Mesenchymal Stem Cell-Extracellular Vesicle Therapy for Stroke: Scalable Production and Imaging Biomarker Studies.

A major clinical hurdle to translate MSC-derived extracellular vesicles (EVs) is the lack of a method to scale-up the production of EVs with customized therapeutic properties. In this study, we tested whether EV production by a scalable 3D-bioprocessing method is feasible and improves neuroplasticity in animal models of stroke using MRI study. MSCs were cultured in a 3D-spheroid using a micro-patterned well. The EVs were isolated with filter and tangential flow filtration and characterized using electron microscopy, nanoparticle tracking analysis, and small RNA sequencing. Compared to conventional 2D culture, the production-reproduction of EVs (the number/size of particles and EV purity) obtained from 3D platform were more consistent among different lots from the same donor and among different donors. Several microRNAs with molecular functions associated with neurogenesis were upregulated in EVs obtained from 3D platform. EVs induced both neurogenesis and neuritogenesis via microRNAs (especially, miR-27a-3p and miR-132-3p)-mediated actions. EV therapy improved functional recovery on behavioral tests and reduced infarct volume on MRI in stroke models. The dose of MSC-EVs of 1/30 cell dose had similar therapeutic effects. In addition, the EV group had better anatomical and functional connectivity on diffusion tensor imaging and resting-state functional MRI in a mouse stroke model. This study shows that clinical-scale MSC-EV therapeutics are feasible, cost-effective, and improve functional recovery following experimental stroke, with a likely contribution from enhanced neurogenesis and neuroplasticity.

Development and application of rTMS device to murine model.

Repetitive transcranial magnetic stimulation (rTMS) is attracting attention as a new treatment technique for brain lesions, and many animal studies showing its effects have been reported. However, the findings of animal application researches cannot directly represent the effects of rTMS in human, mainly due to size difference and mechanistic characteristics of rTMS. Therefore, the authors purposed to develop a mouse rTMS to simulate clinical application and to confirm. Firstly, a virtual head model was created according to magnetic resonance images of murine head. Then, simulations of rTMS stimulation with different coils were performed on the murine head phantom, and an rTMS device for mice was fabricated based on the optimal voltage conditions. Lastly, strengths of magnetic fields generated by the two rTMS devices, for human (conventional clinical use) and mouse (newly fabricated), were measured in air and on mouse head and compared. Resultantly, the magnetic field intensity generated by coil of mouse was lower than human's (p < 0.01), and no differences were found between the predicted simulation values and the measured intensity in vivo (p > 0.05). Further in vivo researches using miniaturized rTMS devices for murine head should be followed to be more meaningful for human.

Brain morphological and connectivity changes on MRI after stem cell therapy in a rat stroke model.

In animal models of stroke, behavioral assessments could be complemented by a variety of neuroimaging studies to correlate them with recovery and better understand mechanisms of improvement after stem cell therapy. We evaluated morphological and connectivity changes after treatment with human mesenchymal stem cells (hMSCs) in a rat stroke model, through quantitative measurement of T2-weighted images and diffusion tensor imaging (DTI). Transient middle cerebral artery occlusion rats randomly received PBS (PBS-only), FBS cultured hMSCs (FBS-hMSCs), or stroke patients' serum cultured hMSCs (SS-hMSCs). Functional improvement was assessed using a modified neurological severity score (mNSS). Quantitative analyses of T2-weighted ischemic lesion and ventricular volume changes were performed. Brain microstructure/connectivity changes were evaluated in the ischemic recovery area by DTI-derived microstructural indices such as relative fractional anisotropy (rFA), relative axial diffusivity (rAD), and relative radial diffusivity (rRD), and relative fiber density (rFD) analyses. According to mNSS results, the SS-hMSCs group showed the most prominent functional improvement. Infarct lesion volume of the SS-hMSCs group was significantly decreased at 2 weeks when compared to the PBS-only groups, but there were no differences between the FBS-hMSCs and SS-hMSCs groups. Brain atrophy was significantly decreased in the SS-hMSCs group compared to the other groups. In DTI, rFA and rFD values were significantly higher and rRD value was significant lower in the SS-hMSCs group and these microstructure/connectivity changes were correlated with T2-weighted morphological changes. T2-weighted volume alterations (ischemic lesion and brain atrophy), and DTI microstructural indices and rFD changes, were well matched with the results of behavioral assessment. These quantitative MRI measurements could be potential outcome predictors of functional recovery after treatment with stem cells for stroke.

