Molecular Factors of the Maintenance and Activation of the Juvenile Phenotype of Cellular Sources for Eye Tissue Regeneration.

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.

Exposure to microgravity for 30 days onboard Bion M1 caused muscle atrophy and impaired regeneration in murine femoral Quadriceps.

Mechanical unloading in microgravity during spaceflight is known to cause muscular atrophy, changes in muscle fiber composition, gene expression, and reduction in regenerative muscle growth. Although some limited data exists for long-term effects of microgravity in human muscle, these processes have mostly been studied in rodents for short periods of time. Here we report on how long-term (30-day long) mechanical unloading in microgravity affects murine muscles of the femoral Quadriceps group. To conduct these studies we used muscle tissue from 6 microgravity mice, in comparison to habitat (7), and vivarium (14) ground control mice from the NASA Biospecimen Sharing Program conducted in collaboration with the Institute for Biomedical Problems of the Russian Academy of Sciences, during the Russian Bion M1 biosatellite mission in 2013. Muscle histomorphology from microgravity specimens showed signs of extensive atrophy and regenerative hypoplasia relative to ground controls. Specifically, we observed a two-fold decrease in the number of myonuclei, compared to vivarium and ground controls, and central location of myonuclei, low density of myofibers in the tissue, and of myofibrils within a fiber, as well as fragmentation and swelling of myofibers. Despite obvious atrophy, muscle regeneration nevertheless appeared to have continued after 30 days in microgravity as evidenced by thin and short newly formed myofibers. Many of them, however, showed evidence of apoptotic cells and myofibril degradation, suggesting that long-term unloading in microgravity may affect late stages of myofiber differentiation. Ground asynchronous and vivarium control animals demonstrated normal, well-developed tissue structure with sufficient blood and nerve supply and evidence of regenerative formation of new myofibers free of apoptotic nuclei. Regenerative activity of satellite cells in muscles was observed both in microgravity and ground control groups, using Pax7 and Myogenin immunolocalization, as well as Myogenin expression analysis. In addition, we have detected positive nuclear immunolocalization of c-Jun and c-Myc proteins indicating their sensitivity to changes in gravitational loading in a given model. In summary, long-term spaceflight in microgravity caused significant atrophy and degeneration of the femoral Quadriceps muscle group, and it may interfere with muscle regenerative processes by inducing apoptosis in newly-formed myofibrils during their differentiation phase.

[Identification of the Gene Encoding Nucleostemin in the Eye Tissues of Pleurodeles waltl].

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.

Preventive and therapeutic effects of SkQ1-containing Visomitin eye drops against light-induced retinal degeneration.

The human retina is constantly affected by light of varying intensity, this being especially true for photoreceptor cells and retinal pigment epithelium. Traditionally, photoinduced damages of the retina are induced by visible light of high intensity in albino rats using the LIRD (light-induced retinal degeneration) model. This model allows study of pathological processes in the retina and the search for retinoprotectors preventing retinal photodamage. In addition, the etiology and mechanisms of retina damage in the LIRD model have much in common with the mechanisms of the development of age-related retinal disorders, in particular, with age-related macular degeneration (AMD). We have studied preventive and therapeutic effects of Visomitin eye drops (based on the mitochondria-targeted antioxidant SkQ1) on albino rat retinas damaged by bright light. In the first series of experiments, rats receiving Visomitin for two weeks prior to illumination demonstrated significantly less expressed atrophic and degenerative changes in the retina compared to animals receiving similar drops with no SkQ1. In the second series, the illuminated rats were treated for two weeks with Visomitin or similar drops without SkQ1. The damaged retinas of the experimental animals were repaired much more effectively than those of the control animals. Therefore, we conclude that Visomitin SkQ1-containing eye drops have pronounced preventive and therapeutic effects on the photodamaged retina and might be recommended as a photoprotector and a pharmaceutical preparation for the treatment of AMD in combination with conventional medicines.

[Study of regeneration in amphibians in age of molecular-genetic approaches and methods].

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 generate 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 oftransgenesis in Urodela, the animal species with the highest regeneration abilities.

[Determination of mRNA-transcripts and heat shock proteins HSP70 and HSP90 in retina of the adult Spanish Ribbed Newt Pleurodeles waltl].

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 immunohistochemistry. 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.

Phenotypic plasticity of retinal pigment epithelial cells from adult human eye 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.

Expression of Ca-binding protein recoverin in normal, surviving, and regenerating retina of Pleurodeles waltl adult triton.

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.

New method of in vitro culturing of pigment retinal epithelium in the structure of the posterior eye sector of adult rat.

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.

In vivo and in vitro proliferative and differentiation activity of human embryonic retinal cells.

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.

Urodelean amphibians in studies on microgravity: effects upon organ and tissue regeneration.

