203 (miR-203) is abundantly expressed in human AESCs and human UCB-MSCs. This miR-203 is predicted to target multiple retina development-relevant genes, particularly DKK1, CRX, RORβ, NEUROD1, NRL and THRB. The inhibition of miR-203 induced a retina differentiation of AESCs and UCB-MSCs. Moreover, successive treatments of anti-miR-203 led to the expression of both mature photoreceptor (PR) markers, rhodopsin and opsin. In addition, we determined that CRX, NRL and DKK1 are direct targets of miR-203 using a luciferase assay. Thus, the work presented here suggests that somatic stem cells can potentially differentiate into neural retina cell types when treated with anti-miR-203. They may prove to be a source of both PR subtypes for future allogeneic stem cell-based therapies of non-regenerative retina diseases.

Progresses of cell-based therapy and tissue engineering in the treatment of age-related macular degeneration

Age-related macular degeneration (AMD) has become one of the leading causes of irreversible blindness worldwide. With the advancement of stem cell technology, tissue engineering and biomaterials, cell-based therapy has been inspiring for many degenerative diseases. For its unique advantages, AMD has become one of the most promising fields for cell-based therapy, which involve retinal pigment epithelium(RPE)cells, induced differentiation of neural retina cells and related cytokine regulations. RPE cells can be derived from human embryonic stem cells (hESC) or Induced pluripotent stem cells (iPS). Recently hESC-derived RPE cells have been applied to patients with dry AMD with initial success in clinical trials. In terms of tissue engineering, studies are focused on factors affecting the long-term survival of transplanted cells, including tissue scaffolds, soluble hybrid materials and scaffold anchoring. This article briefly reviews the RPE differentiation, neural retina differentiation and related cytokines of cell-based therapy and scaffolds, materials, and cell-scaffolds interactions of tissue engineering in AMD treatment. Key words: Macular degeneration/therapy; Tissue therapy; Tissue engineering; Review

A LIM-homeobox gene, Lhx1 is required for neural retina differentiation of the chicken eye. Immunostaining for retinal differentiation markers of an HH stage 29-eye after Lhx1-RNAi, which changes the presumptive neural retina into pigment retina. Signals

A LIM-homeobox gene, Lhx1 is required for neural retina differentiation of the chicken eye. Immunostaining for retinal differentiation markers of an HH stage 29-eye after Lhx1-RNAi, which changes the presumptive neural retina into pigment retina. Signals

The expression of LIM‐homeobox genes, Lhx1 and Lhx5, in the forebrain is essential for neural retina differentiation

Elucidating the mechanisms underlying eye development is essential for advancing the medical treatment of eye-related disorders. The primordium of the eye is an optic vesicle (OV), which has a dual potential for generation of the developing neural retina and retinal pigment epithelium. However, the factors that regulate the differentiation of the retinal primordium remain unclear. We have previously shown that overexpression of Lhx1 and Lhx5, members of the LIM-homeobox genes, induced the formation of a second neural retina from the presumptive pigmented retina of the OV. However, the precise timing of Lhx1 expression required for neural retina differentiation has not been clarified. Moreover, RNA interference of Lhx5 has not been previously reported. Here, using a modified electroporation method, we show that, Lhx1 expression in the forebrain around stage 8 is required for neural retina formation. In addition, we have succeeded in the knockdown of Lhx5 expression, resulting in conversion of the neural retina region to a pigment vesicle-like tissue, which indicates that Lhx5 is also required for neural retina differentiation, which correlates temporally with the activity of Lhx1. These results suggest that Lhx1 and Lhx5 in the forebrain regulate neural retina differentiation by suppressing the development of the retinal pigment epithelium, before the formation of the OV.

Translating induced pluripotent stem cells from bench to bedside: application to retinal diseases.

