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Retinal pigment epithelium (RPE) is a source of cells for retinal regeneration in amphibians in vivo and the development of retinal diseases in mammals and humans. Transdifferentiation of RPE cells into cells of other phenotypes is the basis for both processes: RPE cells transform into neural in the first case and into mesenchymal cells in the second case. The review describes the main stages of RPE cell transdifferentiation: initiation of the process, cell migration and proliferation, dedifferentiation, reprogramming, and specialization of cells into new directions. Information about the molecular and genetic mechanisms that ensure the passage of these stages by cells is given. Molecular participants of the regulation of transdifferentiation on the levels of the whole organism, the local cellular microenvironment (growth factors, signaling cascades), the expression of transcription factors, and the epigenome regulation are presented. Similarities and differences in the molecular and genetic mechanisms of implementation of different strategies for RPE transdifferentiation in amphibians and mammals are noted. The discovery of key molecular regulators of this choice serves both for the development of the theory of cellular reprogramming and approaches for the treatment of proliferative diseases of the human retina associated with RPE pathologies.

The development of the retina and the signaling pathways involved in this process are conserved among vertebrates. Therefore, it was hypothesized that active factors stimulating the retinal regeneration in newts can cause transdifferentiation of human retinal pigment epithelial (RPE) cells. To verify this, we studied the effect of conditioned media (CM) obtained from in vitro regenerating retinas of newts on the phenotype, expression of a number of genes, and localization of cell proteins of human RPE cells of the ARPE-19 line. Within 120 h of cultivation, a change in the morphology of human RPE cells was observed, which manifested in a change in shape and an increase in cell size, which was accompanied by a decrease in proliferative activity. 24 hours after exposure to CM, ARPE-19 showed a short-term increase in the expression of TUBB3 (a panneuronal marker) and a decrease in the expression of BMP2 and BMP4 (neural diffe-rentiation inhibitors). Using immunocytochemical (ICC) methods, we observed an increase in the intensity of staining of cells with antibodies to βIII-tubulin protein and a decrease in the intensity of staining with antibodies to the Cx43 protein of gap junctions (compared to the control) during the period between 24 and 72 h after exposure. At the same time, we observed a decrease in the levels of mRNA expression of the RPE markers OTX2 and KRT18, as well as the KLF4 gene, which is responsible for proliferation and differentiation. Additionally, the shift in the distribution of β-catenin to cytoplasmic localization was recorded using the ICC method. To exclude mesenchymal (and osteogenic in particular) differentiation under the effect of CM, we studied expression of the SPP1 and RUNX2 genes by qPCR. No SPP1 transcription was found in ARPE-19 cells in the control and under the effect of CM, and a decrease in the expression level of RUNX2 mRNA under the effect of CM was observed. Therefore, an attempt to shift to neuronal differentiation was observed in ARPE-19 cells during the period between 24 and 72 h after exposure to the CM of newt retinal regenerates, as indicated by an increase in transcription and translation of βIII-tubulin, a weakening of intercellular adhesion, and a decrease in the expression of inhibitors of neural differentiation BMP2 and BMP4, as well as RPE markers OTX2 and KRT18. However, after that, due to the short-term effect of CM, during the period between 72 and 120 h after exposure, the cells returned to the initial differentiation, as evidenced by a decrease in the expression of neuronal differentiation markers (NES, TUBB3, PAX6) and an increase in the expression of RPE markers (OTX2, MITF).

Retinal pigment epithelium (RPE), whose main functions are to participate directly in light perception and ensure the trophic and functioning of the retina, is a traditional subject of numerous studies. A significant part of them is biomedical and is aimed at identifying the causes of aging and degeneration of RPE, which are, in turn, associated with several serious retinal diseases that lead to the loss of the eyesight. Rodents, in particular, the albino Wistar rats, are often used as the object of these studies. The work is aimed at identifying signs of aging in the RPE in young (from 2 weeks up to 2 months) albino rats. Using the methods of fluorescence analysis and Schmorl staining, the accumulation of the “aging pigment” lipofuscin in the isolated layer of RPE, as well as a high content of polyploid binuclear cells to mononuclear cells, were detected in albino rats of an early age. In parallel, the presence of lipofuscin was detected in the isolated vascular layer (choroid) of the retina that underlies RPE. The relationship between the early appearance of signs of aging in the RPE of albino rats and the absence of the melanin pigment in the cells of this tissue is discussed. The obtained data should be taken into account when conducting experimental ophthalmological studies on this animal model object.