An equal-TE ultrafast 3D gradient-echo imaging method with high tolerance to magnetic susceptibility artifacts: Application to BOLD functional MRI.

PURPOSE: To develop an ultrafast 3D gradient echo-based MRI method with constant TE and high tolerance to B0 inhomogeneity, dubbed ERASE (equal-TE rapid acquisition with sequential excitation), and to introduce its use in BOLD functional MRI (fMRI). THEORY AND METHODS: Essential features of ERASE, including spin behavior, were characterized, and a comparison study was conducted with conventional EPI. To demonstrate high tolerance to B0 inhomogeneity, in vivo imaging of the mouse brain with a fiber-optic implant was performed at 9.4 T, and human brain imaging (including the orbitofrontal cortex) was performed at 3 T and 7 T. To evaluate the performance of ERASE in BOLD-fMRI, the characteristics of SNR and temporal SNR were analyzed for in vivo rat brains at 9.4 T in comparison with multislice gradient-echo EPI. Percent signal changes and t-scores are also presented. RESULTS: For both mouse brain and human brain imaging, ERASE exhibited a high tolerance to magnetic susceptibility artifacts, showing much lower distortion and signal dropout, especially in the regions involving large magnetic susceptibility effects. For BOLD-fMRI, ERASE provided higher temporal SNR and t-scores than EPI, but exhibited similar percent signal changes in in vivo rat brains at 9.4 T. CONCLUSION: When compared with conventional EPI, ERASE is much less sensitive, not only to EPI-related artifacts such as Nyquist ghosting, but also to B0 inhomogeneity including magnetic susceptibility effects. It is promising for use in BOLD-fMRI, providing higher temporal SNR and t-scores with constant TE when compared with EPI, although further optimization is needed for human fMRI.

Exploring the Potential of Mesenchymal Stem Cell-Based Therapy in Mouse Models of Vascular Cognitive Impairment.

Closely linked to Alzheimer's disease (AD), the pathological spectrum of vascular cognitive impairment (VCI) is known to be wide and complex. Considering that multiple instead of a single targeting approach is considered a treatment option for such complicated diseases, the multifaceted aspects of mesenchymal stem cells (MSCs) make them a suitable candidate to tackle the heterogeneity of VCI. MSCs were delivered via the intracerebroventricular (ICV) route in mice that were subjected to VCI by carotid artery stenosis. VCI was induced in C57BL6/J mice wild type (C57VCI) mice by applying a combination of ameroid constrictors and microcoils, while ameroid constrictors alone were bilaterally applied to 5xFAD (transgenic AD mouse model) mice (5xVCI). Compared to the controls (minimal essential medium (MEM)-injected C57VCI mice), changes in spatial working memory were not noted in the MSC-injected C57VCI mice, and unexpectedly, the mortality rate was higher. In contrast, compared to the MEM-injected 5xVCI mice, mortality was not observed, and the spatial working memory was also improved in MSC-injected 5xVCI mice. Disease progression of the VCI-induced mice seems to be affected by the method of carotid artery stenosis and due to this heterogeneity, various factors must be considered to maximize the therapeutic benefits exerted by MSCs. Factors, such as the optimal MSC injection time point, cell concentration, sacrifice time point, and immunogenicity of the transplanted cells, must all be adequately addressed so that MSCs can be appropriately and effectively used as a treatment option for VCI.

Heterogeneous Disease Progression in a Mouse Model of Vascular Cognitive Impairment.

