Dermal skin-derived fibroblasts from rodent and human have been found to exhibit mesenchymal surface antigen immunophenotype and differentiation potential along the three main mesenchymal-derived tissues: bone, cartilage and fat. Human dermal skin-derived mesenchymal stem cells constitute a promising cell source in clinical applications. Therefore, we isolated fibroblastic mesenchymal stem-cell-like cells from human dermis derived from juvenile foreskins, which share a mesenchymal stem cell phenotype and multi-lineage differentiation potential. We could show similar expression patterns for CD14()), CD29(+), CD31()), CD34()), CD44(+), CD45()), CD71(+), CD73 ⁄ SH3-SH4(+), CD90 ⁄ Thy-1(+), CD105 ⁄ SH2(+), CD133()) and CD166 ⁄ ALCAM(+) in wellestablished adipose tissue derived-stem cells and fibroblastic mesenchymal stem-cell-like cells by flow cytometry. Immunostainings showed that fibroblastic mesenchymal stem-celllike cells expressed vimentin, fibronectin and collagen; they were less positive for a-smooth muscle actin and nestin, while they were negative for epithelial cytokeratins. When cultured under appropriate inducible conditions, both cell types could differentiate along the adipogenic and osteogenic lineages. Additionally, fibroblastic mesenchymal stem-cell-like cells demonstrated a high proliferation potential. These findings are of particular importance, because skin or adipose tissues are easily accessible for autologous cell transplantations in regenerative medicine. In summary, these data indicate that dermal fibroblasts with multilineage differentiation potential are present in human dermis and they might play a key role in cutaneous wound healing.
GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.
Brain function is absolutely dependent on an appropriate supply of energy. A shortfall in supply-as occurs, for instance, following stroke-can lead rapidly to irreversible damage to this vital organ. While the consequences of pathophysiological energy depletion have been well documented, much less is known about the physiological energy dynamics of brain cells, although changes in the intracellular concentration of adenosine triphosphate (ATP), the major energy carrier of cells, have been postulated to contribute to cellular signaling. To address this issue more closely, we have investigated intracellular ATP in cultured primary cortical astrocytes by time-lapse microscopy using a genetically encoded fluorescent sensor for ATP. The cytosolic ATP sensor signal decreased after application of the neurotransmitter glutamate in a manner dependent on both glutamate concentration and glutamate transporter activity, but independent of glutamate receptors. The application of dopamine did not affect ATP levels within astrocytes. These results confirm that intracellular ATP levels in astrocytes do indeed respond to changes in physiological activity and pave the way for further studies addressing factors that affect regulation of ATP. V C 2017 Wiley Periodicals, Inc.
Astrocytes are a glial cell type, which is indispensable for brain energy metabolism. Within cells, the NADH/NAD redox state is a crucial node in metabolism connecting catabolic pathways to oxidative phosphorylation and ATP production in mitochondria. To characterize the dynamics of the intracellular NADH/NAD redox state in cortical astrocytes Peredox, a genetically encoded sensor for the NADH/NAD redox state, was expressed in cultured cortical astrocytes as well as in cortical astrocytes in acutely isolated brain slices. Calibration of the sensor in cultured astrocytes revealed a mean basal cytosolic NADH/NAD redox ratio of about 0.01; however, with a broad distribution and heterogeneity in the cell population, which was mirrored by a heterogeneous basal cellular concentration of lactate. Inhibition of glucose uptake decreased the NADH/NAD redox state while inhibition of lactate dehydrogenase or of lactate release resulted in an increase in the NADH/NAD redox ratio. Furthermore, the NADH/NAD redox state was regulated by the extracellular concentration of K , and application of the neurotransmitters ATP or glutamate increased the NADH/NAD redox state dependent on purinergic receptors and glutamate uptake, respectively. This regulation by K , ATP, and glutamate involved NBCe1 mediated sodium-bicarbonate transport. These results demonstrate that the NADH/NAD redox state in astrocytes is a metabolic node regulated by neuronal signals reflecting physiological activity, most likely contributing to adjust astrocytic metabolism to energy demand of the brain.
Inhibitory neurons crucially contribute to shaping the breathing rhythm in the brain stem. These neurons use GABA or glycine as neurotransmitter; or co-release GABA and glycine. However, the developmental relationship between GABAergic, glycinergic and cotransmitting neurons, and the functional relevance of cotransmitting neurons has remained enigmatic. Transgenic mice expressing fluorescent markers or the split-Cre system in inhibitory neurons were developed to track the three different interneuron phenotypes. During late embryonic development, the majority of inhibitory neurons in the ventrolateral medulla are cotransmitting cells, most of which differentiate into GABAergic and glycinergic neurons around birth and around postnatal day 4, respectively. Functional inactivation of cotransmitting neurons revealed an increase of the number of respiratory pauses, the cycle-by-cycle variability, and the overall variability of breathing. In summary, the majority of cotransmitting neurons differentiate into GABAergic or glycinergic neurons within the first 2 weeks after birth and these neurons contribute to fine-tuning of the breathing pattern.
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