Induced pluripotent stem cells (iPSc) are a scientific and medical frontier. Application of reprogrammed somatic cells for clinical trials is in its dawn period; advances in research with animal and human iPSc are paving the way for retinal therapies with the ongoing development of safe animal cell transplantation studies and characterization of patient- specific and disease-specific human iPSc. The retina is an optimal model for investigation of neural regeneration; amongst other advantageous attributes, it is the most accessible part of the CNS for surgery and outcome monitoring. A recent clinical trial showing a degree of visual restoration via a subretinal electronic prosthesis implies that even a severely degenerate retina may have the capacity for repair after cell replacement through potential plasticity of the visual system. Successful differentiation of neural retina from iPSc and the recent generation of an optic cup from human ESc invitro increase the feasibility of generating an expandable and clinically suitable source of cells for human clinical trials. In this review we shall present recent studies that have propelled the field forward and discuss challenges in utilizing iPS cell derived retinal cells as reliable models for clinical therapies and as a source for clinical cell transplantation treatment for patients suffering from genetic retinal disease.

Conditional knockdown of DNA methyltransferase 1 reveals a key role of retinal pigment epithelium integrity in photoreceptor outer segment morphogenesis

Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice. Conditional Dnmt1 knockdown in early eye development mediated by Rx-Cre did not produce lamination or cell fate defects, except in cones; however, the photoreceptors completely lacked outer segments despite near normal expression of phototransduction and cilia genes. We also identified disruption of RPE morphology and polarization as early as E15.5. Defects in outer segment biogenesis were evident with Dnmt1 exon excision only in RPE, but not when excision was directed exclusively to photoreceptors. We detected a reduction in DNA methylation of LINE1 elements (a measure of global DNA methylation) in developing mutant RPE as compared with neural retina, and of Tuba3a, which exhibited dramatically increased expression in mutant retina. These results demonstrate a unique function of DNMT1-mediated DNA methylation in controlling RPE apicobasal polarity and neural retina differentiation. We also establish a model to study the epigenetic mechanisms and signaling pathways that guide the modulation of photoreceptor outer segment morphogenesis by RPE during retinal development and disease.

Otx2 Is Involved in the Regional Specification of the Developing Retinal Pigment Epithelium by Preventing the Expression of Sox2 and Fgf8, Factors That Induce Neural Retina Differentiation

The retinal pigment epithelium (RPE) shares its developmental origin with the neural retina (NR). When RPE development is disrupted, cells in the presumptive RPE region abnormally differentiate into NR-like cells. Therefore, the prevention of NR differentiation in the presumptive RPE area seems to be essential for regionalizing the RPE during eye development. However, its molecular mechanisms are not fully understood. In this study, we conducted a functional inhibition of a transcription factor Otx2, which is required for RPE development, using early chick embryos. The functional inhibition of Otx2 in chick eyes, using a recombinant gene encoding a dominant negative form of Otx2, caused the outer layer of the optic cup (the region forming the RPE, when embryos normally develop) to abnormally form an ectopic NR. In that ectopic NR, the characteristics of the RPE did not appear and NR markers were ectopically expressed. Intriguingly, the repression of Otx2 function also caused the ectopic expression of Fgf8 and Sox2 in the outer layer of the optic cup (the presumptive RPE region of normally developing eyes). These two factors are known to be capable of inducing NR cell differentiation in the presumptive RPE region, and are not expressed in the normally developing RPE region. Here, we suggest that Otx2 prevents the presumptive RPE region from forming the NR by repressing the expression of both Fgf8 and Sox2 which induce the NR cell fate.

