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.

[Laser microdissection for biology and medicine].

For routine extraction of DNA, RNA, proteins and metabolites, small tissue pieces are placed into lysing solution. These tissue pieces in general contain different cell types. For this reason, lysate contains components of different cell types, which complicates the interpretation of molecular analysis results. The laser microdissection allows overcoming this trouble. The laser microdissection is a method to procure tissue samples contained defined cell subpopulations, individual cells and even subsellular components under direct microscopic visualization. Collected samples can be undergone to different downstream molecular assays: DNA analysis, RNA transcript profiling, cDNA library generation and gene expression analysis, proteomic analysis and metabolite profiling. The laser microdissection has wide applications in oncology (research and routine), cellular and molecular biology, biochemistry and forensics. This paper reviews the principles of different laser microdissection instruments, examples of laser microdissection application and problems of sample preparation for laser microdissection.

[Effects of human cultural neuronal and mesenchymal stem cells on the rat learning and brain state after acute hypoxia].

The aim of the study was to compare the effects of neurotransplantation of cultural neural stem cells (NSC) and mesenchymal stem cells (MSC) on the rat behaviour and brain state after acute hypoxia. It was shown that development of two-way avoidance defensive conditioning in a shuttle box improved in rats-recipients with NSC, but not MSC as compared to control. Both the transplants of NSC and transplants of MSC exert neuroprotective influence on the rat brain. NSC both in vitro (before transplantation) and in vivo (on day 27 after transplantation) gave rise to all neural cell types: stem/progenitor cells, precursors of neurons and glia, neurons and glial cells. MSC population in vitro and in vivo (on day 10 after transplantation) consisted of fibroblast-like cells which were eliminated by day 20 after transplantation and were surrounded by reactive glia. We suggest that effects of NSC may be connected with their good survival and potential to differentiate into neurons and with trophic influence on the brain of recipient, whereas MSC only have possible positive trophic effect at early stages after transplantation.

[Immunohistochemical analysis of development of suspension and tissue neurotransplants].

Engraftment and development of suspension and whole tissue allografts from the mouse brain embryonal tissue, in which cells of the green fluorescent protein (GFP) are synthesized, have been studied. The transgenic mouse cells with synthesized GFP can be used for neurotransplantation. Whole tissue and suspension transplants are able to survive more than 30 days without rejection in the brain of adult mice. It was found that the cells with synthesized GFP are able to differentiate in the neuronal and glial directions in both whole tissue and suspension transplants. The results of immunohistochemical analysis showed the reciprocal ingrowth of fibers between the cells of donor and recipient in both cases. A study of proliferation and differentiation of the cells showed the higher ability of tissue transplants for development.

In vitro phenotypic modification of pigmented epithelium cells from human eye at early stages of development.

Multipotent characteristics of human fetal (9-11.5 weeks) pigmented epithelial retinal cells and capacity to transdifferentiation in neuronal direction were studied in vitro under different culturing conditions. The cultures were analyzed using a wide spectrum of antibodies. It was found that pigmented epithelium of human eye is a heterogeneous cell population with three subtypes differing by adhesion characteristics, migration, transdifferentiation potential, and reaction to microenvironmental factors. Subtype 1 cells steadily retain their epithelial characteristics, subtype 2 cells change their morphotype and produce neuroblast and photoreceptor cell proteins, and subtype 3 cells form free floating spheres and are capable to multipotent differentiation.

BLBP-immunoreactive cells in the primary culture of neural precursors from embryonic mouse brain.

The population of neurosphere cells was studied by staining for the radial glial marker BLBP protein. An immunohistochemical study of cultures showed that neurosphere cells proliferate and retain the ability for pluripotent differentiation over 9 passages. BLBP-immunoreactive cells were present in neurospheres at all passages. However, they did not belong to the population of proliferating multipotent precursors or GFAP-positive cells. BLPB expression was detected in S-100b-positive cells of astrocyte differentiation. Our results suggest that BLBP-immunopositive cells of the radial glia in vitro loss the state of multipotent precursors and differentiate into the astroglia. Otherwise, some heterogeneous cells of the radial glia include a previously unknown linear population that differentiates into S-100b-positive astrocytes.

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.

[Study of expression of beta-III tubulin in human eye tissues during prenatal development].

Expression of beta-III tubulin, a marker protein of early neuronal cells, was studied by molecular genetic and immunochemical techniques. The study was performed with human eyes in the 8.5th to 27-28th weeks of prenatal development. Expression of beta-III tubulin was detected immunochemically in the retina and lens fibers in the 8.5 to 22-23 weeks of development. PCR revealed a high level of expression of the gene for beta-III tubulin in the retina of 9.5-week embryos. The level of expression of this gene remained high until the 18th week of prenatal development, slightly decreased to the 24th week, and became negligible in 27- to 28-week embryos. In the 15th to 24th weeks of prenatal development, the level of expression of this gene in the lens was very low and became undetectable in 27- to 28-week embryos. The results of PCR analysis are consistent with immunochemical data.

Analysis of differentiation of stem/progenitor cells in tissue culture of neocortical primordium of mouse brain.

