Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere, the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development, we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca(2+)) imaging indicated dysregulation of Ca(2+) cycling and elevation in intracellular Ca(2+) ([Ca(2+)](i)) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca(2+) homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease.

Muscle LIM protein (MLP, CSRP3) is a key regulator of striated muscle function, and its mutations can lead to both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, due to lack of human models, mechanisms underlining the pathogenesis of MLP defects remain unclear. In this study, we generated a knockout MLP/CSRP3 human embryonic stem cell (hESC) H9 cell line using CRISPR/Cas9 mediated gene disruption. CSRP3 disruption had no impact on the cardiac differentiation of H9 cells and led to confirmed MLP deficiency in hESC-derived cardiomyocytes (ESC-CMs). MLP-deficient hESC-CMs were found to develop phenotypic features of HCM early after differentiation, such as enlarged cell size, multinucleation, and disorganized sarcomeric ultrastructure. Cellular phenotypes of MLP-deficient hESC-CMs subsequently progressed to mimic heart failure (HF) by 30 days post differentiation, including exhibiting mitochondrial damage, increased ROS generation, and impaired Ca2+ handling. Pharmaceutical treatment with beta agonist, such as isoproterenol, was found to accelerate the manifestation of HCM and HF, consistent with transgenic animal models of MLP deficiency. Furthermore, restoration of Ca2+ homeostasis by verapamil prevented the development of HCM and HF phenotypes, suggesting that elevated intracellular Ca2+ concentration is a central mechanism for pathogenesis of MLP deficiency. In summary, MLP-deficient hESC-CMs recapitulate the pathogenesis of HCM and its progression toward HF, providing an important human model for investigation of CSRP3/MLP-associated disease pathogenesis. More importantly, correction of the autonomous dysfunction of Ca2+ handling was found to be an effective method for treating the in vitro development of cardiomyopathy disease phenotype.

Exosomes are attractive as nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and their ability to penetrate physiological barriers that are impermeable to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs, into cell-secreted exosomes leads to low yields. Here we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared with bulk electroporation and other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and a more than 103-fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic phosphatase and tensin homologue (PTEN)-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced inhibition of tumour growth and increased survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.

Regenerative medicine in tissue engineering often relies on stem cells and specific growth factors at a supraphysiological dose. These approaches are costly and may cause severe side effects. Herein, therapeutic small extracellular vesicles (t-sEVs) endogenously loaded with a cocktail of human vascular endothelial growth factor A (VEGF-A) and human bone morphogenetic protein 2 (BMP-2) mRNAs within a customized injectable PEGylated poly (glycerol sebacate) acrylate (PEGS-A) hydrogel for bone regeneration in rats with challenging femur critical-size defects are introduced. Abundant t-sEVs are produced by a facile cellular nanoelectroporation system based on a commercially available track-etched membrane (TM-nanoEP) to deliver plasmid DNAs to human adipose-derived mesenchymal stem cells (hAdMSCs). Upregulated microRNAs associated with the therapeutic mRNAs are enriched in t-sEVs for enhanced angiogenic-osteogenic regeneration. Localized and controlled release of t-sEVs within the PEGS-A hydrogel leads to the retention of therapeutics in the defect site for highly efficient bone regeneration with minimal low accumulation in other organs.

Acute lung injury (ALI) is a life-threatening respiratory condition characterized by severe inflammation and lung tissue damage, frequently causing rapid respiratory failure and long-term complications. The microRNA let-7a-5p is involved in the progression of lung injury, inflammation, and fibrosis by regulating immune cell activation and cytokine production. This study aims to use an innovative cellular electroporation platform to generate extracellular vesicles (EVs) carring let-7a-5p (EV-let-7a-5p) derived from transfected Wharton's jelly-mesenchymal stem cells (WJ-MSCs) as a potential gene therapy for ALI.

S100B is a calcium-binding protein, localized to astroglial cells, which has a variety of neurotrophic functions, including roles in serotonergic neuronal growth, synaptogenesis dendritic branching and apoptosis. In humans, the gene for S100B is found on chromosome 21, within what is considered the obligate region for Down Syndrome (DS) and levels of S100B are increased in brain of both DS and Alzheimer's Disease (AD). We have been characterizing a transgenic mouse overexpressing this protein and have previously found evidence of pathological changes in brains of the mice. In the current study, we have examined the expression of clusterin, a protein expressed in aging neurons, in the mice at two ages. Our findings show increased clusterin expression in the aged S100B mice compared to their CD-1 controls, a finding we have interpreted as further evidence of pathological brain aging.

