Mesenchymal stem cell engineering by ARCA analog-capped mRNA.

We previously have shown that mRNA-based engineering may enhance mesenchymal stem cell (MSC) trafficking. However, optimal conditions for in vitro mRNA engineering of MSCs are unknown. Here, we investigated several independent variables: (1) transfection factor (Lipofectamine 2000 vs. TransIT), (2) mRNA purification method (spin column vs. high-performance liquid chromatography [HPLC] column), and (3) mRNA capping (ARCA vs. ß-S-ARCA D1 and ß-S-ARCA D2). Dependent variables included protein production based on mRNA template (measured by the bioluminescence of reporter gene luciferase over hours), MSC metabolic activity corresponding with their wellbeing measured by CCK-8 over days, and endogenous expression of genes by RT-qPCR related to innate intracellular immune response and decapping at two time points: days 2 and 5. We have found that Lipofectamine 2000 outperforms TransIT, and used it throughout the study. Then, we showed that mRNA must be purified by HPLC to be relatively neutral to MSCs in terms of metabolic activity and endogenous protein production. Ultimately, we demonstrated that ß-S-ARCA D1 enables higher protein production but at the cost of lower MSC metabolic activity, with no impact on RT-qPCR results. Thus Lipofectamine 2000-based in vitro transfection of HPLC-purified and ARCA- or ß-S-ARCA D1-capped mRNA is optimal for MSC engineering.

Glia-Neurotrophic Factor Relationships: Possible Role in Pathobiology of Neuroinflammation-Related Brain Disorders.

Neurotrophic factors (NTFs) play an important role in maintaining homeostasis of the central nervous system (CNS) by regulating the survival, differentiation, maturation, and development of neurons and by participating in the regeneration of damaged tissues. Disturbances in the level and functioning of NTFs can lead to many diseases of the nervous system, including degenerative diseases, mental diseases, and neurodevelopmental disorders. Each CNS disease is characterized by a unique pathomechanism, however, the involvement of certain processes in its etiology is common, such as neuroinflammation, dysregulation of NTFs levels, or mitochondrial dysfunction. It has been shown that NTFs can control the activation of glial cells by directing them toward a neuroprotective and anti-inflammatory phenotype and activating signaling pathways responsible for neuronal survival. In this review, our goal is to outline the current state of knowledge about the processes affected by NTFs, the crosstalk between NTFs, mitochondria, and the nervous and immune systems, leading to the inhibition of neuroinflammation and oxidative stress, and thus the inhibition of the development and progression of CNS disorders.

Modulation of the endoplasmic reticulum stress and unfolded protein response mitigates the behavioral effects of early-life stress.

BACKGROUND: Early-life stress (ELS) affects brain development and increases the risk of mental disorders associated with the dysfunction of the medial prefrontal cortex (mPFC). The mechanisms of ELS action are not well understood. Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are cellular processes involved in brain maturation through the regulation of pro-survival or proapoptotic processes. We hypothesized that ER stress and the UPR in the mPFC are involved in the neurobiology of ELS. METHODS: We performed a maternal separation (MS) procedure from postnatal days 1 to 14 in rats. Before each MS, pups were injected with an inhibitor of ER stress, salubrinal or a vehicle. The mRNA and protein expression of UPR and apoptotic markers were evaluated in the mPFC using RT-qPCR and Western blot methods, respectively. We also estimated the numbers of neurons and glial cells using stereological methods. Additionally, we assessed behavioral phenotypes related to fear, anhedonia and response to psychostimulants. RESULTS: MS slightly enhanced the activation of the UPR in juveniles and modulated the expression of apoptotic markers in juveniles and preadolescents but not in adults. Additionally, MS did not affect the numbers of neurons and glial cells at any age. Both salubrinal and vehicle blunted the expression of UPR markers in juvenile and preadolescent MS rats, often in a treatment-specific manner. Moreover, salubrinal and vehicle generally alleviated the behavioral effects of MS in preadolescent and adult rats. CONCLUSIONS: Modulation of ER stress and UPR processes may potentially underlie susceptibility or resilience to ELS.

