Isolation and Characterization of Small Extracellular Vesicles from Porcine Blood Plasma, Cerebrospinal Fluid, and Seminal Plasma

Extracellular vesicles (EVs) are a highly attractive subject of biomedical research as possible carriers of nucleic acid and protein biomarkers. EVs released to body fluids enable indirect access to inner organs by so-called “liquid biopsies”. Obtaining a high-quality EV sample with minimum contaminants is crucial for proteomic analyses using LC−MS/MS or other techniques. However, the EV content in various body fluids largely differs, which may hamper subsequent analyses. Here, we present a comparison of extracellular vesicle yields from blood plasma, cerebrospinal fluid, and seminal plasma using an experimental pig model. Pigs are widely used in biomedical research as large animal models with anatomy and physiology close to those of humans and enable studies (e.g., of the nervous system) that are unfeasible in humans. EVs were isolated from body fluids by differential centrifugation followed by ultracentrifugation. EVs were characterized according to protein yields and to the quality of the isolated vesicles (e.g., size distribution, morphology, positivity for exosome markers). In our experimental setting, substantial differences in EV amounts were identified among body fluids, with the seminal plasma being the richest EV source. The yields of pellet proteins from ultracentrifugation of 1 mL of porcine body fluids may help to estimate body fluid input volumes to obtain sufficient samples for subsequent proteomic analyses

Potential for Treatment of Glioblastoma: New Aspects of Superparamagnetic Iron Oxide Nanoparticles

Glioblastoma (GB) is a highly aggressive and infiltrative brain tumor characterized by poor outcomes and a high rate of recurrence despite maximal safe resection, chemotherapy, and radiation. Superparamagnetic iron oxide nanoparticles (SPIONs) are a novel tool that can be used for many applications including magnetic targeting, drug delivery, gene delivery, hyperthermia treatment, cell tracking, or multiple simultaneous functions. SPIONs are studied as a magnetic resonance imaging tumor contrast agent by targeting tumor cell proteins or tumor vasculature. Drug delivery to GB tumor has been targeted with SPIONs in murine models. In addition to targeting tumor cells for imaging or drug-delivery, SPION has also been shown to be effective at targeting for hyperthermia. Along with animal models, human trials have been conducted for a number of different modes of SPION utilization, with important findings and lessons for further preclinical and clinical experiments. SPIONs are opening up several new avenues for monitoring and treatment of GB tumors; here, we review the current research and a variety of possible clinical applications

Využití feromagnetických nanočástic s nízkou Curieovou teplotou pro zobrazovací magnetickou rezonancí řízenou termoablaci

Magnetic nanoparticles represent a tool for use in magnetic resonance imaging (MRI)-guided thermoablation of tumors using an external high-frequency mag¬netic field. To avoid local overheating, perovskite nanoparticles with a lower Curie temperature (Tc) were proposed for use in thermotherapy. However, deposited power decreases when approaching the Curie temperature and consequently may not be sufficient for effective ablation. The goal of the study was to test this hypothesis. Perovskite nanoparticles (Tc = 66–74°C) were characterized and tested both in vitro and in vivo. Magnetic particles with low Tc can be tracked in vivo by MRI and heated by a HF field. The particles are capable of inducing cell apoptosis in suspensions in vitro at high concentrations only.Magnetické nanočástice představují nástroj pro použití v magnetickou rezonancí (MRI) řízené termoablaci nádorů pomocí externího vysokofrekvenčního magnetického pole. Aby se zabránilo lokálnímu přehřátí, byly navrženy perovskitové nanočástice s nižší Curieovou teplotou (Tc) pro použití v termoterapii. Nicméně vložená energie se snižuje, když se blíží Curieova teplota a v důsledku toho nemusí být dostatečná pro účinnou ablaci. Cílem této studie bylo otestovat tuto hypotézu. Perovskitové nanočástice (Tc = 66–74 °C) byly charakterizovány a testovány jak in vitro, tak in vivo. Magnetické částice s nízkou Tc mohou být sledovány in vivo pomocí MRI a zahřívány VF polem. Tyto částice jsou schopné indukovat apoptózu buněk v suspenzi in vitro při pouze vysokých koncentracích

Clinically Relevant Solution for the Hypothermic Storage and Transportation of Human Multipotent Mesenchymal Stromal Cells

