Emerging Roles of Exosomes in Stroke Therapy.

Stroke is the number one cause of morbidity in the United States and number two cause of death worldwide. There is a critical unmet medical need for more effective treatments of ischemic stroke, and this need is increasing with the shift in demographics to an older population. Recently, several studies have reported the therapeutic potential of stem cell-derived exosomes as new candidates for cell-free treatment in stoke. This review focuses on the use of stem cell-derived exosomes as a potential treatment tool for stroke patients. Therapy using exosomes can have a clear clinical advantage over stem cell transplantation in terms of safety, cost, and convenience, as well as reducing bench-to-bed latency due to fewer regulatory milestones. In this review article, we focus on (1) the therapeutic potential of exosomes in stroke treatment, (2) the optimization process of upstream and downstream production, and (3) preclinical application in a stroke animal model. Finally, we discuss the limitations and challenges faced by exosome therapy in future clinical applications.

Donor-recipient race-ethnicity concordance and patient survival after liver transplantation.

INTRODUCTION: We assessed the association between patient survival after liver transplantation (LT) and donor-recipient race-ethnicity (R/E) concordance. METHODS: The Scientific Registry of Transplant Recipients (SRTR) was retrospectively analyzed using data collected between 2002 and 2019. Only adults without history of prior organ transplant and recipients of LT alone were included. The primary outcome was patient survival. Donors and recipients were categorized into five R/E groups: White/Caucasian, African American/Black, Hispanic/Latino, Asian, and Others. Statistical analyses were performed using Kaplan-Meier survival curves and Cox Proportional Hazards models, adjusting for donor and recipient covariates. RESULTS: 85,427 patients were included. Among all the R/E groups, Asian patients had the highest 5-year survival (81.3%; 95% CI = 79.9-82.7), while African American/Black patients had the lowest (71.4%; 95% CI = 70.3-72.6) (P < 0.001). Lower survival rates were observed in recipients who received discordant R/E grafts irrespective of their R/E group. The fully adjusted hazard ratio for death was statistically significant in African American/Black (aHR 1.07-1.18-1.31; P < 0.01) and in White∕Caucasian patients (aHR 1.00-1.04-1.07; P = 0.03) in the presence of donor-recipient R/E discordance. CONCLUSION: Disparities in post-LT outcomes might be influenced by biological factors in addition to well-known social determinants of health.

A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes.

Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.

Concise Review: Human-Animal Neurological Chimeras: Humanized Animals or Human Cells in an Animal?

Blastocyst complementation is an emerging methodology in which human stem cells are transferred into genetically engineered preimplantation animal embryos eventually giving rise to fully developed human tissues and organs within the animal host for use in regenerative medicine. The ethical issues surrounding this method have caused the National Institutes of Health to issue a moratorium on funding for blastocyst complementation citing the potential for human cells to substantially contribute to the brain of the chimeric animal. To address this concern, we performed an in-depth review of the neural transplantation literature to determine how the integration of human cells into the nonhuman neural circuitry has altered the behavior of the host. Despite reports of widespread integration of human cell transplants, our review of 150 transplantation studies found no evidence suggestive of humanization of the animal host, and we thus conclude that, at present, concerns over humanization should not prevent research on blastocyst complementation to continue. We suggest proceeding in a controlled and transparent manner, however, and include recommendations for future research with careful consideration for how human cells may contribute to the animal host nervous system. Stem Cells 2019;37:444-452.

Transplants of adult mesenchymal and neural stem cells provide neuroprotection and behavioral sparing in a transgenic rat model of Huntington's disease.

Stem cells have gained significant interest as a potential treatment of neurodegenerative diseases, including Huntington's disease (HD). One source of these cells is adult neural stem cells (aNSCs), which differentiate easily into neuronal lineages. However, these cells are vulnerable to immune responses following transplantation. Another source is bone-marrow-derived mesenchymal stem cells (MSCs), which release neurotrophic factors and anti-inflammatory cytokines following transplantation, and are less vulnerable to rejection. The goal of this study was to compare the efficacy of transplants of MSCs, aNSCs, or cotransplants of MSCs and aNSCs for reducing deficits in a transgenic rat model of HD. HD rats received intrastriatal transplantations of 400,000 MSCs, aNSCs, or a combination of MSCs/aNSCs, while wild-type and HD controls were given vehicle. Rats were tested on the rotarod over the course of 20 weeks. The results indicated that transplants of: (a) aNSCs produced a strong immune response and conferred short-term behavioral benefits; (b) MSCs elicited a relatively weak immune response, and provided a longer term behavioral benefit; and (c) combined MSCs and aNSCs conferred long-term behavioral benefits and increased survival of the transplanted aNSCs. The finding that cotransplanting MSCs with aNSCs can prolong aNSC survival and provide greater behavioral sparing than when the transplants contains only aNSCs suggests that MSCs are capable of creating a more suitable microenvironment for aNSC survival. This cotransplantation strategy may be useful as a future therapeutic option for treating HD, especially if long-term survival of differentiated cells proves to be critically important for preserving lasting functional outcomes.