Both PAX6 and MITF are required for pigment epithelium development in vivo

Both PAX6 and MITF are required for pigment epithelium development <i>in vivo</i>

Role of apoptosis and mitosis during human eye development

The spatial and temporal distribution as well as ultrastructural and biochemical characteristics of apoptotic and mitotic cells during human eye development were investigated in 14 human conceptuses of 4-9 postovulatory weeks, using electron and light microscopy. In the 5th developmental week, apoptotic and mitotic cells were found in the neuroepithelium of the optic cup and stalk, being the most numerous at the borderline between the two layers of the optic cup, and at the place of transition of the optic cup into stalk. They were also found at the region of detachment of the lens pit from the surface ectoderm. In the later developmental stages (the 6th-the 9th week), apoptotic and mitotic cells were observed in the neural retina and the anterior lens epithelium. Throughout all stages examined, mitotic cells were found exclusively adjacent to the lumen either of the intraretinal space or the optic stalk ventricle, or were restricted to the superficial epithelial layer of the lens primordium. Unlike mitotic cells, apoptotic cells occurred throughout the whole width both of the neuroepithelium and the surface epithelium. Ultrastructurally, apoptotic cells were characterised by round- or crescent-shaped condensations of chromatin near the nuclear membrane, while in the more advanced stages of apoptosis by apoptotic bodies. The distribution of caspase-3-positive cells coincided with the location of apoptotic cells described by morphological techniques indicating that the caspase-3-dependent apoptotic pathway operates during the all stages of human eye development. The location of cells positive for anti-apoptotic bcl-2 protein was in accordance with the regions of eye with high mitotic activity, confirming the role of bcl-2 in protecting cells from apoptosis. In the earliest stage of eye development, apoptosis and mitosis might be associated with the sculpturing of the walls of optic cup and stalk, while high mitotic activity along the intraretinal space and optic stalk ventricle indicates its role in the gradual luminal closure. These processes also participate in the detachment of the lens pit epithelium from the surface ectoderm as well as in further closure of the lens vesicle. Later on, both processes seem to be involved in the neural retina differentiation, lens morphogenesis and secondary lens fibre differentiation.

Optic cup morphogenesis requires pre-lens ectoderm but not lens differentiation

The formation of the vertebrate optic cup is a morphogenetic event initiated after the optic vesicle contacts the overlying surface/pre-lens ectoderm. Placodes form in both the optic neuroepithelium and lens ectoderm. Subsequently, both placodes invaginate to form the definitive optic cup and lens, respectively. We examined the role of the lens tissue in inducing and/or maintaining optic cup invagination in ovo. Lens tissue was surgically removed at various stages of development, from pre-lens ectoderm stages to invaginating lens placode. Removal of the pre-lens ectoderm resulted in persistent optic vesicles that initiated neural retinal differentiation but failed to invaginate. In striking contrast, ablation of the lens placode gave rise to optic vesicles that underwent invagination and formed the optic cup. The results suggest that: (1) the optic vesicle neuroepithelium requires a temporally specific association with pre-lens ectoderm in order to undergo optic cup morphogenesis; and (2) the optic cup can form in the absence of lens formation. If ectopic BMP is added, a neural retina does not develop and optic cup morphogenesis fails, although lens formation appears normal. FGF-induced neural retina differentiation in the absence of the pre-lens ectoderm is not sufficient to create an optic cup. We hypothesize the presence of a signal coming from the pre-lens ectoderm that induces the optic vesicle to form an optic cup.

Multiple functions of fibroblast growth factor-8 (FGF-8) in chick eye development

Fibroblast growth factor-8 (FGF-8) is an important signaling molecule in the generation and patterning of the midbrain, tooth, and limb. In this study we show that it is also involved in eye development. In the chick, Fgf-8 transcripts first appear in the distal optic vesicle when it contacts the head ectoderm. Subsequently Fgf-8 expression increases and becomes localized to the central area of the presumptive neural retina (NR) only. Application of FGF-8 has two main effects on the eye. First, it converts presumptive retinal pigment epithelium (RPE) into NR. This is apparent by the failure to express Bmp-7 and Mitf (a marker gene for the RPE) in the outer layer of the optic cup, coupled with the induction of NR genes, such as Rx, Sgx-1 and Fgf-8 itself. The induced retina displays the typical multilayered cytoarchitecture and expresses late neuronal differentiation markers such as synaptotagmin and islet-1. The second effect of FGF-8 exposure is the induction of both lens formation and lens fiber differentiation. This is apparent by the expression of a lens specific marker, L-Maf, and by morphological changes of lens cells. These results suggest that FGF-8 plays a role in the initiation and differentiation of neural retina and lens.

