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Intranasal administration of exosomes derived from mesenchymal stem cells ameliorates autistic-like behaviors of BTBR mice

Intranasal administration of exosomes derived from mesenchymal stem cells ameliorates... Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. They have been steadily increasing in children over the past several years, with no effective treatment. BTBR T+tf/J (BTBR) mice are an accepted model of evaluating autistic-like behaviors as they present all core symptoms of ASD. We have previously shown that transplantation of human bone marrow mesenchymal stem cells (MSC) to the lateral ventricles of BTBR mice results in long lasting improvement in their autistic behavioral phenotypes. Recent studies point exosomes as the main mediators of the therapeutic effect of MSC. Here, we tested whether treatment with the exosomes secreted from MSC (MSC-exo) will show similar beneficial effects. We found that intranasal administration of MSC- exo increased male to male social interaction and reduced repetitive behaviors. Moreover, the treatment led to increases of male to female ultrasonic vocalizations and significant improvement in maternal behaviors of pup retrieval. No negative symptoms were detected following MSC-exo intranasal treatments in BTBR or healthy C57BL mice. The marked beneficial effects of the exosomes in BTBR mice may translate to a novel, non-invasive, and therapeutic strategy to reduce the symptoms of ASD. Introduction Moreover, the behavioral effect of MSC on BTBR mice Autism spectrum disorders (ASD) are neurodevelop- had lasted for 6 months despite the fact that the MSC mental disabilities characterized by three core symp- themselves do not survive in the transplanted tissue for toms: severe impairment of social interactions and such an extended period [5]. Such phenomenon has communication skills, increased repetitive behaviors, been reported in the literature under the title “hit and and cognitive inflexibility [1]. In this study, we used the run” meaning MSC have the ability to leave a lasting inbred mouse strain BTBR T+tf/J (BTBR) that incorpo- “memory” on the tissue, even after the MSC cells have rates multiple behavioral phenotypes relevant to all three been degraded [6]. It has been suggested that the MSC diagnostic symptoms of autism. BTBR present signifi- therapeutic effect is mainly mediated by their secretome cantly reduced social approach, low reciprocal social in- to the tissue [7]. The secretome is referred to as the teractions, and impaired juvenile play in comparison to complete repertoire of molecules and extracellular vesi- the C57BL controls [2, 3]. Using this model, we have re- cles secreted from MSC. Yet, it has been found that the cently shown that MSC transplantation to the lateral nano-vesicles, exosomes, are the major mediators be- ventricles of the brain of BTBR mice has the ability to tween the MSC and the tissue [8, 9]. ameliorate their core autistic-like behaviors [4]. Exosomes were initially thought to be a mechanism for removing unneeded membrane proteins from reticu- * Correspondence: [email protected] locytes. However, recent studies have shown that they Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel are also used for cell to cell communication through the Sacklar School of Medicine, Department of Human Genetics and carrying of genetic information from between cells [10]. Biochemistry, Tel Aviv University, Tel Aviv, Israel Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Perets et al. Molecular Autism (2018) 9:57 Page 2 of 12 Several studies have reported that MSC-exo have func- MSC-exo improves male to male social interaction and tions similar to those of MSC, such as repairing dam- reduces repetitive behaviors during social interaction aged tissue, suppressing inflammatory responses, and Mice were tested for male to male interaction before modulating the immune system [11, 12]. Exosomes con- and after MSC-exo or saline administration. Intra-sub- tain mainly proteins, as well as RNA and a large number ject analysis showed that the BTBR MSC-exo mice of micro RNAs. Approximately, 25% of these proteins group had spent significantly more time in social inter- and RNA play a role in cell growth and maintenance actions after the treatment whereas the BTBR saline and [13, 14]. A key advantage of MSC-exo over MSC is their C57BL saline groups did not show any change (paired t ability to enter the brain with ease following intranasal test, t < 0.0001). Comparison analysis between the administration [15, 16]. We have previously demon- groups showed that the BTBR mice of both groups had strated that exosomes loaded with gold nanoparticles spent significantly less time engaging in social inter- can cross the blood-brain barrier via intranasal adminis- action in comparison to C57BL basal behavior tration and can be visualize in vivo inside the brain [17]. (ANOVA1, F = 14.4, p < 0.01, Bonferroni). Exosome 2,18 Here, we show that BTBR mice that were treated with treatment dramatically increased social interactions in MSC-exo via intranasal administration present signifi- the BTBR MSC-exo group in comparison to BTBR mice cant improvement in the social interaction domain, treated with saline and is comparable to the C57BL mice ultrasonic communication, and repetitive behavior. group (ANOVA1, F = 9.44, p < 0.01, Bonferroni) 2,16 Moreover, we show for the first time that BTBR mothers (Fig. 2a, Additional files 1 and 2). that were treated with MSC-exo presented significant During the social interaction test, the repetitive behav- improvement in maternal pup retrieval behavior. ior was also measured. Intra-subject analysis showed that the BTBR MSC-exo-treated group spent signifi- cantly less time in repetitive behaviors while both the Results BTBR saline and C57BL saline mice groups did not MSC-exo characterization show any changes in comparison to their basal behaviors MSC-exo were characterized by NanoSight. The average (paired t test, t < 0.001). A comparison analysis between size was 114 ± 2.9 nm, and the average concentration groups showed that before treatment, there was no dif- was 3.81 × 10 particles/μL (Fig. 1a, b). Western blot ference between BTBR MSC-exo and BTBR saline basal analysis indicates that the MSC-exo express CD9 and behaviors, and that both groups significantly differed CD63, while we could not detect it in the MSC lysate from the basal behavior of the C57BL saline group (not shown). In contrast, Calnexin was undetectable in (ANOVA1, F = 13.71, p < 0.001, Bonferroni). After 2,18 the MSC-exo and found in the MSC lysate, indicating treatment, The BTBR MSC-exo group was not signifi- for the purity of the exosomes [18] (Fig. 1c). cantly different from the BTBR saline group and the Fig. 1 Characterization of MSC-exo. a Visualization of the exosomes using NanoSight technology. b Analysis of size distribution and concentrations. c Western blot analysis of CD9 which exists in exosomes but not in the MSC lysate in contrast to Calnexin which is absent in exosomes and found in the MSC lysate Perets et al. Molecular Autism (2018) 9:57 Page 3 of 12 Fig. 2 MSC-exo increased male to male social interaction and repetitive behaviors during social interaction. Each group was tested for basal behaviors (gray) and was re-tested 3 weeks after treatment (saline or MSC-exo, black). a Intra-subject comparison showed that the BTBR MSC-exo group had spent a significantly longer time engaging in social interaction with other stranger male mice (paired T-test). Inter-group comparison showed that the BTBR MSC-exo group had spent significantly more time engaging in social interaction compared to BTBR saline (ANOVA1, Bonferroni). b Intra-subject comparison of BTBR MSC-exo group had spent significantly less time in repetitive behaviors (paired T test). Inter-group comparison showed BTBR MSC-exo is not significantly different than both BTBR saline and C57BL saline groups in time spent in repetitive behaviors. (ANOVA1, Bonferroni). The data is presented as mean + SEM. ***p < 0.001 C57BL group (Fig. 2b). In repetitive behavior tests that compared to saline-treated group (ANOVA1, F = 2,14 were outside of the context of social interaction, BTBR 4.28, p < 0.05, Bonferroni). Importantly, MSC-exo and MSC-exo spent significantly less time in repetitive saline groups were also used as a biological replication grooming and digging in intra-subject analysis compared of the social interaction with different seven mice per to their basal behaviors (t < 0.05). In groups, compari- group (Additional file 5: Figure S3). sons showed that they were significantly different from the BTBR saline group but not from the C57BL saline MSC-exo improved male to female ultrasonic group (ANOVA1, F = 13.83, p < 0.01, Bonferroni) vocalizations 2,18 (Additional file 5: Figure S1). Importantly, C57BL In general, male mice emitted a large number of com- MSC-exo mice did not present any behavioral differ- plex ultrasonic vocalizations (USVs) when interacting ences in neither social interactions, antisocial interac- with adult females (Fig. 3a, experimental demonstration). tions, nor repetitive behaviors during social contact As seen qualitatively from the spectrograms in our (Additional file 5: Figure S2A). In comparison between study, BTBR MSC-exo vocalizations became more com- MSC-exo to exosomes isolated from neuronal stem cells plex and longer compared to the BTBR saline group, (NSC-exo), we found that only the MSC-exo-treated making them more similar to C57BL (Fig. 3b). During group presented a significant increase in social inter- the first 5 min of interaction with females, BTBR saline action, a reduction in repetitive behaviors, and a signifi- mice emitted 317 ± 39.4 syllables, BTBR MSC-exo emit- cant increase in communication while NSC-exo-treated ted 571 ± 74 (180% more), and C57BL emitted 854.5 ± mice did not present the same behavioral differences 65.2 syllables (Fig. 