Safety of Patients with Renal Artery Aneurysm to Proceed with Living Donation.

BACKGROUND: Renal artery aneurysm (RAA) is a rare condition that involves dilation of all layers of the arterial wall of the renal artery. The risk of rupture is rare, but intervention is recommended for larger aneurysms. Surgical decision-making regarding live donor renal transplantation (LDRT) centers around safety for the living donor, and laterality of the donated kidney is based on providing the donor with the best longevity pertaining to the remaining kidney. We looked to review our long-term outcomes surrounding live donor transplants from donors with RAA with ex vivo resection and reconstruction prior to implantation. METHODS: A retrospective review was done of all laparoscopic live donor transplant nephrectomies with ex vivo aneurysm resection, reconstruction, and implantation at a single center. RESULTS: Three pairs of patients underwent successful laparoscopic donor nephrectomy, RAA resection, reconstruction, and transplantation of kidney. 2 males and 1 female ages 47 to 58 years of age underwent transplantation. The donors at 5 years of follow-up were noted to be functioning appropriately with no long-term sequelae of their donation and a mean remanent kidney function of 63 mL/min. DISCUSSION: For potential live donors with asymptomatic, unilateral renal artery aneurysm and no systemic disease, live donation with ex vivo resection and reconstruction can be performed with excellent long-term donor and recipient outcomes.

Survival after live donor versus deceased donor liver transplantation: propensity score-matched study.

BACKGROUND: For individuals with advanced liver disease, equipoise in outcomes between live donor liver transplant (LDLT) and deceased donor liver transplant (DDLT) is uncertain. METHODS: A retrospective cohort study was performed using data extracted from the Scientific Registry of Transplant Recipients. Adults who underwent first-time DDLT or LTDL in the United States between 2002 and 2020 were paired using propensity-score matching with 1:10 ratio without replacement. Patient and graft survival were compared using the model for end-stage liver disease (MELD) score for stratification. RESULTS: After propensity-score matching, 31 522 DDLT and 3854 LDLT recipients were included. For recipients with MELD scores ≤15, LDLT was associated with superior patient survival (HR = 0.92; 95% c.i. 0.76 to 0.96; P = 0.013). No significant differences in patient survival were observed for MELD scores between 16 and 30. Conversely, for patients with MELD scores >30, LDLT was associated with higher mortality (HR 2.57; 95% c.i. 1.35 to 4.62; P = 0.003). Graft survival was comparable between the two groups for MELD ≤15 and for MELD between 21 and 30. However, for MELD between 16 and 20 (HR = 1.15; 95% c.i. 1.00 to 1.33; P = 0.04) and MELD > 30 (HR = 2.85; 95% c.i. 1.65 to 4.91; P = 0.001), graft survival was considerably shorter after LDLT. Regardless of MELD scores, re-transplantation rate within the first year was significantly higher after LDLT. CONCLUSIONS: In this large propensity score-matched study using national data, comparable patient survival was found between LDLT and DDLT in recipients with MELD scores between 16 and 30. Conversely, for patients with MELD > 30, LDLT was associated with worse outcomes. These findings underscore the importance of transplant selection for patients with high MELD scores.

Does Frequency of Baseline Testing Influence Concussion Diagnostic Decision Making Among College Athletes.

OBJECTIVE: Concussion is a growing public health concern given the large number of youth and collegiate athletes participating in collision sports. Sport-related concussions can have an adverse impact on student-athletes' health and academic performance. Athletic programs within academic organizations are motivated to employ the most effective and efficient diagnostic and recovery procedures to minimize the duration and impact of these symptoms on student-athletes' functioning. The present study sought to further our understanding regarding the value and frequency of conducting baseline assessments when evaluating sport-related concussions. METHOD: A total of 41 athletes (24 men, 17 women) between the ages of 18 and 22 were evaluated following suspected concussive injury between 2015 and 2018. Post-injury test results were compared to baselines that had been collected either 1 or 2 years prior, and to normative data, to determine consistency in diagnostic outcomes. RESULTS: Baseline test/retest reliability using Pearson's bivariate correlations revealed modest correlations on measures of verbal and visual memory (0.437-0.569) and very strong correlations on measures of reaction time and visual-motor speed (0.811-0.821). Meanwhile, minimal if any differences in clinical decision-making regarding the diagnostic outcome was observed when comparing post-injury test results to different baselines and to normative data. CONCLUSIONS: Findings indicate that yearly baseline testing may not improve diagnostic accuracy, and in many cases, normative data may be adequate for decision-making. Additional research should evaluate the potential benefit of baseline testing in return-to-play decision-making among broader athletic populations.

