Induced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often yields incompletely reprogrammed, functionally compromised cells, raising the question of whether pluripotency is required to achieve a high degree of nuclear reprogramming. Here, we show that transient expression of Gata3, Eomes, and Tfap2c in mouse fibroblasts induces stable, transgene-independent trophoblast stem-like cells (iTSCs). iTSCs possess transcriptional profiles highly similar to blastocyst-derived TSCs, with comparable methylation and H3K27ac patterns and genome-wide H2A.X deposition. iTSCs generate trophoectodermal lineages upon differentiation, form hemorrhagic lesions, and contribute to developing placentas in chimera assays, indicating a high degree of nuclear reprogramming, with no evidence of passage through a transient pluripotent state. Together, these data demonstrate that extensive nuclear reprogramming can be achieved independently of pluripotency.

Neurological disorders are diseases of the central nervous system (CNS), characterized by a progressive degeneration of cells and deficiencies in neural functions. Mesenchymal stem cells (MSCs) are a promising therapy for diseases and disorders of the CNS. Increasing evidence suggests that their beneficial abilities can be attributed to their paracrine secretion of extracellular vesicles (EVs). Administration of EVs that contain a mixture of proteins, lipids, and nucleic acids, resembling the secretome of MSCs, has been shown to mimic most of the effects of the parental cells. Moreover, the small size and safety profile of EVs provide a number of advantages over cell transplantation. Intranasal (IN) administration of EVs has been established as an effective and reliable way to bypass the blood-brain barrier and deliver drugs to the CNS. In addition to pharmacological drugs, EVs can be loaded with a diverse range of cargo designed to modulate gene expression and protein functions in recipient cells, and lead to immunomodulation, neurogenesis, neuroprotection, and degradation of protein aggregates. In this review, we will explore the proposed physiological pathways by which EVs migrate through the nasal route to the CNS where they can actively target a region of injury or inflammation and exert their therapeutic effects. We will summarize the functional outcomes observed in animal models of neurological diseases following IN treatment with MSC-derived EVs. We will also examine key mechanisms that have been suggested to mediate the beneficial effects of EV-based therapy.

Parkinson’s disease is characterized by the gradual appearance of intraneuronal inclusions that are primarily composed of misfolded α-synuclein protein, leading to cytotoxicity and neural death. Recent in vitro and in vivo studies suggest that misfolded α-synuclein may spread transcellularly in a prion-like manner, inducing pathological aggregates in healthy neurons, and is disseminated via secretion of extracellular vesicles. Accordingly, extracellular vesicles derived from brain lysates and cerebrospinal fluid of patients with Parkinson’s disease were shown to facilitate α-synuclein aggregation in healthy cells. Prompted by the hypothesis of Braak et al. that the olfactory bulb is one of the primary propagation sites for the initiation of Parkinson’s disease, we sought to investigate the role of extracellular vesicles in the spread of α-synuclein and progression of Parkinson’s disease through the olfactory bulb. Extracellular vesicles derived from the cerebrospinal fluid of patients diagnosed with Parkinson’s disease or with a nonsynucleinopathy neurodegenerative disorder were administered intranasally to healthy mice, once daily over 4 days. Three months later, mice were subjected to motor and non-motor tests. Functional impairments were elucidated by histochemical analysis of midbrain structures relevant to Parkinson’s disease pathology, 8 months after EVs treatment. Mice treated with extracellular vesicles from the patients with Parkinson’s disease displayed multiple symptoms consistent with prodromal and clinical-phase Parkinson’s disease such as hyposmia, motor behavior impairments, and high anxiety levels. Furthermore, their midbrains showed widespread α-synuclein aggregations, dopaminergic neurodegeneration, neuroinflammation, and altered autophagy activity. Several unconventional pathologies were observed as well, such as α-synuclein aggregations in the red nucleus, growth of premature gray hair, and astrogliosis. Collectively, these data indicate that intranasally administered extracellular vesicles derived from the cerebrospinal fluid of patients with Parkinson’s disease can propagate α-synuclein aggregation in vivo and trigger Parkinson’s disease-like symptoms and pathology in healthy mice.