3c, ANOVA1, F = 19.2, p < 0.001, 2,18 Perets et al. Molecular Autism (2018) 9:57 Page 4 of 12 Fig. 3 Specific improvement of ultrasonic vocalizations after intranasal administration of MSC-exo. a visualization of the experimental set, right panel: male and female mice in courtship meeting. Left panel: real-time spectrogram of ultrasonic vocalizations. b Example of differences in spectrogram of C57BL, saline-treated, and MSC-exo-treated mice suggests that MSC-exo-treated mice improved in complexity of syllables. c BTBR MSC-exo had more syllables of ultrasonic vocalizations compared to saline BTBR, yet less syllables than C57BL mice. d and e Automatic classification of syllables showed significant reduction in the use of simple syllables by BTBR MSC-exo and C57BL saline compared to BTBR saline. Classification also showed increased use in complex and down syllables by BTBR MSC-exo and C57BL saline compared to BTBR saline. f There was not a significant difference between the groups in social contact (nose to nose and nose to genitals), indicating a specific effect on ultrasonic vocalizations. (ANOVA1, Bonferroni). Data is presented as mean + SEM. **p < 0.01, ***p < 0.001 Bonferroni). Classification of syllables revealed signifi- 57%, and C57BL saline used 60% (ANOVA1, F = 2,18 cant differences between the syllable types used by 49.44, p < 0.001, Bonferroni). For complex syllables, BTBR saline and BTBR MSC-exo (Fig. 3d, e). For simple BTBR saline used 2%, BTBR MSC-exo used 23%, and syllables, BTBR saline used 89%, BTBR MSC-exo used C57BL saline used 10% (ANOVA1, F = 30.61, p < 2,18 Perets et al. Molecular Autism (2018) 9:57 Page 5 of 12 0.001, Bonferroni). For down syllables, BTBR saline used was 10.4 s ± 1.08 s. In contrast, only one of eight BTBR 7%, BTBR MSC-exo used 16%, and C57BL saline used saline mothers retrieved two pups (in 156 s), without 26% (ANOVA1, F = 25.23, p < 0.001, Bonferroni). For bringing them back to the nest. Altogether, only 2/24 2,18 up syllables, BTBR saline used 1%, BTBR MSC-exo used pups were retrieved. After treatment, BTBR MSC-exo 1%, and C57BL saline used 6% (ANOVA1, F = 41.22, mothers retrieved all pups (18/18) at a mean time of 2,18 p < 0.001, Bonferroni). 25.24 s ± 5.7 s (ANOVA1 on time of retrieval, F = 2,48 There was no significant difference between groups in 332.5, p < 0.0001). C57BL saline trained virgins retrieved time spent sniffing the female’s genitals or faces, mean- all pups (15/15) with a total average time of 38.3 s ± ing that the effect seen in the ultrasonic vocalizations 3.3 s. BTBR saline trained virgins mostly did not retrieve was not impacted by the pheromones of the females the pups besides for one female that had retrieved two (Fig. 3f). C57BL mice that were treated with MSC-exo pups with a mean time of 152 s. Altogether, 2/21 pups showed similar number of syllables compared to C57BL were retrieved by BTBR saline trained virgins. BTBR saline group (Additional file 5: Figure S2B). In compari- MSC-exo trained virgins retrieved 7/9 pups at a total son between MSC-exo and NSC-exo, only the BTBR averagetimeof83.3±16.02s(ANOVA1ontimeof MSC-exo group presented a significant increase in num- retrieval, F = 29.47, p < 0.0001). Neither the C57BL 2,42 ber of syllables compared to BTBR saline group naïve virgins (0/21) nor the BTBR naïve virgins had (ANOVA1, F = 9.44, p < 0.01, Additional file 5: Figure retrieved the pups (0/21) (Fig. 4c, Additional files 3 2,14 S3B). This result also used as biological replication of and 4). We had assumed that the MSC-exo naïve vir- the vocalizations test. gins would also not retrieve the pups. MSC-EXO improves pup retrieval behavior Visualization of MSC and exosomes after intranasal We have performed pup-retrieval tests for MSC-exo- or delivery saline-treated mothers, naïve virgins, and experienced MAESTRO whole brain imaging was used to visualize virgins [19, 20]. The C57BL saline mothers retrieved all PKH26-labeled MSC and MSC-exo. After intranasal and pups (15/15), and their total average time for retrieval intravenous administration, MSC-exo can be visualized Fig. 4 MSC-exo improves maternal pup retrieval and learning of maternal pup retrieval behaviors. a Experiment’s timeline. b Visualization of normal pup retrieval behavior vs. autistic-like behavior. c C57BL and BTBR mice groups were treated with saline or MSC-exo were tested for pup retrieval. Data is presented as mean + SEM. **p < 0.01, ***p < 0.001 Perets et al. Molecular Autism (2018) 9:57 Page 6 of 12 fluorescently while MSC cannot be detected (Fig. 5a). Discussion Immunostaining indicated that MSC-exo penetrate the We have previously found that surgical transplantation brain parenchyma and are found in the cells of the tissue of MSC to the brain ameliorates the autistic-like behav- (Fig. 5b, c). iors of BTBR mice [4]. Furthermore, we have shown that this effect could last for 6 months post a single treat- MSC-exo migration and efficacy are dependent on their ment, even though MSC does not survive for an ex- membrane proteins tended period in the brain [5]. We have also The ability of MSC-exo to cross the blood-brain barrier demonstrated that MSC-exo efficiently cross the and to be uptake by cells in the brain is critical for the blood-brain barrier after intranasal administration com- therapeutic effect. To block the MSC-exo from being pares to intravenous injection, and can be visualized in uptake by the cells in the brain and to use them as con- vivo when they are loaded with gold nanoparticles [17]. trol, MSC-exo were treated with protease-k (ProtK) in In the current experiment, BTBR mice were treated via order to remove the membrane proteins. Immunostain- intranasal administration of MSC-exo followed by be- ings of the tissue indicated that the MSC-exo-protk de- havioral tests in all the ASD-like phenotypes presented livered intranasally have not been uptake into the cells by this model. Remarkably, the BTBR MSC-exo group at the same efficiency as MSC-exo (Fig. 6a as compared presented significant improvements in all the tested to Fig. 5b). To demonstrate the effectiveness of ProtK in ASD-like phenotypes. degrading the proteins on the exosomes’ membrane, we In social interaction, the BTBR MSC-exo group spent used Western blot analysis to CD9 and CD63 since they a significantly longer time engaging in interaction with are known to be overexpressing on the membrane of the the stranger male compared to the saline-treated mice exosomes and are commonly used for exosomes detec- and their own basal behaviors. BTBR MSC-exo had also tion and characterizations [42]. The Western blot presented a significant decrease in repetitive behaviors showed deletion of the membrane proteins CD9 and a of self-grooming and digging compared to their own reduction of CD63 (Fig. 6b). NanoSight analysis showed basal behavior and to the BTBR saline group. Their no significant change in the number and size of scores had become closer to the C57BL saline group. MSC-exo-protK compared to MSC-exo (Fig. 6c). BTBR Deficits in social communication are a core symptom mice that were treated with MSC-exo-protK did not of ASD in children [1]. It has been reported that BTBR demonstrate behavioral differences in social interaction mice present unusual repertoire of male to female ultra- and repetitive behaviors (Fig. 6d). sonic vocalizations compared to normal C57BL mice Fig. 5 Comparison between MSC-exo and MSC labeled with PKH26 visualization in the brain. a From left to right: MSC-exo intranasal, MSC-exo intravenous, MSC intranasal, and MSC intravenous. MSC-exo cross the blood brain barrier, both intranasally and intravenously, while MSC much less. b Immunostaining of neurons, DAPI and PKH26 labeled MSC-exo after intranasal administration. c Sagittal section of the BTBR MSC-exo brain after intranasal administration of PKH26 labeled MSC-exo (top) and Alan brain atlas (bottom) Perets et al. Molecular Autism (2018) 9:57 Page 7 of 12 Fig. 6 MSC-exo-protK was not found inside the cells and the mice did not present behavioral differences. a MSC-exo-protK were mostly located outside of the cells, unlike MSC-exo (Fig. 5b). b Western blot of MSC-exo-protK show deletion of CD9 and reduce CD63 compared to MSC-exo. c NanoSight analysis of MSC-exo-protK showed no significant change in number and size of MSC-exo-protK compared to MSC-exo (Fig. 1). d BTBR mice treated with MSC-exo-protK did not present behavioral improvement compared to saline treated in social and repetitive behaviors [21]. We reported that BTBR MSC-exo mice had signifi- sexual arousal is an advantage. As compared, the C57BL cant improvement in the number of syllables compared MSC-exo group did not present behavioral changes in to the control BTBR saline group. Their number of sylla- their ultrasonic vocalizations and social interactions to- bles was closer to the C57BL group. To examine other ward both males and females. This finding may suggest features of ultrasonic vocalizations, such as complexity that MSC-exo administration leads to beneficial effects and classification of syllables, we have used an advanced and not to non-specific hyperactivity or behavioral side classification algorithm. This classification revealed sig- effects. nificant improvement in the complexity of syllables of Maternal behavior of BTBR mice has yet to be studied BTBR MSC-exo mice, making their ultrasonic vocaliza- extensively. Here, we observed significant differences be- tions repertoire closer to the C57BL saline group. Im- tween BTBR and C57BL for pup retrieval. C57BL mice portantly, the improvement in male-to-female ultrasonic exhibit “normal” behavior, and they created a nest for vocalizations did not seem to be caused by higher sexual their pups. If the pup was separated, they had quickly re- arousal as there was no significant difference between trieved it [18, 19]. In contrast, BTBR created a nest but the duration of interaction initiated by the males toward reacted slowly to the pup separation