Correspondence to: Giovanni CAMUSSI, Dipartimento di Medicina Interna, Ospedale Maggiore S. Giovanni Battista, Corso Dogliotti 14, 10126, Torino, Italy. Tel.: +39-011-6336708 Fax: +39-011-6631184 E-mail: firstname.lastname@example.org
Several studies indicate that adult stem cells may improve the recovery from acute tissue injury. It has been suggested that they may contribute to tissue regeneration by the release of paracrine factors promoting proliferation of tissue resident cells. However, the factors involved remain unknown. In the present study we found that microvesicles (MVs) derived from human liver stem cells (HLSC) induced in vitro proliferation and apoptosis resistance of human and rat hepatocytes. These effects required internalization of MVs in the hepatocytes by an α4-integrin-dependent mechanism. However, MVs pre-treated with RNase, even if internalized, were unable to induce hepatocyte proliferation and apoptosis resistance, suggesting an RNA-dependent effect. Microarray analysis and quantitative RT-PCR demonstrated that MVs were shuttling a specific subset of cellular mRNA, such as mRNA associated in the control of transcription, translation, proliferation and apoptosis. When administered in vivo, MVs accelerated the morphological and functional recovery of liver in a model of 70% hepatectomy in rats. This effect was associated with increase in hepatocyte proliferation and was abolished by RNase pre-treatment of MVs. Using human AGO2, as a reporter gene present in MVs, we found the expression of human AGO2 mRNA and protein in the liver of hepatectomized rats treated with MVs. These data suggested a translation of the MV shuttled mRNA into hepatocytes of treated rats. In conclusion, these results suggest that MVs derived from HLSC may activate a proliferative program in remnant hepatocytes after hepatectomy by a horizontal transfer of specific mRNA subsets.
Several reports suggest that the administration of in vitro expanded stem cells might promote liver regeneration [1, 2]. Sources of pluripotent cells with hepatic potential include adipose tissue , bone marrow derived-stem cells [4–6] and embryonic stem cells . However, the mechanisms involved in hepatic regeneration are not completely understood and the relative contribution of mature hepatocytes and of resident stem cells is still intensely debated . It has been suggested that bone marrow derived stem cells may engraft in the liver undergoing transdifferentiation or fusion [9–11]. However, more recent studies have suggested that tissue regeneration triggered by exogenous stem cells may depend on the release of paracrine factors rather than on stem cell transdifferentiation [12–15]. The liver is known to have the capacity to regenerate after injury induced by chemicals, partial surgical resection or sepsis [8, 16]. Following partial hepatectomy, most of the quiescent hepatocytes in the remnant liver tissue quickly proliferate leading to rapid restoration of liver mass . Whether resident stem cells are involved in such regeneration remains unknown. We recently described in the adult human liver a population of pluripotent resident liver stem cells (HLSC) able to localize within the injured liver and contribute to liver regeneration when injected in mice with acute liver failure induced by acetoaminophene . Recently, it has been proposed that a dynamic stem cell regulation may occur as result of differentiated cell-stem cell interaction via a microvesicle (MV)-based genetic information transfer . Progenitor/stem cells may re-direct the behaviour of differentiated cells by a horizontal transfer of mRNA shuttled by MVs [20, 21] and conversely differentiated cells may influence the stem cell phenotype . MVs are derived from the endosomal membrane compartment after fusion with the plasma membrane and are shed from the cell surface of activated cells. MVs are now recognized to have an important role in cell to cell communication . Ratajczak and coworkers  demonstrated that MVs derived from embryonic stem cells may contribute to the cell-fate decision and may represent one of the critical components that support self-renewal and expansion of stem cells. We demonstrated that MVs derived from endothelial progenitor cells may activate an angiogenic program in mature quiescent endothelial cells  and that mRNA shuttled by MVs derived from mesenchymal stem cells may induce repair of acute kidney injury . Recently Kostin and Popescu  demonstrated that the interstitial Cajal-like cells that have been described to be present in the heart , communicate with neighbouring cells via shedding of MVs.
In the present study we characterized the MVs derived from HLSC and their potential to induce proliferation and apoptosis resistance in cultured human hepatocytes and to favour liver regeneration in the model of 70% hepatectomy in rats.