Using the literature and the authors’ own data, the review provides information on experiments carried out at different times with organotypic culturing of the retina in vertebrates and humans. The method allows one to maintain the structure and viability of the retina, model a number of its pathological conditions, and observe the processes of retinal development, regeneration, reconstruction, death, and growth of neuron outgrowths. In addition, organotypic culturing makes it possible to affect all indicated processes by different regulating factors as well as protective/damaging agents at strictly specified concentrations and under controlled conditions. Particular attention is paid to the behavior of retinal pigment epithelium cells, photoreceptor cells, Muller glia cells, and ganglion cells and their axons (that is, to those cell populations that are most often affected in cases of different pathological conditions and retinal diseases). A separate section is devoted to the production and culturing of so-called retinal organoids, which is being actively developed at present. The direction promises opportunities for the transplantation of retinal cells, conducting experiments on gene therapy, and testing ophthalmic pharmacological drugs.

We summarize data from our experiments on stem-like cell-dependent regeneration in amphibians in microgravity. Considering its deleterious effect on many tissues, we asked whether microgravity is compatible with reparative processes, specifically activation and proliferation of source cells. Experiments were conducted using tailed amphibians, which combine profound regenerative capabilities with high robustness, allowing an in vivo study of lens, retina, limb, and tail regeneration in challenging settings of spaceflight. Microgravity promoted stem-like cell proliferation to a varying extent (up to 2-fold), and it seemed to speed up source cell dedifferentiation, as well as sequential differentiation in retina, lens, and limb, leading to formation of bigger and more developed regenerates than in 1g controls. It also promoted proliferation and hypertrophy of Müller glial cells, eliciting a response similar to reactive gliosis. A significant increase in stem-like cell proliferation was mostly beneficial for regeneration and only in rare cases caused moderate tissue growth abnormalities. It is important that microgravity yielded a lasting effect even if applied before operations. We hypothesize on the potential mechanisms of gravity-dependent changes in stem-like cell behavior, including fibroblast growth factor 2 signaling pathway and heat shock proteins, which were affected in our experimental settings. Taken together, our data indicate that microgravity does not disturb the natural regenerative potential of newt stem-like cells, and, depending on the system, even stimulates their dedifferentiation, proliferation, and differentiation. We discuss these data along with publications on mammalian stem cell behavior in vitro and invertebrate regeneration in vivo in microgravity. In vivo data are very scarce and require further research using contemporary methods of cell behavior analysis to elucidate mechanisms of stem cell response to altered gravity. They are relevant for both practical applications, such as managing human reparative responses in spaceflight, and fundamental understanding of stem cell biology.

Retinal diseases often cause the loss of photoreceptor cells and, consequently, impairment of vision. To date, several cell populations are known as potential endogenous retinal regeneration cell sources (RRCSs): the eye ciliary zone, the retinal pigment epithelium, the iris, and Müller glia. Factors that can activate the regenerative responses of RRCSs are currently under investigation. The present review considers accumulated data on the relationship between the progenitor properties of RRCSs and the features determining their differentiation. Specialized RRCSs (all except the ciliary zone in low vertebrates), despite their differences, appear to be partially “prepared” to exhibit their plasticity and be reprogrammed into retinal neurons due to the specific gene expression and epigenetic landscape. The “developmental” characteristics of RRCS gene expression are predefined by the pathway by which these cell populations form during eye morphogenesis; the epigenetic features responsible for chromatin organization in RRCSs are under intracellular regulation. Such genetic and epigenetic readiness is manifested in vivo in lower vertebrates and in vitro in higher ones under conditions permissive for cell phenotype transformation. Current studies on gene expression in RRCSs and changes in their epigenetic landscape help find experimental approaches to replacing dead cells through recruiting cells from endogenous resources in vertebrates and humans.