and nearly always the females in any of the groups. This finding was also had not brought the pup within 3 min. Interestingly, observed in our previous studies after MSC transplant- BTBR MSC-exo mothers demonstrated high scores in ation to BTBR mice [5]. Considering the growing recog- the pup retrieval test, with results comparable to C57BL nition that some adolescents and young adults with ASD saline mothers. Furthermore, a naïve C57BL virgin who may exhibit inappropriate sexual behaviors [22, 23], the spent a few days with a mother can learn this “experi- fact that MSC-exo treatment also does not enhance enced” behavior. BTBR saline females did not manage to Perets et al. Molecular Autism (2018) 9:57 Page 8 of 12 learn from other mothers (C57BL mothers) while BTBR (miR-222, miR-21) [39–41]. Yet, we are aware that MSC-exo had presented learning abilities. Maternal be- MSC-exo are complex vesicles containing hundreds of havior, as well as social interaction, acquires high-level proteins and RNA molecules; therefore, we cannot pin- synchronization of sensory input and behavioral out- point on the specific factors that led to the behavioral put. We suggestMSC-exo mayplayarole inthe difference and future study may be needed to uncover mechanisms of sensory integration, making its effect the mechanism of action that led MSC-exo-treated mice influential in the realms of the social domain symp- to behavioral amelioration. Moreover, since BTBR mice toms. Sensory integration and coordination deficits are multifactorial model of autism, it may be worth to have been suggested to be one of the underlying test MSC-exo effect of other models, including genetic- mechanism of the ASD patients [24–26]. ally modified mice such as shank3 mutation as well. Stem cell therapy has been previously used on ASD Altogether, our findings suggest that MSC-exo may be children with long-term beneficial effects [27]. Bone tested as a safe noninvasive treatment to ameliorate be- marrow MSC has been proven to be safe to use in sev- havioral symptoms of patients with ASD. eral clinical trials [28–30]. Mechanistically, bone marrow MSC was found efficient in promoting tissue regener- Materials and methods ation, immunomodulation, and inflammatory reduction Mesenchymal stem cells preparation [31–33]. We reported that the transplantation of MSC Human MSC were purchased from Lonza (cat:PT-2501, to the lateral ventricles of BTBR mice leads to increased Basel, Switzerland). Cells were cultured and expanded as neurogenesis and BDNF in the hippocampus [4]. Al- previously described [42]. Before exosome collection, the though it is clear that MSC have beneficial properties cells were cultured in exosome-free platelets for 3 days that can be used safely for clinical purposes, recent evi- and this medium was then collected. dence shows that the therapeutic effect of MSC is largely mediated via the secretion of exosomes that contain im- Exosomes purification protocol portant molecular information [34, 35]. Our findings The exosomes were purified by taking the culture fluid support this concept, and our study demonstrates that a and centrifuging it for 10 min at 300g. The supernatant remarkable behavioral effect, on all ASD-like phenotypes was recovered and centrifuged for 10 min at 2000g. of BTBR mice, can be achieved by simply using Once again, the supernatant was recovered and centri- MSC-exo rather than MSC. We are aware that small fuged for 30 min at 10,000g. The supernatant was fil- number of mice per group is a limiting factor of our re- trated through a 0.22-μm filter and centrifuged for sults, yet the behavioral difference post MSC-exo treat- 70 min at 100,000g. The pellet containing the exosomes ment is remarkable. Furthermore, in social interaction and proteins was washed in PBS and then centrifuged and repetitive behaviors, each mouse was compared to for 70 min at 100,000g. The pellet containing the puri- its own basal behavior and also to saline-treated group fied exosomes was re-suspended in 200 μm of sterilized post the treatment, this experimental design was chosen PBS. Each centrifugation occurred at 4 °C [43]. to increase the confidence on the results. Mentionable, for the comparison between MSC-exo and NSC-exo, we used another MSC-exo-treated mice; therefore, the USV Neuronal stem cells preparation and social and repetitive behaviors post MSC-exo treat- Human neuronal stem cell lines (CTX0E03) were pur- ment were tested twice independently. chased from ReNeuron, UK. Cells were cultured and ex- The contents of MSC-exo have been characterized panded according to the company’s protocol. Cell lines using proteomics and RNA sequencing. Importantly, the were routinely cultured at 37 °C in tissue culture flasks capacity of the exosomes seems to be selectively pack- freshly coated with mouse laminin (20 μg/ml in aged. Therefore, some of the proteins and miRNA that DMEM:F12). Growth medium supplemented with are over expressed in the cells are packaged in exosomes 4-OHT was changed three times per week. When 70– while others have over-representation [14, 36]. While 90% confluent, cells were passaged using trypsin (0.25%) exosomes contain numerous variations of RNA, their for 5–10 min at 37 °C, followed by treatment with soy- miRNA repertoire has been spotlighted as a major can- bean trypsin inhibitor (0.25 mg/ml). Cells were spun didate for their effect in the host cell and the tissue. For down (800×g for 5 min at room temperature) and example, miRNA-143 was found to be related to the im- re-suspended at an appropriate density in full growth munomodulatory effect of MSC, and miRNA-10b was medium supplemented with 100 nM 4-OHT. The found related to their migration abilities [37, 38]. In NSC-exo were purified at the same protocol as addition, multiple miRNAs highly represented in MSC-exo, and a number of particles were matched per MSC-exo (miR-191, miR-222, miR-21) regulate cell cycle mouse using NANOsight analysis (Merkel technologies progression and proliferation and modulate angiogenesis LTD, Israel). Perets et al. Molecular Autism (2018) 9:57 Page 9 of 12 Exosomes characterization Proteinase-k treatment to MSC-exo NanoSight technology (Merkel technologies LTD, Israel) Two hundred microliters of PKH26 labeled exosomes was used to characterize the size and concentration of were incubated with 7 μL proteinase-K (Roche Diagnos- the exosomes (3.81 × 10 particles/μL). Lysates of tica GmbH, Germany) and 750 μL BPS in 55 °C for MSC-exo were subject for Western blot using SDS poly- 10 min [45]. Proteinase-K inhibitor (7 μL) (phenylmetha- acrylamide gel. Proteins were transferred to Immobilon®-P nesulfonyl fluoride, Sigma) was added to the solution and membranes (Millipore, Amsterdam, The Netherlands), in- was suspended for 2 h in 70 ml BPS for ultracentrifugation. cubated in 5% milk for 1 h, and probed overnight at 4 °C Finally, the pellet (MSC-exo-protk) was re-suspended in with CD9 antibody (ABCAM), CD63 (ABCAM), and Cal- 200ul PBS. For behavioral treatment, non-labeled MSC nexin (ABCAM). After three washes in TBS-Tween 20, -exo-protk were used. Proteinase-k-treated MSC-exo were the membranes were incubated with the secondary anti- characterized with NANO-sight and Western blot. body for 1 h and re-washed. Animals Exosomes labeling Mice were placed under a 12-h light/12-h dark condition Exosomes were labeled with PKH26 (Sigma-Aldrich) and grown in individual ventilated cages with access to [44, 45]. PKH26 (2 μL) in 500 μL diluent was then added food and water ad libitum. All experimental protocols to 50 μL exosomes in PBS for 5 min of incubation. Exo- were approved by the Tel Aviv University Committee of somes were suspended in 70 ml PBS and were centri- Animal Use for Research and Education. All methods fuged for 90 min at 100,000g at 4 °C. The pellet was were performed in accordance with relevant guidelines suspended in 200 μL of PBS. and regulations. BTBR mice were bred from adult pairs originally purchased from The Jackson Laboratory (Bar Ex vivo imaging Harbor, ME). At 4 weeks of age, the first cohort of litter- Adult BTBR male mice (6–7 weeks) were given 5 μLof mate male mice was randomly assigned for basal behav- labeled exosomes or labeled MSC via intranasal adminis- ioral experiments followed by intranasal administration tration (N = 2 per condition). Another adult BTBR male of saline (BTBR saline, N = 7) or MSC-exo (BTBR mice (6–7 weeks) were given 100 μL of intravenously MSC-exo, N = 7). For the pup retrieval experiment, the with labeled exosomes or labeled MSC. (The tail vain first cohort of littermate female mice was randomly was warmed using water and exosomes were directly assigned at 5 weeks of age to saline or to MSC-exo. At injected to the vain, no anesthetics used, N = 2 per con- the end of the treatment, random groups of females dition.) The number of exosomes per mouse was equal were placed at breeding colonies with BTBR male mice between the intranasal/intravenous administrations (1 male 2 females, BTBR mothers). At days 1–2 post (19.05 × 10 particles) as well as the number of MSC. whelping, random groups of virgin females were placed For 24-h post administration, mice were perfused and with the mothers for 4 days to gain experience (BTBR fixated with PBS and 4% paraformaldehyde (PFA). The experienced virgins). The rest of the mice were left at brains were incubated in PFA for 24 h followed by 30% their home cage (BTBR naïve virgins) (Additional file 5: sucrose for 48 h and stored at 4 °C. Whole brain fluores- Table S1 for number of females in each group). cence imaging was taken with Maestro CRi, excitation C57BL mice were bred from adult pairs originally pur- filter 523, and emission filter 560. For immunostaining chased from The Jackson Laboratory (Bar Harbor, ME). analysis, the brains were frozen in chilled At 4 weeks of age, the first cohort of littermate male 2-methylbutane (Sigma-Aldrich), stored at 4 °C, and mice was randomly assigned for basal