Materials and methods
Isolation and characterization of HLSC
HLSC were isolated from human cryopreserved normal adult hepatocytes (Lonza, Basel, Switzerland). The isolation, culture and characterization of HLSC were performed as previously described . By cytofluorimetric analysis HLSC expressed the mesenchymal stem cell markers CD29, CD44, CD73, CD90 but not the haematopoietic and endothelial markers CD34, -CD45, -CD14, -CD117, -CD133, -CD31, -CD144. Moreover, HLSC were positive for α5-integrin, α4-integrin, α6-integrin and -αvβ3-integrin. By immunofluorescence HLSC were positive for the hepatic markers human albumin and α-fetoprotein, for the resident stem cells markers vimentin and nestin and were negative for the oval cell markers CD34, CD117 and cytocheratin19 . In addition, HLSC expressed the embryonic stem cell markers nanog, Oct4, SOX2 and SSEA4. Before use HLSC were shown to undergo osteogenic, endothelial and hepatic differentiation under appropriate culture conditions .
Isolation of MVs
MVs were obtained from supernatants of HLSC cultured overnight in α-MEM deprived of foetal calf serum (FCS). The viability of HLSC at the time of MV collection was >99% as detected by trypan blue exclusion. After centrifugation at 2000 ×g for 20 min. cell-free supernatants were ultracentrifuged at 100,000 ×g (Beckman Coulter Optima L-90K, Optima L-90K, Beckman Coulter, Fullerton, CA, USA) for 1 hr at 4°C, washed in serum-free medium 199 containing Hepes 25 mM (Sigma, St. Louis, MO, USA) and submitted to a second ultracentrifugation in the same conditions [21, 23]. The protein content of MV preparation was quantified by Bradford method (Bio-Rad, Hercules, CA, USA). To trace MVs by fluorescent microscopy or cytofluorimetry, MVs were labelled with the red fluorescent aliphatic chromophore PKH26 (Sigma) . Endotoxin contamination of MVs was excluded by Limulus test (Charles River Laboratories, Inc., Wilmington, MA, USA).
In selected experiments MVs from HLSC were treated with 1 U/ml RNase (Ambion, Inc., Austin, TX, USA) [21, 23] for 1 hr at 37°C, the reaction was stopped by addition of 10 U/ml RNase inhibitor (Ambion) followed by ultracentrifugation. The effectiveness of RNase treatment was evaluated as previously described  by spectrophotometer analysis of total extracted RNA (untreated: 4.1 ± 0.3 μg RNA/mg protein MVs; RNase treated: <0.2 μg RNA/mg protein MVs). As control, MVs were treated with 1 U/ml DNase (Ambion) for 1 hr at 37°C.
Cytofluorimetric analysis of MVs
The size of MVs was determined by cytofluorimetry as previously described [21, 23] using beads of different sizes (1, 2 and 4 μm; Molecular Probes, Leiden, The Netherlands). The analysis was performed using a log scale for forward scatter and side scatter parameters. Moreover, the size of MVs, was evaluated by the Zetasizer Nano (Malvern Instruments, Malvern Worcestershire, UK). Cytofluorimetric analysis for the detection of surface molecules was performed as previously described [21, 23], using the following FITC- or PE-conjugated antibodies directed to: CD44 (Dakocytomation, Copenhagen, Denmark), CD29, α4-integrin, (BD Pharmingen), α5-integrin, α6-integrin (BioLegend, San Diego, CA, USA) and HLA-I (BD Pharmingen). FITC or PE mouse nonimmune isotypic IgG (Dakocytomation, Copenhagen, Denmark) were used as control.
Culture of hepatocytes
Human hepatocytes (Lonza) were cultured in William’s medium supplemented with 10% of FCS, 7.5 μg/ml hydrocortisone, 200 μg/ml streptomycin and 200 U/ml penicillin. Rat hepatocytes were obtained from liver of healthy male Sprague–Dawley rats using a two-step collagenase perfusion procedure and were cultured as previously described . Cell viability was greater than 90% as determined by trypan blue exclusion.
Incorporation of MVs in hepatocytes
To study the incorporation of MVs into hepatocytes, we incubated 50 μg/ml of MVs labelled with PKH-26 dye for 3 hrs at 37°C. We studied the MV incorporation by cytofluorimetry and confocal microscopy (LSM 5 Pascal Confocal Microscope, Carl Zeiss International, Oberkochen, Germany).