Modern achievements in the understanding of tissue regeneration, identification of endogenous cell sources for regeneration, and development of approaches for induction and differentiation of pluripotent stem cells have open broad prospects for regenerative medicine. However, application of the obtained information in medicine is hindered by insufficient knowledge on the molecular factors and their combinations capable of regulating the age and fate of cellular sources for eye tissue reparation as well as on the regenerative responses of these cells. In the review, we present our own and literature data on cells serving as endogenous sources for eye tissue regeneration in lower and higher vertebrates and properties of gene expression that allow these cells to maintain their juvenile phenotype. Transcription factors and signal pathways providing cell juvenile status as well as cell reprogramming and entry into the S-phase are discussed. The role of systemic factors (blood and immune system factors, hormones, oxidative stress products, and cell rejuvenation factors) in these processes and their interaction with local factors of the cell environment are described. Molecular factors and conditions for induction of reprogramming and proliferation of cellular sources involved in regeneration in vitro are analyzed with special attention to the role of epigenetic factors (associated with cell senescence, in particular) in the source cells conversion during eye tissue regeneration.

A number of retinal diseases are known to result in impaired vision and even blindness. In this regard, the major issue that modern biology faces nowadays is to identify endogenous populations of cells that could be a potential source of retinal regeneration and provide their detailed description using advanced methods and various animal models. This will allow for finding the ways to control the behavior and regenerative activity of these cells in order to replace damaged retinal neurons, particularly photoreceptors, either by involving natural mechanisms or via cell transplantation. In this review, the latest data published in literature and results of the authors’ own research on localization, biology, and molecular genetic features of cell sources for retinal regeneration, obtained for all classes of vertebrates, from fish to mammals, are summarized. In the range of these cell populations existing in vertebrates, certain versatility in terms of gene expression has been found. A variety of ways for their mobilization for regeneration of the damaged retinal tissue in different animal classes and species have also been well documented.

The review considers the molecular, cellular, organismal, and ontogenetic properties of Urodela that exhibit the highest regenerative abilities among tetrapods. The genome specifics and the expression of genes associated with cell plasticity are analyzed. The simplification of tissue structure is shown using the examples of the sensory retina and brain in mature Urodela. Cells of these and some other tissues are ready to initiate proliferation and manifest the plasticity of their phenotype as well as the correct integration into the pre-existing or de novo forming tissue structure. Without excluding other factors that determine regeneration, the pedomorphosis and juvenile properties, identified on different levels of Urodele amphibians, are assumed to be the main explanation for their high regenerative abilities. These properties, being fundamental for tissue regeneration, have been lost by amniotes. Experiments aimed at mammalian cell rejuvenation currently use various approaches. They include, in particular, methods that use secretomes from regenerating tissues of caudate amphibians and fish for inducing regenerative responses of cells. Such an approach, along with those developed on the basis of knowledge about the molecular and genetic nature and age dependence of regeneration, may become one more step in the development of regenerative medicine

The highest potencies of regeneration in tailed amphibians in comparison with the abilities of organ and tissue restoration in other vertebrates represent the goal of longstanding and intense studies. Accumulated information can half-open some mysteries of cellular and molecular fundamentals of regeneration in Urodela, but it does not explain the maintenance of regenerative abilities in mature, adult animals. The information summarized in the review suggests that the paedomorphosis inherent in this animal group determines the keeping of the juvenile state on all levels of organization—from organismic to molecular. This, in turn, permits and eases initiation and development of regenerative responses to trauma, right up to the epimorphic regeneration of whole organs. As an example, we have traced paedomorphosis-associated cellular and molecular specificities of urodelean eye and brain tissues, which could possibly play a permissive role in their complete regeneration.

Nucleotide sequences were identified in the eye tissues (lens, retina, and retinal pigment epithelium) of the adult newt Pleurodeles waltl by the polymerase chain reaction with primers for the Ns gene. Sequencing showed that these nucleotide sequences belong to the Ns gene of the newt P. walt, which encodes the nucleolar protein nucleostemin. Structural analysis revealed a high homology of Ns nucleotide sequences of P. walt! with those of newts. Cynops pyrrhogaster and Notophthalmus viridescens. The expression of the Ns gene of P. walt, identified in the specialized eye cells of adult newts of the studied species, indicates that these differentiated cells retain some of the molecular characteristics inherent to the undifferentiated cells.

