Behaviour of adipose-derived canine mesenchymal stem cells after superparamagnetic iron oxide nanoparticles labelling for magnetic resonance imaging

Background: Therapy with mesenchymal stem cells (MSCs) has been reported to provide beneficial effects in the treatment of neurological and orthopaedic disorders in dogs. The exact mechanism of action is poorly understood. Magnetic resonance imaging (MRI) gives the opportunity to observe MSCs after clinical administration. To visualise MSCs with the help of MRI, labelling with an MRI contrast agent is necessary. However, it must be clarified whether there is any negative influence on cell function and viability after labelling prior to clinical administration. Results: For the purpose of the study, seven samples with canine adipose-derived stem cells were incubated with superparamagnetic iron oxide nanoparticles (SPIO: 319.2 µg/mL Fe) for 24 h. The internalisation of the iron particles occurred via endocytosis. SPIO particles were localized as free clusters in the cytoplasm or within lysosomes depending on the time of investigation. The efficiency of the labelling was investigated using Prussian blue staining and MACS assay. After 3 weeks the percentage of SPIO labelled canine stem cells decreased. Phalloidin staining showed no negative effect on the cytoskeleton. Labelled cells underwent osteogenic and adipogenic differentiation. Chondrogenic differentiation occurred to a lesser extent compared with a control sample. MTT-Test and wound healing assay showed no influence of labelling on the proliferation. The duration of SPIO labelling was assessed using a 1 Tesla clinical MRI scanner and T2 weighted turbo spin echo and T2 weighted gradient echo MRI sequences 1, 2 and 3 weeks after labelling. The hypointensity caused by SPIO lasted for 3 weeks in both sequences. Conclusions: An Endorem labelling concentration of 319.2 µg/mL Fe (448 µg/mL SPIO) had no adverse effects on the viability of canine ASCs. Therefore, this contrast agent could be used as a model for iron oxide labelling agents. However, the tracking ability in vivo has to be evaluated in further studies

Multipotency of skeletal muscle stem cells on their native substrate and the expression of Connexin 43 during adoption of adipogenic and osteogenic fate

Skeletal muscle contains a resident stem cell population called Satellite cells (SC) which have a huge capacity to regenerate damaged tissue. Transplantation of a single fiber consisting of less than ten cells is able to generate tens of thousands of myonuclei within a matter of a few weeks (Collins et al., 2005). SC take their name from their peripheral position relative to the muscle fiber (Mauro, 1961). They are located under the basal lamina, in direct contact with the sarcolemma of muscle fibers. In undamaged muscle, they are relatively metabolically inactive as indicated by their low cytoplasmic content and they exist in a quiescent state. However, they express certain markers including Pax7 that aid their identification (Zammit et al., 2006). Upon muscle damage, SC become activated by inducing a number of genes including MyoD that encodes a member of the Myogenic Determination Factor family (MRF) of transcriptions factors (Zammit et al., 2006). Activation of SC permits cell division as well as migration. Initially, SC migrate under the basal lamina but then take up a supra-basal position by remodeling their overlying extra-cellular matrix (Otto et al., 2011). Activated SC can either revert to their quiescent state by down-regulating MyoD while maintaining Pax7 or can commit to myogenic differentiation by shutting off Pax7 expression and inducing the expression o

The effect of caloric restriction on the forelimb skeletal muscle fibers of 1 the hypertrophic myostatin null mice

Skeletal muscle mass loss has a broad impact on body performance and physical activity. Muscle wasting occurs due to genetic mutation as in muscular dystrophy, age-related muscle loss (sarcopenia) as well as in chronic wasting disorders as in cancer cachexia. Food restriction reduces muscle mass underpinned by increased muscle protein break down. However the influence of dietary restriction on the morphometry and phenotype of forelimb muscles in a genetically modified myostatin null mice are not fully characterized. The effect of a five week dietary limitation on five anatomically and structurally different forelimb muscles was examined. C57/BL6 wild type (Mstn+/+) and myostatin null (Mstn-/-) mice were either given a standard rodent normal daily diet ad libitum (ND) or 60% food restriction (FR) for a 5 week period. M. triceps brachii Caput laterale (T.lateral), M. triceps brachii Caput longum (T.long), M. triceps brachii Caput mediale (T.medial), M. extensor carpi ulnaris (ECU) and M. flexor carpi ulnaris (FCU) were dissected, weighted and processed for immunohistochemistry. Muscle mass, fibers cross sectional areas (CSA) and myosin heavy chain types IIB, IIX, IIA and type I were analyzed. We provide evidence that caloric restriction results in muscle specific weight reduction with the fast myofibers being more prone to atrophy. We show that slow fibers are less liable to dietary restriction induced muscle atrophy. The effect of dietary restriction was more pronounced in Mstn-/- muscles to implicate the oxidative fibers compared to Mstn+/+. Furthermore, peripherally located myofibers are more susceptible to dietary induced reduction compared to deep fibers. We additionally report that dietary restriction alters the glycolytic phenotype of the Mstn-/- into the oxidative form in a muscle dependent manner

