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We investigated the effect of homogenization strategy and protein precipitation on downstream protein quantitation using multiple reaction monitoring mass spectrometry (MRM-MS). Our objective was to develop a workflow capable of processing disparate tissue types with high throughput, minimal variability, and maximum purity. Similar abundances of endogenous proteins were measured in nine different mouse tissues regardless of the homogenization method used; however, protein precipitation had strong positive effects on several targets. The best throughput was achieved by lyophilizing tissues to dryness, followed by homogenization via bead-beating without sample buffer. Finally, the effect of tissue perfusion prior to dissection and collection was explored in 20 mouse tissues. MRM-MS showed decreased abundances of blood-related proteins in perfused tissues; however, complete removal was not achieved. Concentrations of nonblood proteins were largely unchanged, although significantly higher variances were observed for proteins from the perfused lung, indicating that perfusion may not be suitable for this organ. We present a simple yet effective tissue processing workflow consisting of harvest of fresh nonperfused tissue, novel lyophilization and homogenization by bead-beating, and protein precipitation. This workflow can be applied to a range of mouse tissues with the advantages of simplicity, minimal manual manipulation of samples, use of commonly available equipment, and high sample quality.

During a pandemic, the availability of specific pathogen free chicken eggs is a major bottleneck for up-scaling response to the demand for influenza vaccine. This has led us to explore the use of Madin-Darby Canine Kidney (MDCK) cells for the manufacture of live attenuated influenza vaccine (LAIV) that provides production flexibility and speed. The present study reports the comparison of the immunogenicity and efficacy of two MDCK-based LAIVs against two egg-based LAIVs prepared from the same pandemic potential strains of H5 and H7 subtypes after a single dose of the vaccine followed by a challenge with a homologous wild type strain. The vaccine strains have been generated by classical method of reassortment using the A/Leningrad/134/17/57 master donor strain. Additionally, a prime-boost regimen of the MDCK-based vaccine followed by a challenge with a homologous wild type strain for H5 and H7 immunized ferrets and also a heterologous wild type strain for the H5 immunized animals was studied. No difference in the hemagglutination inhibition and virus neutralization antibody titers against the homologous virus was observed following a single dose of either egg-based or MDCK-based H5 and H7 LAIV vaccine. A second dose of MDCK-based vaccine significantly boosted antibody titers in the vaccinated animals. Both a single dose or two doses of LAIV provided complete protection from lower respiratory tract infection and resulted in a significant reduction in the virus titers recovered from the throat, nasal turbinates and lungs after challenge with the homologous wild type strain. Protection from a challenge with a heterologous strain of H5 was also observed after two doses of the MDCK-based LAIVs. This data strongly supports the use of MDCK as a substrate for the manufacture of LAIV which ensures reliable quality, safety, production flexibility, speed and breadth of protection, features that are highly critical during a pandemic.

Yellow fever vaccine associated neurovirulence and viscerotropism have been reported by various countries. In this study, the neurovirulence, viscerotropism and immunogenicity of yellow fever vaccine seed lots (master and working) and final product manufactured at Serum Institute of India (SII) were evaluated in cynomolgus monkeys. WHO reference virus 168-73 and Stamaril™ as a control vaccine was used for comparison. Neurovirulence and viscerotropism scores of the seed lots and final product were lower than Stamaril™. The SII seed virus and vaccine complies to the WHO requirement for neurovirulence, viscerotropism and immunogenicity, when tested in comparison to WHO reference seed virus 168/73. All challenged animals showed 100 % seroconversion as early as day 14 and neutralizing antibody titers were sustainable at day 30 in all animals.