The legacy of mRNA engineering: A lineup of pioneers for the Nobel Prize.

mRNA is like Hermes, delivering the genetic code to cellular construction sites, so it has long been of interest, but only to a small group of scientists, and only demonstrating its remarkable efficacy in coronavirus disease 2019 (COVID-19) vaccines allowed it to go out into the open. Therefore, now is the right timing to delve into the stepping stones that underpin this success and pay tribute to the underlying scientists. From this perspective, advances in mRNA engineering have proven crucial to the rapidly growing role of this molecule in healthcare. Development of consecutive generations of cap analogs, including anti-reverse cap analogs (ARCAs), has significantly boosted translation efficacy and maintained an enthusiasm for mRNA research. Nucleotide modification to protect mRNA molecules from the host's immune system, followed by finding appropriate purification and packaging methods, were other links in the chain enabling medical breakthroughs. Currently, vaccines are the central area of mRNA research, but it will reach far beyond COVID-19. Supplementation of missing or abnormal proteins is another large field of mRNA research. Ex vivo cell engineering and genome editing have been expanding recently. Thus, it is time to recognize mRNA pioneers while building upon their legacy.

Mesenchymal stem cells in glioblastoma therapy and progression: How one cell does it all.

Mesenchymal stem cells (MSCs) are among the most investigated and applied somatic stem cells in experimental therapies for the regeneration of damaged tissues. Moreover, as it was recently postulated, MSCs may demonstrate anti-tumor properties. Glioblastoma (GBM) is a grade IV central nervous system tumor with no available effective therapy and an inevitably fatal prognosis. Experimental studies utilizing MSCs in GBM treatment resulted in numerous controversies. Native MSCs were shown to exert anti-GBM activity by controlling angiogenesis, regulating cell cycle, and inducing apoptosis. They also were used as sensitizing factors and vehicles delivering various anti-cancer compounds. On the other hand, some experiments revealed significant risks related to MSC-based therapies for GBM, such as enhancement of tumor cell proliferation, invasion, and aggressiveness. The following review elaborates on all mentioned contradictory data and provides a realistic, current clinical perspective on MSCs' potential in GBM treatment.

Mesenchymal Stem Cells for Neurological Disorders.

Neurological disorders are becoming a growing burden as society ages, and there is a compelling need to address this spiraling problem. Stem cell-based regenerative medicine is becoming an increasingly attractive approach to designing therapies for such disorders. The unique characteristics of mesenchymal stem cells (MSCs) make them among the most sought after cell sources. Researchers have extensively studied the modulatory properties of MSCs and their engineering, labeling, and delivery methods to the brain. The first part of this review provides an overview of studies on the application of MSCs to various neurological diseases, including stroke, traumatic brain injury, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, Parkinson's disease, and other less frequently studied clinical entities. In the second part, stem cell delivery to the brain is focused. This fundamental but still understudied problem needs to be overcome to apply stem cells to brain diseases successfully. Here the value of cell engineering is also emphasized to facilitate MSC diapedesis, migration, and homing to brain areas affected by the disease to implement precision medicine paradigms into stem cell-based therapies.

Neuroinflammation evoked by brain injury in a rat model of lacunar infarct.

Stroke is the leading cause of long-term, severe disability worldwide. Immediately after the stroke, endogenous inflammatory processes are upregulated, leading to the local neuroinflammation and the potentiation of brain tissue destruction. The innate immune response is triggered as early as 24 h post-brain ischemia, followed by adaptive immunity activation. Together these immune cells produce many inflammatory mediators, i.e., cytokines, growth factors, and chemokines. Our study examines the immune response components in the early stage of deep brain lacunar infarct in the rat brain, highly relevant to the clinical scenario. The lesion was induced by stereotactic injection of ouabain into the adult rat striatum. Ouabain is a Na/K ATPase pump inhibitor that causes excitotoxicity and brings metabolic and structural changes in the cells leading to focal brain injury. We have shown a surge of neurodegenerative changes in the peri-infarct area in the first days after brain injury. Immunohistochemical analysis revealed early microglial activation and the gradual infiltration of immune cells with a significant increase of CD4+ and CD8+ T lymphocytes in the ipsilateral hemisphere. In our studies, we identified the higher level of pro-inflammatory cytokines, i.e., interleukin-1α, interleukin-1ß, tumor necrosis factor-α, and interferon-γ, but a lower level of anti-inflammatory cytokines, i.e., interleukin-10 and transforming growth factor-ß2 in the injured brain than in normal rats. Concomitantly focal brain injury showed a significant increase in the level of chemokines, i.e., monocyte chemoattractant protein-1 and CC motif chemokine ligand 5 compared to control. Our findings provide new insights into an early inflammatory reaction in our model of the deep-brain lacunar infarct. The results of this study may highlight future stroke immunotherapies for targeting the acute immune response accompanied by the insult.

