Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs

Abstract

Background: Cell-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 findings: MVs 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.

Conclusions: This 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.

Figure 1. Expression of ribonucleoproteins within MVs.

Panel A. Representative micrographs showing the presence of stress granule expressing Stau2 and TIA ribonucleoproteins as detected by confocal microscopy in MSCs cultured overnight in the absence of serum. Panel B: representative confocal micrographs showing the expression of Stau2 and Ago2 by MVs released from MSCs cultured overnight in the absence of serum. MVs were labeled with the red PKH26 dye. Merge shows the co-localization of Ago2 and Stau2 within MVs. MVs from MSCs expressed also Stau1, TIA, TIAR and HuR (not shown). (Panel A; original magnification X400. Panel B; original magnification X600). Panel C: representative western blot analysis showing the expression of Stau1, Stau2, TIA, TIAR, HuR, Ago2 and RPS29 by MVs derived from MSCs. Head arrows indicate the expected molecular weight. Panel D: Representative micrographs of transmission electron microscopy obtained on purified MVs. Ultrathin sections, stained with lead citrate. Panel E: Immunogold electron microscopy showing staining for Stau2 and Ago2 (see Methods). MVs were viewed by JEOL Jem 1010 electron microscope (black line = 100 nm). All experiments were performed three times with similar results. Panel F: schematic representation of ribonucleoprotein mediated RNA intracellular traffic, suggesting that MVs/exosomes may represent a site of RNA compartmentalization allowing the transfer of genetic material to target cells.

Figure 2. Effect of cytochalasin B on MV release and stress granule accumulation.

Panel A and B: The release of MVs by MSCs (A) and HLSCs (B) was evaluated using the gating strategy described by Wysoczynsk . Size beads were used to define the proper gate of MVs, as events under 1 µm. The stopping gate was set up on 1,000 events collected from the cells region (R1). Panel C: The number of MVs was evaluated by flow cytometry at 6 h for MSCs and 24 hours for HLSCs after 1 hour treatment with cytochalasin B (white bar, cyt B) or with vehicle alone (black bar, CTR). Data are expressed as mean ± 1SD of 5 experiments. Nonparametric Mann-Whitney t test  =  * p<0.05. Panel D: representative confocal micrographs of TIA and Stau2 expression by stress granules in MSCs pre-treated with cytochalasin B (cyt B) or vehicle (CTR) as described in Methods. Five experiments were performed with similar results.

Figure 3. Venn diagram comparing miRNA expression in HLSCs, MSCs and their MVs and expression profile of common miRNAs.

Panel A–C: the number of shared and specific miRNAs for MSCs and HLSCs (A), for MSCs and MSC MVs (B) and for HLSCs and HLSC MVs (C) are shown. Panel D: Heat map demonstrating the expression profile for MSCs, HLSCs and the corresponding MVs is generated for commonly expressed miRNAs.

Figure 4. Bioanalyzer profile of RNA extracted from MSCs, HLSCs and the corresponding MVs and miRNA expression seen by in situ hybridization in cells and MVs.

Panel A and B: Representative bioanalyzer profile of the RNAs contained in MSCs, HLSCs and MVs showing that, whereas the ribosomal subunit 28 and 18S were detectable in cells, they were absent or barely detectable in the corresponding MVs. MVs exhibited a relevant peak of small RNAs at variance with the cells. Panel C: Representative bioanalyzer profile of small RNAs performed on MVs derived from MSCs and HLSCs (MSC MVs and HLSC MVs) showing an enrichment of small RNAs (range: 30–50%) of the size of miRNAs in respect to the cells of origin (range: 4–8%; not shown). Three different samples tested in triplicate were analyzed for each type of cells and MVs with similar results. Panel D–F: Representative micrographs of in situ hybridization on MSCs (D) and MSC-derived MVs (E and F) using a probe for miR-24 or a scramble-miR probe (miR-Scr) as control. Confocal microscopy original magnification X400 (D) and X600 (E). In panel E, MVs were labeled with a red dye PKH26. Panel F: The in situ hybridization was revealed by immunogold transmission electron microscopy as described in Methods (original magnification X50,000). Three different experiments were performed with similar results.