Retinal pigment epithelium (RPE) cells that have the unique ability to reprogram retinal cells in vivo were analyzed in the adult newt. Our own data and that available in the literature on the peculiarities of the biology of these cells (from morphology to molecular profile, which can be associated with the capability of phenotype change) were summarized. It was established that the molecular traits of specialized and poorly differentiated cells are combined in RPE of the adult newt. It was registered that persistent (at a low level) proliferation and rapid change of specific cytoskeleton proteins can contribute to the success of RPE cell reprogramming in the neuronal direction. Each of the considered factors of competence for reprogramming can be found for animal RPE, whose cells are not able in vivo to change the phenotype to a neuronal one; however, their totality (supported by the epigenetic state permissive for conversion) is probably an internal property of only newt RPE.

Regenerating newt tail has recently been found to react to hypergravity in a stable and reproducible way - by curving downwards. Such morphogenetic effect of non-specific physical factor applied to a complex structure of an adult animal is a rare phenomenon with unknown molecular basis. For the first steps of unraveling this basis we've chosen heat shock proteins (HSPs) as promising candidates. Morphometrical analysis of tail regeneration was performed in aquarium (control), on substrate (relative hypergravity) and in aquarium under weekly application of heat shock. HSPs were inhibited pharmacologically during regeneration in aquarium and on substrate. Hsp70, 90 gene expression and protein localization were analyzed in the studied conditions. Weekly application of heat shock to newts regenerating tails in otherwise normal conditions led to development of curved tails (both upwards and downwards), suggesting that similar mechanisms are at play in both hypergravity-altered and heat shock-altered morphogenesis. Heat shock protein inhibitor KNK437 didn't affect tail shape during normal regeneration, but prevented the formation of tail curve in appropriate conditions. It was shown that HSP70 and HSP90 proteins are present in muscle and connective tissue of intact tails as well as regenerates, but only appear in epidermis in hypergravity-altered regenerates and heated tails. Based on our data, we hypothesize, that different external factors (e.g. hypergravity and heat shock) are received, analyzed and transmitted further to affect morphogenesis by similar mechanisms that utilize a set of HSP in epidermal cells.

The FGF2 signaling pathway components in tissues of the posterior wall in the normal and regenerating eye of the adult Pleurodeles waltl newt were detected for the first time. The fgf2 gene expression was found in the retina, retinal pigment epithelium, and choroid using polymerase chain reaction (PCR). A high homology of the mRNA nucleotide sequence of the most conservative fgf2 gene region in the P. waltl with the fgf2 orthologs in other vertebrates was proved. The Fgf2 protein aminoacid sequence of the P. waltl newt demonstrates even more homology with this growth factor in other vertebrates. The Fgf2 protein with a molecular weight 35 kDa was found in the studied eye tissues using Western blot hybridization. Localization of the Fgf2 protein and its Fgfr receptors was immunohistochemically studied in the pigment epithelium, choroid, central and growth retina regions of the newt native eye, and in the connective cilium of photoreceptors. Using real-time PCR and immunohistochemistry methods, it was found that the fgf2 gene down-regulation and a decrease in the intensity of the immunochemical reaction of its protein product (Fgf2) occur in the early period after the retina removal (in 4-8 days) (as compared with those in the same department of the unoperated eye).

Expression of genes and heat shock proteins in normal intact retina of the Spanish Ribbed Newt Pleurodeles waltl was studied using polymerase chain reaction, Western blot hybridization, and immunohis-tochemistry. It was shown that the proteins HSP70 and HSP90, as well as their encoding transcripts of relevant genes, are constitutively expressed in eye tissues. These proteins were distributed differentially, and they were characterized by expression of different levels in the retina: HSP70 dominated in the external retina, while HSP90 dominated in the internal one, in particular, in Muller glial cells and the optic nerve. Transcripts and heat shock proteins HSP70 and HSP90 were also found in the retinal pigment epithelium and eye growth zone.