We proteotyped blood plasma from 30 mouse knockout strains and corresponding wild-type mice from the International Mouse Phenotyping Consortium. We used targeted proteomics with internal standards to quantify 375 proteins in 218 samples. Our results provide insights into the manifested effects of each gene knockout at the plasma proteome level. We first investigated possible contamination by erythrocytes during sample preparation and labeled, in one case, up to 11 differential proteins as erythrocyte originated. Second, we showed that differences in baseline protein abundance between female and male mice were evident in all mice, emphasizing the necessity to include both sexes in basic research, target discovery, and preclinical effect and safety studies. Next, we identified the protein signature of each gene knockout and performed functional analyses for all knockout strains. Further, to demonstrate how proteome analysis identifies the effect of gene deficiency beyond traditional phenotyping tests, we provide in-depth analysis of two strains, C8a−/− and Npc2+/−. The proteins encoded by these genes are well-characterized providing good validation of our method in homozygous and heterozygous knockout mice. Ig alpha chain C region, a poorly characterized protein, was among the differentiating proteins in C8a−/−. In Npc2+/− mice, where histopathology and traditional tests failed to differentiate heterozygous from wild-type mice, our data showed significant difference in various lysosomal storage disease-related proteins. Our results demonstrate how to combine absolute quantitative proteomics with mouse gene knockout strategies to systematically study the effect of protein absence. The approach used here for blood plasma is applicable to all tissue protein extracts.

Cell culture based live attenuated influenza vaccines (LAIV) as an alternative to egg-based LAIV have been explored because of lack of easy access to SPF eggs for large scale production. In this study, feasibility of MDCK platform was assessed by including multiple LAIV strains covering both type A (H1 and H3) and type B seasonal strains as well as the candidate pandemic potential strains like A/H5 and A/H7 for the growth in MDCK cells. A risk assessment study was conducted on the cell banks to evaluate safety concerns related to tumorigenicity with a regulatory perspective. Tumorigenic potential of the MDCK cells was evaluated in nude mice (107cells/mouse) model system. The 50% tumor producing dose (TPD50) of MDCK cells was studied in SCID mice to determine the amount of cells required for induction of tumors. Further, we conducted an oncogenicity study in three sensitive rodent species as per the requirements specified in the WHO guidelines. We determined TPD50 value of 1.9 X 104 cells/mice through subcutaneous route. Our results suggest that, the intranasal route of administration of the cell culture based LAIV pose minimal to no risk of tumorigenicity associated with the host cells. Also, non-oncogenic nature of MDCK cells was demonstrated. Host cell DNA in the vaccine formulations was The present study clearly establishes the suitability of MDCK cells as a substrate for the manufacture of a safe and viable LAIV.

Peptide-receptor imaging and therapy with radiolabeled somatostatin analogs such as 68Ga-DOTA-TATE and 177Lu-DOTA-TATE have become an effective treatment option for SSTR-positive neuroendocrine tumors. The purpose of this study was to evaluate the correlation of somatostatin receptor-2 (SSTR2) expression with 68Ga-DOTA-TATE uptake and 177Lu-DOTA-TATE therapy in neuroblastoma (NB) xenograft models. We demonstrated variable SSTR2 expression profiles in eight NB cell lines. From micro-PET imaging and autoradiography, a higher uptake of 68Ga-DOTA-TATE was observed in SSTR2 high-expressing NB xenografts (CHLA-15) compared to SSTR2 low-expressing NB xenografts (SK-N-BE(2)). Combined autoradiography-immunohistochemistry revealed histological colocalization of SSTR2 and 68Ga-DOTA-TATE uptake in CHLA-15 tumors. With a low dose of 177Lu-DOTA-TATE (20 MBq/animal), tumor growth inhibition was achieved in the CHLA-15 high SSTR2 expressing xenograft model. Although, in vitro, NB cells showed variable expression levels of norepinephrine transporter (NET), a molecular target for 131I-MIBG therapy, low 123I-MIBG uptake was observed in all selected NB xenografts. In conclusion, SSTR2 expression levels are associated with 68Ga-DOTA-TATE uptake and antitumor efficacy of 177Lu-DOTA-TATE. 68Ga-DOTA-TATE PET is superior to 123I-MIBG SPECT imaging in detecting NB tumors in our model. Radiolabeled DOTA-TATE can be used as an agent for NB tumor imaging to potentially discriminate tumors eligible for 177Lu-DOTA-TATE therapy.