Figure 5. MVs containing miRNAs.

Fold change analyses of selected miRNAs in MVs from MSCs and HLSCs in respect to their cell of origin was detected by qRT-PCR and expressed as Log of 2^−ΔCt (MVs versus cells). miRNA comparisons between cells and MVs were based on the relative expression data normalized using the geometric mean value of four of the most stable miRNAs identified in the profiling between cells and MVs (see Methods). The following miRNAs were tested: miR-221 (line1), miR-99a (line 2), miR-222 (line 3), miR-24 (line 4), miR-410 (line 5), miR-21 (line 6), miR-100 (line 7), miR-214 (line 8), miR-31 (line9), miR-223 (line 12), miR-122 (line 13) and miR-451 (line 14). As control snoRNAs, RNU 44 (line 10) and RNU 48 (line 11) were used. In panel A, the dark dotted box indicate the miRNAs co-expressed by MVs and the cells of origin and the grey dotted box indicate miRNAs enriched within MVs. Panel B and C are a magnification of the dark (B) and grey (C) boxes respectively. The boxed areas represent the mean ± quartile and the whiskers extend out to the minimum and maximum values. Anova with Newman-Keuls multicomparison test was performed and all the reported miRNAs exhibited a significance <0.001 in respect to the control snoRNAs.

Figure 6. Cellular processes overrepresented by validated and predicted mRNA targets of miRNAs co-expressed by MVs and cells.

A GO tree obtained from BiNGO, showing the hierarchy of gene ontology biological processes overrepresented by the validated and predicted targets of miRNAs co-expressed by MVs and cells. (p<0.01, see the color bar). Node colors represent the statistical significance. The white nodes (p>0.01) showed the relationships among their downstream nodes. The boxes indicated the overrepresented biological processes categorized into development, catalytic activity, cell fate differentiation and metabolic processes.

Figure 7. miRNA transfer and MV uptake by mTEC.

Panel A: mTEC were incubated with MVs derived from MSCs for 12, 24 and 48 hours (h) at 37°C in the presence of 50 µg/ml α-amanitin to inhibit transcriptional activation in mTEC and the transfer of selected miRNAs was evaluated by qRT-PCR. The difference in Ct values between α-amanitin treated mTEC alone or stimulated with MVs is shown for each miRNA. The snoRNA, RNU48 and the miR-410, that was not present in MVs, were used as controls. Data are the mean ± SD of four experiments. Panel B: Micrograph representative of the incorporation of MVs labeled with PKH26 in mTEC evaluated by confocal microscopy after incubation for 12 and 24 hours at 37°C. Three experiments were performed with similar results (original magnification X400). Panel C: To evaluate the transfer of miRNAs within mTEC we used two reporter miRNAs, the Alexa-488 labeled siRNA or the FAM labeled microRNA Mimic (hsa-miR-21). mTEC were incubated for 3 hours at 37°C with MVs isolated from MSCs transfected with Alexa-488 labeled siRNA (MV Alexa-488 siRNA) or with FAM labeled microRNA Mimic (MV FAM hsa-miR-21). The uptake of MVs was evaluated by confocal microscopy. miRNAs incorporated within mTEC were detected as green fluorescence signal of Alexa-488 or FAM fluorophores (Original magnification X400). Panel D: Downregulation in mTEC of proteins targeted by some of the enriched miRNAs present in MVs, was evaluated by western blot analysis. mTEC were incubated at 37°C with 30 µg/ml of MVs derived from MSCs for 3, 12, 24 and 48 hours and the cell lysates were submitted to Western Blot as described in Methods for detection of PTEN, cyclin D1, Bcl-2, AKT and as control Actin. mTEC incubated for 48 hours in the absence of MVs were also used as control (Ctrl 48 h). Cell viability evaluated as trypan blue exclusion was 98±0.7%. Three experiments were conducted with similar results.