Although chronic wounds are common and continue to be a major cause of morbidity and mortality, treatments for these conditions are lacking and often ineffective. A large body of evidence exists demonstrating the therapeutic potential of mesenchymal stem cells (MSCs) for repair and regeneration of damaged tissue, including acceleration of cutaneous wound healing. However, the exact mechanisms of wound healing mediated by MSCs are unclear. In this study, we examined the role of MSC exosomes in wound healing. We found that MSC exosomes ranged from 30 to 100-nm in diameter and internalization of MSC exosomes resulted in a dose-dependent enhancement of proliferation and migration of fibroblasts derived from normal donors and chronic wound patients. Uptake of MSC exosomes by human umbilical vein endothelial cells also resulted in dose-dependent increases of tube formation by endothelial cells. MSC exosomes were found to activate several signaling pathways important in wound healing (Akt, ERK, and STAT3) and induce the expression of a number of growth factors [hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF1), nerve growth factor (NGF), and stromal-derived growth factor-1 (SDF1)]. These findings represent a promising opportunity to gain insight into how MSCs may mediate wound healing.

Mesenchymal Stem Cell Exosomes Induce Proliferation and Migration of Normal and Chronic Wound Fibroblasts, and Enhance Angiogenesis In Vitro

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.

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

https://www.fertstert.org/article/S0015-0282(14)02371-1/pdf

Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health

Dormant breast cancers resurge as metastatic disease after a long dormancy period in the bone marrow, where cancer cells interact with mesenchymal stem cells (MSC). However, the nature of early interactions between breast cancer cells and MSCs in the bone marrow microenvironment that facilitate adaptation to a quiescent state remains poorly understood. Here, we report that breast cancer cells prime MSC to release exosomes containing distinct miRNA contents, such as miR-222/223, which in turn promotes quiescence in a subset of cancer cells and confers drug resistance. Building on these results, we developed a novel, nontoxic therapeutic strategy to target dormant breast cancer cells based on systemic administration of MSC loaded with antagomiR-222/223. In an immunodeficient mouse model of dormant breast cancer, this therapy sensitized breast cancer cells to carboplatin-based therapy and increased host survival. Overall, our findings illuminate the nature of the regulatory interactions between breast cancer cells and MSCs in the evolution of tumor dormancy and resurgence in the micrometastatic microenvironment of the bone marrow. Cancer Res; 76(19); 5832-44. ©2016 AACR.

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Mesenchymal Stem Cell–Derived Exosomes Stimulate Cycling Quiescence and Early Breast Cancer Dormancy in Bone Marrow

Author(s): Machtinger, Ronit; Laurent, Louise C; Baccarelli, Andrea A | Abstract: BackgroundExtracellular vesicles (EVs) are membrane-bound vesicles, found in biofluids, that carry and transfer regulatory molecules, such as microRNAs (miRNAs) and proteins, and may mediate intercellular communication between cells and tissues. EVs have been isolated from a wide variety of biofluids, including plasma, urine, and, relevant to this review, seminal, follicular and uterine luminal fluid. We conducted a systematic search of the literature to review and present the currently available evidence on the possible roles of EVs in follicular growth, resumption of oocyte development and maturation (meiosis), sperm maturation, fertilization and embryo implantation.MethodsMEDLINE, Embase and Web of Science databases were searched using keywords pertaining to EVs, including 'extracellular vesicles', 'microvesicles', 'microparticles' and 'exosomes', combined with a range of terms associated with the period of development between fertilization and implantation, including 'oocyte', 'sperm', 'semen', 'fertilization', 'implantation', 'embryo', 'follicular fluid', 'epididymal fluid' and 'seminal fluid'. Relevant research articles published in English (both animal and human studies) were reviewed with no restrictions on publication date (i.e. from earliest database dates to July 2015). References from these articles were used to obtain additional articles.ResultsA total of 1556 records were retrieved from the three databases. After removing duplicates and irrelevant titles, we reviewed the abstracts of 201 articles, which included 92 relevant articles. Both animal and human studies unequivocally identified various types of EVs in seminal, follicular and ULFs. Several studies provided evidence for the roles of EVs in these biofluids. In men, EVs in seminal fluid were linked with post-testicular sperm maturation, including sperm motility acquisition and reduction of oxidative stress. In women, EVs in follicular fluid were shown to contain miRNAs with potential roles in follicular growth, resumption of oocyte meiosis, steroidogenesis and prevention of polyspermy after fertilization. EVs were also detected in the media of cultured embryos, suggesting that EVs released from embryos and the uterus may mediate embryo-endometrium cross-talk during implantation. It is important to note that many of the biologically plausible functions of EVs in reproduction discussed in the current literature have not yet been substantiated by conclusive experimental evidence.ConclusionsA detailed understanding of the contributions of EVs in the series of events from gametogenesis to fertilization and then on to implantation, in both normal and pathological cases, may enable the development of valuable tools to advance reproductive health. Because of the early stage of the field, it is unsurprising that the current literature includes not only growing experimental evidence, but also as-yet unproven hypotheses pertaining to the roles of EVs in key reproductive processes. In this review, we present a comprehensive survey of the rapidly expanding literature on this subject, highlighting both relevant findings and gaps in knowledge.

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Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.

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Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence.

Despite its role in increasing the number of offspring during the lifetime of an individual animal, controlled ovarian hyperstimulation (COH) may have detrimental effects on oocyte development, embryo quality and endometrial receptivity. Circulating miRNAs in bio-fluids have been shown to be associated with various pathological conditions including cancers. Here we aimed to investigate the effect of COH on the level of extracellular miRNAs in bovine follicular fluid and blood plasma and elucidate their mode of circulation and potential molecular mechanisms to be affected in the reproductive tract. Twelve simmental heifers were estrous synchronized and six of them were hyperstimulated using FSH. Follicular fluid samples from experimental animals were collected using ovum pick up technique at day 0 of the estrous cycle and blood samples were collected at day 0, 3 and 7 of post ovulation. The expression profile of circulatory miRNAs in follicular fluid and blood plasma were performed using the human miRCURY LNA™ Universal RT miRNA PCR array system. A comparative threshold cycle method was used to determine the relative abundance of the miRNAs. A total of 504 and 402 miRNAs were detected in both bovine follicular fluid and blood plasma, respectively. Of these 57 and 21 miRNAs were found to be differentially expressed in follicular fluid and blood plasma, respectively derived from hyperstimulated versus unstimulated heifers. Bioinformatics analysis of those circulating miRNAs indicated that their potential target genes are involved in several pathways including TGF-beta signaling pathway, MAPK signaling pathway, pathways in cancer and Oocyte meiosis. Moreover, detail analysis of the mode of circulation of some candidates showed that most of the miRNA were found to be detected in both exosomal and Ago2 protein complex fraction of both follicular fluid and blood plasma. Our data provide the consequence of hyperstimulation induced changes of extracellular miRNAs in bovine follicular fluid and blood plasma, which may have a potential role in regulating genes associated not only with bovine ovarian function but also involved in altering various physiological in bovine oocytes, embryos and modulating reproductive tract environment.

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Sina Seifi Noferesti

Papers

Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence.

Controlled ovarian hyperstimulation induced changes in the expression of circulatory miRNA in bovine follicular fluid and blood plasma.