Modulating endothelial adhesion and migration impacts stem cell therapies efficacy.

BACKGROUND: Limited knowledge of stem cell therapies` mechanisms of action hampers their sustainable implementation into the clinic. Specifically, the interactions of transplanted stem cells with the host vasculature and its implications for their therapeutic efficacy are not elucidated. We tested whether adhesion receptors and chemokine receptors on stem cells can be functionally modulated, and consequently if such modulation may substantially affect therapeutically relevant stem cell interactions with the host endothelium. METHODS: We investigated the effects of cationic molecule polyethylenimine (PEI) treatment with or without nanoparticles on the functions of adhesion receptors and chemokine receptors of human bone marrow-derived Mesenchymal Stem Cells (MSC). Analyses included MSC functions in vitro, as well as homing and therapeutic efficacy in rodent models of central nervous system´s pathologies in vivo. FINDINGS: PEI treatment did not affect viability, immunomodulation or differentiation potential of MSC, but increased the CCR4 expression and functionally blocked their adhesion receptors, thus decreasing their adhesion capacity in vitro. Intravenously applied in a rat model of brain injury, the homing rate of PEI-MSC in the brain was highly increased with decreased numbers of adherent PEI-MSC in the lung vasculature. Moreover, in comparison to untreated MSC, PEI-MSC featured increased tumour directed migration in a mouse glioblastoma model, and superior therapeutic efficacy in a murine model of stroke. INTERPRETATION: Balanced stem cell adhesion and migration in different parts of the vasculature and tissues together with the local microenvironment impacts their therapeutic efficacy. FUNDING: Robert Bosch Stiftung, IZEPHA grant, EU grant 7 FP Health.

3D visualization and quantification of microvessels in the whole ischemic mouse brain using solvent-based clearing and light sheet microscopy.

The visualization of cerebral microvessels is essential for understanding brain remodeling after stroke. Injection of dyes allows for the evaluation of perfused vessels, but has limitations related either to incomplete microvascular filling or leakage. In conventional histochemistry, the analysis of microvessels is limited to 2D structures, with apparent limitations regarding the interpretation of vascular circuits. Herein, we developed a straight-forward technique to visualize microvessels in the whole ischemic mouse brain, combining the injection of a fluorescent-labeled low viscosity hydrogel conjugate with 3D solvent clearing followed by automated light sheet microscopy. We performed transient middle cerebral artery occlusion in C57Bl/6j mice and acquired detailed 3D vasculature images from whole brains. Subsequent image processing, rendering and fitting of blood vessels to a filament model was employed to calculate vessel length density, resulting in 0.922 ± 0.176 m/mm3 in healthy tissue and 0.329 ± 0.131 m/mm3 in ischemic tissue. This analysis showed a marked loss of capillaries with a diameter ≤ 10 µm and a more moderate loss of microvessels in the range > 10 and ≤ 20 µm, whereas vessels > 20 µm were unaffected by focal cerebral ischemia. We propose that this protocol is highly suitable for studying microvascular injury and remodeling post-stroke.

Genetic Dissection of Aversive Associative Olfactory Learning and Memory in Drosophila Larvae.

Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes-besides other forms-a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. Here we deconstruct the larval mnemonic organization after aversive olfactory conditioning. We show that after odor-high salt conditioning larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3'5'-monophosphate (cAMP) signaling and synapsin gene function. In addition, we show for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution.

Characterization of Seeding Conditions for Studies on Differentiation Patterns of Subventricular Zone Derived Neurospheres.