Mesenchymal stem cells injected into carotid artery to target focal brain injury home to perivascular space.

Rationale: The groundbreaking discovery of mesenchymal stem cells (MSCs) with their multifaceted benefits led to their widespread application in experimental medicine, including neurology. Efficient delivery of MSCs to damaged regions of the central nervous system may be a critical factor in determining outcome. Integrin VLA-4 (α4ß1) coded by ITGA4 and ITGB1 genes is an adhesion molecule expressed by leukocytes, which is responsible for initiation of their diapedesis through cell docking to the inflamed vessel wall expressing VCAM1 receptor. This function of VLA-4 has been recapitulated in neural stem cells and glial progenitors. Thus, it was prudent to investigate this tool as a vehicle driving extravasation of MSCs. Since MSCs naturally express ITGB1 subunit, we decided to supplement them with ITGA4 only. The purpose of our current study is to investigate the eventual fate of IA delivered ITGA4 engineered and naive MSCs. Methods: mRNA-ITGA4 transfected and naive MSCs were injected to right internal carotid artery of rats with focal brain injury. Through next three days MSC presence in animals' brain was navigated by magnetic resonance imaging. Transplanted cell location relative to the brain blood vessels and host immunological reaction were analyzed post-mortem by immunohistochemistry. The chemotaxis of modified and naive MSCs was additionally examined in in vitro transwell migration assay. Results: Both naïve and ITGA4-overexpressing cells remained inside the vascular lumen over the first two days after IA infusion. On the third day, 39% of mRNA-ITGA4 modified and 51% naïve MSCs homed to perivascular space in the injury region (p=NS). The gradual decrease of both naive and mRNA-ITGA4 transfected hBM-MSCs in the rat brain was observed. mRNA-ITGA4 transfected MSCs appeared to be more vulnerable to phagocytosis than naïve cells. Moreover, in vitro study revealed that homogenate from the injured brain repels migration of MSCs, corroborating the incomplete extravasation observed in vivo. Conclusions: In summary, IA transplanted MSCs are capable of homing to the perivascular space, an integral part of neurovascular unit, which might contribute to the replacement of injured pericytes, a critical element facilitating restoration of CNS function. The mRNA-ITGA4 transfection improves cell docking to vessel but this net benefit vanishes over the next two days due to fast clearance from cerebral vessels of the majority of transplanted cells, regardless of their engineering status. The drawbacks of mRNA-ITGA4 transfection become apparent on day 3 post transplantation due to the lower survival and higher vulnerability to host immune attack.

Microfluidic Systems in CNS Studies.

Current technological progress facilitates the introduction of micro-devices into biotechnology research including studies on central nervous system. Wide range of micro-chambers with diversity of channel systems and multiple compartments enable users to create models which closely mimic nervous tissue structure which nowadays is often called as brain-on-a-chip technology. Heretofore experiments showing the influence of substance gradients, cell interactions, spatial conditions, neuroinflammation, stem cells migration, drug delivery, mechanisms controlling progression of diseases like Alzheimer, Parkinson, multiple sclerosis or nerve injury were performed in microfluidic devices. Moreover, the integration of bio-sensors and development of dedicated software for microfluidic studies can enable performing high throughput and good quality automated experiments investigating regeneration and degeneration processes in models well emulating central nervous system structures.

Immunomodulatory and Regenerative Effects of Mesenchymal Stem Cells and Extracellular Vesicles: Therapeutic Outlook for Inflammatory and Degenerative Diseases.