Spiral Ganglion Stem Cells Can Be Propagated and Differentiated Into Neurons and Glia

Abstract The spiral ganglion is an essential functional component of the peripheral auditory system. Most types of hearing loss are associated with spiral ganglion cell degeneration which is irreversible due to the inner ear's lack of regenerative capacity. Recent studies revealed the existence of stem cells in the postnatal spiral ganglion, which gives rise to the hope that these cells might be useful for regenerative inner ear therapies. Here, we provide an in-depth analysis of sphere-forming stem cells isolated from the spiral ganglion of postnatal mice. We show that spiral ganglion spheres have characteristics similar to neurospheres isolated from the brain. Importantly, spiral ganglion sphere cells maintain their major stem cell characteristics after repeated propagation, which enables the culture of spheres for an extended period of time. In this work, we also demonstrate that differentiated sphere-derived cell populations not only adopt the immunophenotype of mature spiral ganglion cells but also develop distinct ultrastructural features of neurons and glial cells. Thus, our work provides further evidence that self-renewing spiral ganglion stem cells might serve as a promising source for the regeneration of lost auditory neurons

The effect of high-fat diet on the morphological properties of the forelimb musculature in hypertrophic myostatin null mice

Obesity is a worldwide nutritional disorder affecting body performance including 29 skeletal muscle. Inhibition of myostatin not only increases the muscle mass but also it reduces body fat accumulation. We examined the effect of high-fat diet on the phenotypic properties of forelimb muscles from myostatin null mice. Male wild-type and myostatin null mice were fed on either normal diet or high-fat diet (45 % fat) for ten weeks. M. triceps brachii Caput longum; M. triceps brachii Caput laterale; M. triceps brachii Caput mediale; M. extensor carpi ulnaris and M. flexor carpi ulnaris were processed for fiber type composition using immunohistochemistry and morphometric analysis. Although the muscle mass revealed no change under high-fat diet, there were morphometric alterations in the absence of myostatin. We show that high-fat diet reduces the cross-sectional area of the fast (IIB and IIX) fibers in M. triceps brachii Caput longum and M. triceps brachii Caput laterale of both genotypes. In contrast, increases of fast fibers area were observed in both M. extensor carpi ulnaris of wild- type and M. flexor carpi ulnaris of myostatin null. Meanwhile, a high-fat diet increases the area of the fast IIA fibers in wild-type, myostatin null displays a muscle-dependent alteration in the area of the same fiber type. The combined high-fat diet and myostatin deletion shows no effect on the area of slow type I fibers. Despite, a high-fat diet causes a reduction in the area of the peripheral IIB fibers in both genotypes, only myostatin null shows an increase in the area of the central IIB fibers. We provide evidence that a high-fat diet induces a muscle-dependent fast to slow myofiber shift in the absence of myostatin. Taken together, the data suggest that the morphological alterations of muscle fibers under combined high-fat diet and myostatin deletion reflect a functional adaptation of the muscle to utilize the high energy intake

Integration and potential of teaching communication skills in the study of veterinary medicine in Germany

Goal: Presentation of the current range of courses regarding communication at the five German educational institutions for veterinary medicine. In addition to learning objectives and individual solutions, possible potential for future developments are presented. Methods: Interviews with communication educators at the five German education institutions and subsequent synopsis. Results: To date, there are no binding education guidelines regarding communication in veterinary medicine. Nevertheless, communication education has been introduced at all five education institutions, albeit depth and formats vary considerably. The learning objectives are largely consistent and based on the recommendations for day-one-skills made by the European Association of Establishments for Veterinary Education. Communication is not recognized as a fully-fledged subject in the curricula of any of the education institutions. All education institutions clearly fall short of teaching the recommended 150 lecture hours. Conclusion: To ensure communication skills in veterinary medicine graduates, binding education guidelines should be agreed upon. Communication education should be integrated into all veterinary curricula as a fully-fledged subject with longitudinally increasing depth

Odorant-Dependent Generation of Nitric Oxide in Mammalian Olfactory Sensory Neurons