Recently, an asymmetric vascular compromise approach that replicates many aspects of human vascular cognitive impairment (VCI) has been reported. The present study aimed to first investigate on the reproducibility in the disease progression of this newly reported VCI model using wild-type C57BL6/J mice. The second aim was to assess how this approach will affect the disease progression of transgenic Alzheimer's disease (AD) 5XFAD mice subjected to VCI. C57BL6/J and 5XFAD mice were subjected to VCI by placing an ameroid constrictor on the right CCA and a microcoil on the left CCA. Infarcts and hippocampal neuronal loss did not appear predominantly in the right (ameroid side) as expected but randomly in both hemispheres. The mortality rate of C57BL6/J mice was unexpectedly high. Inducing VCI reduced amyloid burden in the hippocampi of 5XFAD mice. Since VCI is known to be complex and complicated, the heterogeneous disease progression observed from this current study shares close resemblance to the clinical manifestation of VCI. This heterogeneity, however, makes it challenging to test novel treatment options using this model. Further study is warranted to tackle the heterogeneous nature of VCI.

Application of Mesenchymal Stem Cell-Derived Extracellular Vesicles for Stroke: Biodistribution and MicroRNA Study.

Mesenchymal stem cells (MSCs) exert their therapeutic capability through a variety of bioactive substances, including trophic factors, microRNAs, and extracellular vesicles (EVs) in infarcted tissues. We therefore hypothesized that MSC-derived EVs (MSC-EVs) possess therapeutic molecules similar to MSCs. Moreover, given their nature as nanosized and lipid-shielded particles, the intravenous infusion of MSC-EVs would be advantageous over MSCs as a safer therapeutic approach. In this study, we investigated the biodistribution, therapeutic efficacy, and mode of action of MSC-EVs in a rat stroke model. MSC-EVs successfully stimulated neurogenesis and angiogenesis in vivo. When compared to the MSC-treated group, rats treated with MSC-EVs exhibited greater behavioral improvements than the control group (p < 0.05). Our biodistribution study using fluorescence-labeled MSC-EVs and MSCs demonstrated that the amounts of MSC-EVs in the infarcted hemisphere increased in a dose-dependent manner, and were rarely found in the lung and liver. In addition, MSC-EVs were highly inclusive of various proteins and microRNAs (miRNAs) associated with neurogenesis and/or angiogenesis compared to fibro-EVs. We further analyzed those miRNAs and found that miRNA-184 and miRNA-210 were essential for promoting neurogenesis and angiogenesis of MSC-EVs, respectively. MSC-EVs represent an ideal alternative to MSCs for stroke treatment, with similar medicinal capacity but an improved safety profile that overcomes cell-associated limitations in stem cell therapy.

Gradient-echo and spin-echo blood oxygenation level-dependent functional MRI at ultrahigh fields of 9.4 and 15.2 Tesla.

PURPOSE: Sensitivity and specificity of blood oxygenation level-dependent (BOLD) functional MRI (fMRI) is sensitive to magnetic field strength and acquisition methods. We have investigated gradient-echo (GE)- and spin-echo (SE)-BOLD fMRI at ultrahigh fields of 9.4 and 15.2  Tesla. METHODS: BOLD fMRI experiments responding to forepaw stimulation were performed with 3 echo times (TE) at each echo type and B0 in α-chloralose-anesthetized rats. The contralateral forelimb somatosensory region was selected for quantitative analyses. RESULTS: At 9.4 T and 15.2 T, average baseline T2* (n = 9) was 26.6 and 17.1 msec, whereas baseline T2 value (n = 9) was 35.7 and 24.5 msec, respectively. Averaged stimulation-induced ΔR2* was -1.72 s-1 at 9.4 T and -3.09 s-1 at 15.2 T, whereas ΔR2 was -1.19 s-1 at 9.4 T and -1.97 s-1 at 15.2 T. At the optimal TE of tissue T2* or T2 , BOLD percent changes were slightly higher at 15.2 T than at 9.4 T (GE: 7.4% versus 6.4% and SE: 5.7% versus 5.4%). The ΔR2* and ΔR2 ratio of 15.2 T to 9.4 T was 1.8 and 1.66, respectively. The ratio of the macrovessel-containing superficial to microvessel-dominant parenchymal BOLD signal was 1.73 to 1.76 for GE-BOLD versus 1.13 to 1.19 for SE-BOLD, indicating that the SE-BOLD contrast is less sensitive to macrovessels than GE-BOLD. CONCLUSION: SE-BOLD fMRI improves spatial specificity to microvessels compared to GE-BOLD at both fields. BOLD sensitivity is similar at the both fields and can be improved at ultrahigh fields only for thermal-noise-dominant ultrahigh-resolution fMRI.