Background: Partial or an entire deletion of SHANK3 are considered as major drivers in the Phelan-McDermid syndrome, in which 75% of patients are diagnosed with autism spectrum disorder (ASD). During the recent years, there was an increasing interest in stem cell therapy in ASD, and specifically, mesenchymal stem cells (MSC). Moreover, it has been suggested that the therapeutic effect of the MSC is mediated mainly via the secretion of small extracellular vesicle that contains important molecular information of the cell and are used for cell-to-cell communication. Within the fraction of the extracellular vesicles, exosomes were highlighted as the most effective ones to convey the therapeutic effect. Methods: Exosomes derived from MSC (MSC-exo) were purified, characterized, and given via intranasal administration to Shank3B KO mice (in the concentration of 10 7 particles/ml). Three weeks post treatment, the mice were tested for behavioral scoring, and their results were compared with saline-treated control and their wild-type littermates. Results: Intranasal treatment with MSC-exo improves the social behavior deficit in multiple paradigms, increases vocalization, and reduces repetitive behaviors. We also observed an increase of GABARB1 in the prefrontal cortex. Conclusions: Herein, we hypothesized that MSC-exo would have a direct beneficial effect on the behavioral autistic-like phenotype of the genetically modified Shank3B KO mouse model of autism. Taken together, our data indicate that intranasal treatment with MSC-exo improves the core ASD-like deficits of this mouse model of autism and therefore has the potential to treat ASD patients carrying the Shank3 mutation.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been employed in the past decade as therapeutic agents in various diseases, including central nervous system (CNS) disorders. We currently aimed to use MSC-EVs as potential treatment for cerebral small vessel disease (CSVD), a complex disorder with a variety of manifestations. MSC-EVs were intranasally administrated to salt-sensitive hypertension prone SBH/y rats that were DOCA-salt loaded (SBH/y-DS), which we have previously shown is a model of CSVD. MSC-EVs accumulated within brain lesion sites of SBH/y-DS. An in vitro model of an inflammatory environment in the brain demonstrated anti-inflammatory properties of MSC-EVs. Following in vivo MSC-EV treatment, gene set enrichment analysis (GSEA) of SBH/y-DS cortices revealed downregulation of immune system response-related gene sets. In addition, MSC-EVs downregulated gene sets related to apoptosis, wound healing and coagulation, and upregulated gene sets associated with synaptic signaling and cognition. While no specific gene was markedly altered upon treatment, the synergistic effect of all gene alternations was sufficient to increase animal survival and improve the neurological state of affected SBH/y-DS rats. Our data suggest MSC-EVs act as microenvironment modulators, through various molecular pathways. We conclude that MSC-EVs may serve as beneficial therapeutic measure for multifactorial disorders, such as CSVD.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder with main core symptoms including deficits in social-communication abilities and repetitive behaviors/restricted interests. ASD affects 1 of 88 children worldwide and currently there is no sufficiently effective treatment that alleviates its core deficits. In our previous studies, we have shown that both MSC and MSC-exo can ameliorate core ASD-like symptoms of the BTBR multifactorial mouse model of autism. Furthermore, we have demonstrated that the MSC-exo migrate to distinct neuropathological areas in several mouse models, including the frontal cortex and cerebellum in BTBR mice. In contrast to BTBR mice, which is a multifactorial model of autism, the Shank3B KO mouse is used to study ASD which develops due to a specific genetic mutation. Here we demonstrate that intranasal treatment with MSC-exo improves the social behavior deficit in multiple paradigms, increases vocalization and reduces repetitive behaviors. We also observed an increase of GABRB1 in the prefrontal cortex. Taken together, our data indicate that intranasal treatment with MSC-exo improves the core ASD-like deficits of in this mouse model autism and therefore has the potential to treat ASD patients carrying the Shank3 mutation.

Background: Partial or an entire deletion of SHANK3 are considered as major drivers in the Phelan McDermid syndrome, in which 75% of patients are diagnosed with autism spectrum disorder (ASD). During the recent years, there was an increasing interest in stem cell therapy in ASD, and specifically, mesenchymal stem cells (MSC). Moreover, it has been suggested that the therapeutic effect of the MSC is mediated mainly via the secretion of small extracellular-vesicle that contain important molecular information of the cell and are used for cell-to cell communication. Within the fraction of the extracellular-vesicles, exosomes were highlighted as the most effective ones to convey the therapeutic effect.Methods: Exosomes derived from MSC (MSC-exo) were purified, characterized and given via intranasal administration to Shank3B KO mice (in concentration of 107 particals/ml). Three weeks post treatment the mice were tested for behavioral scoring and their results were compared to control saline treated and to their wild type littermates. Results: Intranasal treatment with MSC-exo improves the social behavior deficit in multiple paradigms, increases vocalization and reduces repetitive behaviors. We also observed an increase of GABARB1 in the prefrontal cortex.Conclusions: Herein, we hypothesized that MSC-exo would have a direct beneficial effect on the behavioral autistic-like phenotype of the genetically modified Shank3B KO mice model of autism. Taken together, our data indicate that intranasal treatment with MSC-exo improves the core ASD-like deficits of this mouse model autism and therefore has the potential to treat ASD patients carrying the Shank3 mutation.

The CRISPR-Cas system holds great promise in the treatment of diseases caused by genetic variations. As wildtype SpyCas9 is known to generate many off-target effects, its use in the clinic remains an elusive goal. To date, several high-fidelity Cas9 variants with improved specificity over wildtype SpyCas9 have been described. Each of the variants was engineered either by rational design or experimental directed evolution. However, the potential of improved specificity using both methods is constrained to specific residues due to the requirement of a priori knowledge and challenges in selecting engineered proteins, which arise from factors such as selection pressure, assay limitations, host organism compatibility, and library size and diversity. Therefore, SpyCas9 modifications through in-silico protein engineering allow an efficient specificity improvement while overcoming those limitations. Nevertheless, the lack of living models to test the evolved protein remains a major challenge of computational protein engineering methods. We recently demonstrated the advantage of normal mode analysis to simulate and predict the enzymatic function of SpyCas9 in the presence of mismatches. Here, we report ComPE, a novel computational protein engineering method to modify the protein and measure the vibrational entropy of wildtype or variant SpyCas9 in complex with its sgRNA and target DNA. Using this platform, we discovered novel high-fidelity Cas9 variants with improved specificity. We functionally validated the improved specificity of four variants, and the intact on-target activity in one of them. Lastly, we demonstrate their reduced off-target editing and non-specific gRNA-independent DNA damage, highlighting their advantages for clinical applications. The described method could be applied to a wide range of proteins, from CRISPR-Cas orthologs to distinct proteins in any field where engineered proteins can improve biological processes.