The gaseous signalling molecule nitric oxide (NO) is involved in various physiological processes including regulation of blood pressure, immunocytotoxicity and neurotransmission. In the mammalian olfactory bulb (OB), NO plays a role in the formation of olfactory memory evoked by pheromones as well as conventional odorants. While NO generated by the neuronal isoform of NO synthase (nNOS) regulates neurogenesis in the olfactory epithelium, NO has not been implicated in olfactory signal transduction. We now show the expression and function of the endothelial isoform of NO synthase (eNOS) in mature olfactory sensory neurons (OSNs) of adult mice. Using NO-sensitive micro electrodes, we show that stimulation liberates NO from isolated wild-type OSNs, but not from OSNs of eNOS deficient mice. Integrated electrophysiological recordings (electro-olfactograms or EOGs) from the olfactory epithelium of these mice show that NO plays a significant role in modulating adaptation. Evidence for the presence of eNOS in mature mammalian OSNs and its involvement in odorant adaptation implicates NO as an important new element involved in olfactory signal transduction. As a diffusible messenger, NO could also have additional functions related to cross adaptation, regeneration, and maintenance of MOE homeostasis

Speech Illusions in People at Clinical High Risk for Psychosis Linked to Clinical Outcome

BACKGROUND AND HYPOTHESIS: Around 20% of people at clinical high risk (CHR) for psychosis later develop a psychotic disorder, but it is difficult to predict who this will be. We assessed the incidence of hearing speech (termed speech illusions [SIs]) in noise in CHR participants and examined whether this was associated with adverse clinical outcomes. STUDY DESIGN: At baseline, 344 CHR participants and 67 healthy controls were presented with a computerized white noise task and asked whether they heard speech, and whether speech was neutral, affective, or whether they were uncertain about its valence. After 2 years, we assessed whether participants transitioned to psychosis, or remitted from the CHR state, and their functioning. STUDY RESULTS: CHR participants had a lower sensitivity to the task. Logistic regression revealed that a bias towards hearing targets in stimuli was associated with remission status (OR = 0.21, P = 042). Conversely, hearing SIs with uncertain valence at baseline was associated with reduced likelihood of remission (OR = 7.72. P = .007). When we assessed only participants who did not take antipsychotic medication at baseline, the association between hearing SIs with uncertain valence at baseline and remission likelihood remained (OR = 7.61, P = .043) and this variable was additionally associated with a greater likelihood of transition to psychosis (OR = 5.34, P = .029). CONCLUSIONS: In CHR individuals, a tendency to hear speech in noise, and uncertainty about the affective valence of this speech, is associated with adverse outcomes. This task could be used in a battery of cognitive markers to stratify CHR participants according to subsequent outcomes

Neural differentiated embryonic stem cells : from cell biology to cell replacement therapy