During life human eye is constantly exposed to sunlight and artificial light, the sources of reactive oxygen species (ROS)—the main cause of age-related eye pathology. A novel mitochondria-targeted antioxidant SkQ1 has recently been invented to reduce mitochondrial ROS by cleaning the mitochondria matrix, “the dirtiest place in the cell” in respect of ROS production and accumulation. Earlier we studied SkQ1 effects upon retinal pigment epithelium and choroid in the rat eye posterior cups exposed to long-term 3D organotypic culturing. It was found that under in vitro conditions 20 nM SkQ1 effectively reduced cell death in retinal pigment epithelium and choroid and protected the tissues from disintegration and cell withdrawal. In the present study we used same ex vivo conditions to examine the effect of SkQ1 upon the rat neural retina kept in the content of the posterior eye cup. Eye cups were isolated and cultured in vitro during 7, 14, and 30 days under rotation in the presence and absence of 20 nM SkQ1 in the culture medium. Serial sections of cultivated eye cups were subjected to histology, computer morphometry and immunohistochemistry. Obtained results show that SkQ1 operates as a strong protective agent, preventing neuronal cell death and other degenerative processes in the neural retina. Cell rescue by SkQ1 was more vivid in the central part of the retina than at the periphery. That, in turn, suggests SkQ1 effectiveness in treatment of some age-related eye diseases when central part of the retina, including macula, is most susceptible to degeneration.

Many regeneration processes in animals are based on the phenomenon of cell reprogramming followed by proliferation and differentiation in a different specialization direction. An insight into what makes natural (in vivo) cell reprogramming possible can help to solve a number of biomedical problems. In particular, the first problem is to reveal the intrinsic properties of the cells that are necessary and sufficient for reprogramming; the second, to evaluate these properties and, on this basis, to reveal potential endogenous sources for cell substitution in damaged tissues; and the third, to use the acquired data for developing approaches to in vitro cell reprogramming in order to obtain a cell reserve for damaged tissue repair. Normal cells of the retinal pigment epithelium (RPE) in newts (Urodela) can change their specialization and transform into retinal neurons and ganglion cells (i.e., actualize their retinogenic potential). Therefore, they can serve as a model that provides the possibility to identify factors of the initial competence of vertebrate cells for reprogramming in vivo. This review deals mainly with the endogenous properties of native newt RPE cells themselves and, to a lesser extent, with exogenous mechanisms regulating the process of reprogramming, which are actively discussed.

Gravity-dependent shape alterations in newt tail regenerates are described, which were previously noticed in experiments onboard satellites Foton M2, M3 and in corresponding laboratory controls. Laboratory conditions were developed that allow reproducing this phenomenon persistently in the adult newts Pleurodeles waltl (Michahelles, 1830). The newts kept in an aquarium (in partial weightlessness) after 1/3 tail amputation developed normal lanceolate regenerates, while those that stayed on a moist mat (exposed to greater gravity than in aquarium) developed curved tail regenerates. Dynamics of the shape alterations were described using computer morphometric analysis. The curve was shown to develop at stage III of regeneration and to be caused by bending of the developing axial structures: the ependymal tube and the cartilage rode. Cellular processes were described that accompany the tail shape changes, such as cell migration and formation of dense aggregates. Unequal proliferation throughout the wound epidermis and blastema was revealed using BrdU assay. Proliferation increased within dorsal and apical regions of the regenerates in the newts kept on the mat cell compared with the aquarian animals.

The effects of real and simulated microgravity on the eye tissue regeneration of newts were investigated. For the first time changes in Muller glial cells in the retina of eyes regenerating after retinal detachment were detected in newts exposed to clinorotation. The cells divided, were hypertrophied, and their processes were thickened. Such changes suggested reactive gliosis and were more significant in animals exposed to rotation when compared with desk-top controls. Later experiments onboard the Russian biosatellite Bion-11 showed similar changes in the retinas that were regenerating in a two-week spaceflight. In the Bion-11 animals, GFAP, the major structural protein of retinal macroglial cells, was found to be upregulated. In a more recent experiment onboard Foton-M3 (2007), GFAP expression in retinas of space-flown, ground control (kept at 1 g), and basal control (sacrificed on launch day) newts was quantified, using microscopy, immunohistochemistry, and digital image analysis. A low level of immunoreactivity was observed in basal controls. In contrast, retinas of space-flown animals showed greater GFAP immunoreactivity associated with both an increased cell number and a higher thickness of intermediate filaments. This, in turn, was accompanied by up-regulation of stress protein (HSP90) and growth factor (FGF2) expressions. It can be postulated that such a response of Muller cells was to mitigate the retinal stress in newts exposed to microgravity. Taken together, the data suggest that the retinal population of macroglial cells could be sensitive to gravity changes and that in space it can react by enhancing its neuroprotective function.