For neural systems to function effectively, the numbers of each cell type must be proportioned properly during development. We found that conditional knockout of the mouse homeobox genes En1 and En2 in the excitatory cerebellar nuclei neurons (eCN) leads to reduced postnatal growth of the cerebellar cortex. A subset of medial and intermediate eCN are lost in the mutants, with an associated cell non-autonomous loss of their presynaptic partner Purkinje cells by birth leading to proportional scaling down of neuron production in the postnatal cerebellar cortex. Genetic killing of embryonic eCN throughout the cerebellum also leads to loss of Purkinje cells and reduced postnatal growth but throughout the cerebellar cortex. Thus, the eCN play a key role in scaling the size of the cerebellum by influencing the survival of their Purkinje cell partners, which in turn regulate production of granule cells and interneurons via the amount of sonic hedgehog secreted.

Background: Although engaging in physical exercise has been shown to reduce the incidence of cardiovascular events, the molecular mechanisms by which exercise mediates these benefits remain unclear. Based on epidemiological evidence, reductions in traditional risk factors only accounts for 50% of the protective effects of exercise, leaving the remaining mechanisms unexplained. The objective of this study was to determine whether engaging in a regular exercise program in a real world clinical setting mediates cardiovascular protection via modulation of non-traditional risk factors, such as those involved in coagulation, inflammation and metabolic regulation. Methods and Results: We performed a prospective, cohort study in 52 sedentary patients with cardiovascular disease or cardiovascular risk factors at two tertiary medical centers between January 1, 2016 and December 31, 2019. Prior to and at the completion of an 8-week exercise program, we collected information on traditional cardiovascular risk factors, exercise capacity, and physical activity and performed plasma analysis to measure levels of fibrinolytic, inflammatory and metabolic biomarkers to assess changes in non-traditional cardiovascular risk factors. The median weight change, improvement in physical fitness, and change in physical activity for the entire cohort were: -4.6 pounds (IQR: +2 pounds, -11.8 pounds), 0.37 METs (IQR: -0.076 METs, 1.06 METs), and 252.7 kcals/week (IQR: -119, 921.2 kcals/week). In addition to improvement in blood pressure and cholesterol, patients who lost at least 5 pounds, expended at least 1,000 additional kcals/week, and/or achieved ≥0.5 MET increase in fitness had a significant reduction in plasminogen activator inhibitor-1 [9.07 ng/mL (95% CI: 2.78-15.35 ng/mL); P = 0.026], platelet derived growth factor beta [376.077 pg/mL (95% CI: 44.69-707.46 pg/mL); P = 0.026); and angiopoietin-1 [(1104.11 pg/mL (95% CI: 2.92-2205.30 pg/mL); P = 0.049)]. Conclusion: Modest improvements in physical fitness, physical activity, and/or weight loss through a short-term exercise program was associated with decreased plasma levels of plasminogen activator inhibitor, platelet derived growth factor beta, and angiopoietin, which have been associated with impaired fibrinolysis and inflammation.

The recent success of mRNA therapeutics against pathogenic infections has increased interest in their use for other human diseases including cancer. However, the precise delivery of the genetic cargo to cells and tissues of interest remains challenging. Here, we show an adaptive strategy that enables the docking of different targeting ligands onto the surface of mRNA-loaded small extracellular vesicles (sEVs). This is achieved by using a microfluidic electroporation approach in which a combination of nano- and milli-second pulses produces large amounts of IFN-γ mRNA-loaded sEVs with CD64 overexpressed on their surface. The CD64 molecule serves as an adaptor to dock targeting ligands, such as anti-CD71 and anti-programmed cell death-ligand 1 (PD-L1) antibodies. The resulting immunogenic sEVs (imsEV) preferentially target glioblastoma cells and generate potent antitumour activities in vivo, including against tumours intrinsically resistant to immunotherapy. Together, these results provide an adaptive approach to engineering mRNA-loaded sEVs with targeting functionality and pave the way for their adoption in cancer immunotherapy applications.

