Survival of syngeneic and allogeneic iPSC–derived neural precursors after spinal grafting in minipigs

The use of autologous (or syngeneic) cells derived from induced pluripotent stem cells (iPSCs) holds great promise for future clinical use in a wide range of diseases and injuries. It is expected that cell replacement therapies using autologous cells would forego the need for immunosuppression, otherwise required in allogeneic transplantations. However, recent studies have shown the unexpected immune rejection of undifferentiated autologous mouse iPSCs after transplantation. Whether similar immunogenic properties are maintained in iPSC-derived lineage-committed cells (such as neural precursors) is relatively unknown. We demonstrate that syngeneic porcine iPSC-derived neural precursor cell (NPC) transplantation to the spinal cord in the absence of immunosuppression is associated with long-term survival and neuronal and glial differentiation. No tumor formation was noted. Similar cell engraftment and differentiation were shown in spinally injured transiently immunosuppressed swine leukocyte antigen (SLA)–mismatched allogeneic pigs. These data demonstrate that iPSC-NPCs can be grafted into syngeneic recipients in the absence of immunosuppression and that temporary immunosuppression is sufficient to induce long-term immune tolerance after NPC engraftment into spinally injured allogeneic recipients. Collectively, our results show that iPSC-NPCs represent an alternative source of transplantable NPCs for the treatment of a variety of disorders affecting the spinal cord, including trauma, ischemia, or amyotrophic lateral sclerosis

Densitometric patterns of NADPH diaphorase staining in the spinal cord of dog

Mar ala, M., Densitometric patterns of NADPH diaphorase staining in the spinal cord of dog. Biologia, Bratislava, 56: 685-693, 2001; ISSN 0006-3088 (Biologia). ISSN 1335-6399 (Biologia. Section Cellular and Molecular Biology). Segmental and laminar distribution of NADPHd activity was studied in the normal spinal cord of the dog and basic densitometric patterns of somatic, fiber-like and punctuate, non-somatic NADPHd staining were described in the gray and white matter. Prominent NADPHd activity was noted in the superficial and deep dorsal horn, pericentral region, intermediolateral cell column, Lissauer's tract and in the vertical and horizontal limbs of the medial longitudinal bundle of the ventral column in the cervical and upper thoracic segments. Key words: densitometry, NADPH diaphorase, spinal cord, dog. Introduction The use of nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry alone or combined with the nitric oxide synthase immunoreactivity (NOS-IR) allowed for a morphologically distinct and topographically precise localization of small neuronal pools synthesizing, releasing and transporting NOS, an enzyme responsible for nitric oxide (NO) synthesis. The discrete loci, nuclei or solitary NOS-IR neurons have been identified not only in the cortex, brain stem and spinal cord, but also in the peripheral nervous system (Vincent & Johanson, 1983; Immunocytochemistry of the neuronal nitric oxide synthase (nNOS) showed that the occurrence of this enzyme is almost completely homotopic with the localization of neurons stained for NADPHd In the present study an attempt was made to specify the differences of somatic, fiber-like, and punctuate NADPHd staining in the gray and white matter of the spinal cord in the normal dog, including different segments and layers using the densitometric analysis. Densitometric patterns of NADPHd positivity in the undamaged spinal cord may be helpful in experimental studies aimed at a causal interpretation of changes affecting the NOS-containing neuronal pools in various experimental and pathologic conditions. Material and methods Tissue sampling, sectioning, examination of sections and the performance of the densitometric analysis Adult dogs (n = 6) of both sexes weighing 12-18 kg were used in this study. The animals were deeply anesthetized with pentobarbital (50 mg/kg, i.v.) and perfused transcardially with saline followed by freshly prepared 4% paraformaldehyde +0.1% glutaraldehyde buffered with 1M sodium phosphate, pH = 7.4. Following perfusion fixation, the spinal cords were carefully dissected out and stored in toto in the same fixative for 3-4 hours. After postfixation, the spinal cord was divided into cervical, thoracic, lumbar, sacral and coccygeal segments, and each segment was then secondarily divided into three small blocks comprising the upper, middle, and lower segmental levels, respectively. Specimens were then cryoprotected in an ascending concentration of sucrose (15-30%) with the same phosphate buffer and stored overnight at 4 • C. Frozen transverse sections (50 µm thick) were cut from all segments studied and processed for NADPH-d activity by using a modified histochemical procedure The densitometric analysis was performed using transverse sections stained for NADPHd histochemistry. Precise loci identified in the gray and white matter on transverse sections were used for the assessment of the densitometric patterns in both compartments of the spinal cor

Multi-instrumental Analysis of Tissues of Sunflower Plants Treated with Silver(I) Ions – Plants as Bioindicators of Environmental Pollution

The aim of this work is to investigate sunflower plants response on stressinduced by silver(I) ions. The sunflower plants were exposed to silver(I) ions (0, 0.1, 0.5,and 1 mM) for 96 h. Primarily we aimed our attention to observation of basic physiologicalparameters. We found that the treated plants embodied growth depression, coloured changes and lack root hairs. Using of autofluorescence of anatomical structures, such aslignified cell walls, it was possible to determine the changes of important shoot and rootstructures, mainly vascular bungles and development of secondary thickening. Thedifferences in vascular bundles organisation, parenchymatic pith development in the rootcentre and the reduction of phloem part of vascular bundles were well observable.Moreover with increasing silver(I) ions concentration the vitality of rhizodermal cellsdeclined; rhizodermal cells early necrosed and were replaced by the cells of exodermis.Further we employed laser induced breakdown spectroscopy for determination of spatialdistribution of silver(I) ions in tissues of the treated plants. The Ag is accumulated mainlyin near-root part of the sample. Moreover basic biochemical indicators of environmentalstress were investigated. The total content of proteins expressively decreased withincreasing silver(I) ions dose and the time of the treatment. As we compare the resultsobtained by protein analysis – the total protein contents in shoot as well as root parts – wecan assume on the transport of the proteins from the roots to shoots. This phenomenon canbe related with the cascade of processes connecting with photosynthesis. The secondbiochemical parameter, which we investigated, was urease activity. If we compared theactivity in treated plants with control, we found out that presence of silver(I) ions markedlyenhanced the activity of urease at all applied doses of this toxic metal. Finally we studiedthe effect of silver(I) ions on activity of urease in in vitro conditions