Cells have intrinsic mechanisms for cleaning harmful oxidants represented mainly by reactive oxygen species (ROS). Despite the antioxidant defense, ROS can cause serious damage to the retina that with age leads to various eye diseases and even blindness. Among numerous cell sites of ROS generation, mitochondrial electron transport is of crucial importance. Recently, for the purpose of cleaning ROS in the mitochondrial matrix, powerful mitochondria- targeted antioxidant “SkQ1” has been invented. We studied SkQ1 effects upon tissues of rat posterior eye cup that consisted: retinal pigment epithelium (RPE) ? choroidal coat ? scleral coat. The eye cups were isolated from the eyes of adult albino rats and cultivated in rotary tissue culture system in the presence of 20 nM SkQ1 or without this compound. After 7 days - 1 month in vitro eye cup samples were studied by immunohistochemistry, routine histology, morphometry, and digital image analysis. We have found that under chosen, “in vitro like in vivo” conditions 20 nM SkQ1 effectively reduced cell death in RPE and choroid, protected RPE from disintegration caused by cell phenotypic transformation and withdrawal from the layer, suppressed transmigration of choroidal coat cells. In the ex vivo model we used degenerative processes were more pronounced in the eye cup center where SkQ1 effect was most vivid. All this give us hopes for effectiveness of SkQ1 treatment of retinal central part that is very susceptible to light-induced over-oxidation injury and mostly suffering in many age-related diseases, AMD, in particular.

The retinal pigment epithelium (RPE), as well as the neural retina, develops from the neuroectoderm and plays a key role in photoreceptor functions. Several degenerative eye diseases, e.g., macular degeneration or retinitis pigmentosa, associated with an impaired RPE function cause the loss of the photoreceptor and partial or complete blindness. Cultured RPE cells obtained from human cadaver eyes could be a valuable source for transplantation to cure retinal degenerative diseases. The paper describes RPE cell isolation, maintenance in culture, and immunohistochemical characteristics of dedifferentiated cells. It was found that RPE cells from human adults exhibit neural cell properties in vitro.

Phenotypic plasticity of retinal pigment epithelial cells from adult human eye was studied by immunohistochemical methods under different culturing conditions. It was found that retinal pigment epithelium in adult human eye is a heterogeneous population of cells demonstrating different behavior in vitro. Some cells retain epithelial morphology for a long time in culture, while others are rapidly transformed into fibroblast-like cells and synthesize proteins typical of proneural, neural, glial, and photoreceptor cells. However, irrespective of initial morphological features differentiation of retinal pigment cells can be modulated by varying culturing conditions.

For the first time immune-chemical analysis of transcriptional factor Pitx2 localization during triton retina regeneration after its removal and also in tissues of a nonoperated eye of an adult triton has been carried out. Protein Pitx2 has been found in the nucleus of the earliest neuroblasts that form the germ of the retina. At a later stage of retina regeneration, Pitx2 was found in the nucleus of differentiating cells of ganglionic layers that correspond to Pitx2 protein localization in the native retina. Protein Ptix2 has also been found in the nucleus of less differentiated cells of the peripheral region of regenerative and native retina. It was demonstrated that protein Pitx2 is expressed not only in retina but also in other tissues of the posterior sector of the eye (pigment epithelium, choroid) using immune-histochemical and Western blot hybridization. It is supposed that transcriptional factor Pitx2 has been involved in the control of subsequent stages of retina regeneration from pigment epithelium cells.