Ectopic expression of Msx2 in chick retinal pigmented epithelium cultures suggests a role in patterning the optic vesicle

During the initial stages of vertebrate retinogenesis, cells of the optic vesicle adopt one of two alternate cell fates. Cells in the distal-most part of the vesicle, immediately beneath the surface ectoderm, undergo neural differentiation; cells in the proximal part differentiate into retinal pigmented epithelial cells. The mechanisms that establish this pattern of differentiation are poorly understood. In the mouse embryo, Msx2, a homeobox-containing transcription factor, is expressed in cells of the optic vesicle that will form the neural retina, whilst the developing retinal pigmented epithelium (RPE) does not express this gene. Msx2 could therefore be involved in patterning the optic vesicle into neural and pigmented domains. To explore this possibility we ectopically expressed mouse Msx2 in cultures of chick RPE cells. Compared with cultures transfected with a control construct, Msx2-transfected cultures contained fewer cells expressing the RPE marker, Mitf, and more cells expressing class III β-tubulin, a neuronal marker. In addition a small proportion of Msx2-transfected cells acquired a neural-like morphology. These results show that Msx2 can suppress the differentiated state of RPE cells and promote their differentiation into neural cell types. We suggest that Msx2 may pattern the optic vesicle into neural and pigmented domains by affecting the balance between RPE and neural retina differentiation.

Implication of OTX2 in Pigment Epithelium Determination and Neural Retina Differentiation

The expression pattern of Otx2, a homeobox-containing gene, was analyzed from the beginning of eye morphogenesis until neural retina differentiation in chick embryos. Early on, Otx2 expression was diffuse throughout the optic vesicles but became restricted to their dorsal part when the vesicles contacted the surface ectoderm. As the optic cup forms, Otx2 was expressed only in the outer layer, which gives rise to the pigment epithelium. This early Otx2 expression pattern was complementary to that of PAX2, which localizes to the ventral half of the developing eye and optic stalk. Otx2 expression was always observed in the pigment epithelium at all stages analyzed but was extended to scattered cells located in the central portion of the neural retina around stage 22. The number of cells expressing Otx2 transcripts increased with time, following a central to peripheral gradient. Bromodeoxyuridine labeling in combination with immunohistochemistry with anti-OTX2 antiserum and different cell-specific markers were used to determine that OTX2-positive cells are postmitotic neuroblasts undergoing differentiation into several, if not all, of the distinct cell types present in the chick retina. These data indicate that Otx2 might have a double role in eye development. First, it might be necessary for the early specification and subsequent functioning of the pigment epithelium. Later, OTX2 expression might be involved in retina neurogenesis, defining a differentiation feature common to the distinct retinal cell classes.

Fibroblast growth factors are necessary for neural retina but not pigmented epithelium differentiation in chick embryos.

During eye development, optic vesicles evaginate laterally from the neural tube and develop into two bilayered eye cups that are composed of an outer pigment epithelium layer and an inner neural retina layer. Despite their similar embryonic origin, the pigment epithelium and neural retina differentiate into two very distinct tissues. Previous studies have demonstrated that the developmental potential of the pigmented epithelial cells is not completely restricted; until embryonic day 4.5 in chick embryos, the cells are able to switch their phenotype and differentiate into neural retina when treated with fibroblast growth factors (FGF) (Park, C. M., and Hollenberg, M. J. (1989). Dev. Biol. 134, 201-205; Pittack, C., Jones, M., and Reh, T. A. 1991). Development 113, 577-588; Guillemot, F. and Cepko, C. L. (1992). Development 114, 743-754). These studies motivated us to test whether FGF is necessary for neural retina differentiation during the initial stages of eye cup development. Optic vesicles from embryonic day 1.5 chick were cultured for 24 hours as explants in the presence of FGF or neutralizing antibodies to FGF2. The cultured optic vesicles formed eye cups that contained a lens vesicle, neural retina and pigmented epithelium, based on morphology and expression of neural and pigmented epithelium-specific antigens. Addition of FGF to the optic vesicles caused the presumptive pigmented epithelium to undergo neuronal differentiation and, as a consequence, a double retina was formed. By contrast, neutralizing antibodies to FGF2 blocked neural differentiation in the presumptive neural retina, without affecting pigmented epithelial cell differentiation. These data, along with evidence for expression of several FGF family members and their receptors in the developing eye, indicate that members of the FGF family may be required for establishing the distinction between the neural retina and pigmented epithelium in the optic vesicle.