behavioral experi- subsequently sectioned into slices measuring 10 lm. ments followed by intranasal administration of saline Slides were incubated with blocking solution (5% goat/ (C57BL saline, N = 7) or MSC-exo (C57BL MSC-exo). donkey serum, 1% BSA, 0.5% Triton X-100 in PBS) for For the pup retrieval experiment, the first cohort of lit- 1 h. Thereafter, slides were incubated overnight at 4 °C termate female mice was randomly assigned to saline at with primary antibody in blocking solution (mouse 5 weeks of age. At the end of the treatment, random anti-NueN, 1:500, Abcam) and secondary antibody in groups of females were placed at breeding colonies with blocking solution (goat anti-mouse Alexa 488, 1:500, C57BL male mice (1 male 2 females, C57BL mothers). Molecular Probes, Invitrogen) for 1–2 h at room On days 1–2 post-whelping, random groups of virgin fe- temperature. Next, nuclei were counterstained with males were placed with the mothers for 4 days to gain DAPI (1:500; Sigma-Aldrich). Sections were ultimately experience (C57BL experienced virgins). The rest of the mounted with fluorescent mounting solution (Fluoro- mice remained at their home cage (C57BL naïve virgins) mount-G, Southern Biotech), covered with a cover slide, (Additional file 5: Table S1 for number of females in and sealed with nail polish. each group). Perets et al. Molecular Autism (2018) 9:57 Page 10 of 12 For comparison between NSC-exo and MSC-exo, an- encounter to prevent extremely high sexual arousal and other group of mice was randomly assigned. They were mating behaviors. The encounters were filmed for raised as previously described and treated with intranasal male-female interaction analysis. In the study, all males administration of MSC-exo (N = 7), NSC-exo (N = 5), or and females were sexually naïve. Females were in the saline (N = 7). For comparison between saline and same cage in order to synchronize their estrus cycle and MSC-exo+protK in behavioral experiments, another had met the males on the same day. Vocalizations were group of mice was randomly assigned. They were raised recorded with Avisoft-RECORDER v. 4.2.21 recording as previously described and treated with intranasal ad- program. The settings included a sampling rate of ministration of MSC-exo+protK (N = 8) or saline (N = 6). 250 kHz and a format of 16 bit. For spectrogram gener- Complete summary of all the mice in all the experiments ation, recordings were transferred to Avisoft-SASLab Pro is found in Table S2. Version 5.2.07 and a fast Fourier transformation (FFT) Mice were treated with intranasal MSC-exo or saline was conducted. Spectrograms were generated with an FFT for 12 days, 5 μL a day, and every other day (total of length of 256 points and a time window overlap of 50% 30 μL per mouse). Administration was done using a gen- (100% Frame, FlatTop window). The number of syllables tle pipette of 2.5 μL per nostril with no anesthetics. was quantified automatically by Avisoft-SASLab, and syl- lable classification was done by MATLAB using a classifi- Behavioral tests cation algorithm [47] (Additional file 5: Supplementary Reciprocal dyadic social interaction test Materials and Methods). The reciprocal dyadic social interaction test [46]was conducted using a 5-week-old male C57BL/6 stranger Pup retrieval mouse as the social stimulus. The stranger mouse was C57BL and BTBR females were tested for their latency placed in a 40 × 40 × 20 cm cage with the test mouse. to retrieve pups and bring them back to the nest. Each Prior to the test, both mice were isolated for 1 h. In one was tested with three pups, and the duration of each addition, both mice were recorded for 20 min, with the trial was 180 s. In addition, the number of retrieved pups last 10 min quantified by an observer blind to treatment. and time required for each pup retrieval was measured. Cowlog V3 software was used to score the social contact During the test, all females were taken out of the home initiated by the test mouse (Helsinki University, Helsinki, cage and were placed back one at a time for a 5-min Finland). Scoring was determined by the duration that acclimation with the pups prior to testing. Each of the the mice had engaged the stranger mouse in social be- pups was between 1 and 3 days old [18, 19]. The tests haviors. The social behaviors that were quantified in- were filmed with a SAMSUNG 11mp camera. cluded nose to nose sniffing (i.e., approach to the front of the stranger), nose to genital sniffing (i.e., approach to Additional files the back of the stranger), and attacking (i.e., test mouse initiates a fight with the stranger mouse). Active avoid- Additional file 1: BTBR male control male to male social interaction. ance (i.e., test mouse deliberately avoids interaction (AVI 5543 kb) when the stranger mouse initiates it) was considered an Additional file 2: BTBR male MSC-exo male to male social interaction. antisocial behavior. During social interactions, the time (mov 6092 kb) spent in repetitive behaviors (i.e., self-grooming and dig- Additional file 3: BTBR mother control pup retrieval. (AVI 5543 kb) ging) was also observed and quantified. Additional file 4: BTBR mother MSC-exo pup retrieval. (AVI 6092 kb) Additional file 5: Figure S1. MSC-exo decreases repetitive behavior of self-grooming. Intra-subject comparison shows BTBR MSC-exo group Repetitive behaviors not during social interaction spent significantly less time in repetitive behaviors, while BTBR saline and Mice were placed alone in an arena with dimensions C57BL saline showed no difference between basal and post treatment 40 × 40 × 20 cm for 20 min. The last 10 min was quanti- behavior (paired T test). Inter-group comparison shows BTBR MSC-exo is significantly different than BTBR saline group in time spent in repetitive fied for grooming and digging. While observing the behaviors. (ANOVA1, Bonferroni). Data is presented as mean + SEM. **p < grooming behavior, the mice were placed in a clean cage 0.05. Figure S2. MSC-exo had no significant effect on C57BL behavior. A. absent from wood-chips in order to prevent digging. C57BL mice were tested for baseline behavior (baseline) and after MSC- exo intranasal administration (post-treatment) in the tests of social, Also, self-grooming was not measured while observing antisocial interaction, and repetitive behaviors. No significant differences the digging behavior. were found in any of the behaviors (paired T test, p > 0.05). B. C57BL MSC-exo mice presented no difference in their number of USV compared to saline-treated group (unpaired T test, p > 0.05). Data is presented as Ultrasonic vocalizations mean + SEM. Figure S3. MSC-exo but not NSC-exo significantly ameliorates Both BTBR and C57BL males met C57BL/6 females. male to male social interaction, repetitive behaviors, and male to female Each male was placed in a separate cage for 1 h, and a ultrasonic vocalizations of BTBR mice. A. male to male social interaction. B. repetitive behaviors. C. male to female ultrasonic vocalizations (ANOVA 1, female was then placed in the cage. Ultrasonic vocaliza- Bonfferoni *p <0.05, **p < 0.01, ***p < 0.001). Data is presented as mean + tions were recorded for the first five minutes of Perets et al. Molecular Autism (2018) 9:57 Page 11 of 12 4. Segal-Gavish H, Karvat G, Barak N, et al. Mesenchymal stem cell SEM. Figure S4. Full Western blot gels: A. Calnexin as negative marker for transplantation promotes neurogenesis and ameliorates autism related MSC-exo B. CD9 as positive marker of MSC-exo. C. CD63 as positive marker behaviors in BTBR mice. Autism Res. 2016;9(1):17–32. https://doi.org/10. of MSC-exo and for reduction after protK treatment. D. CD9 as positive 1002/aur.1530. marker of MSC-exo and for reduction after ProtK treatment. Table S1. Number 5. Perets N, Segal-Gavish H, Gothelf Y, Barzilay R, Barhum Y, Abramov N, Hertz of females tested in maternal behavioral experiment. Table S2. Number of S, Morozov D, London M, Offen D. Long term beneficial effect of mice at each group in the behavioral experiments. (DOCX 1010 kb) neurotrophic factorssecreting mesenchymal stem cells transplantation in the BTBR mouse model of autism. Behav Brain Res. 2017. https://doi.org/10. Abbreviations 1016/j.bbr.2017.03.047. ASD: Autism spectrum disorders; BTBR: BTBR T+tf/J; MSC: Mesenchymal stem 6. Ankrum J a, Ong JF, Karp JM. Mesenchymal stem cells: immune evasive, not cells; MSC-exo: Exosomes secreted from MSC immune privileged. Nat Biotechnol. 2014;32(3):252–60. https://doi.org/10. 1038/nbt.2816. Acknowledgements 7. Ng KS, Kuncewicz TM, Karp JM. Beyond hit-and-run: stem cells leave a The authors would like to thank the following for their contributions in the lasting memory. Cell Metab. 2015;22(4):541–3. https://doi.org/10.1016/j.cmet. research: Shai Israel for his kind help in the MATLAB code and Michael Anbar 2015.09.019. for his kind help in consulting and helping with Western blots. 8. Zhang Y, Chopp M, Liu XS, Katakowski M, Wang X. Exosomes derived from mesenchymal stromal cells promote axonal growth of cortical neurons. Funding 2016. https://doi.org/10.1007/s12035-016-9851-0. N.P. received funding scholarship from Sagol School of Neuroscience, Tel 9. Braccioli L, Van Velthoven C, Heijnen CJ. Exosomes: a new weapon to treat Aviv University. the central nervous system. 2014:113–9. https://doi.org/10.1007/s12035-013- 8504-9. Availability of data and materials 10. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome- The datasets used and/or analyzed during the current study are available mediated transfer of mRNAs and microRNAs is a novel mechanism of from the corresponding author on reasonable request. genetic exchange between cells. Nat Cell Biol. 2007;9(6):654–9. https://doi. org/10.1038/ncb1596. Authors’ contributions 11. Yu B, Zhang X, Li X. Exosomes derived from mesenchymal stem cells. 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Intranasal administration of exosomes derived from mesenchymal stem cells ameliorates autistic-like behaviors of BTBR mice