Dependence induced increases in intragastric alcohol consumption in mice.

Three experiments used the intragastric alcohol consumption (IGAC) procedure to examine the effects of variations in passive ethanol exposure on withdrawal and voluntary ethanol intake in two inbred mouse strains, C57BL/6J (B6) and DBA/2J (D2). Experimental treatments were selected to induce quantitative differences in ethanol dependence and withdrawal severity by: (1) varying the periodicity of passive ethanol exposure (three, six or nine infusions/day); (2) varying the dose per infusion (low, medium or high); and (3) varying the duration of passive exposure (3, 5 or 10 days). All experiments included control groups passively exposed to water. B6 mice generally self-infused more ethanol than D2 mice, but passive ethanol exposure increased IGAC in both strains, with D2 mice showing larger relative increases during the first few days of ethanol access. Bout data supported the characterization of B6 mice as sippers and D2 mice as gulpers. Three larger infusions per day produced a stronger effect on IGAC than six or nine smaller infusions, especially in D2 mice. Increased IGAC was strongly predicted by cumulative ethanol dose and intoxication during passive exposure in both strains. Withdrawal during the passive exposure phase was also a strong predictor of increased IGAC in D2 mice. However, B6 mice showed little withdrawal, precluding analysis of its potential role. Overall, these data support the hypothesis that dependence-induced increases in IGAC are jointly determined by two processes that might vary across genotypes: (1) tolerance to aversive postabsorptive ethanol effects and (2) negative reinforcement (i.e. alleviation of withdrawal by self-administered ethanol).

Confronting the Business Models of Modern Slavery.

Despite growing attention from companies and regulators looking to eradicate modern slavery, we know little about how slavery works from a business perspective. We address this gap by empirically examining innovations in the business models of modern slavery, focusing on how the business models of slavery in advanced economies have evolved since slavery was legally abolished. While continuities exist, novel business models have emerged based on new actors, activities, and linkages. We categorize these as four innovative models per actors involved (producer/intermediary) and how value is created and captured (revenue generation/cost reduction), and discuss implications for research, policy, and practice.

The general ability measure for adults underrepresents true IQ in a high-functioning aviation population.

The General Ability Measure for Adults (GAMA) is a brief nonverbal assessment of general intellectual ability. Presently, there is a lack of research involving the use of the GAMA in a high-functioning population. Aviation pilots (n = 59) were referred for a neuropsychological evaluation and were concluded to be cognitively intact. They were administered a battery that included the GAMA and Wechsler Adult Intelligence Scale - 4th edition (WAIS-IV). A paired samples t-test revealed that IQ scores on the GAMA were significantly lower than IQ scores on the WAIS-IV, with WAIS-IV IQ being 7.54 points higher on average. Fisher's exact test determined there was significant disagreement in score classification between the tests, with individuals classified as Above Average on the WAIS-IV often being classified as Average on the GAMA. The GAMA appears to significantly underrepresent true IQ in both score and classification when compared to a more robust measure of intellectual assessment. Clinically, this suggests that IQ screeners, such as the GAMA, may not be appropriate for assessing patients who are suspected to be of higher premorbid functioning. Future research should expand on these results to assess the validity of verbal-based IQ screening measures in high-functioning populations.

ssDNA nanotubes for selective targeting of glioblastoma and delivery of doxorubicin for enhanced survival.

Effective treatment of glioblastoma remains a daunting challenge. One of the major hurdles in the development of therapeutics is their inability to cross the blood-brain tumor barrier (BBTB). Local delivery is an alternative approach that can still suffer from toxicity in the absence of target selectivity. Here, we show that nanotubes formed from self-assembly of ssDNA-amphiphiles are stable in serum and nucleases. After bilateral brain injections, nanotubes show preferential retention by tumors compared to normal brain and are taken up by glioblastoma cells through scavenger receptor binding and macropinocytosis. After intravenous injection, they cross the BBTB and internalize in glioblastoma cells. In a minimal residual disease model, local delivery of doxorubicin showed signs of toxicity in the spleen and liver. In contrast, delivery of doxorubicin by the nanotubes resulted in no systemic toxicity and enhanced mouse survival. Our results demonstrate that ssDNA nanotubes are a promising drug delivery vehicle to glioblastoma.

