Promoting and Orienting Axon Extension Using Scaffold-Free Dental Pulp Stem Cell Sheets.

Current treatments of facial nerve injury result in poor functional outcomes due to slow and inefficient axon regeneration and aberrant reinnervation. To address these clinical challenges, bioactive scaffold-free cell sheets were engineered using neurotrophic dental pulp stem/progenitor cells (DPCs) and their aligned extracellular matrix (ECM). DPCs endogenously supply high levels of neurotrophic factors (NTFs), growth factors capable of stimulating axonal regeneration, and an aligned ECM provides guidance cues to direct axon extension. Human DPCs were grown on a substrate comprising parallel microgrooves, inducing the cells to align and deposit a linearly aligned, collagenous ECM. The resulting cell sheets were robust and could be easily removed from the underlying substrate. DPC sheets produced NTFs at levels previously shown capable of promoting axon regeneration, and, moreover, inducing DPC alignment increased the expression of select NTFs relative to unaligned controls. Furthermore, the aligned DPC sheets were able to stimulate functional neuritogenic effects in neuron-like cells in vitro. Neuronally differentiated neuroblastoma SH-SY5Y cells produced neurites that were significantly more oriented and less branched when cultured on aligned cell sheets relative to unaligned sheets. These data demonstrate that the linearly aligned DPC sheets can biomechanically support axon regeneration and improve axonal guidance which, when applied to a facial nerve injury, will result in more accurate reinnervation. The aligned DPC sheets generated here could be used in combination with commercially available nerve conduits to enhance their bioactivity or be formed into stand-alone scaffold-free nerve conduits capable of facilitating improved facial nerve recovery.

Gender-specific differential expression of exosomal miRNA in synovial fluid of patients with osteoarthritis.

The pathogenesis of osteoarthritis (OA) is poorly understood, and therapeutic approaches are limited to preventing progression of the disease. Recent studies have shown that exosomes play a vital role in cell-to-cell communication, and pathogenesis of many age-related diseases. Molecular profiling of synovial fluid derived exosomal miRNAs may increase our understanding of OA progression and may lead to the discovery of novel biomarkers and therapeutic targets. In this article we report the first characterization of exosomes miRNAs from human synovial fluid. The synovial fluid exosomes share similar characteristics (size, surface marker, miRNA content) with previously described exosomes in other body fluids. MiRNA microarray analysis showed OA specific exosomal miRNA of male and female OA. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified gender-specific target genes/signaling pathways. These pathway analyses showed that female OA specific miRNAs are estrogen responsive and target TLR (toll-like receptor) signaling pathways. Furthermore, articular chondrocytes treated with OA derived extracellular vesicles had decreased expression of anabolic genes and elevated expression of catabolic and inflammatory genes. In conclusion, synovial fluid exosomal miRNA content is altered in patients with OA and these changes are gender specific.

MicroRNA-183-5p Increases with Age in Bone-Derived Extracellular Vesicles, Suppresses Bone Marrow Stromal (Stem) Cell Proliferation, and Induces Stem Cell Senescence.

Microvesicle- and exosome-mediated transport of microRNAs (miRNAs) represents a novel cellular and molecular pathway for cell-cell communication. In this study, we tested the hypothesis that these extracellular vesicles (EVs) and their miRNAs might change with age, contributing to age-related stem cell dysfunction. EVs were isolated from the bone marrow interstitial fluid (supernatant) of young (3-4 months) and aged (24-28 months) mice to determine whether the size, concentration, and miRNA profile of EVs were altered with age in vivo. Results show that EVs isolated from bone marrow are CD63 and CD9 positive, and the concentration and size distribution of bone marrow EVs are similar between the young and aged mice. Bioanalyzer data indicate that EVs from both young and aged mice are highly enriched in miRNAs, and the miRNA profile of bone marrow EVs differs significantly between the young and aged mice. Specifically, the miR-183 cluster (miR-96/-182/-183) is highly expressed in aged EVs. In vitro assays demonstrate that aged EVs are endocytosed by primary bone marrow stromal cells (BMSCs), and these aged EVs inhibit the osteogenic differentiation of young BMSCs. Transfection of BMSCs with miR-183-5p mimic reduces cell proliferation and osteogenic differentiation, increases senescence, and decreases protein levels of the miR-183-5p target heme oxygenase-1 (Hmox1). In vitro assays utilizing H2O2-induced oxidative stress show that H2O2 treatment of BMSCs increases the abundance of miR-183-5p in BMSC-derived EVs, and Amplex Red assays demonstrate that H2O2 is elevated in the bone marrow microenvironment with age. Together, these data indicate that aging and oxidative stress can significantly alter the miRNA cargo of EVs in the bone marrow microenvironment, which may in turn play a role in stem cell senescence and osteogenic differentiation by reducing Hmox1 activity.

A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents.

Exosomes play a role in cell-to-cell signaling and serve as possible biomarkers. Isolating exosomes with reliable quality and substantial concentration is a major challenge. Our purpose is to compare the exosomes extracted by three different exosome isolation kits (miRCURY, ExoQuick, and Invitrogen Total Exosome Isolation Reagent) and differential ultracentrifugation (UC) using six different volumes of a non-cancerous human serum (5 ml, 1 ml, 500 µl, 250 µl, 100 µl, and 50 µl) and three different volumes (1 ml, 500 µl and 100 µl) of six individual commercial serum samples collected from human donors. The smaller starting volumes (100 µl and 50 µl) are used to mimic conditions of limited availability of heterogeneous biological samples. The isolated exosomes were characterized based upon size, quantity, zeta potential, CD63 and CD9 protein expression, and exosomal RNA (exRNA) quality and quantity using several complementary methods: nanoparticle tracking analysis (NTA) with ZetaView, western blot, transmission electron microscopy (TEM), the Agilent Bioanalyzer system, and droplet digital PCR (ddPCR). Our NTA results showed that all isolation techniques produced exosomes within the expected size range (40-150 nm). The three kits, though, produced a significantly higher yield (80-300 fold) of exosomes as compared to UC for all serum volumes, except 5 mL. We also found that exosomes isolated by the different techniques and serum volumes had similar zeta potentials to previous studies. Western blot analysis and TEM immunogold labelling confirmed the expression of two common exosomal protein markers, CD63 and CD9, in samples isolated by all techniques. All exosome isolations yielded high quality exRNA, containing mostly small RNA with a peak between 25 and 200 nucleotides in size. ddPCR results indicated that exosomes isolated from similar serum volumes but different isolation techniques rendered similar concentrations of two selected exRNA: hsa-miR-16 and hsa-miR-451. In summary, the three commercial exosome isolation kits are viable alternatives to UC, even when limited amounts of biological samples are available.