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Medicine & Public Health; Neurology; Neurosciences; Neuropsychology; Psychiatry; Pediatrics; Human Genetics
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Abstract

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. They have been steadily increasing in children over the past several years, with no effective treatment. BTBR T+tf/J (BTBR) mice are an accepted model of evaluating autistic-like behaviors as they present all core symptoms of ASD. We have previously shown that transplantation of human bone marrow mesenchymal stem cells (MSC) to the lateral ventricles of BTBR mice results in long lasting improvement in their autistic behavioral phenotypes. Recent studies point exosomes as the main mediators of the therapeutic effect of MSC. Here, we tested whether treatment with the exosomes secreted from MSC (MSC-exo) will show similar beneficial effects. We found that intranasal administration of MSC- exo increased male to male social interaction and reduced repetitive behaviors. Moreover, the treatment led to increases of male to female ultrasonic vocalizations and significant improvement in maternal behaviors of pup retrieval. No negative symptoms were detected following MSC-exo intranasal treatments in BTBR or healthy C57BL mice. The marked beneficial effects of the exosomes in BTBR mice may translate to a novel, non-invasive, and therapeutic strategy to reduce the symptoms of ASD. Introduction Moreover, the behavioral effect of MSC on BTBR mice Autism spectrum disorders (ASD) are neurodevelop- had lasted for 6 months despite the fact that the MSC mental disabilities characterized by three core symp- themselves do not survive in the transplanted tissue for toms: severe impairment of social interactions and such an extended period [5]. Such phenomenon has communication skills, increased repetitive behaviors, been reported in the literature under the title “hit and and cognitive inflexibility [1]. In this study, we used the run” meaning MSC have the ability to leave a lasting inbred mouse strain BTBR T+tf/J (BTBR) that incorpo- “memory” on the tissue, even after the MSC cells have rates multiple behavioral phenotypes relevant to all three been degraded [6]. It has been suggested that the MSC diagnostic symptoms of autism. BTBR present signifi- therapeutic effect is mainly mediated by their secretome cantly reduced social approach, low reciprocal social in- to the tissue [7]. The secretome is referred to as the teractions, and impaired juvenile play in comparison to complete repertoire of molecules and extracellular vesi- the C57BL controls [2, 3]. Using this model, we have re- cles secreted from MSC. Yet, it has been found that the cently shown that MSC transplantation to the lateral nano-vesicles, exosomes, are the major mediators be- ventricles of the brain of BTBR mice has the ability to tween the MSC and the tissue [8, 9]. ameliorate their core autistic-like behaviors [4]. Exosomes were initially thought to be a mechanism for removing unneeded membrane proteins from reticu- * Correspondence: [email protected] locytes. However, recent studies have shown that they Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel are also used for cell to cell communication through the Sacklar School of Medicine, Department of Human Genetics and carrying of genetic information from between cells [10]. Biochemistry, Tel Aviv University, Tel Aviv, Israel Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Perets et al. Molecular Autism (2018) 9:57 Page 2 of 12 Several studies have reported that MSC-exo have func- MSC-exo improves male to male social interaction and tions similar to those of MSC, such as repairing dam- reduces repetitive behaviors during social interaction aged tissue, suppressing inflammatory responses, and Mice were tested for male to male interaction before modulating the immune system [11, 12]. Exosomes con- and after MSC-exo or saline administration. Intra-sub- tain mainly proteins, as well as RNA and a large number ject analysis showed that the BTBR MSC-exo mice of micro RNAs. Approximately, 25% of these proteins group had spent significantly more time in social inter- and RNA play a role in cell growth and maintenance actions after the treatment whereas the BTBR saline and [13, 14]. A key advantage of MSC-exo over MSC is their C57BL saline groups did not show any change (paired t ability to enter the brain with ease following intranasal test, t < 0.0001). Comparison analysis between the administration [15, 16]. We have previously demon- groups showed that the BTBR mice of both groups had strated that exosomes loaded with gold nanoparticles spent significantly less time engaging in social inter- can cross the blood-brain barrier via intranasal adminis- action in comparison to C57BL basal behavior tration and can be visualize in vivo inside the brain [17]. (ANOVA1, F = 14.4, p < 0.01, Bonferroni). Exosome 2,18 Here, we show that BTBR mice that were treated with treatment dramatically increased social interactions in MSC-exo via intranasal administration present signifi- the BTBR MSC-exo group in comparison to BTBR mice cant improvement in the social interaction domain, treated with saline and is comparable to the C57BL mice ultrasonic communication, and repetitive behavior. group (ANOVA1, F = 9.44, p < 0.01, Bonferroni) 2,16 Moreover, we show for the first time that BTBR mothers (Fig. 2a, Additional files 1 and 2). that were treated with MSC-exo presented significant During the social interaction test, the repetitive behav- improvement in maternal pup retrieval behavior. ior was also measured. Intra-subject analysis showed that the BTBR MSC-exo-treated group spent signifi- cantly less time in repetitive behaviors while both the Results BTBR saline and C57BL saline mice groups did not MSC-exo characterization show any changes in comparison to their basal behaviors MSC-exo were characterized by NanoSight. The average (paired t test, t < 0.001). A comparison analysis between size was 114 ± 2.9 nm, and the average concentration groups showed that before treatment, there was no dif- was 3.81 × 10 particles/μL (Fig. 1a, b). Western blot ference between BTBR MSC-exo and BTBR saline basal analysis indicates that the MSC-exo express CD9 and behaviors, and that both groups significantly differed CD63, while we could not detect it in the MSC lysate from the basal behavior of the C57BL saline group (not shown). In contrast, Calnexin was undetectable in (ANOVA1, F = 13.71, p < 0.001, Bonferroni). After 2,18 the MSC-exo and found in the MSC lysate, indicating treatment, The BTBR MSC-exo group was not signifi- for the purity of the exosomes [18] (Fig. 1c). cantly different from the BTBR saline group and the Fig. 1 Characterization of MSC-exo. a Visualization of the exosomes using NanoSight technology. b Analysis of size distribution and concentrations. c Western blot analysis of CD9 which exists in exosomes but not in the MSC lysate in contrast to Calnexin which is absent in exosomes and found in the MSC lysate Perets et al. Molecular Autism (2018) 9:57 Page 3 of 12 Fig. 2 MSC-exo increased male to male social interaction and repetitive behaviors during social interaction. Each group was tested for basal behaviors (gray) and was re-tested 3 weeks after treatment (saline or MSC-exo, black). a Intra-subject comparison showed that the BTBR MSC-exo group had spent a significantly longer time engaging in social interaction with other stranger male mice (paired T-test). Inter-group comparison showed that the BTBR MSC-exo group had spent significantly more time engaging in social interaction compared to BTBR saline (ANOVA1, Bonferroni). b Intra-subject comparison of BTBR MSC-exo group had spent significantly less time in repetitive behaviors (paired T test). Inter-group comparison showed BTBR MSC-exo is not significantly different than both BTBR saline and C57BL saline groups in time spent in repetitive behaviors. (ANOVA1, Bonferroni). The data is presented as mean + SEM. ***p < 0.001 C57BL group (Fig. 2b). In repetitive behavior tests that compared to saline-treated group (ANOVA1, F = 2,14 were outside of the context of social interaction, BTBR 4.28, p < 0.05, Bonferroni). Importantly, MSC-exo and MSC-exo spent significantly less time in repetitive saline groups were also used as a biological replication grooming and digging in intra-subject analysis compared of the social interaction with different seven mice per to their basal behaviors (t < 0.05). In groups, compari- group (Additional file 5: Figure S3). sons showed that they were significantly different from the BTBR saline group but not from the C57BL saline MSC-exo improved male to female ultrasonic group (ANOVA1, F = 13.83, p < 0.01, Bonferroni) vocalizations 2,18 (Additional file 5: Figure S1). Importantly, C57BL In general, male mice emitted a large number of com- MSC-exo mice did not present any behavioral differ- plex ultrasonic vocalizations (USVs) when interacting ences in neither social interactions, antisocial interac- with adult females (Fig. 3a, experimental demonstration). tions, nor repetitive behaviors during social contact As seen qualitatively from the spectrograms in our (Additional file 5: Figure S2A). In comparison between study, BTBR MSC-exo vocalizations became more com- MSC-exo to exosomes isolated from neuronal stem cells plex and longer compared to the BTBR saline group, (NSC-exo), we found that only the MSC-exo-treated making them more similar to C57BL (Fig. 3b). During group presented a significant increase in social inter- the first 5 min of interaction with females, BTBR saline action, a reduction in repetitive behaviors, and a signifi- mice emitted 317 ± 39.4 syllables, BTBR MSC-exo emit- cant increase in communication while NSC-exo-treated ted 571 ± 74 (180% more), and C57BL emitted 854.5 ± mice did not present the same behavioral differences 65.2 syllables (Fig. 3c, ANOVA1, F = 