To investigate the role of adhesion molecules expressed by MVs in the incorporation in hepatocytes, MVs were pre-incubated (15 min. at 4°C) with blocking antibodies (1 μg/ml) against the identified adhesion molecules anti-α4-integrin (Biolegend), α6-integrin (Biolegend), CD29 (β1- integrin) (BD Pharmingen) or with soluble hyaluronic acid (sHA) (100 μg/ml from Rooster comb; Sigma) before the incubation with the cells. In selected experiments labelled MVs were treated with 0.5 mM trypsin for 30 min. at 37°C and the integrity of MVs and removal of surface molecules was checked by cytofluorimetric analysis.
Cell proliferation and apoptosis
Hepatocyte were seeded at 50,000 cells/well into 48-well plates in William’s medium E (Gibco, Paisley, UK) deprived of FCS. DNA synthesis was detected as incorporation of 5-bromo-2′-deoxy-uridine (BrdU) into the cellular DNA after 48 hrs of culture. This time point was chosen on the base of preliminary comparative experiments with [3H] thymidine incorporation. Cells were then fixed with 0.5 M ethanol/HCl and incubated with nuclease to digest the DNA. BrdU incorporated into the DNA was detected using an anti-BrdU peroxidise conjugated antibody (Roche Applied Science, Mannheim, Germany). Optical density was measured with an ELISA reader at 405 nm.
Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) technology (Chemicon, CA, USA) as previously described . To induce apoptosis hepatocytes were incubated with 5 mM of D-galactosamine (GalN) for 24 hrs in DMEM supplemented with 2% FCS . Apoptosis was also evaluated in paraffin-embedded liver sections by TUNEL.
Gene array analysis
RNA extraction, samples labelling and hybridization on BeadChips
RNA was extracted from MVs using TRIZOL reagent (Invitrogen, Paisley, UK) following the procedure suggested by the manufacturer. Total RNA was quantified spectrophotometrically (Nanodrop ND-1000, Wilmington DE, USA). cRNA was synthesized using three different quantities of total RNA (0.25, 0.5 and 1 μg). cRNA synthesis and labelling was done using Illumina RNA Amplification Kit (Ambion) following the procedure suggested by manufacturer. Sentrix® Human-6 Expression BeadChip hybridization, washing and staining were also done as suggested by the manufacturer. Arrays were scanned on Illumina BeadStation 500 (Illumina, San Diego, CA, USA).
Microarray data analysis
BeadChip array data quality control was performed with Illumina BeadStudio software version 3.0. Transcript average intensity signals were calculated with BeadStudio without background correction. Raw data were analysed using Bioconductor . Average transcript intensities were log2 transformed and normalized by loess method .
A simple statistical linear model  was used to identify transcript signals linearly correlated to the increment of total RNA concentration used to prepare cRNA. In equation (1) yij is the observed expression level for transcript i in sample j (j= 1, …, 6). μ is the average expression level of transcript i and βRNA represents the effect of total RNA concentration on the expression level of transcript i. ε represents random error for transcript i and sample j, and it is assumed to be independent for each transcript and sample, and normally distributed with mean 0 and variance σ2.
Transcripts characterized by a model with P≤ 0.05, r2≥ 0.8, and a positive slope were selected (120; see additional information 1).
Quantitative real time polymerase chain reaction (Q-PCR)
Q-PCR was performed as previously described  on four different MV preparations and on livers of Sprague–Dawley rats treated with MVs. Cell cDNA was used to evaluate primers efficiency. The primers for the human genes used for real time PCR were: (AGO2; forward 5′ CTAGACCCGACTTTGGGACCT-3′, reverse 5′-GGGCACTTCTCTGGCTTGATA-3; CHECK2; forward 5′-AGAAAGCTCTGGACCTTGTCAAGA-3′, reverse 5′-TGGATTCATTTTCCTCAGACAGAA-3′; CDK2; forward 5′-AATCCGCCTGGACACTGAGA -3′, reverse 5′-TCCAGCAGCTTGACAATATTAGGA-3′, MATK forward 5′-CCGCGACGTCATCCACTAC-3′, reverse 5′-TGTAATGCTCCACCATGTCCAT-3′ and the primers for the rat CCNA1 gene were: forward primer 5′-AAGTCCTGGCATTCGATCTGA-3′, reverse primer 5′-TGCAACATACTTGGCCAAATT-3′. First-strand cDNA was produced from 200 ng of mRNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). 20 μl of RT-PCR mix, containing 1× SYBR GREEN PCR Master Mix (Applied Biosystems), 200 nM of each primer and, respectively, 1 μl of MV cDNA, were assembled using a 48-wells StepOneTM Real Time System (Applied Biosystems). Negative cDNA controls (no cDNA) were cycled in parallel with each run.