QR1, a retina-specific gene, encodes an extracellular matrix protein exclusively expressed during neural retina differentiation

Neural retina development results from growth arrest of neuroectodermal precursors and differentiation of postmitotic cells. The QR1 gene is specifically expressed in Mu¨ller retinal glial cells. Its expression coincides with the stage of withdrawal from the cell cycle and establishment of differentiation and is repressed upon induction of retinal cell proliferation by the v-src gene product. In this report, we show that the QR1 gene encodes several glycosylated proteins that are secreted and can either associate with the extracellular matrix or remain diffusible in the medium. By using pulse-chase experiments, the 100–103 kDa forms seem to appear first and are specifically incorporated into the extracellular matrix, whereas the 108 and 60 kDa polypeptides appear later and are detected as soluble forms in the culture medium. We also report that expression of the QR1 gene is developmentally regulated in the chicken. Its mRNA is first detectable at embryonic day 10, reaches a maximal level at embryonic day 15 and is no longer detected at embryonic day 18. Immunolocalization of the QR1 protein in chicken retina sections during development shows that expression of the protein parallels the differentiation pattern of post-mitotic cells (in particular Mu¨ller cells and rods), corresponding to the two differentiation gradients in the retina: from the ganglion cell layer to the inner nuclear layer and outer nuclear layer, and from the optic nerve to the iris. At embryonic day 10, expression of the QR1 protein(s) is restricted to the optic nerve region and the inner nuclear layer, colocalizing with Mu¨ller cell bodies. As development proceeds, QR1 protein localization spreads towards the iris and towards the outer nuclear layer, following Mu¨ller cell elongations towards the photoreceptors. Between embryonic days 16 and 18, the QR1 protein is no longer detectable in the optic nerve region and is concentrated around the basal segment of the photoreceptors in the peripheral retina. Our results suggest a role for the QR1 gene product in the process of growth arrest and establishment of photoreceptor differentiation.

Transcriptional stimulation of the retina-specific QR1 gene upon growth arrest involves a Maf-related protein.

The avian neural retina (NR) is derived from proliferating neuroectodermal precursors which differentiate after terminal mitosis and become organized in cell strata. Proliferation of postmitotic NR cells can be induced by infection with Rous sarcoma virus (RSV) and requires the expression of a functional v-Src protein. QR1 is a retina-specific gene expressed exclusively at the stage of growth arrest and differentiation during retinal development. In NR cells infected with tsPA101, an RSV mutant conditionally defective in pp60v-src mitogenic capacity, QR1 expression is downregulated in proliferating cells at 37 degrees C and is fully restored when the cells become quiescent as a result of pp60v-src inactivation at 41 degrees C. We were able to arrest proliferation of tsPA101-infected quail NR cells expressing an active v-Src protein by serum starvation at 37 degrees C. This allowed us to investigate the role of cell growth in regulating QR1 transcription. We report that QR1 transcription is stimulated in growth-arrested cells at 37 degrees C compared with that in proliferating cells maintained at the same temperature. Growth arrest-dependent stimulation of QR1 transcription requires the integrity of the A box, a previously characterized cis-acting element responsible for QR1 transcriptional stimulation upon v-Src inactivation and during retinal differentiation. We also show that formation of the C1 complex on the A box is increased upon growth arrest by serum starvation in the presence of an active v-Src oncoprotein. Thus, the C1 complex represents an important link between cell cycle and developmental control of QR1 gene transcription during NR differentiation and RSV infection. By using antibodies directed against different Maf proteins of the leucine zipper family and competition with Maf consensus site-containing oligonucleotides in a gel shift assay, we show that the C1 complex is likely to contain a Maf-related protein. We also show that a purified bacterially expressed v-Maf protein is able to bind the A box and that the level of a 43-kDa Maf-related protein is increased upon growth arrest in infected retinal cells. Moreover, ectopic expression of c-mafI, c-mafII, and mafB cDNAs in quiescent tsPA101-infected quail NR cells is able to stimulate transcription of a QR1 reporter gene through the A box. Therefore, QR1 appears to be the first target gene for a Maf-related protein(s) in the NR.

