Aquaporin-4 expression in the human choroid plexus.

The choroid plexus (CP) consists of specialized ependymal cells and underlying blood vessels and stroma producing the bulk of the cerebrospinal fluid (CSF). CP epithelial cells are considered the site of the internal blood-cerebrospinal fluid barrier, show epithelial characteristics (basal lamina, tight junctions), and express aquaporin-1 (AQP1) apically. In this study, we analyzed the expression of aquaporins in the human CP using immunofluorescence and qPCR. As previously reported, AQP1 was expressed apically in CP epithelial cells. Surprisingly, and previously unknown, many cells in the CP epithelium were also positive for aquaporin-4 (AQP4), normally restricted to ventricle-lining ependymal cells and astrocytes in the brain. Expression of AQP1 and AQP4 was found in the CP of all eight body donors investigated (3 males, 5 females; age 74-91). These results were confirmed by qPCR, and by electron microscopy detecting orthogonal arrays of particles. To find out whether AQP4 expression correlated with the expression pattern of relevant transport-related proteins we also investigated expression of NKCC1, and Na/K-ATPase. Immunostaining with NKCC1 was similar to AQP1 and revealed no particular pattern related to AQP4. Co-staining of AQP4 and Na/K-ATPase indicated a trend for an inverse correlation of their expression. We hypothesized that AQP4 expression in the CP was caused by age-related changes. To address this, we investigated mouse brains from young (2 months), adult (12 months) and old (30 months) mice. We found a significant increase of AQP4 on the mRNA level in old mice compared to young and adult animals. Taken together, we provide evidence for AQP4 expression in the CP of the aging brain which likely contributes to the water flow through the CP epithelium and CSF production. In two alternative hypotheses, we discuss this as a beneficial compensatory, or a detrimental mechanism influencing the previously observed CSF changes during aging.

Generation of pluripotent stem cells from adult human testis.

Human primordial germ cells and mouse neonatal and adult germline stem cells are pluripotent and show similar properties to embryonic stem cells. Here we report the successful establishment of human adult germline stem cells derived from spermatogonial cells of adult human testis. Cellular and molecular characterization of these cells revealed many similarities to human embryonic stem cells, and the germline stem cells produced teratomas after transplantation into immunodeficient mice. The human adult germline stem cells differentiated into various types of somatic cells of all three germ layers when grown under conditions used to induce the differentiation of human embryonic stem cells. We conclude that the generation of human adult germline stem cells from testicular biopsies may provide simple and non-controversial access to individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells.

Aquaporin-1 and Aquaporin-4 Expression in Ependyma, Choroid Plexus and Surrounding Transition Zones in the Human Brain.

The choroid plexus (CP) is a structure in the brain ventricles that produces the main part of the cerebrospinal fluid (CSF). It is covered with specialized cells which show epithelial characteristics and are the site of the blood-CSF barrier. These cells form a contiguous cell sheet with ventricle-lining ependymal cells which are known to express aquaporin-4 (AQP4). In contrast, CP epithelial cells express aquaporin-1 (AQP1) apically. We investigated the expression patterns of aquaporins in the CP-ependyma transition from human body donors using immunofluorescence and electron microscopy. Ependymal cells and subependymal astrocytes at the base of the CP showed a particularly high AQP4 immunoreactivity. Astrocytic processes formed a dense meshwork or glial plate around the blood vessels entering the CP. Interestingly, some of these astrocytic processes were in direct contact with the CP stroma, which contains fenestrated blood vessels, separated only by a basal lamina. Electron microscopy confirmed the continuity of the subastrocytic basal lamina with the CP epithelium. We also probed for components of the AQP4 anchoring dystrophin-dystroglycan complex. Immunolabeling for dystrophin and AQP4 showed an overlapping staining pattern in the glial plate but not in previously reported AQP4-positive CP epithelial cells. In contrast, dystroglycan expression was associated with laminin staining in the glial plate and the CP epithelium. This suggests different mechanisms for AQP4 anchoring in the cell membrane. The high AQP4 density in the connecting glial plate might facilitate the transport of water in and out of the CP stroma and could possibly serve as a drainage and clearing pathway for metabolites.

Distribution of corpora amylacea in the human midbrain: using synchrotron radiation phase-contrast microtomography, high-field magnetic resonance imaging, and histology.

