Visualization and Identification of Bioorthogonally Labeled Exosome Proteins Following Systemic Administration in Mice.

Exosomes transport biologically active cargo (e.g., proteins and microRNA) between cells, including many of the paracrine factors that mediate the beneficial effects associated with stem-cell therapy. Stem cell derived exosomes, in particular mesenchymal stem cells (MSCs), have been shown previously to largely replicate the therapeutic activity associated with the cells themselves, which suggests that exosomes may be a useful cell-free alternative for the treatment of cardiovascular disorders. However, the mechanisms that govern how exosomes home to damaged cells and tissues or the uptake and distribution of exosomal cargo are poorly characterized, because techniques for distinguishing between exosomal proteins and proteins in the targeted tissues are lacking. Here, we report the development of an in vivo model that enabled the visualization, tracking, and quantification of proteins from systemically administered MSC exosomes. The model uses bioorthogonal chemistry and cell-selective metabolic labeling to incorporate the non-canonical amino acid azidonorleucine (ANL) into the MSC proteome. ANL incorporation is facilitated via expression of a mutant (L274G) methionyl-tRNA-synthetase (MetRS∗) and subsequent incubation with ANL-supplemented media; after which ANL can be covalently linked to alkyne-conjugated reagents (e.g., dyes and resins) via click chemistry. Our results demonstrate that when the exosomes produced by ANL-treated, MetRS∗-expressing MSCs were systemically administered to mice, the ANL-labeled exosomal proteins could be accurately and reliably identified, isolated, and quantified from a variety of mouse organs, and that myocardial infarction (MI) both increased the abundance of exosomal proteins and redistributed a number of them from the membrane fraction of intact hearts to the cytosol of cells in infarcted hearts. Additionally, we found that Desmoglein-1c is enriched in MSC exosomes and taken up by ischemic myocardium. Collectively, our results indicate that this newly developed bioorthogonal system can provide crucial insights into exosome homing, as well as the uptake and biodistribution of exosomal proteins.

Characterization and analysis of long non-coding rna (lncRNA) in In Vitro- and Ex Vivo-derived cardiac progenitor cells.

Recent advancements in cell-based therapies for the treatment of cardiovascular disease (CVD) show continuing promise for the use of transplanted stem and cardiac progenitor cells (CPCs) to promote cardiac restitution. However, a detailed understanding of the molecular mechanisms that control the development of these cells remains incomplete and is critical for optimizing their use in such therapy. Long non-coding (lnc) RNA has recently emerged as a crucial class of regulatory molecules involved in directing a variety of critical biological processes including development, homeostasis and disease. As such, a rising body of evidence suggests that they also play key regulatory roles in CPC development, though many questions remain regarding the expression landscape and specific identity of lncRNA involved in this process. To address this, we performed whole transcriptome sequencing of two murine CPC populations-Nkx2-5 EmGFP reporter-sorted embryonic stem (ES) cell-derived and ex vivo, cardiosphere-derived-in an effort to characterize their lncRNA profiles and potentially identify novel CPC regulators. The resulting sequencing data revealed an enrichment in both CPC populations for a panel of previously-identified lncRNA genes associated with cardiac differentiation. Additionally, a total of 1,678 differentially expressed and as-of-yet unannotated, putative lncRNA genes were found to be enriched for in the two CPC populations relative to undifferentiated ES cells.

Foxc1 Regulates Early Cardiomyogenesis and Functional Properties of Embryonic Stem Cell Derived Cardiomyocytes.

Embryonic Stem Cells (ESCs) hold great potential for regeneration of damaged myocardium, however the molecular circuitry that guides ESC differentiation into cardiomyocytes remains poorly understood. This is exemplified by the elusive role of the transcription factor, Foxc1, during cardiac development. The only known Foxc1 target during heart development is Tbx1. Because Foxc1 null mice contain heart mutations that are far more severe than Tbx1 null mice, it is likely that Foxc1 has additional regulatory roles during heart development. The goal of our study was to test whether Foxc1 is critical for ESC differentiation into functional cardiomyocytes through proper regulation of specific downstream gene networks. Converging evidence from Foxc1 deficient and overexpression ESC models reveals a close relationship between Foxc1 levels and early cardiomyogenic factors Isl1, Mef2c, and Nkx2.5 and also the production of functional cardiomyocytes. We show Foxc1 regulates early cardiomyogenesis during a specific window of differentiation, D4-D6. Through whole transcriptome RNA-sequencing analysis, we report pathways regulated by Foxc1 involved in cardiac function including actin cytoskeleton, cell adhesion, tight and gap junctions, and calcium signaling. Our data indicate a novel Foxc1 direct gene target, Myh7, which encodes the predominant myosin heavy chain isoform, MHCß, expressed during cardiac development. These data lead us to conclude that Foxc1 regulates both early cardiomyogenesis and the functional properties of ESC-derived cardiomyocytes. Our findings shed light on the molecular circuitry governing cardiomyogenesis that may lead to the development of better translational strategies for the use of pluripotent stem cells in regenerative medicine towards repairing damaged myocardium. Stem Cells 2016;34:1487-1500.

Intestinal growth factors: potential use in the treatment of inflammatory bowel disease and their role in mucosal healing.

BACKGROUND: A key feature of inflammatory bowel disease (IBD) is impaired epithelial repair. Human growth factors comprise an array of signaling molecules that lead to ligand-specific signal transduction. Their downstream effects are associated with several cellular functions including epithelial healing in response to injury. Several studies have described specific growth factor deficiencies in patients with IBD, implicating their role in disease pathophysiology. The aim of this review was to describe currently known enterocyte-targeted growth factors, their mechanisms of action, and their potential therapeutic utility. METHODS: The National Library of Medicine (http://www.pubmed.gov) and meeting abstracts were searched using the following terms: growth factor, intestine, colon, inflammatory bowel disease, Crohn's disease, ulcerative colitis, colitis, animal model, transforming growth factor, bone morphogenetic protein, activins, growth hormone, fibroblast growth factor, epidermal growth factor (EGF), keratinocyte growth factor (KGF), glucagon-like peptide II, granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF) inhibitors, and trefoil factors. RESULTS: Several growth factors are therapeutic candidates in IBD. Growth hormone, KGF, EGF, teduglutide, GM-CSF/G-CSF have entered early clinical trials, whereas others are currently in preclinical evaluation. CONCLUSIONS: There are several growth factors responsible for epithelial repair. Preliminary studies using recombinant growth factors seem promising in IBD preclinical and clinical trials.