The wide use of human multipotent mesenchymal stromal cells (MSCs) in clinical trials requires a full-scale safety and identity evaluation of the cellular product and subsequent transportation between research/medical centres. This necessitates the prolonged hypothermic storage of cells prior to application. The development of new, nontoxic, and efficient media, providing high viability and well-preserved therapeutic properties of MSCs during hypothermic storage, is highly relevant for a successful clinical outcome. In this study, a simple and effective trehalose-based solution was developed for the hypothermic storage of human bone marrow MSC suspensions for further clinical applications. Human bone marrow MSCs were stored at 4°C for 24, 48, and 72 hrs in the developed buffered trehalose solution and compared to several research and clinical grade media: Plasma-Lyte® 148, HypoThermosol® FRS, and Ringer’s solution. After the storage, the preservation of viability, identity, and therapeutically associated properties of MSCs were assessed. The hypothermic storage of MSCs in the new buffered trehalose solution provided significantly higher MSC recovery rates and ability of cells for attachment and further proliferation, compared to Plasma-Lyte® 148 and Ringer’s solution, and was comparable to research-grade HypoThermosol® FRS. There were no differences in the immunophenotype, osteogenic, and adipogenic differentiation and the immunomodulatory properties of MSCs after 72 hrs of cold storage in these solutions. The obtained results together with the confirmed therapeutic properties of trehalose previously described provide sufficient evidence that the developed trehalose medium can be applied as a low-cost and efficient solution for the hypothermic storage of MSC suspensions, with a high potential for translation into clinical practice

Human conditionally immortalized neural stem cells improve locomotor function after spinal cord injury in the rat

INTRODUCTION: A growing number of studies have highlighted the potential of stem cell and more-differentiated neural cell transplantation as intriguing therapeutic approaches for neural repair after spinal cord injury (SCI). METHODS: A conditionally immortalized neural stem cell line derived from human fetal spinal cord tissue (SPC-01) was used to treat a balloon-induced SCI. SPC-01 cells were implanted into the lesion 1 week after SCI. To determine the feasibility of tracking transplanted stem cells, a portion of the SPC-01 cells was labeled with poly-L-lysine-coated superparamagnetic iron-oxide nanoparticles, and the animals grafted with labeled cells underwent magnetic resonance imaging. Functional recovery was evaluated by using the BBB and plantar tests, and lesion morphology, endogenous axonal sprouting and graft survival, and differentiation were analyzed. Quantitative polymerase chain reaction (qPCR) was used to evaluate the effect of transplanted SPC-01 cells on endogenous regenerative processes. RESULTS: Transplanted animals displayed significant motor and sensory improvement 2 months after SCI, when the cells robustly survived in the lesion and partially filled the lesion cavity. qPCR revealed the increased expression of rat and human neurotrophin and motor neuron genes. The grafted cells were immunohistologically positive for glial fibrillary acidic protein (GFAP); however, we found 25% of the cells to be positive for Nkx6.1, an early motor neuron marker. Spared white matter and the robust sprouting of growth-associated protein 43 (GAP43)(+) axons were found in the host tissue. Four months after SCI, the grafted cells matured into Islet2(+) and choline acetyltransferase (ChAT)(+) neurons, and the graft was grown through with endogenous neurons. Grafted cells labeled with poly-L-lysine-coated superparamagnetic nanoparticles before transplantation were detected in the lesion on T(2)-weighted images as hypointense spots that correlated with histologic staining for iron and the human mitochondrial marker MTCO2. CONCLUSIONS: The transplantation of SPC-01 cells produced significant early functional improvement after SCI, suggesting an early neurotrophic action associated with long-term restoration of the host tissue, making the cells a promising candidate for future cell therapy in patients with SCI

Involvement of mTOR Pathways in Recovery from Spinal Cord Injury by Modulation of Autophagy and Immune Response

Traumatic spinal cord injury (SCI) is untreatable and remains the leading cause of disability. Neuroprotection and recovery after SCI can be partially achieved by rapamycin (RAPA) treatment, an inhibitor of mTORC1, complex 1 of the mammalian target of rapamycin (mTOR) pathway. However, mechanisms regulated by the mTOR pathway are not only controlled by mTORC1, but also by a second mTOR complex (mTORC2). Second-generation inhibitor, pp242, inhibits both mTORC1 and mtORC2, which led us to explore its therapeutic potential after SCI and compare it to RAPA treatment. In a rat balloon-compression model of SCI, the effect of daily RAPA (5 mg/kg; IP) and pp242 (5 mg/kg; IP) treatment on inflammatory responses and autophagy was observed. We demonstrated inhibition of the mTOR pathway after SCI through analysis of p-S6, p-Akt, and p-4E-BP1 levels. Several proinflammatory cytokines were elevated in pp242-treated rats, while RAPA treatment led to a decrease in proinflammatory cytokines. Both RAPA and pp242 treatments caused an upregulation of LC3B and led to improved functional and structural recovery in acute SCI compared to the controls, however, a greater axonal sprouting was seen following RAPA treatment. These results suggest that dual mTOR inhibition by pp242 after SCI induces distinct mechanisms and leads to recovery somewhat inferior to that following RAPA treatment