Administration of mesenchymal stem cells (MSCs) improves the recovery from acute kidney injury (AKI).The mechanism may involve paracrine factors promoting proliferation of surviving intrinsic epithelial cells, but these factors remain unknown. In the current study, we found that microvesicles derived from human bone marrow MSCs stimulated proliferation in vitro and conferred resistance of tubular epithelial cells to apoptosis. The biologic action of microvesicles required their CD44-and ␤1-integrin-dependent incorporation into tubular cells. In vivo, microvesicles accelerated the morphologic and functional recovery of glycerol-induced AKI in SCID mice by inducing proliferation of tubular cells. The effect of microvesicles on the recovery of AKI was similar to the effect of human MSCs. RNase abolished the aforementioned effects of microvesicles in vitro and in vivo, suggesting RNA-dependent biologic effects. Microarray analysis and quantitative real time PCR of microvesicle-RNA extracts indicate that microvesicles shuttle a specific subset of cellular mRNA, such as mRNAs associated with the mesenchymal phenotype and with control of transcription, proliferation, and immunoregulation. These results suggest that microvesicles derived from MSCs may activate a proliferative program in surviving tubular cells after injury via a horizontal transfer of mRNA.

Recent studies suggest that tumor-derived microvesicles (MV) act as a vehicle for exchange of genetic information between tumor and stromal cells, engendering a favorable microenvironment for cancer development. Within the tumor mass, all cell types may contribute to MV shedding, but specific contributions to tumor progression have yet to be established. Here we report that a subset of tumor-initiating cells expressing the mesenchymal stem cell marker CD105 in human renal cell carcinoma releases MVs that trigger angiogenesis and promote the formation of a premetastatic niche. MVs derived only from CD105-positive cancer stem cells conferred an activated angiogenic phenotype to normal human endothelial cells, stimulating their growth and vessel formation after in vivo implantation in immunocompromised severe combined immunodeficient (SCID) mice. Furthermore, treating SCID mice with MVs shed from CD105-positive cells greatly enhanced lung metastases induced by i.v. injection of renal carcinoma cells. Molecular characterization of CD105-positive MVs defines a set of proangiogenic mRNAs and microRNAs implicated in tumor progression and metastases. Our results define a specific source of cancer stem cell-derived MVs that contribute to triggering the angiogenic switch and coordinating metastatic diffusion during tumor progression. Cancer Res; 71(15); 5346-56. Ó2011 AACR.

BackgroundCell-derived microvesicles (MVs) have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs) and liver resident stem cells (HLSCs) were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs).Methodology/Principal FindingsMVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation.ConclusionsThis study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem cells may, at least in part, depend on MV-shuttled miRNAs. Data generated from this study, stimulate further functional investigations on the predicted target genes and pathways involved in the biological effect of human adult stem cells.

Several studies demonstrated that treatment with mesenchymal stem cells (MSCs) reduces cisplatin mortality in mice. Microvesicles (MVs) released from MSCs were previously shown to favor renal repair in non lethal toxic and ischemic acute renal injury (AKI). In the present study we investigated the effects of MSC-derived MVs in SCID mice survival in lethal cisplatin-induced AKI. Moreover, we evaluated in vitro the effect of MVs on cisplatin-induced apoptosis of human renal tubular epithelial cells and the molecular mechanisms involved. Two different regimens of MV injection were used. The single administration of MVs ameliorated renal function and morphology, and improved survival but did not prevent chronic tubular injury and persistent increase in BUN and creatinine. Multiple injections of MVs further decreased mortality and at day 21 surviving mice showed normal histology and renal function. The mechanism of protection was mainly ascribed to an anti-apoptotic effect of MVs. In vitro studies demonstrated that MVs up-regulated in cisplatin-treated human tubular epithelial cells anti-apoptotic genes, such as Bcl-xL, Bcl2 and BIRC8 and down-regulated genes that have a central role in the execution-phase of cell apoptosis such as Casp1, Casp8 and LTA. In conclusion, MVs released from MSCs were found to exert a pro-survival effect on renal cells in vitro and in vivo, suggesting that MVs may contribute to renal protection conferred by MSCs.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture “what the community needs in a tool”. Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