Corpora amylacea (CA) are polyglucosan aggregated granules that accumulate in the human body throughout aging. In the cerebrum, CA have been found in proximity to ventricular walls, pial surfaces, and blood vessels. However, studies showing their three-dimensional spatial distribution are sparse. In this study, volumetric images of four human brain stems were obtained with MRI and phase-contrast X-ray microtomography, followed up by Periodic acid Schiff stain for validation. CA appeared as hyperintense spheroid structures with diameters up to 30 µm. An automatic pipeline was developed to segment the CA, and the spatial distribution of over 200,000 individual corpora amylacea could be investigated. A threefold-or higher-density of CA was detected in the dorsomedial column of the periaqueductal gray (860-4,200 CA count/mm3) than in the superior colliculus (150-340 CA count/mm3). We estimated that about 2% of the CA were located in the immediate vicinity of the vessels or in the peri-vascular space. While CA in the ependymal lining of the cerebral aqueduct was rare, the sub-pial tissue of the anterior and posterior midbrain contained several CA. In the sample with the highest CA density, quantitative maps obtained with MRI revealed high R2* values and a diamagnetic shift in a region which spatially coincided with the CA dense region.

Organization of radial glia reveals growth pattern in the telencephalon of a percomorph fish Astatotilapia burtoni.

In the brain of teleost fish, radial glial cells are the main astroglial cell type. To understand how radial glia structures are adapting to continuous growth of the brain, we studied the astroglial cells in the telencephalon of the cichlid fish Astatotilapia burtoni in small fry to large specimens. These animals grow to a standard length of 10-12 cm in this fish species, corresponding to a more than 100-fold increase in brain volume. Focusing on the telencephalon where glial cells are arranged radially in the everted (dorsal) pallium, immunocytochemistry for glial markers revealed an aberrant pattern of radial glial fibers in the central division of the dorsal pallium (DC, i.e., DC4 and DC5). The main glial processes curved around these nuclei, especially in the posterior part of the telencephalon. This was verified in tissue-cleared brains stained for glial markers. We further analyzed the growth of radial glia by immunocytochemically applied stem cell (proliferating cell nuclear antigen [PCNA], Sox2) and differentiation marker (doublecortin) and found that these markers were expressed at the ventricular surface consistent with a stacking growth pattern. In addition, we detected doublecortin and Sox2 positive cells in deeper nuclei of DC areas. Our data suggest that radial glial cells give rise to migrating cells providing new neurons and glia to deeper pallial regions. This results in expansion of the central pallial areas and displacement of existing radial glial. In summary, we show that radial glial cells can adapt to morphological growth processes in the adult fish brain and contribute to this growth.

A Cleared View on Retinal Organoids.

Human induced pluripotent stem cell (hiPSC)-derived organoids mimicking tissues and organs in vitro have advanced medical research, as they opened up new possibilities for in-depth basic research on human organ development as well as providing a human in vitro model for personalized therapeutic approaches. hiPSC-derived retinal organoids have proven to be of great value for modeling the human retina featuring a very similar cellular composition, layering, and functionality. The technically challenging imaging of three-dimensional structures such as retinal organoids has, however, raised the need for robust whole-organoid imaging techniques. To improve imaging of retinal organoids we optimized a passive clearing technique (PACT), which enables high-resolution visualization of fragile intra-tissue structures. Using cleared retinal organoids, we could greatly enhance the antibody labeling efficiency and depth of imaging at high resolution, thereby improving the three-dimensional microscopy output. In that course, we were able to identify the spatial morphological shape and organization of, e.g., photoreceptor cells and bipolar cell layers. Moreover, we used the synaptic protein CtBP2/Ribeye to visualize the interconnection points of photoreceptor and bipolar cells forming the retinal-specific ribbon synapses.

Large-scale tissue clearing (PACT): Technical evaluation and new perspectives in immunofluorescence, histology, and ultrastructure.

Novel techniques, like CLARITY and PACT, render large tissue specimens transparent and thereby suitable for microscopic analysis. We used these techniques to evaluate their potential in the intestine as an exemplary organ with a complex tissue composition. Immunohistochemistry, light sheet-, and confocal scanning-microscopy enabled us to follow complex three-dimensional structures, like nerve fibers, vessels, and epithelial barriers throughout the entire organ. Moreover, in a systematic electron microscopic study, we analyzed the morphology and preservation of tissue on ultrastructural level during the clearing process. We also connect tissue clearing with classical histology and demonstrate that cleared tissues can be stained with Hematoxylin-Eosin and Heidenhain's Azan stain, suggesting potential use in histopathology. These experiments showed that a neutral pH during the clearing process results in much better preservation of tissue ultrastructure and standard stainability. Volume changes of specimens were monitored and quantified during the course of the protocol. Additionally, we employed the technique to visualize the enteric nervous system and the epithelial barrier in post mortem human gut preparations. Our data show the high potential of tissue clearing throughout different tissue types supporting its usefulness in research and diagnosis, and contribute to the technical discussion of ultrastructural tissue-retention.