Multimodal MRI-Based Triage for Acute Stroke Therapy: Challenges and Progress.

Revascularization therapies have been established as the treatment mainstay for acute ischemic stroke. However, a substantial number of patients are either ineligible for revascularization therapy, or the treatment fails or is futile. At present, non-contrast computed tomography is the first-line neuroimaging modality for patients with acute stroke. The use of magnetic resonance imaging (MRI) to predict the response to early revascularization therapy and to identify patients for delayed treatment is desirable. MRI could provide information on stroke pathophysiologies, including the ischemic core, perfusion, collaterals, clot, and blood-brain barrier status. During the past 20 years, there have been significant advances in neuroimaging as well as in revascularization strategies for treating patients with acute ischemic stroke. In this review, we discuss the role of MRI and post-processing, including machine-learning techniques, and recent advances in MRI-based triage for revascularization therapies in acute ischemic stroke.

Serum-mediated Activation of Bone Marrow-derived Mesenchymal Stem Cells in Ischemic Stroke Patients: A Novel Preconditioning Method.

Stroke induces complex and dynamic, local and systemic changes including inflammatory reactions, immune responses, and repair and recovery processes. Mesenchymal stem cells (MSCs) have been shown to enhance neurological recovery after stroke. We hypothesized that serum factors play a critical role in the activation of bone marrow (BM) MSCs after stroke such as by increasing proliferation, paracrine effects, and rejuvenation. Human MSCs (hMSCs) were grown in fetal bovine serum (FBS), normal healthy control serum (NS), or stroke patient serum (SS). MSCs cultured in growth medium with 10% SS or NS exhibited higher proliferation indices than those cultured with FBS ( P < 0.01). FBS-, NS-, and SS-hMSCs showed differences in the expression of trophic factors; vascular endothelial growth factor, glial cell-derived neurotrophic factor, and fibroblast growth factor were densely expressed in samples cultured with SS ( P < 0.01). In addition, SS-MSCs revealed different cell cycle- or aging-associated messenger RNA expression in a later passage, and ß-galactosidase staining showed the senescence of MSCs observed during culture expansion was lower in MSCs cultured with SS than those cultured with NS or FBS ( P < 0.01). Several proteins related to the activity of receptors, growth factors, and cytokines were more prevalent in the serum of stroke patients than in that of normal subjects. Neurogenesis and angiogenesis were markedly increased in rats that had received SS-MSCs ( P < 0.05), and these rats showed significant behavioral improvements ( P < 0.01). Our results indicate that stroke induces a process of recovery via the activation of MSCs. Culture methods for MSCs using SS obtained during the acute phase of a stroke could constitute a novel MSC activation method that is feasible and efficient for the neurorestoration of stroke.

Stroke Induces Mesenchymal Stem Cell Migration to Infarcted Brain Areas Via CXCR4 and C-Met Signaling.

Mesenchymal stem cells circulate between organs to repair and maintain tissues. Mesenchymal stem cells cultured with fetal bovine serum have therapeutic effects when intravenously administered after stroke. However, only a small number of mesenchymal stem cells reach the brain. We hypothesized that the serum from stroke patients increases mesenchymal stem cells trophism toward the infarcted brain area. Mesenchymal stem cells were grown in fetal bovine serum, normal serum from normal rats, or stroke serum from ischemic stroke rats. Compared to the fetal bovine serum group, the stroke serum group but not the normal serum group showed significantly greater migration toward the infarcted brain area in the in vitro and in vivo models (p < 0.05). Both C-X-C chemokine receptor type 4 and c-Met expression levels significantly increased in the stroke serum group than the others. The enhanced mesenchymal stem cells migration of the stroke serum group was abolished by inhibition of signaling. Serum levels of chemokines, cytokines, matrix metalloproteinase, and growth factors were higher in stroke serum than in normal serum. Behavioral tests showed a significant improvement in the recovery after stroke in the stroke serum group than the others. Stroke induces mesenchymal stem cells migration to the infarcted brain area via C-X-C chemokine receptor type 4 and c-Met signaling. Culture expansion using the serum from stroke patients could constitute a novel preconditioning method to enhance the therapeutic efficiency of mesenchymal stem cells.