Mesenchymal stem cells (MSCs) are non-hematopoietic, multipotent stem cells derived from mesoderm, which can be easily isolated from many sources such as bone marrow, umbilical cord or adipose tissue. MSCs provide support for hematopoietic stem cells and have an ability to differentiate into multiple cell lines. Moreover, they have proangiogenic, protective and immunomodulatory properties. MSCs have the capacity to modulate both innate and adaptive immune responses, which accompany many diseases, by inhibiting pro-inflammatory reactions and stimulating anti-inflammatory activity. Recent findings revealed that the positive effect of MSCs is at least partly associated with the production of extracellular vesicles (EVs). EVs are small membrane structures, containing proteins, lipids and nuclei acids, which take part in intra-cellular communication. Many studies indicate that EVs contain protective and pro-regenerative properties and can modulate an immune response that is activated in various diseases such as CNS diseases, myocardial infarction, liver injury, lung diseases, ulcerative colitis or kidney injury. Thus, EVs have similar functions as their cells of origin and since they do not carry the risk of cell transplantation, such as tumor formation or small vessel blockage, they can be considered a potential therapeutic tool for cell-free therapy.

Human bone marrow mesenchymal stem cell-derived extracellular vesicles attenuate neuroinflammation evoked by focal brain injury in rats.

BACKGROUND: Ischemic stroke is the major cause of long-term severe disability and death in aged population. Cell death in the infarcted region of the brain induces immune reaction leading to further progression of tissue damage. Immunomodulatory function of mesenchymal stem cells (MSCs) has been shown in multiple preclinical studies; however, it has not been successfully translated to a routine clinical practice due to logistical, economical, regulatory, and intellectual property obstacles. It has been recently demonstrated that therapeutic effect of intravenously administered MSCs can be recapitulated by extracellular vesicles (EVs) derived from them. However, in contrast to MSCs, EVs were not capable to decrease stroke-induced neuroinflammation. Therefore, the aim of the study was to investigate if intra-arterial delivery of MSC-derived EVs will have stronger impact on focal brain injury-induced neuroinflammation, which mimics ischemic stroke, and how it compares to MSCs. METHODS: The studies were performed in adult male Wistar rats with focal brain injury induced by injection of 1 µl of 50 nmol ouabain into the right hemisphere. Two days after brain insult, 5 × 105 human bone marrow MSCs (hBM-MSCs) labeled with Molday ION or 1.3 × 109 EVs stained with PKH26 were intra-arterially injected into the right hemisphere under real-time MRI guidance. At days 1, 3, and 7 post-transplantation, the rats were decapitated, the brains were removed, and the presence of donor cells or EVs was analyzed. The cellular immune response in host brain was evaluated immunohistochemically, and humoral factors were measured by multiplex immunoassay. RESULTS: hBM-MSCs and EVs transplanted intra-arterially were observed in the rat ipsilateral hemisphere, near the ischemic region. Immunohistochemical analysis of brain tissue showed that injection of hBM-MSCs or EVs leads to the decrease of cell activation by ischemic injury, i.e., astrocytes, microglia, and infiltrating leucocytes, including T cytotoxic cells. Furthermore, we observed significant decrease of pro-inflammatory cytokines and chemokines after hBM-MSC or EV infusion comparing with non-treated rats with focal brain injury. CONCLUSIONS: Intra-arterially injected EVs attenuated neuroinflammation evoked by focal brain injury, which mimics ischemic stroke, and this effect was comparable to intra-arterial hBM-MSC transplantation. Thus, intra-arterial injection of EVs might be an attractive therapeutic approach, which obviates MSC-related obstacles.

Neuroinflammation as a target for treatment of stroke using mesenchymal stem cells and extracellular vesicles.