Differentiation of neural stem/progenitor cells from neocortical primordium of the brain from 14-day mouse embryos was studied by immunohistochemical methods during their culturing. Non-differentiated cells expressing nestin and vimentin persisted in freely floating neurospheres throughout the experiment. Glioblasts, neuroblasts, and differentiated neurons were found in neurospheres cultured in differentiating medium. However, neurons disappeared with increasing the number of passages, the formation of neuroblasts was terminated, and only astrocytes and nestin-positive cells were seen in the culture. It was found that cells of mouse embryonic neocortex lose the capacity for spontaneous multipotent differentiation during culturing.

Functional activity of mitochondria in cultured neural precursor cells.

We studied mitochondrial transmembrane potential of neural precursor cells forming neurospheres in culture. Uneven energization of mitochondria in neurosphere cells was detected. Heterogeneity of cells by the mitochondrial potential increased with neurosphere enlargement during culturing. Decrease in the mitochondrial potential in the central cells in large spheres, presumably caused by insufficient diffusion of oxygen and nutrients, can provoke their damage and death. Population of cells with high mitochondrial potential responded to addition of the nuclear dye by a decrease in mitochondrial potential, which can indicate functioning of ABCG2 complex in these cells, characteristic of undifferentiated stem cells. These data will help to create optimum conditions for culturing of neural stem cells for the maintenance of their maximum functional and proliferative activity.

Comparative analysis of differentiation and behavior of human neural and mesenchymal stem cells in vitro and in vivo.

Comparative analysis of differentiation of human neural and mesenchymal stem cells in tissue culture and after transplantation into the brain was carried out using the same antibody set. Neural stem cells differentiated into all types of neural cells, are retained after transplantation, migrate, and form reciprocal relationships with the recipient brain. Mesenchymal stem cells were incapable of neural development under conditions of common culturing or after transplantation and retained the fibroblast-like status. Recipient filaments grew into mesenchymal stem cell transplants containing no neural cells due to local changes in the extracellular matrix at the site of transplantation.

Structure and cell composition of spheres cultured from human fetal retina.

The structure and cell composition of spheres obtained by culturing human fetal retinal cells after 15, 18, 22-23, and 24 weeks of gestation were studied. The cells were cultured as neurospheres: in serum-free medium with growth factors, in which they formed floating spheres. Immunocytochemical analysis showed that cell proliferation in the spheres decreased with increasing fetal age. Stem/progenitor cells, neuroblasts, and photoreceptors were detected in the spheres. Glial cells were detected only in spheres originating from 22- and 24-week fetuses. All spheres, irrespective of age and duration of culturing, consisted of numerous cell rosettes, each histotypically similar to the neuroblastic layer of the developing retina.

Formation of neuroepithelial structures in culture of neural stem cells from human brain.

Dissociated fetal brain cells in a floating culture form clusters and then neurospheres, some of which contain structures shaped as cell "rosettes". The cells in these "rosettes" are arranged radially around the central cavity, in which their apical processes form desmosome-like contacts. Mitotic division of cells in the "rosettes" is associated with migration of the nuclei, similarly to division of neuroepithelial cells in the neural tube during normal embryogenesis. These cells express nestin, a marker of neural stem cells. The cells in "rosettes" found after transplantation have similar characteristics.

Characteristics of human neural stem cells in vitro and after transplantation into rat brain.

We studied the effect of culturing conditions on the fate of human neural stem cells after transplantation into rat brain. Human neural stem cells cultured in the presence of mitogens without LIF migrated along the ependyma and cerebral vessels of recipients, but to a great extent degenerated by the 20th day after transplantation. Neural stem cells cultured with LIF migrated, apart from the above mentioned pathways, in the cortex and hippocampus, well survived; proliferating cells were retained 30 days after transplantation.

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.

Human fetal neural stem cells in rat brain: effects of preculturing and transplantation.

The fate of human fetal stem/progenitor cells transplanted into rat brain depends on conditions of preculturing (long or short) and state and site of transplantation. Human nestin-positive stem cells cultured according to the short protocol did not migrate into hypoxic and normal brain after transplantation, but actively migrated in damaged spinal cord. After transplantation of long-cultured cells into the brain mainly committed neuroblasts and solitary nestin-positive cells migrated from the site of transplantation into the brain.

Human neural stem cells normalize rat behavior after hypoxia.

Transplants of cultured neural stem cells from human brain survived, retained multipotent activity, and produced a neuroprotective effect on degenerating neurons in the brain of adult rats subjected to hypoxic hypoxia. They normalized animal behavior and improved conditioning in two-way avoidance response paradigm in a shuttle box.

Immunohistochemical study of fetal stem/progenitor cells from human brain transplanted into traumatized spinal cord of adult rats.

Neural stem/progenitor cells from human fetal brain were grown in a tissue culture and transplanted into traumatized spinal cord of adult rats. The behavior and differentiation of transplanted cells were studied morphologically by means of histological and immunohistochemical methods and confocal microscopy. Human neural stem/progenitor cells were viable for not less than 3 months. They migrated and differentiated into neurons and glia in the traumatized spinal cord of adult rats.

Transplantation of cultured neural cells from human fetuses into the brain of rats exposed to acute hypoxia.

Neural stem cells of human brain were cultured for a long time and successfully transplanted into the brain of rats exposed to acute hypoxia. Stem and committed cells, neuroblasts, and astrocytes were revealed in transplants by immunohistochemical assay. The transplants and brain tissue were not separated with a glial barrier. Human neuroblasts widely migrated into regions of neuronal degeneration in the host brain.