Change in Balance Performance Predicts Neurocognitive Dysfunction and Symptom Endorsement in Concussed College Athletes.

OBJECTIVE: Following concussion, there is an array of sequelae including symptom burden, neurocognitive dysfunction, and balance impairment. However, the magnitude of change in balance performance has yet to be explored fully regarding its relationship with neurocognitive functioning or symptom endorsement. The present study hypothesized that the magnitude of change in balance performance from baseline to post-trauma would have unique predictive power in identifying acute clinical outcomes. METHOD: Sixty-eight college athletes completed annual preparticipation baseline testing and were later diagnosed with a concussion. RESULTS: Linear regressions determined that the magnitude of change in balance performance was a better predictor of neurocognitive dysfunction and endorsement of "balance problems" than post-trauma balance performance alone. CONCLUSIONS: These findings highlight the importance of incorporating balance measurements during preparticipation baseline assessment.

Hepatic Differentiation of Marmoset Embryonic Stem Cells and Functional Characterization of ESC-Derived Hepatocyte-Like Cells.

BACKGROUND: Primary human hepatocytes (PHHs) are the ideal candidates for studying critical liver functions such as drug metabolism and toxicity. However, as they are isolated from discarded livers that are unsuitable for transplantation, they possess limited expansion ability in vitro and their enzymatic functions deteriorate rapidly because they are often of poor quality. Therefore, there is a compelling reason to find reliable alternative sources of hepatocytes. METHODS: In this study, we report on efficient and robust differentiation of embryonic stem cells (ESC) from the common marmoset Callithrix jacchus into functional hepatocyte-like cells (HLC) using a simple, and reproducible three-step procedure. ESC-derived HLCs were examined by morphological analysis and tested for their expression of hepatocyte-specific markers using a combination of immunohistochemistry, RT-PCR, and biochemical assays. Primary human hepatocytes were used as controls. RESULTS: ESC-derived HLCs expressed each of the hepatocyte-specific markers tested, including albumin; α-fetoprotein; asialoglycoprotein receptor 1; α-1 antitrypsin; hepatocyte nuclear factors 1α and 4; cytokeratin 18; hepatocyte growth factor receptor; transferrin; tyrosine aminotransferase; alkaline phosphatase; c-reactive protein; cytochrome P450 enzymes CYP1A2, CYP2E1 and CYP3A4; and coagulation factors FVII and FIX. They were functionally competent as demonstrated by biochemical assays in addition to producing urea. CONCLUSION: Our data strongly suggest that marmoset HLCs possess characteristics similar to those of PHHs. They could, therefore, be invaluable for studies on drug metabolism and cell transplantation therapy for a variety of liver disorders. Because of the similarities in the anatomical and physiological features of the common marmoset to that of humans, Callithrix jacchus is an appropriate animal model to study human disease conditions and cellular functions.

The American Public Is Ready to Accept Human-Animal Chimera Research.

We report findings from a new survey of US public attitudes toward human-animal chimeric embryo (HACE) research, designed to compare with recently reported Japanese survey data. We find that 59% of the US public can personally accept the process of injecting human induced pluripotent stem cells into genetically modified swine embryos and having human tissues produced in a pig's body transplanted into a human. This is greater acceptance than in Japan, and there is even strong acceptance among those with strong religious affiliations and who self-identify as conservatives. We argue that strong public support for HACE research, as well as the emerging literature suggesting that humanization of research animals is very unlikely, should compel the NIH to lift its current moratorium on HACE research.

Zika virus-based immunotherapy enhances long-term survival of rodents with brain tumors through upregulation of memory T-cells.

Zika virus (ZIKV) exhibits a tropism for brain tumor cells and has been used as an oncolytic virus to target brain tumors in mice with modest effects on extending median survival. Recent studies have highlighted the potential for combining virotherapy and immunotherapy to target cancer. We postulated that ZIKV could be used as an adjuvant to enhance the long-term survival of mice with malignant glioblastoma and generate memory T-cells capable of providing long-term immunity against cancer remission. To test this hypothesis mice bearing malignant intracranial GL261 tumors were subcutaneously vaccinated with irradiated GL261 cells previously infected with the ZIKV. Mice also received intracranial injections of live ZIKV, irradiation attenuated ZIKV, or irradiated GL261 cells previously infected with ZIKV. Long-term survivors were rechallenged with a second intracranial tumor to examine their immune response and look for the establishment of protective memory T-cells. Mice with subcutaneous vaccination plus intracranial irradiation attenuated ZIKV or intracranial irradiated GL261 cells previously infected with ZIKV exhibited the greatest extensions to overall survival. Flow cytometry analysis of immune cells within the brains of long-term surviving mice after tumor rechallenge revealed an increase in the number of T-cells, including CD4+ and tissue-resident effector/ effector memory CD4+ T-cells, in comparison to long-term survivors that were mock-rechallenged, and in comparison to naïve untreated mice challenged with intracranial gliomas. These results suggest that ZIKV can serve as an adjuvant to subcutaneous tumor vaccines that enhance long-term survival and generate protective tissue-resident memory CD4+ T-cells.