Phenotypic changes induced by extracellular vesicles have been implicated in mesenchymal stromal cellpromoted recovery of AKI. MicroRNAs are potential candidates for cell reprogramming toward a proregenerative phenotype. The aim of this study was to evaluate whether microRNA deregulation inhibits the regenerative potential of mesenchymal stromal cells and derived extracellular vesicles in a model of glycerol-induced AKI in severe combined immunodeficient mice. We generated mesenchymal stromal cells depleted of Drosha to alter microRNA expression. Drosha-knockdown cells produced extracellular vesicles that did not differ from those of wild-type cells in quantity, surface molecule expression, and internalization within renal tubular epithelial cells. However, these vesicles showed global downregulation of microRNAs. Whereas wild-type mesenchymal stromal cells and derived vesicles administered intravenously induced morphologic and functional recovery in AKI, the Drosha-knockdown counterparts were ineffective. RNA sequencing analysis showed that kidney genes deregulated after injury were restored by treatment with mesenchymal stromal cells and derived vesicles but not with Drosha-knockdown cells and vesicles. Gene ontology analysis showed in AKI an association of downregulated genes with fatty acid metabolism and upregulated genes with inflammation, matrix-receptor interaction, and cell adhesion molecules. These alterations reverted after treatment with wild-type mesenchymal stromal cells and extracellular vesicles but not after treatment with the Drosha-knockdown counterparts. In conclusion, microRNA depletion in mesenchymal stromal cells and extracellular vesicles significantly reduced their intrinsic regenerative potential in AKI, suggesting a critical role of microRNAs in recovery after AKI.

Mesenchymal stem cells (MSCs) have opposite effects on tumor growth, being able either to favor angiogenesis and tumor initiation or to inhibit progression of established tumors. Factors produced by MSCs within the tumor microenvironment may be relevant for their biological effects. Recent studies demonstrated that microvesicles (MVs) are an integral component of inter-cellular communication within the tumor microenvironment. In the present study, we evaluated whether MVs derived from human bone marrow MSCs may stimulate or inhibit in vitro and in vivo growth of HepG2 hepatoma, Kaposi's sarcoma, and Skov-3 ovarian tumor cell lines. We found that MVs inhibited cell cycle progression in all cell lines and induced apoptosis in HepG2 and Kaposi's cells and necrosis in Skov-3. The observed activation of negative regulators of cell cycle may explain these biological effects. In vivo intra-tumor administration of MVs in established tumors generated by subcutaneous injection of these cell lines in SCID mice significantly inhibited tumor growth. In conclusion, MVs from human MSCs inhibited in vitro cell growth and survival of different tumor cell lines and in vivo progression of established tumors.

Microvesicles (MVs) play a pivotal role in cell-to-cell communication. Recent studies demonstrated that MVs may transfer genetic information between cells. Here, we show that MVs derived from human adult liver stem cells (HLSC) may reprogram in vitro HepG2 hepatoma and primary hepatocellular carcinoma cells by inhibiting their growth and survival. In vivo intratumor administration of MVs induced regression of ectopic tumors developed in SCID mice. We suggest that the mechanism of action is related to the delivery of microRNAs (miRNAs) from HLSC-derived MVs (MV-HLSC) to tumor cells on the basis of the following evidence: (a) the rapid, CD29-mediated internalization of MV-HLSC in HepG2 and the inhibition of tumor cell growth after MV uptake; (b) the transfer by MV-HLSC of miRNAs with potential antitumor activity that was downregulated in HepG2 cells with respect to normal hepatocytes; (c) the abrogation of the MV-HLSC antitumor effect after MV pretreatment with RNase or generation of MVs depleted of miRNAs; (d) the relevance of selected miRNAs was proven by transfecting HepG2 with miRNA mimics. The antitumor effect of MV-HLSC was also observed in tumors other than liver such as lymphoblastoma and glioblastoma. These results suggest that the delivery of selected miRNAs by MVs derived from stem cells may inhibit tumor growth and stimulate apoptosis. Stem Cells2012;30:1985–1998