The procedure for aspiration of bone marrow from the femur of patients undergoing total knee arthroplasty (TKA) or total hip arthroplasty (THA) may vary from an OR (operating room) to OR based on the surgeon’s skill and may lead to varied extent of clotting of the marrow and this, in turn, presents difficulty in the isolation of mesenchymal stem cells (MSCs) from such clotted bone marrow. We present a simple detailed protocol for aspirating bone marrow from such patients, isolation, and characterization of MSCs from the aspirated bone marrow specimens and show that the bone marrow presented no clotting or exhibited minimal clotting. This represents an economical source and convenient source of MSCs from bone marrow for use in regenerative medicine. Also, we presented the detailed protocol and showed that the MSCs derived from such bone marrow specimens exhibited MSCs characteristics and generated micromass cartilages, the recipe for regenerative medicine for osteoarthritis. The protocols we presented can be used as standard operating procedures (SOPs) by researchers and clinicians.

Targeted and localized mass spectrometry imaging allows faster characterization of cancer compared to conventional methods., While mass spectrometers can detect chemical signatures within milliseconds of data acquisition time, the non-targeted nature of mass spectrometry imaging (MSI) necessitates probing the entire surface of the sample to reveal molecular composition even if the information is only sought from a sample subsection. This leads to long analysis times. Here, we used polarimetry to identify, within a biological tissue, areas of polarimetric heterogeneity indicative of cancer. We were then able to target our MS analysis using polarimetry results to either the cancer region itself or to the cancer margin. A tandem of polarimetry and Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI) enables fast (10 fold compared to non-targeted imaging), and accurate pathology assessment (cancer typification in less than 2 minutes compared to 30 minutes for histopathology) of ex vivo tissue slices, without additional sample preparation. This workflow reduces the overall analysis time of MSI as a research tool.

A ferret challenge study was conducted to address the efficacy of the egg-based and Madin-Darby canine kidney (MDCK)-based live attenuated influenza vaccine (LAIV) strains. Vaccines derived as 6:2 reassortants from the A/Leningrad/134/17/57 master donor strain and the HA and NA components from the A/California/07/2009 (A/Cal)- and A/Michigan/45/2015 (A/Mich)-like strains of type A H1N1 influenza virus were used in the study. Monovalent, trivalent and quadrivalent formulations of the LAIV containing either of the two H1N1 strains were analysed. A total of ten groups of six animals each were immunised intranasally (i.n.) with a single dose of 0.5-ml vaccine formulation or placebo and challenged on day 28 with the homologous wild-type A/Cal or A/Mich strain. Immune response post immunisation and virus replication post challenge were studied. Both the strains derived from embryonated eggs or MDCK cells, irrespective of the vaccine valency, were capable of rendering complete protection from virus replication in the lung. The A/Mich vaccine strain showed higher immune titres and efficacy than the A/Cal vaccine strain in all the vaccine formulations. The haemagglutination inhibition and virus neutralisation antibody titres were induced, and the reduction in the virus load in the respiratory tract was observed to be higher in animals treated with the monovalent formulation compared to the trivalent and quadrivalent formulations. Overall, it appears that the monovalent formulations render better protection from infection and would therefore be the best candidate during a pandemic.

Natural killer (NK) cells are cytotoxic lymphocytes involved in innate immunity. NK-92, a human NK cell line, may be targeted to tumor-associated antigens in solid malignancies where it exhibits antitumor efficacy, but its clinical utility for treating brain tumors is limited by an inability to cross the blood–brain barrier (BBB). We investigated the potential for focused ultrasound (FUS) to deliver targeted NK-92 cells to the brain using a model of metastatic breast cancer. HER2-expressing human breast tumor cells were implanted into the brain of nude rats. The NK-92-scFv(FRP5)-zeta cell line expressing a chimeric HER2 antigen receptor was transfected with superparamagnetic iron oxide nanoparticles before intravenous injection, before and following BBB disruption using focused ultrasound (551.5 kHz focused transducer, 0.33 MPa average peak rarefaction pressure) in the presence of a microbubble contrast agent. Baseline and posttreatment 1.5T and 7T MR imaging was done, and histology used to identify NK-92 cells post-mortem. Contrast-enhanced MRI showed reproducible and consistent BBB disruption. 7T MR images obtained at 16 hours posttreatment revealed a significant reduction in signal indicating the presence of iron-loaded NK-92 cells at the tumor site. The average ratio of NK-92 to tumor cells was 1:100 when NK cells were present in the vasculature at the time of sonication, versus 2:1,000 and 1:1,000 when delivered after sonication and without BBB disruption, respectively. Our results offer a preclinical proof-of-concept that FUS can improve the targeting of immune cell therapy of brain metastases. Cancer Res; 73(6); 1892–9. ©2012 AACR.