How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

Microglia can shift into different complex morphologies depending on the microenvironment of the central nervous system (CNS). The distinct morphologies correlate with specific functions and can indicate the pathophysiological state of the CNS. Previous postmortem studies of autism spectrum disorder (ASD) showed neuroinflammation in ASD indicated by increased microglial density. These changes in the microglia density can be accompanied by changes in microglia phenotype but the individual contribution of different microglia phenotypes to the pathophysiology of ASD remains unclear. Here, we used an unbiased stereological approach to quantify six structurally and functionally distinct microglia phenotypes in postmortem human temporal cortex, which were immuno-stained with Iba1. The total density of all microglia phenotypes did not differ between ASD donors and typically developing individual donors. However, there was a significant decrease in ramified microglia in both gray matter and white matter of ASD, and a significant increase in primed microglia in gray matter of ASD compared to typically developing individuals. This increase in primed microglia showed a positive correlation with donor age in both gray matter and white of ASD, but not in typically developing individuals. Our results provide evidence of a shift in microglial phenotype that may indicate impaired synaptic plasticity and a chronic vulnerability to exaggerated immune responses.

Imaging studies of serotonin transporter binding or tryptophan retention in autistic patients suggest that the brain serotonin system is decreased. However, treatment with drugs which increase serotonin (5-HT) levels, specific serotonin reuptake inhibitors (SSRIs), commonly produce a worsening of the symptoms. In this study we examined 5-HT axons that were immunoreactive to a serotonin transporter (5-HTT) antibody in a number of postmortem brains from autistic patients and controls with no known diagnosis who ranged in age from 2 to 29 years. Fine, highly branched, and thick straight fibers were found in forebrain pathways (e.g. medial forebrain bundle, stria terminalis and ansa lenticularis). Many immunoreactive varicose fine fibers were seen in target areas (e.g. globus pallidus, amygdala and temporal cortex). Morphometric analysis of the stained axons at all ages studied indicated that the number of serotonin axons was increased in both pathways and terminal regions in cortex from autism donors. Our findings provide morphological evidence to warrant caution when using serotonin enhancing drugs (e.g. SSRIs and receptor agonist) to treat autistic children. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Tumour cell phagocytosis by antigen presenting cells (APCs) is critical to the generation of antitumour immunity. However, cancer cells can evade phagocytosis by upregulating anti-phagocytosis molecule CD47. Here, we show that CD47 blockade alone is inefficient in stimulating glioma cell phagocytosis. However, combining CD47 blockade with temozolomide results in a significant pro-phagocytosis effect due to the latter's ability to induce endoplasmic reticulum stress response. Increased tumour cell phagocytosis subsequently enhances antigen cross-presentation and activation of cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) in APCs, resulting in more efficient T cell priming. This bridging of innate and adaptive responses inhibits glioma growth, but also activates immune checkpoint. Sequential administration of an anti-PD1 antibody overcomes this potential adaptive resistance. Together, these findings reveal a dynamic relationship between innate and adaptive immune regulation in tumours and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.

Targeted protein degradation methods offer a unique avenue to assess a protein's function in a variety of model systems. Recently, these approaches have been applied to mammalian cell culture models, enabling unprecedented temporal control of protein function. However, the efficacy of these systems at the tissue and organismal levels in vivo is not well established. Here, we tested the functionality of the degradation tag (dTAG) degron system in mammalian development. We generated a homozygous knock-in mouse with a FKBP12F36V tag fused to negative elongation factor b (Nelfb) locus, a ubiquitously expressed regulator of transcription. In our validation of targeted endogenous protein degradation across mammalian development and adulthood, we demonstrate that irrespective of the route of administration the dTAG system is non-toxic, rapid, and efficient in embryos from the zygote-to-mid-gestation stages. Additionally, acute depletion of NELFB revealed a specific role in zygote-to-2-cell development and zygotic genome activation (ZGA).

Peripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia.

Pancreatic ductal adenocarcinoma (PDAC) tumours carry multiple gene mutations and respond poorly to treatments. There is currently an unmet need for drug carriers that can deliver multiple gene cargoes to target high solid tumour burden like PDAC. Here, we report a dual targeted extracellular vesicle (dtEV) carrying high loads of therapeutic RNA that effectively suppresses large PDAC tumours in mice. The EV surface contains a CD64 protein that has a tissue targeting peptide and a humanized monoclonal antibody. Cells sequentially transfected with plasmid DNAs encoding for the RNA and protein of interest by Transwell®-based asymmetric cell electroporation release abundant targeted EVs with high RNA loading. Together with a low dose chemotherapy drug, Gemcitabine, dtEVs suppress large orthotopic PANC-1 and patient derived xenograft tumours and metastasis in mice and extended animal survival. Our work presents a clinically accessible and scalable way to produce abundant EVs for delivering multiple gene cargoes to large solid tumours.