19.2, p < 0.001, 2,18 Perets et al. Molecular Autism (2018) 9:57 Page 4 of 12 Fig. 3 Specific improvement of ultrasonic vocalizations after intranasal administration of MSC-exo. a visualization of the experimental set, right panel: male and female mice in courtship meeting. Left panel: real-time spectrogram of ultrasonic vocalizations. b Example of differences in spectrogram of C57BL, saline-treated, and MSC-exo-treated mice suggests that MSC-exo-treated mice improved in complexity of syllables. c BTBR MSC-exo had more syllables of ultrasonic vocalizations compared to saline BTBR, yet less syllables than C57BL mice. d and e Automatic classification of syllables showed significant reduction in the use of simple syllables by BTBR MSC-exo and C57BL saline compared to BTBR saline. Classification also showed increased use in complex and down syllables by BTBR MSC-exo and C57BL saline compared to BTBR saline. f There was not a significant difference between the groups in social contact (nose to nose and nose to genitals), indicating a specific effect on ultrasonic vocalizations. (ANOVA1, Bonferroni). Data is presented as mean + SEM. **p < 0.01, ***p < 0.001 Bonferroni). Classification of syllables revealed signifi- 57%, and C57BL saline used 60% (ANOVA1, F = 2,18 cant differences between the syllable types used by 49.44, p < 0.001, Bonferroni). For complex syllables, BTBR saline and BTBR MSC-exo (Fig. 3d, e). For simple BTBR saline used 2%, BTBR MSC-exo used 23%, and syllables, BTBR saline used 89%, BTBR MSC-exo used C57BL saline used 10% (ANOVA1, F = 30.61, p < 2,18 Perets et al. Molecular Autism (2018) 9:57 Page 5 of 12 0.001, Bonferroni). For down syllables, BTBR saline used was 10.4 s ± 1.08 s. In contrast, only one of eight BTBR 7%, BTBR MSC-exo used 16%, and C57BL saline used saline mothers retrieved two pups (in 156 s), without 26% (ANOVA1, F = 25.23, p < 0.001, Bonferroni). For bringing them back to the nest. Altogether, only 2/24 2,18 up syllables, BTBR saline used 1%, BTBR MSC-exo used pups were retrieved. After treatment, BTBR MSC-exo 1%, and C57BL saline used 6% (ANOVA1, F = 41.22, mothers retrieved all pups (18/18) at a mean time of 2,18 p < 0.001, Bonferroni). 25.24 s ± 5.7 s (ANOVA1 on time of retrieval, F = 2,48 There was no significant difference between groups in 332.5, p < 0.0001). C57BL saline trained virgins retrieved time spent sniffing the female’s genitals or faces, mean- all pups (15/15) with a total average time of 38.3 s ± ing that the effect seen in the ultrasonic vocalizations 3.3 s. BTBR saline trained virgins mostly did not retrieve was not impacted by the pheromones of the females the pups besides for one female that had retrieved two (Fig. 3f). C57BL mice that were treated with MSC-exo pups with a mean time of 152 s. Altogether, 2/21 pups showed similar number of syllables compared to C57BL were retrieved by BTBR saline trained virgins. BTBR saline group (Additional file 5: Figure S2B). In compari- MSC-exo trained virgins retrieved 7/9 pups at a total son between MSC-exo and NSC-exo, only the BTBR averagetimeof83.3±16.02s(ANOVA1ontimeof MSC-exo group presented a significant increase in num- retrieval, F = 29.47, p < 0.0001). Neither the C57BL 2,42 ber of syllables compared to BTBR saline group naïve virgins (0/21) nor the BTBR naïve virgins had (ANOVA1, F = 9.44, p < 0.01, Additional file 5: Figure retrieved the pups (0/21) (Fig. 4c, Additional files 3 2,14 S3B). This result also used as biological replication of and 4). We had assumed that the MSC-exo naïve vir- the vocalizations test. gins would also not retrieve the pups. MSC-EXO improves pup retrieval behavior Visualization of MSC and exosomes after intranasal We have performed pup-retrieval tests for MSC-exo- or delivery saline-treated mothers, naïve virgins, and experienced MAESTRO whole brain imaging was used to visualize virgins [19, 20]. The C57BL saline mothers retrieved all PKH26-labeled MSC and MSC-exo. After intranasal and pups (15/15), and their total average time for retrieval intravenous administration, MSC-exo can be visualized Fig. 4 MSC-exo improves maternal pup retrieval and learning of maternal pup retrieval behaviors. a Experiment’s timeline. b Visualization of normal pup retrieval behavior vs. autistic-like behavior. c C57BL and BTBR mice groups were treated with saline or MSC-exo were tested for pup retrieval. Data is presented as mean + SEM. **p < 0.01, ***p < 0.001 Perets et al. Molecular Autism (2018) 9:57 Page 6 of 12 fluorescently while MSC cannot be detected (Fig. 5a). Discussion Immunostaining indicated that MSC-exo penetrate the We have previously found that surgical transplantation brain parenchyma and are found in the cells of the tissue of MSC to the brain ameliorates the autistic-like behav- (Fig. 5b, c). iors of BTBR mice [4]. Furthermore, we have shown that this effect could last for 6 months post a single treat- MSC-exo migration and efficacy are dependent on their ment, even though MSC does not survive for an ex- membrane proteins tended period in the brain [5]. We have also The ability of MSC-exo to cross the blood-brain barrier demonstrated that MSC-exo efficiently cross the and to be uptake by cells in the brain is critical for the blood-brain barrier after intranasal administration com- therapeutic effect. To block the MSC-exo from being pares to intravenous injection, and can be visualized in uptake by the cells in the brain and to use them as con- vivo when they are loaded with gold nanoparticles [17]. trol, MSC-exo were treated with protease-k (ProtK) in In the current experiment, BTBR mice were treated via order to remove the membrane proteins. Immunostain- intranasal administration of MSC-exo followed by be- ings of the tissue indicated that the MSC-exo-protk de- havioral tests in all the ASD-like phenotypes presented livered intranasally have not been uptake into the cells by this model. Remarkably, the BTBR MSC-exo group at the same efficiency as MSC-exo (Fig. 6a as compared presented significant improvements in all the tested to Fig. 5b). To demonstrate the effectiveness of ProtK in ASD-like phenotypes. degrading the proteins on the exosomes’ membrane, we In social interaction, the BTBR MSC-exo group spent used Western blot analysis to CD9 and CD63 since they a significantly longer time engaging in interaction with are known to be overexpressing on the membrane of the the stranger male compared to the saline-treated mice exosomes and are commonly used for exosomes detec- and their own basal behaviors. BTBR MSC-exo had also tion and characterizations [42]. The Western blot presented a significant decrease in repetitive behaviors showed deletion of the membrane proteins CD9 and a of self-grooming and digging compared to their own reduction of CD63 (Fig. 6b). NanoSight analysis showed basal behavior and to the BTBR saline group. Their no significant change in the number and size of scores had become closer to the C57BL saline group. MSC-exo-protK compared to MSC-exo (Fig. 6c). BTBR Deficits in social communication are a core symptom mice that were treated with MSC-exo-protK did not of ASD in children [1]. It has been reported that BTBR demonstrate behavioral differences in social interaction mice present unusual repertoire of male to female ultra- and repetitive behaviors (Fig. 6d). sonic vocalizations compared to normal C57BL mice Fig. 5 Comparison between MSC-exo and MSC labeled with PKH26 visualization in the brain. a From left to right: MSC-exo intranasal, MSC-exo intravenous, MSC intranasal, and MSC intravenous. MSC-exo cross the blood brain barrier, both intranasally and intravenously, while MSC much less. b Immunostaining of neurons, DAPI and PKH26 labeled MSC-exo after intranasal administration. c Sagittal section of the BTBR MSC-exo brain after intranasal administration of PKH26 labeled MSC-exo (top) and Alan brain atlas (bottom) Perets et al. Molecular Autism (2018) 9:57 Page 7 of 12 Fig. 6 MSC-exo-protK was not found inside the cells and the mice did not present behavioral differences. a MSC-exo-protK were mostly located outside of the cells, unlike MSC-exo (Fig. 5b). b Western blot of MSC-exo-protK show deletion of CD9 and reduce CD63 compared to MSC-exo. c NanoSight analysis of MSC-exo-protK showed no significant change in number and size of MSC-exo-protK compared to MSC-exo (Fig. 1). d BTBR mice treated with MSC-exo-protK did not present behavioral improvement compared to saline treated in social and repetitive behaviors [21]. We reported that BTBR MSC-exo mice had signifi- sexual arousal is an advantage. As compared, the C57BL cant improvement in the number of syllables compared MSC-exo group did not present behavioral changes in to the control BTBR saline group. Their number of sylla- their ultrasonic vocalizations and social interactions to- bles was closer to the C57BL group. To examine other ward both males and females. This finding may suggest features of ultrasonic vocalizations, such as complexity that MSC-exo administration leads to beneficial effects and classification of syllables, we have used an advanced and not to non-specific hyperactivity or behavioral side classification algorithm. This classification revealed sig- effects. nificant improvement in the complexity of syllables of Maternal behavior of BTBR mice has yet to be studied BTBR MSC-exo mice, making their ultrasonic vocaliza- extensively. Here, we observed significant differences be- tions repertoire closer to the C57BL saline group. Im- tween BTBR and C57BL for pup retrieval. C57BL mice portantly, the improvement in male-to-female ultrasonic exhibit “normal” behavior, and they created a nest for vocalizations did not seem to be caused by higher sexual their pups. If the pup was separated, they had quickly re- arousal as there was no significant difference between trieved it [18, 19]. In contrast, BTBR created a nest but the duration of interaction initiated by the males toward reacted slowly to the pup separation and nearly