Identification of necrosis in tumors is of prognostic value in treatment planning, as necrosis is associated with aggressive forms of cancer and unfavourable outcomes. To facilitate rapid detection of necrosis with Mass Spectrometry (MS), we report the lipid MS profile of necrotic breast cancer with Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) imaging validated with statistical analysis and correlating pathology. This MS profile is characterized by (1) the presence of the ion of m/z 572.48 [Cer(d34:1) + Cl]− which is a ceramide absent from the viable cancer subregions; (2) the absence of the ion of m/z 391.25 which is present in small abundance only in viable cancer subregions; and (3) a slight increase in the relative intensity of known breast cancer biomarker ions of m/z 281.25 [FA(18:1)-H]− and 303.23 [FA(20:4)-H]−. Necrosis is accompanied by alterations in the tissue optical depolarization rate, allowing tissue polarimetry to guide DESI-MS analysis for rapid MS profiling or targeted MS imaging. This workflow, in combination with the MS profile of necrosis, may permit rapid characterization of necrotic tumors from tissue slices. Further, necrosis-specific biomarker ions are detected in seconds with single MS scans of necrotic tumor tissue smears, which further accelerates the identification workflow by avoiding tissue sectioning and slide preparation.

In the event of a highly pathogenic influenza pandemic, the Indian subcontinent would need 1.2 billion doses of vaccine to immunize its entire population, double if two doses were required to assure immunity. Serum Institute of India Limited (SII) thus became one of six initial grantees of the World Health Organization (WHO) technology transfer initiative to create capacity in developing countries to manufacture H5N1 pandemic influenza vaccine. At the outbreak of the A(H1N1) 2009 influenza pandemic, experience gained from the H5N1 project was used to develop a live attenuated influenza vaccine (LAIV), since this was the only option for the level of surge capacity required for a large-scale immunization campaign in India. SII took

Blood-brain barrier disruption (BBBD) shows promise for drug delivery in the brain; however, optimal parameters for disruption have yet to be firmly established. Previous work has shown that BBBD can be achieved using bursts comprised of microsecond-length pulses at 50% duty cycle to eliminate standing waves and variability. The capabilities and limitations of this sort of pulse sequence comprising short bursts were examined. Ultrasound-induced BBBD was performed in 28 rats using Definity contrast agent. The spacing between 3-μs pulses at 1.18 MHz was either 6 μs, 60 μs, 300 μs or 600 μs during a 10-ms pulse, or 1 s for a single-pulse burst. The rate of infusion of the microbubbles was also examined, as well as the burst pulse repetition frequency (PRF) under infusion conditions. A semi-log relationship between enhancement mean and the number of cycles in a burst was discovered, with a one-pulse burst (i.e., a 3-μs burst at 1 Hz) still capable of disrupting the BBB. No increased efficacy or safety benefit over bursts containing more cycles was found, however. Microbubble infusions showed no improvement in T1w enhancement, but did improve consistency. Increased burst PRF combined with infusion improved T1w enhancement but without statistical significance, whereas a decrease in burst PRF showed a statistically significant decrease in enhancement. (E-mail: moreilly@sir.utoronto.ca )

Necrosis is a form of cell death that is often associated with highly aggressive forms of cancer, is of prognostic value in treatment planning. Mass Spectrometry (MS) is a highly sensitive analytic platform capable of providing a molecular profile of cancer on the basis of mass to charge (m/z) ratio of tissue constituent molecules. MS analysis of ex vivo tissue slices from metastatic murine xenograft tumors from LM2-4 cell line with Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) allowed direct comparisons with histology images to determine the molecular profile of necrotic tissues. The necrotic tissue is characterized by the presence of a ceramide absent from the viable cancer regions. The spatial distribution of this ion fully correlated to necrotic areas from pathology in additional independent tumor samples examined. The same ion was detected from in situ necrotic tissue using tissue aerosols generated by hand-held ablation probes coupled to evaporative ionization interface in only a few seconds of sampling. These developments further establish MS as a novel tool for rapid pathology that is highly complementary to current histology based methods widely used in characterization of cancer in both imaging mode (to provide spatial information on cancer border) and profiling mode (to provide information on cancer type and subtype); all based on unique molecular profile associated with each cancer type and subtype. Current efforts in creating cancer molecular profile libraries will facilitate translation. Citation Format: Arash Zarrine-Afsar, Bindesh Shrestha, Alessandra Tata, Michael Woolman, Manuela Ventura, Nicholas Bernards, Milan Ganguly, Howard Ginsberg, Jinzi Zheng, Emma Bluemke. Rapid detection of necrosis in breast cancer with ex vivo and in situ mass spectrometry analysis methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 741. doi:10.1158/1538-7445.AM2017-741