Cek5, a Tyrosine Kinase of the Eph Subclass, Is Activated during Neural Retina Differentiation

The expression of Cek5, a receptor-type tyrosine kinase of the Eph subclass, and its variant form Cek5+ were examined in the chick neural retina during development. Cek5 is present at high levels at all stages of retinal development examined, while Cek5+ is most abundant during differentiation. Cek5 mRNA expression and immunoreactivity are evenly distributed in the undifferentiated retina. With differentiation, Cek5 becomes concentrated in the inner and outer plexiform layers. While only moderate changes in Cek5 protein expression are observed throughout retinal development, Cek5 phosphorylation on tyrosine in vivo is dramatically increased during differentiation. This suggests that the Cek5 ligand is expressed at high levels and causes Cek5 activation. Thus, Cek5 is likely to play an active role in retinal morphogenesis, particularly during the establishment of interneuronal contacts.

Alpha-Fetoprotein Uptake by Differentiating Neuroretinal Structures of the Chick Embryo

Internalization of exogenous fluoresceinated alpha-fetoprotein (FITC-AFP) was studied at different stages of development on embryonic chick neural retinas maintained, for short periods, in organ cultures. Cellular localization of endogenous, native AFP was carried out by immunohistoperoxidase methods. Cells which specifically internalized exogenous FITC-AFP (neurons and their processes) were precisely those showing positive immunolabeling for the endogenous, native protein. Such a result supports the hypothesis of a predominantly exogenous origin of intracellular neuroretinal AFP. A precise topography and temporal sequence of AFP labeling after internalization in retinal structures is given. AFP uptake was not displayed by undifferentiated cell precursors or germinal cell layers but was apparent in cells with phenotypic characteristics of maturing neurons. Nerve fibers and synaptic layers actively internalized FITC-AFP at specific stages of development. Fully differentiated neurons and processes did not internalize AFP. The possible role of AFP, a carrier of biologically active substances such as fatty acids, in neural retina differentiation is discussed.

Cell commitment and differentiation in explants of embryonic rat neural retina. Comparison with the developmental potential of dissociated retina

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.

Ultracytochemical study on in vitro differentiation of neural retinal cells of chick embryos

In order to study cell differentiation and morphogenesis of neural retina, ultracytochemical examination for acetylcholine esterase (AChE) was carried out on neural retinal cells from 6-day-old chick embryos cultured in monolayer for 20 days. AChE is a suitable marker for identifying cell specificity and structure of cultured neural retinal cells, because its specific localization in the intact chick neural retina has been established. After about 2 weeks of culturing a number of cell aggregates formed on the monolayer sheet of glial cells, in which cell bodies were generally located on the periphery regions while their cellular processes were in the center, forming neuropil structures. Among such peripherally located cells presumptive ganglion, amacrine, bipolar, and photoreceptor cells could be distinguished. In the neuropil structures, some cellular processes had typical ribbon synapses indicating that these structures correspond to the plexiform layers of the retina. We could also classify the neuropils into two types of both from the AChE activity and from the structure of the nerve terminals. These findings indicate that our cell culture system can be used for the study of cell differentiation and histogenesis of retinal cells.