Impact of Slow Blood Filling via Collaterals on Infarct Growth: Comparison of Mismatch and Collateral Status.

BACKGROUND AND PURPOSE: Perfusion-diffusion mismatch has been evaluated to determine whether the presence of a target mismatch helps to identify patients who respond favorably to recanalization therapies. We compared the impact on infarct growth of collateral status and the presence of a penumbra, using magnetic resonance perfusion (MRP) techniques. METHODS: Consecutive patients who were candidates for recanalization therapy and underwent serial diffusion-weighted imaging (DWI) and MRP were enrolled. A collateral flow map derived from MRP source data was generated by automatic post-processing. The impact of a target mismatch (Tmax>6 s/apparent diffusion coefficient (ADC) volume≥1.8, ADC volume<70 mL; and Tmax>10 s for ADC volume<100 mL) on infarct growth was compared with MR-based collateral grading on day 7 DWI, using multivariate linear regression analysis. RESULTS: Among 73 patients, 55 (75%) showed a target mismatch, whereas collaterals were poor in 14 (19.2%), intermediate in 36 (49.3%), and good in 23 (31.5%) patients. After adjusting for initial severity of stroke, early recanalization (P<0.001) and the MR-based collateral grading (P=0.001), but not the presence of a target mismatch, were independently associated with infarct growth. Even in patients with a target mismatch and successful recanalization, the degree of infarct growth depended on the collateral status. Perfusion status at later Tmax time points (beyond the arterial phase) was more closely correlated with collateral status. CONCLUSIONS: Patients with good collaterals show a favorable outcome in terms of infarct growth, regardless of the presence of a target mismatch pattern. The presence of slow blood filling predicts collateral status and infarct growth.

Infarct Pattern and Collateral Status in Adult Moyamoya Disease: A Multimodal Magnetic Resonance Imaging Study.

BACKGROUND AND PURPOSE: Moyamoya disease (MMD) is a unique cerebrovascular disease characterized by the progressive stenosis of large intracranial arteries and a hazy network of basal collaterals, called moyamoya vessels. Although hemodynamic studies have been applied in MMD patients, the mechanisms of stroke in MMD are still unclear. The present study evaluated the infarct pattern and collateral status using multimodal magnetic resonance imaging in MMD patients. METHODS: Adult MMD patients with acute ischemic stroke were prospectively recruited, and infarct pattern on diffusion-weighted imaging was evaluated. A collateral flow map, derived from magnetic resonance perfusion-weighted imaging data, was generated through automatic postprocessing, and collateral status was assigned into 3 grades. Transcranial Doppler monitoring was performed to detect microembolic signals in selected patients. RESULTS: A total of 67 hemispheres (31 patients with bilateral and 5 patients with unilateral MMD) were analyzed. Most patients (83.7%) showed embolic pattern and rarely deep (9.3%) or hemodynamic infarct pattern (7.0%) on diffusion-weighted imaging. Most cases (86%) showed good collateral status, and few patients with acute infarcts of embolic pattern showed poor collateral status (n=7). One third (31.6%) of patients who underwent transcranial Doppler monitoring showed microembolic signals. CONCLUSIONS: In the studied population of adult MMD patients, embolic phenomenon played an important role in ischemic stroke. Therapeutic strategies against thromboembolism, as well as collateral enhancing strategies targeting improvement of hemodynamic status or increased washout of emboli, are warranted.