Ischemic stroke is the third cause of death in the developed countries and the main reason of severe disability. Brain ischemia leads to the production of damage-associated molecular patterns (DAMPs) by neurons and glial cells which results in astrocyte and microglia activation, pro-inflammatory cytokines and chemokines production, blood-brain barrier (BBB) disruption, infiltration of leukocytes from the peripheral blood into the infarcted area, and further exacerbation of tissue damage. However, some immune cells such as microglia or monocytes are capable to change their phenotype to anti-inflammatory, produce anti-inflammatory cytokines, and protect injured nervous tissue. In this situation, therapies, which will modulate the immune response after brain ischemia, such as transplantation of mesenchymal stem cells (MSCs) are catching interest. Many experimental studies of ischemic stroke revealed that MSCs are able to modulate immune response and act neuroprotective, through stimulation of neurogenesis, oligodendrogenesis, astrogenesis, and angiogenesis. MSCs may also have an ability to replace injured cells, but the release of paracrine factors directly into the environment or via extracellular vesicles (EVs) seems to play the most pronounced role. EVs are membrane structures containing proteins, lipids, and nucleic acids, and they express similar properties as the cells from which they are derived. However, EVs have lower immunogenicity, do not express the risk of vessel blockage, and have the capacity to cross the blood-brain barrier. Experimental studies of ischemic stroke showed that EVs have immunomodulatory and neuroprotective properties; therefore, they can stimulate neurogenesis and angiogenesis. Up to now, 20 clinical trials with MSC transplantation into patients after stroke were performed, from which two concerned on only hemorrhagic stroke and 13 studied only on ischemic stroke. There is no clinical trial with EV injection into patients after brain ischemia so far, but the case with miR-124-enriched EVs administration is planned and probably there will be more clinical studies with EV transplantation in the near future.

Labeling of human mesenchymal stem cells with different classes of vital stains: robustness and toxicity.

BACKGROUND: Mesenchymal stem cell (MSC) transplantation has been explored as a new clinical approach to repair injured tissues. However, in order to evaluate the therapeutic activity of MSC, cell tracking techniques are required to determine the fate of transplanted cells in both preclinical and clinical studies. In these aspects, different vital stains offer the potential for labeling and monitoring of grafted cells in vivo. It is desirable to have tracking agents which have long-term stability, are not toxic to the cells, and do not affect cell function. METHODS: Here, we selected three different labels: CellTracker™ Green CMFDA, eGFP-mRNA (genetic pre-tag), and Molday ION Rhodamine B™ (nanoparticle-based fluorescent and magnetic label) and performed extensive analysis of their influence on in vitro expansion of human bone marrow-derived mesenchymal stem cells (hBM-MSCs), as well as potential of affecting therapeutic activity and the impact on the durability of staining. RESULTS: Our study showed that basic hBM-MSC characteristics and functions might be affected by labeling. We observed strong alterations of metabolic activity and morphology after eGFP and CellTracker™ Green CMFDA hBM-MSC staining. Molday ION Rhodamine B™ labeling revealed superior properties relatively to other vital stains. The relative expression level of most of the investigated growth factors remained stable after cell labeling, but we have observed some changes in the case of EGF, GDNF, HGF, and IGF gene expression. CONCLUSIONS: Taken together, we suggest performing similar to ours extensive analysis prior to using any cell label to tag MSC in experiments, as it can thoroughly bias results.

Concise Review: Mesenchymal Stem Cells: From Roots to Boost.

It was shown as long as half a century ago that bone marrow is a source of not only hematopoietic stem cells, but also stem cells of mesenchymal tissues. Then the term "mesenchymal stem cells" (MSCs) was coined in the early 1990s, and more than a decade later, the criteria for defining MSCs have been released by the International Society for Cellular Therapy. The easy derivation from a variety of fetal and adult tissues and undemanding cell culture conditions made MSCs an attractive research object. It was followed by the avalanche of reports from preclinical studies on potentially therapeutic properties of MSCs, such as immunomodulation, trophic support and capability for a spontaneous differentiation into connective tissue cells, and differentiation into the majority of cell types upon specific inductive conditions. Although ontogenesis, niche, and heterogeneity of MSCs are still under investigation, there is a rapid boost of attempts at clinical applications of MSCs, especially for a flood of civilization-driven conditions in so quickly aging societies, not only in the developed countries, but also in the populous developing world. The fields of regenerative medicine and oncology are particularly extensively addressed by MSC applications, in part due to the paucity of traditional therapeutic options for these highly demanding and costly conditions. There are currently almost 1,000 clinical trials registered worldwide at ClinicalTrials.gov, and it seems that we are starting to witness the snowball effect with MSCs becoming a powerful global industry; however, the spectacular effects of MSCs in the clinic still need to be shown. Stem Cells 2019;37:855-864.