To aid in development of chronic stage treatments for sensorimotor deficits induced by ischemic stroke, we investigated the effects of GABA antagonism on brain structure and fine skilled reaching in a rat model of focal ischemia induced via cortical microinjections of endothelin-1 (ET-1). Beginning 7 days after stroke, animals were administered a gamma-aminobutyric acid (GABAA) inverse agonist, L-655,708, at a dose low enough to afford α5-GABAA receptor specificity. A week after stroke, the ischemic lesion comprised a small hypointense necrotic core (6 ± 1 mm3) surrounded by a large (62 ± 11 mm3) hyperintense perilesional region; the skilled reaching ability on the Montoya staircase test was decreased to 34% ± 2% of the animals' prestroke performance level. On L-655,708 treatment, animals showed a progressive decrease in total stroke volume (13 ± 4 mm3 per week), with no change in animals receiving placebo. Concomitantly, treated animals' skilled reaching progressively improved by 9% ± 1% per week, so that after 2 weeks of treatment, these animals performed at 65% ± 6% of their baseline ability, which was 25% ± 11% better than animals given placebo. These data indicate beneficial effects of delayed, sustained low-dose GABAA antagonism on neuroanatomic injury and skilled reaching in the chronic stage of stroke recovery in an ET-1 rat model of focal ischemia.

The efficacy of anticancer drugs in solid tumours is impaired by their inability to reach all cancer cells in sufficient concentration to cause cytotoxicity. Hyperthermia-triggered release of drugs from thermosensitive liposomes can increase tumour drug concentration, but tumour-specific drug delivery requires precise temperature control, and effects on microregional distribution of anticancer drugs in tumours are unknown. Here we evaluate thermally triggered release of doxorubicin in a rabbit tumour model by comparing free versus thermosensitive liposomal doxorubicin administered systemically during magnetic resonance imaging (MRI)-controlled focused ultrasound hyperthermia.Twelve rabbits with a transplanted VX2 tumour in each thigh had a 10 mm diameter region in one tumour heated to 43°C using focused ultrasound with temperature control by MRI thermometry. Delivery of doxorubicin to tumours and normal tissues was quantified by fluorescence in tissue homogenates, and by fluorescence microscopy.Using thermosensitive liposomal doxorubicin (2.5 mg/kg), doxorubicin concentrations in heated tumours were 26.7 times higher than in unheated tumours (n = 7, p = 0.017, two-sided Wilcoxon signed-rank test). There was no significant enhancement with free doxorubicin in heated versus unheated tumours (n = 3, p = 0.5). With thermosensitive liposomes (8.3 mg/kg), fluorescence microscopy demonstrated increased doxorubicin fluorescence in heated versus unheated tumours, co-localised with nuclear staining throughout the tumour.Localised image-guided delivery of high concentrations of doxorubicin to cancer cells was achieved non-invasively in implanted tumours with temperature-sensitive drug carriers and a preclinical MRI-controlled focused ultrasound hyperthermia system.

Blood-brain barrier (BBB) disruption can be achieved with ultrasound (US) and circulating microbubble (MB) contrast agent. Using dorsal US sonication and Definity, an MB contrast agent, responses of the cortical cerebral vasculature to BBB opening were observed with varying acoustic peak negative pressure (0.071 to 0.25 MPa) under two-photon microscope. Wistar rats with a craniotomy were sonicated with a single piezoelectric transducer following the intravenous injection of Texas Red for visualization of vasculature and leakage from BBB opening. Based on time-dependent intensity change in the extravascular area, the leakage was classified into three types: fast, sustained, and slow. Fast leakage was characterized by a rapid increase to peak intensity during sonication, but a decrease afterwards, occurring at all pressures and vessels sizes analyzed in our study. Sustained leakage was indicated by a similar, immediate increase to peak intensity but one that remained elevated for the duration of imaging, occurring at low-to-intermediate pressures. Slow leakage began 5 to 15 minutes after sonication, dominating at low pressures, and was more prevalent among smaller vessels than fast and sustained leakage. Our study showed the possibility of controlling leakage type and vessel size in US-induced BBB opening through varying acoustic pressure.