Total RNA extracted from livers of rats was submitted to RT-PCR using the following primer for human AGO2 forward 5′ CTAGACCCGACTTTGGGACCT-3′ and reverse 5′-GGGCACTTCTCTGGCTTGATA-3. Bands of the expected size (90 pb) were detected in a 4% agarose gel after electrophoresis. cDNA from a preparation of HLSC was used as positive control.
Immunoprecipitation and Western blot
Rat liver tissue was homogenized (TissueRuptor, Qiagen, Valencia, CA, USA). Proteins were lysated with 1 μl of lysis buffer containing 25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, protease and phosphatase inhibitor cocktails (all from Sigma) as previously described . The lysates after pre-clearing with protein A–coupled Sepharose (Amersham, Buckinghamshire, UK) were incubated with human anti-AGO2 antibody (Abcam, Cambridge, MA, USA) overnight at 4°C, under agitation. Samples were subjected to SDS-PAGE in 8% acrylamide gel and transferred to a nitrocellulose membrane as described . Membrane was revealed with a chemiluminescence reagent and analysed with Chemidoc (Biorad, Hercules, CA, USA). Western blot was also performed on MV lysates for detection of Staufen 2 using 8–16% linear gradient acrylamide gels (Biorad) and anti-STAU2 mouse monoclonal antibody (Abcam).
Rat model of 70% hepatectomy
All experiments were performed in accordance with the Principles of Laboratory Animal Care and approved by the Local Committee for Experimental Animal Research. Adult Sprague–Dawley rats (Charles River, Calco, Italy) weighing 180–200 g were anesthetized and subjected to 70% hepatectomy as previously described . In sham-operated animals, livers were briefly exposed outside the peritoneal cavity. Immediately after hepatectomy, animals received an intravenous injection of 30 μg HLSC-derived MVs either untreated or RNase treated. Control rats received an equal volume of saline i.p. To measure hepatocyte proliferation in excised livers, rats received an i.p. injection of BrdU (100 mg/kg) 2 hrs before being killed as suggested by Lanier et al. for the evaluation of hepatocellular proliferation in rodents. Rats were killed at 8, 24, 48 or 72 hrs after surgery to collect the remnant liver. Blood was also withdrawn from the abdominal aorta and serum was obtained by centrifugation (3000 rpm, 20 min.) to measure alanine transaminase (ALT), aspartate transaminase (AST) and albumin. Six animals were used for each time point. Liver samples were snap frozen in liquid nitrogen and stored at −80°C until used in molecular biology studies. Additional liver samples were paraffin embedded for histology, immunohistochemistry and immunofluorescence analysis. Body and liver weights were recorded at each post-operative time to assess liver regeneration rate that was expressed as the ratio wet liver weight to body weight (%) .
For hepatic histology, 5-μm-thick paraffin liver sections were routinely stained with haematoxylin and eosin. Immunohistochemistry for detection of hepatocyte proliferation was performed as previously described  using anti-BrdU (Dakocytomation) or anti-PCNA (Santa Cruz Biotechnology, Santa Cruz CA, USA) monoclonal antibodies. Ten non-consecutive sections were counted for BrdU+ and PCNA+ hepatocytes at 200× magnification. Confocal microscopy analysis was performed on frozen sections for detection of specific human AGO2 protein. Sections were blocked and labelled with rabbit anti-human AGO2 (Abcam) (1:300 dilution). Omission of the primary antibodies or substitution with non-immune rabbit IgG was used as controls. Alexa Fluor 488 anti-rabbit (Molecular Probes) was used as secondary antibody. Hoechst 33258 dye (Sigma) was added for nuclear staining.