Single-cell, high-throughput analysis of cell docking to vessel wall.

Therapeutic potential of mesenchymal stem cells (MSCs) has been reported consistently in animal models of stroke, with mechanism mainly through immunomodulation and paracrine activity. Intravenous injection has been a prevailing route for MSCs administration, but cell quantities needed when scaling-up from mouse to human are extremely high putting into question feasibility of that approach. Intra-arterial delivery directly routes the cells to the brain thus lowering the required dose. Cell engineering may additionally improve cell homing, further potentiating the value of intra-arterial route. Therefore, our goal was to create microfluidic platform for screening and fast selection of molecules that enhance the docking of stem cells to vessel wall. We hypothesized that our software will be capable of detecting distinct docking properties of naïve and ITGA4-engineered MSCs. Indeed, the cell flow tracker analysis revealed positive effect of cell engineering on docking frequency of MSCs (42% vs. 9%, engineered vs. control cells, p < 0.001). These observations were then confirmed in an animal model of focal brain injury where cell engineering resulted in improved homing to the brain. To conclude, we developed a platform to study the docking of cells to the vessel wall which is highly relevant for intraarterial cell targeting or studies on neuroinflammation.

Usefulness of high-speed digital imaging (HSDI) in the diagnosis of oedematous - hypertrophic changes of the larynx in people using voice occupationally.

INTRODUCTION: The aim of the study is the evaluation of the usefulness of High-Speed Digital Imaging (HSDI) in the diagnosis of organic dysphonia in a form of oedematous-hypertrophic changes of vocal fold mucosa, morphologically confirmed by Transmission Electron Microscopy (TEM) method in patients working with voice occupationally. MATERIAL AND METHODS: The group consisted of 30 patients working with voice occupationally with oedematous-hypertrophic changes of vocal fold mucosa. Parameters of vocal folds vibrations were evaluated using HSDI technique with a digital HS camera, HRES Endocam Richard Wolf GmbH. The image of vocal folds was recorded with a rate of 4000 frames per second. Postoperative material of the larynx was prepared in a routine way and observed in transmission electron microscope OPTON 900-PC. RESULTS: HSDI technique allows to assess the real vibrations of vocal folds and determine many parameters. The results of TEM in the postoperative material showed destruction of epithelial cells with severe vacuolar degeneration, the enlargement of intercellular spaces and a large number of blood vessels in the stroma, which indicates the presence of oedematous-hypertrophic changes of the larynx. DISCUSSION: The ultrastructural assessment confirm the particular usefulness of HSDI method in the diagnosis of organic dysphonia in a form of oedematous-hypertrophic changes. Key words: High-Speed Digital Imaging, oedematous-hypertrophic changes, vocal fold mucosa, larynx.

Translation, but not transfection limits clinically relevant, exogenous mRNA based induction of alpha-4 integrin expression on human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) represent promising resource of cells for regenerative medicine in neurological disorders. However, efficient and minimally invasive methods of MSCs delivery to the brain still have to be developed. Intra-arterial route is very promising, but MSCs are missing machinery for diapedesis through blood-brain barrier. Thus, here we have tested a mRNA-based method to induce transient expression of ITGA4, an adhesion molecule actively involved in cell extravasation. We observed that transfection with an ITGA4-mRNA construct bearing a conventional cap analogue (7-methylguanosine) failed to produce ITGA4 protein, but exogenous ITGA4-mRNA was detected in transfected MSCs. This indicates that not transfection, but rather translation being the major roadblock. Stabilization of ITGA4-mRNA with SSB proteins resulted in ITGA4 protein synthesis in HEK293 cells only, whereas in MSCs, satisfactory results were obtained only after using an anti-reverse-cap-analogue (ARCA). The presence of ITGA4 protein in MSCs was transient and lasted for up to 24 h after transfection. Membranous location was confirmed by flow cytometry of viable non-permeabilized cells using anti-ITGA4 antibody. The mRNA-based expression of itga4 transgene is potentially sufficient for diapedesis after intra-arterial delivery. To conclude, mRNA-based engineering of stem cells is a rapid and integration-free method and attractive from the perspective of potential future clinical application.