To understand why the retinal pigment epithelium (RPE) has different potentials for neural differentiation in lower and higher vertebrates, the RPEs of adult newts and rats were compared under similar in vitro cultivation conditions. The RPEs of both animal species were organotypically cultivated within the posterior eye wall under constant rotation in the serum medium free of growth factors. Comparison of the cell morphology, proliferation, and expression of pan-neural markers demonstrated that the RPE cells of adult newts and rats under similar in vitro conditions displayed both similarities and differemces. They were able to synthesize DNA but rarely divided mitotically. In addition, part of the RPE cells of both the newt and the rat were dislodged from the layer, migrated, and acquired a macrophage phenotype. However, the majority of the cells retained the initial morphology and remained within the layer. In several cases, these cells displayed the initial characteristics of neural differentiation, namely, expression of pan-neural proteins. The difference between the newt and rat RPE cells was in the ability of the former to generate in vitro an additional row of dedifferentiated NF-200-positive cells, characteristic of in vivo newt retinal regeneration. These data demonstrate that the RPE cells of the adult newt and rat retain the potential of manifesting neural cell traits; however, more advanced changes towards differentiation are characteristic of only the newt RPE.

Immunohistochemical study of the expression of recoverin (photoreceptor protein) in the retina of Pleurodeles waltl adult triton was carried out in health, during regeneration after removal, and under conditions of long-lasting detachment. Studies with polyclonal (monospecific) antibodies to recoverin showed that normally it is present in the internal segment, connective cilium, in distal portions of the external segments of cones and rods, and in Landolt clubs of displaced bipolar cells. Detachment of the retina is associated with translocation of recoverin from the photoreceptor processes to perikaryons, and the content of recoverin-positive displaced bipolar cells increases. During regeneration of the retina after its excision via conversion of the pigmented epithelial cells, recoverin is synthesized in the prospective photoreceptor perikaryons and then accumulates in the forming inner segments. Hence, recoverin can serve as a reliable marker in studies of photoreceptor differentiation and functioning during regeneration or survival of the retina.

This work continues the studies of the proliferative ability of cells in the adult newt retina. The model of experimental detachment of the retina from pigment epithelium and two techniques to saturate the ocular tissues in vivo with precursors of DNA synthesis were used: (1) the method of repeated [3H]-thymidine labeling and subsequent autoradiographic analysis of semithin sections and (2) an original method for continuous labeling of thymidine analog bromodeoxyuridine and subsequent immunochemical detection. The data obtained confirm and extend our previous data on the localization of DNA-synthesizing cells in the neural retina and expose the pattern of S-phase cell accumulation after retinal detachment for each proliferation-competent cell population. In addition to cells in the growth zone of the retina, Muller glia, microglia, and minor cell population in the vitreal part of interneurons, DNA-synthesizing cells included astrocytes of the optic nerve and cells of its vascular network. Four weeks after detachment, the number of S-phase cells in the growth zone could reach 15-20%, while the above-mentioned DNA-synthesizing cells in the differentiated retina have low reproductive rate and could produce only one generation within the same period.

The effect of the adhesion protein isolated from the bovine cornea was studied on the model of mechanical injury (cross cutting of the cornea). In the concentration of 10−12 mg/ml, the protein influenced the proliferation of corneal epithelial cells in newt Pleurodeles waltl in vivo. Experiments were conducted using autoradiography, and the nuclear labeling index (NLI) was determined at different times after surgery and in different corneal regions. This adhesion protein significantly induced proliferation of corneal epithelial cells relative to control groups with the injured eyes treated with the serum adhesion protein at the same concentration or water. The differences between the experimental and control animals were most pronounced 7 days after surgery. By day 14, they were less pronounced but still significant. On day 28, no significant differences in NLI were observed between the three groups, although these values remained higher than in intact animals. An increased pool of proliferating cells in the corneal epithelium was observed both in the affected and intact areas. The data obtained indicate that the biological activity of this protein is not species specific and that it can be a proliferation factor for corneal epithelial cells.