Efficacy of stem cell-based therapies for stroke.

Stroke remains a prevalent disease with limited treatment options. Available treatments offer little in the way of enhancing neurogenesis and recovery. Because of the limitations of available treatments, new therapies for stroke are needed. Stem cell-based therapies for stroke offer promise because of their potential to provide neurorestorative benefits. Stem cell-based therapies aim to promote neurogenesis and replacement of lost neurons or protect surviving neurons in order to improve neurological recovery. The mechanism through which stem cell treatments mediate their therapeutic effect is largely dependent on the type of stem cell and route of administration. Neural stem cells have been shown in pre-clinical and clinical trials to promote functional recovery when used in intracerebral transplantations. The therapeutic effects of neural stem cells have been attributed to their formation of new neurons and promotion of neuroregeneration. Bone marrow stem cells (BMSC) and mesenchymal stem cells (MSC) have been shown to enhance neurogenesis in pre-clinical models in intracerebral transplantations, but lack clinical evidence to support this therapeutic approach in patients and appear to be less effective than neural stem cells. Intravenous and intra-arterial administration of BMSC and MSC have shown more promise, where their effects are largely mediated through neuroprotective mechanisms. The immune system has been implicated in exacerbating initial damage caused by stroke, and BMSC and MSC have demonstrated immunomodulatory properties capable of dampening post-stroke inflammation and potentially improving recovery. While still in development, stem cell therapies may yield new treatments for stroke which can improve neurological recovery.

Efficacy of Cell-Based Therapies for Traumatic Brain Injuries.

Traumatic brain injuries (TBIs) are a leading cause of death and disability. Additionally, growing evidence suggests a link between TBI-induced neuroinflammation and neurodegenerative disorders. Treatments for TBI patients are limited, largely focused on rehabilitation therapy, and ultimately, fail to provide long-term neuroprotective or neurorestorative benefits. Because of the prevalence of TBI and lack of viable treatments, new therapies are needed which can promote neurological recovery. Cell-based treatments are a promising avenue because of their potential to provide multiple therapeutic benefits. Cell-based therapies can promote neuroprotection via modulation of inflammation and promote neurorestoration via induction of angiogenesis and neurogenesis. Neural stem/progenitor cell transplantations have been investigated in preclinical TBI models for their ability to directly contribute to neuroregeneration, form neural-like cells, and improve recovery. Mesenchymal stem cells (MSCs) have been investigated in clinical trials through multiple different routes of administration. Intravenous administration of MSCs appears most promising, demonstrating a robust safety profile, correlation with neurological improvements, and reductions in systemic inflammation following TBI. While still preliminary, evidence suggests cell-based therapies may become a viable treatment for TBI based on their ability to promote neuroregeneration and reduce inflammation.

Immunomodulation with Human Umbilical Cord Blood Stem Cells Ameliorates Ischemic Brain Injury - A Brain Transcriptome Profiling Analysis.

Our group previously demonstrated that administration of a CD34-negative fraction of human non- hematopoietic umbilical cord blood stem cells (UCBSC) 48 h after ischemic injury could reduce infarct volume by 50% as well as significantly ameliorate neurological deficits. In the present study, we explored possible mechanisms of action using next generation RNA sequencing to analyze the brain transcriptome profiles in rats with ischemic brain injury following UCBSC therapy. Two days after ischemic injury, rats were treated with UCBSC. Five days after administration, total brain mRNA was then extracted for RNAseq analysis using Illumina Hiseq 2000. We found 275 genes that were significantly differentially expressed after ischemic injury compared with control brains. Following UCBSC treatment, 220 of the 275 differentially expressed genes returned to normal levels. Detailed analysis of these altered transcripts revealed that the vast majority were associated with activation of the immune system following cerebral ischemia which were normalized following UCBSC therapy. Major alterations in gene expression profiles after ischemia include blood-brain-barrier breakdown, cytokine production, and immune cell infiltration. These results suggest that UCBSC protect the brain following ischemic injury by down regulating the aberrant activation of innate and adaptive immune responses.