always the females in any of the groups. This finding was also had not brought the pup within 3 min. Interestingly, observed in our previous studies after MSC transplant- BTBR MSC-exo mothers demonstrated high scores in ation to BTBR mice [5]. Considering the growing recog- the pup retrieval test, with results comparable to C57BL nition that some adolescents and young adults with ASD saline mothers. Furthermore, a naïve C57BL virgin who may exhibit inappropriate sexual behaviors [22, 23], the spent a few days with a mother can learn this “experi- fact that MSC-exo treatment also does not enhance enced” behavior. BTBR saline females did not manage to Perets et al. Molecular Autism (2018) 9:57 Page 8 of 12 learn from other mothers (C57BL mothers) while BTBR (miR-222, miR-21) [39–41]. Yet, we are aware that MSC-exo had presented learning abilities. Maternal be- MSC-exo are complex vesicles containing hundreds of havior, as well as social interaction, acquires high-level proteins and RNA molecules; therefore, we cannot pin- synchronization of sensory input and behavioral out- point on the specific factors that led to the behavioral put. We suggestMSC-exo mayplayarole inthe difference and future study may be needed to uncover mechanisms of sensory integration, making its effect the mechanism of action that led MSC-exo-treated mice influential in the realms of the social domain symp- to behavioral amelioration. Moreover, since BTBR mice toms. Sensory integration and coordination deficits are multifactorial model of autism, it may be worth to have been suggested to be one of the underlying test MSC-exo effect of other models, including genetic- mechanism of the ASD patients [24–26]. ally modified mice such as shank3 mutation as well. Stem cell therapy has been previously used on ASD Altogether, our findings suggest that MSC-exo may be children with long-term beneficial effects [27]. Bone tested as a safe noninvasive treatment to ameliorate be- marrow MSC has been proven to be safe to use in sev- havioral symptoms of patients with ASD. eral clinical trials [28–30]. Mechanistically, bone marrow MSC was found efficient in promoting tissue regener- Materials and methods ation, immunomodulation, and inflammatory reduction Mesenchymal stem cells preparation [31–33]. We reported that the transplantation of MSC Human MSC were purchased from Lonza (cat:PT-2501, to the lateral ventricles of BTBR mice leads to increased Basel, Switzerland). Cells were cultured and expanded as neurogenesis and BDNF in the hippocampus [4]. Al- previously described [42]. Before exosome collection, the though it is clear that MSC have beneficial properties cells were cultured in exosome-free platelets for 3 days that can be used safely for clinical purposes, recent evi- and this medium was then collected. dence shows that the therapeutic effect of MSC is largely mediated via the secretion of exosomes that contain im- Exosomes purification protocol portant molecular information [34, 35]. Our findings The exosomes were purified by taking the culture fluid support this concept, and our study demonstrates that a and centrifuging it for 10 min at 300g. The supernatant remarkable behavioral effect, on all ASD-like phenotypes was recovered and centrifuged for 10 min at 2000g. of BTBR mice, can be achieved by simply using Once again, the supernatant was recovered and centri- MSC-exo rather than MSC. We are aware that small fuged for 30 min at 10,000g. The supernatant was fil- number of mice per group is a limiting factor of our re- trated through a 0.22-μm filter and centrifuged for sults, yet the behavioral difference post MSC-exo treat- 70 min at 100,000g. The pellet containing the exosomes ment is remarkable. Furthermore, in social interaction and proteins was washed in PBS and then centrifuged and repetitive behaviors, each mouse was compared to for 70 min at 100,000g. The pellet containing the puri- its own basal behavior and also to saline-treated group fied exosomes was re-suspended in 200 μm of sterilized post the treatment, this experimental design was chosen PBS. Each centrifugation occurred at 4 °C [43]. to increase the confidence on the results. Mentionable, for the comparison between MSC-exo and NSC-exo, we used another MSC-exo-treated mice; therefore, the USV Neuronal stem cells preparation and social and repetitive behaviors post MSC-exo treat- Human neuronal stem cell lines (CTX0E03) were pur- ment were tested twice independently. chased from ReNeuron, UK. Cells were cultured and ex- The contents of MSC-exo have been characterized panded according to the company’s protocol. Cell lines using proteomics and RNA sequencing. Importantly, the were routinely cultured at 37 °C in tissue culture flasks capacity of the exosomes seems to be selectively pack- freshly coated with mouse laminin (20 μg/ml in aged. Therefore, some of the proteins and miRNA that DMEM:F12). Growth medium supplemented with are over expressed in the cells are packaged in exosomes 4-OHT was changed three times per week. When 70– while others have over-representation [14, 36]. While 90% confluent, cells were passaged using trypsin (0.25%) exosomes contain numerous variations of RNA, their for 5–10 min at 37 °C, followed by treatment with soy- miRNA repertoire has been spotlighted as a major can- bean trypsin inhibitor (0.25 mg/ml). Cells were spun didate for their effect in the host cell and the tissue. For down (800×g for 5 min at room temperature) and example, miRNA-143 was found to be related to the im- re-suspended at an appropriate density in full growth munomodulatory effect of MSC, and miRNA-10b was medium supplemented with 100 nM 4-OHT. The found related to their migration abilities [37, 38]. In NSC-exo were purified at the same protocol as addition, multiple miRNAs highly represented in MSC-exo, and a number of particles were matched per MSC-exo (miR-191, miR-222, miR-21) regulate cell cycle mouse using NANOsight analysis (Merkel technologies progression and proliferation and modulate angiogenesis LTD, Israel). Perets et al. Molecular Autism (2018) 9:57 Page 9 of 12 Exosomes characterization Proteinase-k treatment to MSC-exo NanoSight technology (Merkel technologies LTD, Israel) Two hundred microliters of PKH26 labeled exosomes was used to characterize the size and concentration of were incubated with 7 μL proteinase-K (Roche Diagnos- the exosomes (3.81 × 10 particles/μL). Lysates of tica GmbH, Germany) and 750 μL BPS in 55 °C for MSC-exo were subject for Western blot using SDS poly- 10 min [45]. Proteinase-K inhibitor (7 μL) (phenylmetha- acrylamide gel. Proteins were transferred to Immobilon®-P nesulfonyl fluoride, Sigma) was added to the solution and membranes (Millipore, Amsterdam, The Netherlands), in- was suspended for 2 h in 70 ml BPS for ultracentrifugation. cubated in 5% milk for 1 h, and probed overnight at 4 °C Finally, the pellet (MSC-exo-protk) was re-suspended in with CD9 antibody (ABCAM), CD63 (ABCAM), and Cal- 200ul PBS. For behavioral treatment, non-labeled MSC nexin (ABCAM). After three washes in TBS-Tween 20, -exo-protk were used. Proteinase-k-treated MSC-exo were the membranes were incubated with the secondary anti- characterized with NANO-sight and Western blot. body for 1 h and re-washed. Animals Exosomes labeling Mice were placed under a 12-h light/12-h dark condition Exosomes were labeled with PKH26 (Sigma-Aldrich) and grown in individual ventilated cages with access to [44, 45]. PKH26 (2 μL) in 500 μL diluent was then added food and water ad libitum. All experimental protocols to 50 μL exosomes in PBS for 5 min of incubation. Exo- were approved by the Tel Aviv University Committee of somes were suspended in 70 ml PBS and were centri- Animal Use for Research and Education. All methods fuged for 90 min at 100,000g at 4 °C. The pellet was were performed in accordance with relevant guidelines suspended in 200 μL of PBS. and regulations. BTBR mice were bred from adult pairs originally purchased from The Jackson Laboratory (Bar Ex vivo imaging Harbor, ME). At 4 weeks of age, the first cohort of litter- Adult BTBR male mice (6–7 weeks) were given 5 μLof mate male mice was randomly assigned for basal behav- labeled exosomes or labeled MSC via intranasal adminis- ioral experiments followed by intranasal administration tration (N = 2 per condition). Another adult BTBR male of saline (BTBR saline, N = 7) or MSC-exo (BTBR mice (6–7 weeks) were given 100 μL of intravenously MSC-exo, N = 7). For the pup retrieval experiment, the with labeled exosomes or labeled MSC. (The tail vain first cohort of littermate female mice was randomly was warmed using water and exosomes were directly assigned at 5 weeks of age to saline or to MSC-exo. At injected to the vain, no anesthetics used, N = 2 per con- the end of the treatment, random groups of females dition.) The number of exosomes per mouse was equal were placed at breeding colonies with BTBR male mice between the intranasal/intravenous administrations (1 male 2 females, BTBR mothers). At days 1–2 post (19.05 × 10 particles) as well as the number of MSC. whelping, random groups of virgin females were placed For 24-h post administration, mice were perfused and with the mothers for 4 days to gain experience (BTBR fixated with PBS and 4% paraformaldehyde (PFA). The experienced virgins). The rest of the mice were left at brains were incubated in PFA for 24 h followed by 30% their home cage (BTBR naïve virgins) (Additional file 5: sucrose for 48 h and stored at 4 °C. Whole brain fluores- Table S1 for number of females in each group). cence imaging was taken with Maestro CRi, excitation C57BL mice were bred from adult pairs originally pur- filter 523, and emission filter 560. For immunostaining chased from The Jackson Laboratory (Bar Harbor, ME). analysis, the brains were frozen in chilled At 4 weeks of age, the first cohort of littermate male 2-methylbutane (Sigma-Aldrich), stored at 4 °C, and mice was randomly assigned for basal behavioral