Stem cell research depends on extensive in vitro research. Poly-D-lysine (PDL) and polyornithine (PornT) are chemically synthesized amino acid chains promoting cell adhesion to solid substrates. Although, PDL and PornT are extensively used, there is no common agreement regarding the most optimal substance and its concentration. We therefore aimed at testing the effect of increasing concentrations (10, 50, and 100 µg/ml) for each compound and their corresponding mixtures (5+5 and 10+10 µg/ml) on the differentiation patterns of subventricular zone derived neurospheres. The latter were cultured for 24 h for protein and morphological analysis or for 8 h for migration analysis. No significant differences were found between increasing concentrations of PDL and PornT alone and the 10+10 condition in Western blots and immunocytochemistry. However, the mixed condition of 5+5 showed decreased glial fibrillary acidic protein and nestin expression with no changes in Akt, pAkt, GSK-3-beta, and pGSK-3-beta expression patterns. The various coating conditions also had no influence on migration of cells emerging from the neurosphere. Nevertheless, stimulation with recombinant human Erythropoietin (rhEpo) reduced migration by 20% regardless of the coating condition. We therefore conclude that a minimal concentration of 10 µg/ml of either compound should be used to produce reliable results with no alterations in protein levels as found for the 5+5 groups, and that the coating has no effect on the response of cells to chemical interventions. As such, a concentration of 10 µg/ml for either substance is sufficient when studying cellular processes of neurospheres in an in vitro or ex vivo environment.

Extracellular Vesicles Improve Post-Stroke Neuroregeneration and Prevent Postischemic Immunosuppression.

UNLABELLED: Although the initial concepts of stem cell therapy aimed at replacing lost tissue, more recent evidence has suggested that stem and progenitor cells alike promote postischemic neurological recovery by secreted factors that restore the injured brain's capacity to reshape. Specifically, extracellular vesicles (EVs) derived from stem cells such as exosomes have recently been suggested to mediate restorative stem cell effects. In order to define whether EVs indeed improve postischemic neurological impairment and brain remodeling, we systematically compared the effects of mesenchymal stem cell (MSC)-derived EVs (MSC-EVs) with MSCs that were i.v. delivered to mice on days 1, 3, and 5 (MSC-EVs) or on day 1 (MSCs) after focal cerebral ischemia in C57BL6 mice. For as long as 28 days after stroke, motor coordination deficits, histological brain injury, immune responses in the peripheral blood and brain, and cerebral angiogenesis and neurogenesis were analyzed. Improved neurological impairment and long-term neuroprotection associated with enhanced angioneurogenesis were noticed in stroke mice receiving EVs from two different bone marrow-derived MSC lineages. MSC-EV administration closely resembled responses to MSCs and persisted throughout the observation period. Although cerebral immune cell infiltration was not affected by MSC-EVs, postischemic immunosuppression (i.e., B-cell, natural killer cell, and T-cell lymphopenia) was attenuated in the peripheral blood at 6 days after ischemia, providing an appropriate external milieu for successful brain remodeling. Because MSC-EVs have recently been shown to be apparently safe in humans, the present study provides clinically relevant evidence warranting rapid proof-of-concept studies in stroke patients. SIGNIFICANCE: Transplantation of mesenchymal stem cells (MSCs) offers an interesting adjuvant approach next to thrombolysis for treatment of ischemic stroke. However, MSCs are not integrated into residing neural networks but act indirectly, inducing neuroprotection and promoting neuroregeneration. Although the mechanisms by which MSCs act are still elusive, recent evidence has suggested that extracellular vesicles (EVs) might be responsible for MSC-induced effects under physiological and pathological conditions. The present study has demonstrated that EVs are not inferior to MSCs in a rodent stroke model. EVs induce long-term neuroprotection, promote neuroregeneration and neurological recovery, and modulate peripheral post-stroke immune responses. Also, because EVs are well-tolerated in humans, as previously reported, the administration of EVs under clinical settings might set the path for a novel and innovative therapeutic stroke concept without the putative side effects attached to stem cell transplantation.

The indirect NMDAR antagonist acamprosate induces postischemic neurologic recovery associated with sustained neuroprotection and neuroregeneration.