Extracellular Vesicles in Physiology, Pathology, and Therapy of the Immune and Central Nervous System, with Focus on Extracellular Vesicles Derived from Mesenchymal Stem Cells as Therapeutic Tools.

Extracellular vesicles (EVs) are membrane-surrounded structures released by most cell types. They are characterized by a specific set of proteins, lipids and nucleic acids. EVs have been recognized as potent vehicles of intercellular communication to transmit biological signals between cells. In addition, pathophysiological roles of EVs in conditions like cancer, infectious diseases and neurodegenerative disorders are well established. In recent years focus has been shifted on therapeutic use of stem cell derived-EVs. Use of stem cell derived-EVs present distinct advantage over the whole stem cells as EVs do not replicate and after intravenous administration, they are less likely to trap inside the lungs. From the therapeutic perspective, the most promising cellular sources of EVs are mesenchymal stem cells (MSCs), which are easy to obtain and maintain. Therapeutic activity of MSCs has been shown in numerous animal models and the beneficial paracrine effect of MSCs may be mediated by EVs. The various components of MSC derived-EVs such as proteins, lipids, and RNA might play a specific therapeutic role. In this review, we characterize the role of EVs in immune and central nervous system (CNS); present evidences for defective signaling of these vesicles in neurodegeneration and therapeutic role of EVs in CNS.

Pre- and postmortem imaging of transplanted cells.

Therapeutic interventions based on the transplantation of stem and progenitor cells have garnered increasing interest. This interest is fueled by successful preclinical studies for indications in many diseases, including the cardiovascular, central nervous, and musculoskeletal system. Further progress in this field is contingent upon access to techniques that facilitate an unambiguous identification and characterization of grafted cells. Such methods are invaluable for optimization of cell delivery, improvement of cell survival, and assessment of the functional integration of grafted cells. Following is a focused overview of the currently available cell detection and tracking methodologies that covers the entire spectrum from pre- to postmortem cell identification.

The cerebral embolism evoked by intra-arterial delivery of allogeneic bone marrow mesenchymal stem cells in rats is related to cell dose and infusion velocity.

INTRODUCTION: Intra-arterial cell infusion is an efficient delivery route with which to target organs such as the ischemic brain. However, adverse events including microembolisms and decreased cerebral blood flow were recently reported after intra-arterial cell delivery in rodent models, raising safety concerns. We tested the hypothesis that cell dose, infusion volume, and velocity would be related to the severity of complications after intra-arterial cell delivery. METHODS: In this study, 38 rats were subjected to a sham middle cerebral artery occlusion (sham-MCAO) procedure before being infused with allogeneic bone-marrow mesenchymal stem cells at different cell doses (0 to 1.0 × 10(6)), infusion volumes (0.5 to 1.0 ml), and infusion times (3 to 6 minutes). An additional group (n = 4) was infused with 1.0 × 10(6) cells labeled with iron oxide for in vivo tracking of cells. Cells were infused through the external carotid artery under laser Doppler flowmetry monitoring 48 hours after sham-MCAO. Magnetic resonance imaging (MRI) was performed 24 hours after cell infusion to reveal cerebral embolisms or hemorrhage. Limb placing, cylinder, and open field tests were conducted to assess sensorimotor functions before the rats were perfused for histology. RESULTS: A cell dose-related reduction in cerebral blood flow was noted, as well as an increase in embolic events and concomitant lesion size, and sensorimotor impairment. In addition, a low infusion velocity (0.5 ml/6 minutes) was associated with high rate of complications. Lesions on MRI were confirmed with histology and corresponded to necrotic cell loss and blood-brain barrier leakage. CONCLUSIONS: Particularly cell dose but also infusion velocity contribute to complications encountered after intra-arterial cell transplantation. This should be considered before planning efficacy studies in rats and, potentially, in patients with stroke.