Die vorliegende kumulative Habilitationsschrift stellt die Differenzierungskapazität von murinen embryonale Stammzellen in die neurale Richtung und deren Verwendung für embryologische Studien zur Neurogenese dar. Darüber hinaus wird ihre Eignung als Ausgangsmaterial für Zellersatztherapien bei neurodegenerativen Erkrankungen modellhaft charakterisiert. Der erste Abschnitt der Arbeit befasst sich mit der Differenzierungspotenz der Zellen zu Neuronen, unter Verwendung eines FCS- und Retinsäure-haltigem Kultivierungsmediums. Die entstehende Vielfalt von differenzierenden neuronalen Phänotypen wird immunzytochemisch beurteilt und die Neurone zusätzlich anhand ihres Rezeptorprofiles elektrophysiologisch charakterisiert. Die Übertragbarkeit der Befunde auf die in vivo- Situation wird ausführlich diskutiert. Aufgrund der immensen Bedeutung des Kalziumhaushaltes während der neuronalen Differenzierung, wird die Ausstattung der differenzierenden Neurone hinsichtlich spannungsabhängiger Kalziumkanäle sowie Kalzium-bindender Proteine untersucht. Dabei kann eine differenzierungsabhängige Verschiebung des Kalziumkanalmusters beobachtet werden. Dagegen ist die Expression der verschiedenen Kalzium-bindenden Proteine auf bestimmte neuronale Reifungsstadien beschränkt. Die Untersuchung der Differenzierungskapazität in Makro- und Mikrogliazellen ergänzt die Charakterisierung der neuronalen Differenzierung. In einem experimentellen Abschnitt wird die Bedeutung der NO-Synthase II für die neuronale Differenzierung im Vergleich mit der neuronalen kortikalen Primärkultur demonstriert. Die Daten korrelieren mit in vivo Befunden zur Rezeptorgenese der Riechschleimhaut und des Vestibulocochlearorgans sowie mit Befunden der Kortex- und Retinaentwicklung an embryonalen Schnittserien. Parallel dazu kann mit Hilfe einer Knock-out- ES-Zellinie, die eine Defizienz für das ß1-Integrin aufweist, die Notwendigkeit dieses transmembranären Moleküles für eine regelgerecht ablaufende Differenzierung gezeigt werden. Schließlich demonstriert der experimentelle Abschnitt unter Verwendung von pharmakologischen Methoden und mit Hilfe einer Vitalbeobachtung der differenzierenden Neurone den Effekt einer Glutamatintoxikation auf sich entwickelnde Nervenzellen. Somit eignet sich das Modell der embryonalen Stammzellen auch für die Evaluation von neurotoxikologischen Fragestellungen. Im letzten Abschnitt der Arbeit steht die modellhafte Verwendung von neural differenzierten ES-Zellen für Zellersatztherapien im Vordergrund. Dazu werden neural selektionierte ES-Zellen eines Klones mit einer GFP-Expression unter der Kontrolle des ß-Aktinpromoters, zur sicheren Identifizierung der Zellen im Empfängergewebe, in das Striatum adulter Ratten transplantiert und ihre Differenzierungskapazität in Neurone und Gliazellen beurteilt. Unter Verwendung eines weiteren ES-Zellklones, bei dem die GFP-Expression unter der Kontrolle des 2. Introns des humanen Nestingens steht, kann die Selektion der neuralen Vorläuferzellen in vitro unmittelbar verfolgt werden. Damit wird die Voraussetzung für eine nahezu 100%ige Aufreinigung der Zellen mittels präparativen FACS geschaffen. Hinsichtlich einer zukünftigen Therapie des M. Parkinson unter Verwendung von humanen ES-Zellen kann anhand dieser murinen Zellinie, eine Erhöhung des Prozentsatzes dopaminerger Neurone mit Hilfe eines Inhibitors der Phosphatidylinositokinase 3 in Kultur modellhaft dargestellt werden. Tatsächlich wird anhand des Tiermodelles für die humane Makuladegeneration gezeigt, daß neural differenzierte ES-Zellen im Rahmen einer Zellersatztherapie die Verbesserung des krankhaften Zustandes bewirken. Als Lieferanten protektiver neurotropher Faktoren halten sie fortschreitende Degenerationsprozesse auf.The presented thesis deals with the differentiation capacity of murine embryonic stem cells into the neural lineage and their use in embryological studies regarding neurogenesis as well as their use as cell material for cell replacement therapies in neurodegenerative diseases. In the first section of this work the differentiation potency of ES-cells into the neuronal direction in a cultivation medium supplemented with FCS and retinoic acid is elucidated. The variety of differentiating neuronal phenotypes is evaluated immunocytochemically and additionally generated neurons are characterized electrophysiologically regarding their receptor profiles for the main excitatory and inhibitory neurotransmitters. The conveyance of the obtained data to the situation in vivo is thoroughly discussed. In order to meet the extraordinary significance of the calcium homeostasis during neuronal differentiation the occurrence of voltage gated calcium channels as well as calcium binding proteins is investigated. While there is a differentiation-dependent shift in the expression of calcium channels subtypes in all maturation stages, the expression of calcium binding proteins is restricted to certain neuronal subtypes. In addition to the thorough characterisation of neuronal differentiation in the ES-cell system, the differentiation capacity into macro- and microglial cells is evaluated. In the following experimental section the significance of the NO-synthase II for neuronal differentiation is studied in comparison with the primary culture of embryonic cortex neurons. Furthermore, data are correlated with in vivo findings concerning olfactory and vestibulocochlear receptorgenesis as well as with findings from cortical and retinal development. In a second experimental approach using a ß1-integrin knock out ES-cell line, the necessity of this transmebrane molecule is demonstrated for an undisturbed neuronal differentiation by modulating cell-cell and cell-matrix interaction. Additionally, using computer assisted vital microscopy the effect of a glutamate intoxication is evaluated on differentiated neurons, indicating the use of ES-cells as a model system for neuro- and embryo-toxicological studies, respectively. In the final section of this work the use of neurally differentiated embryonic stem cells for cell replacement therapies is evaluated. In order to determine the differentiation capacity of neurally selected stem cells in vivo after transplantation, cells of an ES-cell clone with a GFP-expression under the control of a ß-actin promoter are applied. Thus, transplanted cells can be unequivocally identified after stereotactic injection into the adult striatum and furthermore, their differentiation into neurons and astrocytes can be investigated immunocytochemically. With a second genetically modified ES-cell clone with a GFP expression under the control of the second intron of the human nestin gene, the selection of neural precursor cells can be directly monitored in vitro. This procedure offers a tool to obtain a near pure population of neural precursor cells by preparative flow cytometry (FACS). In order to prepare neural differentiated ES-cells for a specific treatment of Parkinson`s Disease it is demonstrated how the percentage of TH positive dopaminergic neurons can be markedly increased by incubating the cells in the presence of the inhibitor for the phosphatidylinositolkinase 3. Finally, an actual benefit using neurally selected ES-cells is shown in an animal model for the human macula degeneration. In this model neurally selected ES-cells have the potency to delay continuous degeneration processes by supplying neuroptotective factors