Cerebral ischemia stimulates N-methyl-d-aspartate receptors (NMDARs) resulting in increased calcium concentration and excitotoxicity. Yet, deactivation of NMDAR failed in clinical studies due to poor preclinical study designs or toxicity of NMDAR antagonists. Acamprosate is an indirect NMDAR antagonist used for patients with chronic alcohol dependence. We herein analyzed the therapeutic potential of acamprosate on brain injury, neurologic recovery and their underlying mechanisms. Mice were exposed to cerebral ischemia, treated with intraperitoneal injections of acamprosate or saline (controls), and allowed to survive until 3 months. Acamprosate yielded sustained neuroprotection and increased neurologic recovery when given no later than 12 hours after stroke. The latter was associated with increased postischemic angioneurogenesis, albeit acamprosate did not stimulate angioneurogenesis itself. Rather, increased angioneurogenesis was due to inhibition of calpain-mediated pro-injurious signaling cascades. As such, acamprosate-mediated reduction of calpain activity resulted in decreased degradation of p35, increased abundance of the pro-survival factor STAT6, and reduced N-terminal-Jun-kinase activation. Inhibition of calpain was associated with enhanced stability of the blood-brain barrier, reduction of oxidative stress and cerebral leukocyte infiltration. Taken into account its excellent tolerability, its sustained effects on neurologic recovery, brain tissue survival, and neural remodeling, acamprosate is an intriguing candidate for adjuvant future stroke treatment.

Neural precursor cells in the ischemic brain - integration, cellular crosstalk, and consequences for stroke recovery.

After an ischemic stroke, neural precursor cells (NPCs) proliferate within major germinal niches of the brain. Endogenous NPCs subsequently migrate toward the ischemic lesion where they promote tissue remodeling and neural repair. Unfortunately, this restorative process is generally insufficient and thus unable to support a full recovery of lost neurological functions. Supported by solid experimental and preclinical data, the transplantation of exogenous NPCs has emerged as a potential tool for stroke treatment. Transplanted NPCs are thought to act mainly via trophic and immune modulatory effects, thereby complementing the restorative responses initially executed by the endogenous NPC population. Recent studies have attempted to elucidate how the therapeutic properties of transplanted NPCs vary depending on the route of transplantation. Systemic NPC delivery leads to potent immune modulatory actions, which prevent secondary neuronal degeneration, reduces glial scar formation, diminishes oxidative stress and stabilizes blood-brain barrier integrity. On the contrary, local stem cell delivery allows for the accumulation of large numbers of transplanted NPCs in the brain, thus achieving high levels of locally available tissue trophic factors, which may better induce a strong endogenous NPC proliferative response. Herein we describe the diverse capabilities of exogenous (systemically vs. locally transplanted) NPCs in enhancing the endogenous neurogenic response after stroke, and how the route of transplantation may affect migration, survival, bystander effects and integration of the cellular graft. It is the authors' claim that understanding these aspects will be of pivotal importance in discerning how transplanted NPCs exert their therapeutic effects in stroke.

Fetal brain genomic reprogramming following asphyctic preconditioning.

BACKGROUND: Fetal asphyctic (FA) preconditioning is effective in attenuating brain damage incurred by a subsequent perinatal asphyctic insult. Unraveling mechanisms of this endogenous neuroprotection, activated by FA preconditioning, is an important step towards new clinical strategies for asphyctic neonates. Genomic reprogramming is thought to be, at least in part, responsible for the protective effect of preconditioning. Therefore we investigated whole genome differential gene expression in the preconditioned rat brain. FA preconditioning was induced on embryonic day 17 by reversibly clamping uterine circulation. Male control and FA offspring were sacrificed 96 h after FA preconditioning. Whole genome transcription was investigated with Affymetrix Gene1.0ST chip. RESULTS: Data were analyzed with the Bioconductor Limma package, which showed 53 down-regulated and 35 up-regulated transcripts in the FA-group. We validated these findings with RT-qPCR for adh1, edn1, leptin, rdh2, and smad6. Moreover, we investigated differences in gene expression across different brain regions. In addition, we performed Gene Set Enrichment Analysis (GSEA) which revealed 19 significantly down-regulated gene sets, mainly involved in neurotransmission and ion transport. 10 Gene sets were significantly up-regulated, these are mainly involved in nucleosomal structure and transcription, including genes such as mecp2. CONCLUSIONS: Here we identify for the first time differential gene expression after asphyctic preconditioning in fetal brain tissue, with the majority of differentially expressed transcripts being down-regulated. The observed down-regulation of cellular processes such as neurotransmission and ion transport could represent a restriction in energy turnover which could prevent energy failure and subsequent neuronal damage in an asphyctic event. Up-regulated transcripts seem to exert their function mainly within the cell nucleus, and subsequent Gene Set Enrichment Analysis suggests that epigenetic mechanisms play an important role in preconditioning induced neuroprotection.