Transfer of MicroRNAs by Embryonic Stem Cell Microvesicles

Citation: Yuan A, Farber EL, Rapoport AL, Tejada D, Deniskin R, et al. ( Transfer of MicroRNAs by Embryonic Stem Cell Microvesicles Alex Yuan 0 Erica L. Farber 0 Ana Lia Rapoport 0 Desiree Tejada 0 Roman Deniskin 0 Novrouz B. Akhmedov 0 Debora B. Farber 0 Alfred Lewin, University of Florida, United States of America 0 Jules Stein Eye Institute, UCLA School of Medicine , Los Angeles, California , United States of America Microvesicles are plasma membrane-derived vesicles released into the extracellular environment by a variety of cell types. Originally characterized from platelets, microvesicles are a normal constituent of human plasma, where they play an important role in maintaining hematostasis. Microvesicles have been shown to transfer proteins and RNA from cell to cell and they are also believed to play a role in intercellular communication. We characterized the RNA and protein content of embryonic stem cell microvesicles and show that they can be engineered to carry exogenously expressed mRNA and protein such as green fluorescent protein (GFP). We demonstrate that these engineered microvesicles dock and fuse with other embryonic stem cells, transferring their GFP. Additionally, we show that embryonic stem cells microvesicles contain abundant microRNA and that they can transfer a subset of microRNAs to mouse embryonic fibroblasts in vitro. Since microRNAs are short (21-24 nt), naturally occurring RNAs that regulate protein translation, our findings open up the intriguing possibility that stem cells can alter the expression of genes in neighboring cells by transferring microRNAs contained in microvesicles. Embryonic stem cell microvesicles may be useful therapeutic tools for transferring mRNA, microRNAs, protein, and siRNA to cells and may be important mediators of signaling within stem cell niches. - Funding: This work was supported by grants from NIH (EY018739), The Vision of Children, and Hope for Vision. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Circulating platelet-derived vesicles were first identified in human plasma in the 1960s [1]. These vesicles, called microvesicles or microparticles, are heterogeneous in size and range from ,30 nm to 1 mm. Previously believed to be inert cellular debris, microvesicles are now gaining acceptance as important mediators of intercellular communication [29]. For example, microvesicles may mediate intercellular communication by transporting bioactive lipids, mRNA, or proteins between cells. Microvesicles have been identified from many cellular sources including monocytes, macrophages, endothelial cells, leukocytes, polymorphonuclear leukocytes, and tumor cells [2,4,5,10]. More recently, microvesicles were isolated from embryonic stem cells. These embryonic stem cell microvesicles (ESMVs) were capable of reprogramming hematopoietic progenitors [6], suggesting that microvesicles are capable of providing stem cells with extrinsic cues, which may regulate stem cell proliferation, and fate in stem cell niches. Shedding of microvesicles is a normal physiological process and is interestingly related to high rates of cellular proliferation. Cellular stress and damage, however, can also result in the release of membrane microvesicles [7,1113]. In humans, microvesicles play an essential role in maintaining hematostasis. A reduction in the number of platelet microvesicles causes a bleeding disorder called Scott Syndrome [14]. Elevated levels of microvesicles are also associated with a variety of disorders including acute coronary syndrome, hypertension, diabetes, and pulmonary embolism (reviewed in [15]). In addition to maintaining hematostasis, microvesicles have also been implicated in carrying membrane bound morphogens in Drosophila [9], and they may influence the behavior and survival of hematopoietic progenitors [6,10]. The mechanism by which ESMVs may mediate intercellular signaling could involve the activation of receptors on the recipient cell by ligands in the ESMV. In this manner, ESMVs would be able to carry membrane bound ligands considerable distances from their stem cell origin. Alternatively, ESMVs may be able to mediate signaling by the direct transfer of proteins, RNA, or bioactive lipids to the recipient cell, serving as physiological liposomes [6,7]. If ESMVs can indeed serve as physiological liposomes, transferring RNA and proteins to cells, they can perhaps be used to deliver exogenously expressed genes for therapeutic purposes. To explore this possibility, we first characterized the RNA and protein content of ESMVs and whether they carry the mRNA and protein expressed from a transgene present in embryonic stem cells (ESCs) encoding green fluorescent protein (GFP). We then investigated the ability of ESMVs to transfer GFP to recipient cells. We also explored the possibility that ESMVs can transport and transfer microRNAs (miRNAs) to cells. MiRNAs are a group of small (2124nt) noncoding RNAs that regulate gene expression in mammals by binding to the 39 untranslated region (UTR) of mRNAs to repress translation [1618]. miRNAs have been found in peripheral blood microvesicles of healthy individuals [19] and in ovarian tumor microvesicles circulating in the peripheral blood of patients [20]. We show that ESMVs are capable of transferring a subset of miRNAs to mouse embryonic fibroblasts (MEFs), suggesting a tightly regulated transfer process. Transfer of miRNAs by microvesicles represents a novel method of paracrine signaling, potentially making microvesicles important components of stem cell niches. It also opens up the possibility of transferring siRNAs via microvesicles. The delivery of siRNAs by microvesicles may be particularly useful in the treatment of retinal disorders such as age related macular degeneration, since injection of naked siRNAs into the eye may have deleterious effects [19]. ESMV RNA profile Although ESMVs have been reported to contain mRNA [6], little was known about their total RNA profile or the quality and types of RNA that they contain. Thus, we isolated mouse ESMVs and determined their total RNA profile by gel electrophoresis and capillary electrophoresis (Figures 1A and 1B). 4.7760.7 mg (n = 10) of total RNA can be obtained over a 48 hour period from ESMVs released by 3.56106 ESCs plated on a T175 culture flask and cultured to ,70% confluence in serum-free media. Unlike total RNA collected from ESCs, the total RNA from ESMVs does not contain 28S and 18S rRNA (Figures 1A and 1B). Instead, the majority of the total RNA in ESMVs is concentrated below 2 kb, which may represent mRNAs and small RNAs such as miRNAs. ESMVs contain mRNA Consistent with previous studies [6,10,21], we detected the presence of mRNA in microvesicles. Purified polyA-containing RNA from ESMVs (0.59%60.2% of total RNA, n = 4) was found to be less (...truncated)