Results obtained from nine experiments performed onboard Russian biosatellites have shown that microgravity promotes tissue regeneration in the newt, Pleurodeles waltl. The effect has been reproduced in all flights and on a clinostat as well for eye tissues (lens and retina), limbs and tail. The effect was demonstrated in 1.5- to 2-fold increase in cell proliferation in the early stages of regeneration in space flight. Animals "flown" intact and operated after flight regenerated faster than control ones and showed long-lasting micro-"g" effect. The most recent experiment flew aboard the Bion-11 biosatellite. This test was performed for study on microgravity effect on neural retina regeneration after optic nerve lesioning in the newt. Obtained results confirmed our previous information about intensification of regenerative processes in detached neural retina in urodela exposed to simulated weightlessness (Grigoryan et al., 1998). In particular, we found the increase and activation of cell populations participating in neural retina restoration and maintenance of retinal structure. Our findings suggest that promoting effect of microgravity upon regeneration could be influenced by several factors, largely influenced by a response of the whole organism to changed gravity vector. We hypothesized the synthesis of the specific range of stress proteins induced by micro-"g" and their regulative role in cell proliferation. Such a hypothesis for the existence of "altered gravity stress proteins" is discussed.

Changes in the numbers of osteoclasts in newts under conditions of microgravity.

Intensity of osteoclastic resorption and calcium content were investigated in intact limb bones of the newts flown on board of a biosatellite Cosmos-2229 after amputation of their forelimbs and tail. Using X-ray microanalysis it was shown an increase in calcium content in the bones on 20th day after operation. Histological study revealed an activation of osteoclastic resorption on endosteal surface of long bones. The newts exposed after surgery on a biosatellite had the same level of bone mineralisation as operated ground control ones, but the increase in number of polynuclear osteoclasts was lower.

Influence of clinorotation and fettering stress on tail regeneration of Triturus vulgaris (Urodela).

Tail-amputated adult Triturus vulgaris, fettered in cuvettes of a fast-rotating clinostat were exposed to simulated weightlessness (60 rpm; equiv. to 10(-3)-10(-4) g), during a 14-day period. To feed and clean the animals rotation was stopped once a day for approx. 10 min. To test the influence of the fettering stress, a second series of animals was kept separately under normal earth conditions without rotation. A further control series was kept in a dark container without any handicap. While tail regeneration of the rotated animals was markedly accelerated, the fettered-only animals showed a considerably less marked acceleration effect. At the end of the 14-day period, all regenerates were reamputated together with an additional 5 mm of the tail stump. Although this second level of amputation was distant from the first, the regenerative growth rate of the rotated series was accelerated 123% in contrast to both the control and the fettered-only series. Our results demonstrate that the growth acceleration is induced by clinorotation. Fettering stress has no comparable influence. The growth promoting effect is not limited to the regenerating area.

Microgravity effects on neural retina regeneration in the newt.

Data on forelimb and eye lens regeneration in urodeles under spaceflight conditions (SFC) have been obtained in our previous studies. Today, evidence is available that SFC stimulate regeneration in experimental animals rather than inhibit it. The results of control on-ground experiments with simulated microgravity suggest that the stimulatory effect of SFC is due largely to weightlessness. An original experimental model is proposed, which is convenient for comprehensively analyzing neural regeneration under SFC. The initial results described here concern regeneration of neural retina in Pleurodeles waltl newts exposed to microgravity simulated in radial clinostat. After clinorotation for seven days (until postoperation day 16), a positive effect of altered gravity on structural restoration of detached neural retina was confirmed by a number of criteria. Specifically, an increased number of Mullerian glial cells, an increased relative volume of the plexiform layers, reduced cell death, advanced redifferentiation of retinal pigment epithelium, and extended areas of neural retina reattachment were detected in experimental newts. Moreover, cell proliferation in the inner nuclear layer of neural retina increased as compared with control. Thus, low gravity appears to intensify natural cytological and molecular mechanisms of neural retina regeneration in lower vertebrates.

Regeneration of organs and tissues in lower vertebrates during and after space flight.

In this paper most important data obtained in studies on the effect of space flight conditions on regeneration in the adult newt are summarized. We demonstrate a phenomenon of synchronization of limb and lens regeneration and increase in its rate during and after space flight. We also describe a peculiarities of cell proliferation in lens, limb and tail regenerates and of the process of minced muscle regeneration.

Influence of longitudinal whole animal clinorotation on lens, tail, and limb regeneration in urodeles.

Two species of newts (Urodela) and two types of clinostats for fast clinorotation (60 rpm) were used to investigate the influence of simulated weightlessness on regeneration and to compare results obtained with data from spaceflight experiments. Seven or fourteen days of weightlessness in Russian biosatellites caused acceleration of lens and limb regeneration by an increase in cell proliferation, differentiation, and rate of morphogenesis in comparison with ground controls. After a comparable time of clinorotation the results obtained with Triturus vulgaris using a horizontal clinostat were similar to those found in spaceflight. In contrast, in Pleurodeles waltl using both horizontal and radial clinostats the results were contradictory compared to Triturus. We speculate that different levels of gravity or/and species specific thresholds for gravitational sensitivity could be responsible for these contradictory results.