The neuropeptide somatostatin is found in the retina of many species, yet its role in the visual process remains to be elucidated. The aim of the present study was to examine the expression and cellular localization of somatostatin receptor subtypes (sst; sst 2A , sst 2B and sst 3 ) in the eye of the adult newt Pleurodeles waltlii using immunohistochemistry. sst 2A immunoreactivity was observed in bipolar cells, in the inner segments of cone photoreceptors, as well as in the region corresponding to connecting cilia of rods. sst 2B immunoreactivity was not detected. sst 3 immunostaining was localized most intensely in the inner segments of cones, and in cilia of rods. These results suggest that somatostatin acting via sst 2A and sst 3 receptors may play an important role in retinal physiology of the lower vertebrates.

Data on the use of various immunochemical markers specifically indicating cell types of the neural retina and pigment epithelium are reviewed. It is demonstrated how this approach can be applied to the analysis of specific features of vertebrate retinal development, including the order and timing of differentiation of the main cell types, their interdependence in the course of this process, and factors controlling the latter. Problems concerning the state of differentiation and its change in the cells of retinal pigment epithelium and glial cells are discussed in respect to their analysis with the aid of specific protein markers. The current state of retina regeneration research involving the use of labeled cell sources and regenerated cells in lower vertebrates is analyzed. Problems in the search for new markers of retinal photoreceptor, macroglial, and microglial cells and their use in experiments are addressed.

Two groups of proteins were isolated from the retina and pigment epithelium of eight-day-old chick embryos. Experiments with suspension cultures of retinal cells demonstrated that only the retinal extracts and the fraction of its acidic proteins can stimulate cell aggregation in vitro. Analysis by the method of high-performance liquid chromatography showed that fractions of acidic and basic retinal proteins, which markedly differ in their electric charge and biological activity, have similar composition. To study the effect of these proteins on the morphological and functional state of pigment epitheliumin vitro, a new experimental model is proposed, with the posterior segment of the newt (Pleurodeles waltl) eye used as a test tissue. The fraction of basic proteins isolated from the chick embryonic pigment epithelium stabilized cell differentiation in the newt pigment epithelium. The analyzed proteins proved to be biologically active at extremely low doses, corresponding to 10−12 M solutions.

We propose a new method of organotypic roller 3D-culturing of the posterior sector of the eye. The method allows maintaining tissue viability in vitro for 14 days (which considerably surpasses the capacities of stationary culturing) and studying of the behavior, of pigment retinal epithelial cells and choriocapillary membrane. Using this method we demonstrated phenotypic transformation, migration, and proliferation of pigment retinal epithelial cells under conditions of roller organotypic culture. In the absence of the retina, these cells exhibit properties of scavenger cells (phagocytes) both within and outside the layer. Under conditions of roller culturing in vitro, cells of the pigment retinal epithelium undergo changes similar to those observed in various retinal pathologies in vivo, including age-associated changes. Here we discuss the possibility of using the proposed method for evaluation of the effect of various factors added to the culture medium on the pigment epithelium, for modeling of processes developing in damaged pigment epithelium or under conditions of various pathologies, and for the study of regeneration responses in cells of pigment retinal epithelium in adult vertebrates.

Differentiation of human embryonic retinal cells (20–22 weeks’ gestation) was studied using morphological, immunohistochemical, and biomolecular approaches. The retina included several regions differing by the degree of cell differentiation. Mitoses were rarely found in the marginal zone. This zone contained low differentiated cells. The central retinal area consisted of typical layers with differentiated cells. Culturing was accompanied by the formation of aggregates and neurospheres, where mitoses and progenitor or differentiated cells expressing markers of photoreceptors, neurons, and glia were found.

The results of molecular-genetic mechanisms of regeneration in amphibians are reviewed. Based on the examples of traditional and well-studied models of the restoration of the retinas and lenses of eyes, as well as limbs and tails in amphibians, we analyze the current state of regeneration problems and questions linked to cell reprogramming, growth, and morphogenesis. The development of the Kol’tsov school of thought in the age of molecular-genetic approaches and methods are monitored. The contemporary interpretation of organ regeneration in terms of molecular-genetic regulation and a new look at the definition of regeneration as repeated development is proposed. We also emphasize the current problems that exist in that field of developmental biology and are caused by the many difficulties of genome sequencing and the introduction of transgenesis in Urodela, the animal species with the highest regeneration abilities.