An evolutionary statistical learning method was applied to classify drugs according to their biological target and also to discriminate between a compilation of oral and nonoral drugs. The emphasis was placed not only on how well the models predict but also on their interpretability. In an enhancement to previous studies, the consistency of the model weights over several runs of the genetic algorithm was considered with the goal of producing comprehensible models. Via this approach, the descriptors and their ranges that contribute most to class discrimination were identified. Selecting a bin step size that enables the average descriptor properties of the class being trained to be captured improves the interpretability and discriminatory power of a model. The performance, consistency, and robustness of such models were further enhanced by using two novel approaches that reduce the variability between individual solutions: consensus and splice modeling. Finally, the ability of the genetic algorithm to discriminate between activity classes was compared with a similarity searching method, while naive Bayes classifiers and support vector machines were applied in discriminating the oral and nonoral drugs.

It is estimated that only 2-6% of patients receive thrombolytic therapy for acute ischemic stroke suggesting that alternative therapies are necessary. In this study, we investigate the potential for high intensity focused ultrasound (HIFU) to initiate thrombolysis in an embolic model of stroke. Iron-loaded blood clots were injected into the middle cerebral artery (MCA) of New Zealand White rabbits, through the internal carotid artery and blockages were confirmed by angiography. MRI was used to localize the iron-loaded clot and target the HIFU beam for treatment. HIFU pulses (1.5 MHz, 1 ms bursts, 1 Hz pulse repetition frequency, 20 s duration) were applied to initiate thrombolysis. Repeat angiograms and histology were used to assess reperfusion and vessel damage. Using 275 W of acoustic power, there was no evidence of reperfusion in post-treatment angiograms of 3 rabbits tested. In a separate group of animals, 415 W of acoustic power was applied and reperfusion was observed in 2 of the 4 (50%) animals treated. In the last group of animals, acoustic power was further increased to 550 W, which led to the reperfusion in 5 of 7 (∼70%) animals tested. Histological analysis confirmed that the sonicated vessels remained intact after HIFU treatment. Hemorrhage was detected outside of the sonication site, likely due to the proximity of the target vessel with the base of the rabbit skull. These results demonstrate the feasibility of using HIFU, as a stand-alone method, to cause effective thrombolysis without immediate damage to the targeted vessels. HIFU, combined with imaging modalities used to identify and assess stroke patients, could dramatically reduce the time to achieve flow restoration in patients thereby significantly increasing the number of patients which benefit from thrombolysis treatments.

Stem cell therapy is a promising strategy to treat neurodegenerative diseases, traumatic brain injury, and stroke. For stem cells to progress towards clinical use, the risks associated with invasive intracranial surgery used to deliver the cells to the brain, needs to be reduced. Here, we show that MRI-guided focused ultrasound (MRIgFUS) is a novel method for non-invasive delivery of stem cells from the blood to the brain by opening the blood brain barrier (BBB) in specific brain regions. We used MRI guidance to target the ultrasound beam thereby delivering the iron-labeled, green fluorescent protein (GFP)-expressing neural stem cells specifically to the striatum and the hippocampus of the rat brain. Detection of cellular iron using MRI established that the cells crossed the BBB to enter the brain. After sacrifice, 24 hours later, immunohistochemical analysis confirmed the presence of GFP-positive cells in the targeted brain regions. We determined that the neural stem cells expressed common stem cell markers (nestin and polysialic acid) suggesting they survived after transplantation with MRIgFUS. Furthermore, delivered stem cells expressed doublecortin in vivo indicating the stem cells were capable of differentiating into neurons. Together, we demonstrate that transient opening of the BBB with MRIgFUS is sufficient for transplantation of stem cells from the blood to targeted brain structures. These results suggest that MRIgFUS may be an effective alternative to invasive intracranial surgery for stem cell transplantation.