Human Dermal Fibroblasts Demonstrate Positive Immunostaining for Neuron- and Glia- Specific Proteins.

In stem cell cultures from adult human tissue, undesirable contamination with fibroblasts is frequently present. The presence of fibroblasts obscures the actual number of stem cells and may result in extracellular matrix production after transplantation. Identification of fibroblasts is difficult because of the lack of specific fibroblast markers. In our laboratory, we isolate and expand neural-crest-derived stem cells from human hair follicle bulges and investigate their potential to differentiate into neural cells. To establish cellular identities, we perform immunohistochemistry with antibodies specific for glial and neuronal markers, and use fibroblasts as negative control. We frequently observe that human adult dermal fibroblasts also express some glial and neuronal markers. In this study, we have sought to determine whether our observations represent actual expression of these markers or result from cross-reactivity. Immunohistochemistry was performed on human adult dermal fibroblasts using acknowledged glial and neuronal antibodies followed by verification of the data using RT-qPCR. Human adult dermal fibroblasts showed expression of the glia-specific markers SOX9, glial fibrillary acidic protein and EGR2 (KROX20) as well as for the neuron-specific marker class III ß-tubulin, both at the protein and mRNA level. Furthermore, human adult dermal fibroblasts showed false-positive immunostaining for S100ß and GAP43 and to a lower extent for OCT6. Our results indicate that immunophenotyping as a tool to determine cellular identity is not as reliable as generally assumed, especially since human adult dermal fibroblasts may be mistaken for neural cells, indicating that the ultimate proof of glial or neuronal identity can only be provided by their functionality.

Hair follicle bulge cultures yield class III ß-tubulin-positive melanoglial cells.

Class III ß-tubulin (TUBB3)-positive cells from the hair follicle bulge are thought to be neuronal cells derived from a local neural crest stem cell. However, TUBB3 has recently been shown to be expressed in the melanocytic lineage. To evaluate the neural-crest-associated immunophenotype of TUBB3-positive cells from hair follicle bulge explants, we dissected hair follicle bulges out from mouse whisker pads and cultured for 1 month and assessed outgrowing cells by means of immunocytochemistry using the biomarkers TUBB3, nestin, NGFR, SOX9, TYRP1 and laminin. Large amounts of TUBB3-positive cells could be cultured that co-expressed nestin, NGFR, SOX9 and, to a lesser degree, TYRP1, matching a melanoglial phenotype. In addition, a small population of TUBB3-negative but laminin-positive cells was found, which presumably are of glial origin. It can be concluded that cells of melanoglial origin can easily be obtained from hair follicle bulge explants. These cells may be of use in experimental animal or human disease and wound healing models. Notably, the TUBB3-positive cells are of melanoglial rather than neuronal origin.

Effects of aging on inner ear morphology in dogs in relation to brainstem responses to toneburst auditory stimuli.

BACKGROUND: Age-related hearing loss (ARHL) is the most common form of hearing loss in humans and is increasingly recognized in dogs. HYPOTHESIS: Cochlear lesions in dogs with ARHL are similar to those in humans and the severity of the histological changes is reflected in tone audiograms. ANIMALS: Ten geriatric dogs (mean age: 12.7 years) and three 9-month-old dogs serving as controls for histological analysis. METHODS: Observational study. Auditory thresholds were determined by recording brainstem responses (BERA) to toneburst auditory stimuli (1, 2, 4, 8, 12, 16, 24, and 32 kHz). After euthanasia and perfusion fixation, the temporal bones were harvested and processed for histological examination of the cochleas. The numbers of outer hair cells (OHCs) and inner hair cells (IHCs) were counted and the spiral ganglion cell (SGC) packing density and stria vascularis cross-sectional area (SVCA) were determined. RESULTS: A combination of cochlear lesions was found in all geriatric dogs. There were significant reductions (P .001) in OHC (42%, 95% confidence interval [CI]; 24-64%) and IHC counts (21%, 95% CI; 62-90%) and SGC packing densities (323, 95% CI; 216-290) in the basal turn, SVCA was smaller in all turns. The greatest reduction in auditory sensitivity was at 8-32 kHz. CONCLUSIONS AND CLINICAL IMPORTANCE: ARHL in this specific population of geriatric dogs was comparable histologically to the mixed type of ARHL in humans. The predominance of histological changes in the basal cochlear turn was consistent with the large threshold shifts observed in the middle- to high-frequency region.

Reduced expression of sialoglycoconjugates in the outer hair cell glycocalyx after systemic aminoglycoside administration.

In this study we investigated the effect of systemic aminoglycoside administration on the expression of sialoglycoconjugates in the outer hair cell (OHC) glycocalyx of the adult guinea pig. Sialoglycoconjugates were visualized by means of ultrastructural lectin cytochemistry, using Limax flavus agglutinin (LFA) and wheat germ agglutinin (WGA) as probes. Labelling densities were determined for the apical membranes (including the stereocilia and stereociliary cross-links) and basolateral membranes of OHCs in the respective (basal, middle and apical) cochlear turns from animals that had been treated with gentamicin or neomycin for 5 or 15 consecutive days. Our results indicate that: (1) sialoglycoconjugate expression in the OHC glycocalyx demonstrates an intracochlear gradient decreasing towards the apical turn; (2) OHCs demonstrate a polarity in sialoglycoconjugate expression, in that the basolateral membranes contain more sialoglycoconjugates per surface area than the apical membranes; (3) aminoglycoside administration results in reduced expression of sialoglycoconjugates in the OHC glycocalyx; in this respect, basal-turn OHCs are more susceptible than those in the middle and apical turns; (4) reduction in sialoglycoconjugate expression after aminoglycoside administration is more prominent in the basolateral membranes; and (5) the difference in ototoxic potencies between gentamicin and neomycin is not reflected at the level of sialoglycoconjugate expression. The present data support our earlier hypothesis that aminoglycosides, already at an early phase of intoxication, interfere with the function of the endoplasmic reticulum and/or the Golgi apparatus, implying that these organelles play a crucial role in the initial phase of aminoglycoside-induced OHC degeneration.