Effect of transplants of retinal pigment epithelial cells from adult human eye on degenerative processes in the brain of rats with experimental acute hypoxia.

Stimulation of cell regeneration in the brain and eye retina in various degenerative processes is a pressing problem in neurobiology. A promising approach is transplantation of somatic cells reprogrammed towards neural lineage. We studied the effect of transplantation of retinal pigment epithelial cells from adult human eye transdifferentiated in culture on degenerative processes in the brain of rats subjected to acute hypoxia. Immunohistochemical and molecular genetic analysis suggests that retinal pigment epithelial cells transdifferentiate in vitro and express markers of low-differentiated neural cells. The cells transplanted into rat brain survive for at least 20 days. During this period, they stimulate compensatory and reparative processes that protected cortical neurons in the recipients from hypoxia-induced degeneration.

Comparative analysis of the expression of neural stem cell-associated genes during neocortex and retina development in human.

We compared the expression of Sox2, Oct4, Nanog, Pax6, Prox1 genes associated with plasticity of neural stem and progenitor cells during human neocortex and retina development and in cell cultures. At the analyzed stages of neurogenesis, Pax6 gene is expressed in the neocortex and retina at constant levels, the expression is by one order of magnitude higher in the retina. The dynamics of Sox2 and Pax6 expression in the neocortex was similar. The expression of Oct4 and Nanog genes during neurogenesis in the neocortex and human fetal retina reflects the existence of a high-plasticity cell pool. The dynamics of ßIII-tubulin expression indicates that the retina develops more rapidly than the neocortex. Our experiments showed that genetically determined cell potencies typical of native cells are realized in primary cultures without specific stimulation.

Expression of multipotent and retinal markers in pigment epithelium of adult human in vitro.

Immunoperoxidase and molecular genetic analysis showed that retinal pigment epithelial cells from adult human eye undergo morphogenetic changes in vitro. They lose expression of tissue-specific protein RPE65 and start to express stem cell markers: Oct4 (POU5F1), Nanog, Prox1, Musashi 1, and Pax6, which indicates their differentiation. Expression of Musashi 1 and Pax6 attest to neural differentiation, which is also confirmed by the expression of ßIII-tubulin, a neuroblast marker, and markers of differentiated neuronal cells, tyrosine hydroxylase and neurofilament proteins. These findings attest to the capacity of retinal pigment epithelium from adult human eye to transdifferentiation into neural lineage cells, which makes them an interesting object for cell therapy in neurodegeneration.

Molecular genetic and immunophenotypical analysis of Pax6 transcription factor and neural differentiation markers in human fetal neocortex and retina in vivo and in vitro.

Neurotransplantation of various cells, including heterotransplantation of fetal cerebral stem/progenitor cells into the eye is used in experimental studies of central nervous tissue repair during neurodegeneration. For evaluation of this approach, human fetal (weeks 9-20) stem/progenitor cells of the neocortex and retina were studied in vivo and in vitro by quantitative PCR and immunohistochemical staining. Native tissues and cultures were characterized by expression of Pax6 transcription factor (critical for the development of the retina and neocortex) and differentiation markers (nestin, betaIII-tubulin, glial fibrillary acidic protein, recoverin, NeuN, neurofilaments, Ki-67). The expression of Pax6 gene in the retina during active neurogenesis was stable and much higher than in the neocortex. In primary cultures, the pattern of Pax6 gene expression is retained and repeats that in native tissues. Immunohistochemical analysis revealed similarity of nestin and betaIII-tubulin expression in the neocortex and retina during the early (9-10 weeks) and later (20 weeks) periods and differences in cell phenotypes and their distribution. Culture studies showed that neocortical and retinal stem/progenitor cells are determined and exhibit specific differentiation characteristic of the corresponding native tissues. It can be hypothesized that heterotransplantation of the cerebral progenitor cells into the retina of experimental animals can lead to realization of their neurotrophic effect, but not to their functional integration.

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.