Volume and morphology of left atrial appendage as determinants of stroke subtype in patients with atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is a leading cause of stroke, but not all cases of stroke in patients with AF are due to AF. OBJECTIVE: The purpose of this study was to determine whether morphometric or volumetric parameters of left atrial appendage (LAA) would be related to the development of cardioembolism in subjects with AF. METHODS: A total of 433 consecutive patients with acute ischemic stroke underwent multidetector cardiac computed tomography (MDCT). Of these patients, 88 with AF were divided into cardioembolic stroke (CES; n = 57) and non-CES (n = 31) groups, and 95 age- and sex-matched patients with non-CES without AF served as controls. Clinical factors, echocardiographic findings, and MDCT parameters were evaluated. RESULTS: Brain infarct volume, LAA orifice diameter, and LAA volume were larger in patients with CES with AF than in those with non-CES with AF (P<.05 in all cases), but no difference was observed between patients with non-CES with AF and those with non-CES without AF. MDCT and echocardiographic parameters of left atrial (LA) dysfunction were different depending on the presence of AF but not between patients with CES with AF vs non-CES with AF. After adjusting for covariates, LAA orifice diameter (odds ratio 1.19, 95% confidence interval 1.06-1.33, P = .004) and LAA volume (odds ratio 12.20, 95% confidence interval 2.58-57.79, P = .002) were independently associated with CES with AF, as was infarct volume. CONCLUSION: In patients with AF, LAA orifice diameter and LAA volume, but not left atrial dysfunction, were determinants of CES and were useful for stratifying noncardioembolic risk in patients with AF.

Predicting Collateral Status With Magnetic Resonance Perfusion Parameters: Probabilistic Approach With a Tmax-Derived Prediction Model.

BACKGROUND AND PURPOSE: Good collateral flow is an important predictor for favorable responses to recanalization therapy and successful outcomes after acute ischemic stroke. Magnetic resonance perfusion-weighted imaging (MRP) is widely used in patients with stroke. However, it is unclear whether the perfusion parameters and thresholds would predict collateral status. The present study evaluated the relationship between hypoperfusion severity and collateral status to develop a predictive model for good collaterals using MRP parameters. METHODS: Patients who were eligible for recanalization therapy that underwent both serial diffusion-weighted imaging and serial MRP were enrolled into the study. A collateral flow map derived from MRP source data was generated through automatic postprocessing. Hypoperfusion severity, presented as proportions of every 2-s Tmax strata to the entire hypoperfusion volume (Tmax≥2 s), was compared between patients with good and poor collaterals. Prediction models for good collaterals were developed with each Tmax strata proportion and cerebral blood volumes. RESULTS: Among 66 patients, 53 showed good collaterals based on MRP-based collateral grading. Although no difference was noted in delays within 16 s, more severe Tmax delays (Tmax16-18 s, Tmax18-22 s, Tmax22-24 s, and Tmax>24 s) were associated with poor collaterals. The probability equation model using Tmax strata proportion demonstrated high predictive power in a receiver operating characteristic analysis (area under the curve=0.9303; 95% confidence interval, 0.8682-0.9924). The probability score was negatively correlated with the volume of infarct growth (P=0.030). CONCLUSIONS: Collateral status is associated with more severe Tmax delays than previously defined. The present Tmax severity-weighted model can determine good collaterals and subsequent infarct growth.

A novel magnetic resonance imaging approach to collateral flow imaging in ischemic stroke.

OBJECTIVE: Dedicated magnetic resonance (MR) imaging (MRI) sequences for evaluation of collaterals can be generated using MR perfusion (MRP) source data. We compared a novel collateral flow imaging technique with digital subtraction angiography (DSA) for determining collateral circulation in acute stroke and evaluated the ability of MR-based collateral flow maps to predict outcomes after recanalization therapy. METHODS: Consecutive patients who were candidates for endovascular treatment were enrolled. A collateral flow map derived from MRP source data was generated by manual or automatic postprocessing. Collateral grading based on the collateral flow map was performed and compared with grading based on DSA. Clinical and radiological outcomes were evaluated according to MR-based collateral grading and early reperfusion (ER) status. RESULTS: There was good correlation between MRI-based and DSA-based collateral grades (weighted κ-coefficient = 0.70). Collateral status and achievement of ER were the 2 main determinants of a favorable functional outcome and neurological improvement, in addition to infarct growth. Regardless of achievement of ER, better collaterals were significantly associated with a lower modified Rankin score at day 90 (p < 0.001 for trend in both ER(-) and ER(+) ). Most symptomatic intracranial hemorrhages occurred in patients with a poor collateral grade and ER(+) , whereas no patient with excellent collaterals suffered symptomatic intracranial hemorrhage or died. INTERPRETATION: MRI techniques to assess collaterals are rapidly being developed, and may provide insight into collateral perfusion. The combination of collateral images derived from pretreatment MRP source data and reperfusion status is a robust predictor of outcomes in acute ischemic stroke.