experi- subsequently sectioned into slices measuring 10 lm. ments followed by intranasal administration of saline Slides were incubated with blocking solution (5% goat/ (C57BL saline, N = 7) or MSC-exo (C57BL MSC-exo). donkey serum, 1% BSA, 0.5% Triton X-100 in PBS) for For the pup retrieval experiment, the first cohort of lit- 1 h. Thereafter, slides were incubated overnight at 4 °C termate female mice was randomly assigned to saline at with primary antibody in blocking solution (mouse 5 weeks of age. At the end of the treatment, random anti-NueN, 1:500, Abcam) and secondary antibody in groups of females were placed at breeding colonies with blocking solution (goat anti-mouse Alexa 488, 1:500, C57BL male mice (1 male 2 females, C57BL mothers). Molecular Probes, Invitrogen) for 1–2 h at room On days 1–2 post-whelping, random groups of virgin fe- temperature. Next, nuclei were counterstained with males were placed with the mothers for 4 days to gain DAPI (1:500; Sigma-Aldrich). Sections were ultimately experience (C57BL experienced virgins). The rest of the mounted with fluorescent mounting solution (Fluoro- mice remained at their home cage (C57BL naïve virgins) mount-G, Southern Biotech), covered with a cover slide, (Additional file 5: Table S1 for number of females in and sealed with nail polish. each group). Perets et al. Molecular Autism (2018) 9:57 Page 10 of 12 For comparison between NSC-exo and MSC-exo, an- encounter to prevent extremely high sexual arousal and other group of mice was randomly assigned. They were mating behaviors. The encounters were filmed for raised as previously described and treated with intranasal male-female interaction analysis. In the study, all males administration of MSC-exo (N = 7), NSC-exo (N = 5), or and females were sexually naïve. Females were in the saline (N = 7). For comparison between saline and same cage in order to synchronize their estrus cycle and MSC-exo+protK in behavioral experiments, another had met the males on the same day. Vocalizations were group of mice was randomly assigned. They were raised recorded with Avisoft-RECORDER v. 4.2.21 recording as previously described and treated with intranasal ad- program. The settings included a sampling rate of ministration of MSC-exo+protK (N = 8) or saline (N = 6). 250 kHz and a format of 16 bit. For spectrogram gener- Complete summary of all the mice in all the experiments ation, recordings were transferred to Avisoft-SASLab Pro is found in Table S2. Version 5.2.07 and a fast Fourier transformation (FFT) Mice were treated with intranasal MSC-exo or saline was conducted. Spectrograms were generated with an FFT for 12 days, 5 μL a day, and every other day (total of length of 256 points and a time window overlap of 50% 30 μL per mouse). Administration was done using a gen- (100% Frame, FlatTop window). The number of syllables tle pipette of 2.5 μL per nostril with no anesthetics. was quantified automatically by Avisoft-SASLab, and syl- lable classification was done by MATLAB using a classifi- Behavioral tests cation algorithm [47] (Additional file 5: Supplementary Reciprocal dyadic social interaction test Materials and Methods). The reciprocal dyadic social interaction test [46]was conducted using a 5-week-old male C57BL/6 stranger Pup retrieval mouse as the social stimulus. The stranger mouse was C57BL and BTBR females were tested for their latency placed in a 40 × 40 × 20 cm cage with the test mouse. to retrieve pups and bring them back to the nest. Each Prior to the test, both mice were isolated for 1 h. In one was tested with three pups, and the duration of each addition, both mice were recorded for 20 min, with the trial was 180 s. In addition, the number of retrieved pups last 10 min quantified by an observer blind to treatment. and time required for each pup retrieval was measured. Cowlog V3 software was used to score the social contact During the test, all females were taken out of the home initiated by the test mouse (Helsinki University, Helsinki, cage and were placed back one at a time for a 5-min Finland). Scoring was determined by the duration that acclimation with the pups prior to testing. Each of the the mice had engaged the stranger mouse in social be- pups was between 1 and 3 days old [18, 19]. The tests haviors. The social behaviors that were quantified in- were filmed with a SAMSUNG 11mp camera. cluded nose to nose sniffing (i.e., approach to the front of the stranger), nose to genital sniffing (i.e., approach to Additional files the back of the stranger), and attacking (i.e., test mouse initiates a fight with the stranger mouse). Active avoid- Additional file 1: BTBR male control male to male social interaction. ance (i.e., test mouse deliberately avoids interaction (AVI 5543 kb) when the stranger mouse initiates it) was considered an Additional file 2: BTBR male MSC-exo male to male social interaction. antisocial behavior. During social interactions, the time (mov 6092 kb) spent in repetitive behaviors (i.e., self-grooming and dig- Additional file 3: BTBR mother control pup retrieval. (AVI 5543 kb) ging) was also observed and quantified. Additional file 4: BTBR mother MSC-exo pup retrieval. (AVI 6092 kb) Additional file 5: Figure S1. MSC-exo decreases repetitive behavior of self-grooming. Intra-subject comparison shows BTBR MSC-exo group Repetitive behaviors not during social interaction spent significantly less time in repetitive behaviors, while BTBR saline and Mice were placed alone in an arena with dimensions C57BL saline showed no difference between basal and post treatment 40 × 40 × 20 cm for 20 min. The last 10 min was quanti- behavior (paired T test). Inter-group comparison shows BTBR MSC-exo is significantly different than BTBR saline group in time spent in repetitive fied for grooming and digging. While observing the behaviors. (ANOVA1, Bonferroni). Data is presented as mean + SEM. **p < grooming behavior, the mice were placed in a clean cage 0.05. Figure S2. MSC-exo had no significant effect on C57BL behavior. A. absent from wood-chips in order to prevent digging. C57BL mice were tested for baseline behavior (baseline) and after MSC- exo intranasal administration (post-treatment) in the tests of social, Also, self-grooming was not measured while observing antisocial interaction, and repetitive behaviors. No significant differences the digging behavior. were found in any of the behaviors (paired T test, p > 0.05). B. C57BL MSC-exo mice presented no difference in their number of USV compared to saline-treated group (unpaired T test, p > 0.05). Data is presented as Ultrasonic vocalizations mean + SEM. Figure S3. MSC-exo but not NSC-exo significantly ameliorates Both BTBR and C57BL males met C57BL/6 females. male to male social interaction, repetitive behaviors, and male to female Each male was placed in a separate cage for 1 h, and a ultrasonic vocalizations of BTBR mice. A. male to male social interaction. B. repetitive behaviors. C. male to female ultrasonic vocalizations (ANOVA 1, female was then placed in the cage. Ultrasonic vocaliza- Bonfferoni *p <0.05, **p < 0.01, ***p < 0.001). Data is presented as mean + tions were recorded for the first five minutes of Perets et al. Molecular Autism (2018) 9:57 Page 11 of 12 4. Segal-Gavish H, Karvat G, Barak N, et al. Mesenchymal stem cell SEM. Figure S4. Full Western blot gels: A. Calnexin as negative marker for transplantation promotes neurogenesis and ameliorates autism related MSC-exo B. CD9 as positive marker of MSC-exo. C. CD63 as positive marker behaviors in BTBR mice. Autism Res. 2016;9(1):17–32. https://doi.org/10. of MSC-exo and for reduction after protK treatment. D. CD9 as positive 1002/aur.1530. marker of MSC-exo and for reduction after ProtK treatment. Table S1. Number 5. Perets N, Segal-Gavish H, Gothelf Y, Barzilay R, Barhum Y, Abramov N, Hertz of females tested in maternal behavioral experiment. Table S2. Number of S, Morozov D, London M, Offen D. Long term beneficial effect of mice at each group in the behavioral experiments. (DOCX 1010 kb) neurotrophic factorssecreting mesenchymal stem cells transplantation in the BTBR mouse model of autism. Behav Brain Res. 2017. https://doi.org/10. Abbreviations 1016/j.bbr.2017.03.047. ASD: Autism spectrum disorders; BTBR: BTBR T+tf/J; MSC: Mesenchymal stem 6. Ankrum J a, Ong JF, Karp JM. Mesenchymal stem cells: immune evasive, not cells; MSC-exo: Exosomes secreted from MSC immune privileged. Nat Biotechnol. 2014;32(3):252–60. https://doi.org/10. 1038/nbt.2816. Acknowledgements 7. Ng KS, Kuncewicz TM, Karp JM. Beyond hit-and-run: stem cells leave a The authors would like to thank the following for their contributions in the lasting memory. Cell Metab. 2015;22(4):541–3. https://doi.org/10.1016/j.cmet. research: Shai Israel for his kind help in the MATLAB code and Michael Anbar 2015.09.019. for his kind help in consulting and helping with Western blots. 8. Zhang Y, Chopp M, Liu XS, Katakowski M, Wang X. Exosomes derived from mesenchymal stromal cells promote axonal growth of cortical neurons. Funding 2016. https://doi.org/10.1007/s12035-016-9851-0. N.P. received funding scholarship from Sagol School of Neuroscience, Tel 9. Braccioli L, Van Velthoven C, Heijnen CJ. Exosomes: a new weapon to treat Aviv University. the central nervous system. 2014:113–9. https://doi.org/10.1007/s12035-013- 8504-9. Availability of data and materials 10. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome- The datasets used and/or analyzed during the current study are available mediated transfer of mRNAs and microRNAs is a novel mechanism of from the corresponding author on reasonable request. genetic exchange between cells. Nat Cell Biol. 2007;9(6):654–9. https://doi. org/10.1038/ncb1596. Authors’ contributions 11. Yu B, Zhang X, Li X. Exosomes derived from mesenchymal stem cells. 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Molecular Autism – Springer Journals

Published: Nov 21, 2018

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