Oil red O staining for hepatic triglyceride detection
Frozen liver sections were assayed by a modification of the Folch method for triglyceride content using Oil red O staining . Frozen sections of liver were fixed for 10 min. in neutral buffered 10% formalin and stained with Oil red O (Sigma) for 10 min., counterstained with haematoxylin for 2 min., and cover slipped with a water-based mounting medium.
Serum ALT and AST levels were measured using a standard clinical automatic analyser. Serum rat albumin was determined by ELISA (Alpco diagnostic, Salem, NH, USA).
Data were analysed using the t-tests, ANOVA with Newmann–Keuls’ or ANOVA with Dunnet’s multicomparison tests as appropriate. A P-value of <0.05 was considered significant.
Characterization of HLSC-derived MVs
By cytofluorimetric analyses MVs produced by HLSC were detected mainly below the forward scatter signal corresponding to 1-μm beads (Fig. 1A). When determined by Zetasizer, the size of MVs ranged from 80 nm to 1μm, with a mean value of 195 nm. Cytofluorimetric analyses showed the presence of several adhesion molecules also present on HLSC plasma membrane such as α4-integrin, CD29 and CD44. MVs did not express α5- and α6-integrins and HLA-class I (Fig. 1A) at variance with the cells of origin .
Gene array analyses of HLSC-derived MVs
mRNA extracted from MVs was submitted to microarray analysis to define which transcripts were present . A total of 65 transcripts were found with this procedure; 60 were associated to Entrez gene identifiers  by IPA 6.0 analysis (see additional information). This observation indicated that MVs shuttled a specific subset rather than a random sample of cellular mRNA. MVs contained mRNA related to several cell functions involved in the control of transcription and metabolism (Table 1). In particular the following genes, that were detected in MVs, are known to be involved in cell proliferation: MATK, MRE11A, CHECK2, MYH11, VASP and CDK2. In particular, CDK2 gene has been shown to be involved in liver regeneration . Moreover, MVs contained the AGO2 (EIF2C2) gene which is a translation regulator with a critical role in the control of microRNA production . Quantitative real time PCR confirmed the presence in MVs of genes randomly chosen from those detected by Microarray (AGO2, CHEK2, CDK2, MATK).
Table 1. MV-shuttled mRNA involved in transcription, translation and cell proliferation
CASP8 associated protein 2
Doublesex and mab-3 related transcription factor 2
MRE11 meiotic recombination 11 homolog A (S. cerevisiae)
ST3 β-galactoside α-2,3-sialyltransferase 4
Serine/threonine kinase 40
Acid phosphatase, prostate
Progastricsin (pepsinogen C)
Cyclin-dependent kinase 2
Cell cycle checkpoint regulator
Leucocyte receptor tyrosine kinase
Megakaryocyte-associated tyrosine kinase
Phosphatase and tensin homolog
Protein tyrosine phosphatase
Adaptor-related protein complex 3, mu 2 subunit
ATPase, Na+/K+ transporting, α2+ polypeptide
Organic cation/carnitine transporter
Calcium channel, voltage-dependent, γ subunit 1
G-protein coupled receptor
Melanocortin 3 receptor
Transmembrane protein 179
Torsin A interacting protein 2
Moreover, MVs were shown to contain Staufen 2 (Fig. 1B) a ribonucleoprotein involved in the transport and stability of mRNA .
Incorporation of MVs in cultured hepatocytes
MVs labelled with PKH26 dye were incorporated by cultured human hepatocytes after 3 hrs of incubation at 37°C as shown by FACS analysis and confocal microscopy (Fig. 1A, C). In order to investigate the role of adhesion molecules expressed by the MV surface in the incorporation in target cells, MVs were pre-incubated (15 min., at 4°C) with blocking antibodies against the identified adhesion molecules or with sHA to block CD44 (Fig. 1C, D). MV treatment with anti-α4-integrin blocking antibody inhibited MV incorporation in human hepatocytes. Blockade of CD44 or CD29 only slightly reduced incorporation of MVs (Fig. 1D). The anti-α6-integrin blocking antibody used as negative control did not prevent MV internalization. Moreover, removal of surface molecules by trypsin treatment of MVs inhibited their incorporation in human hepatocytes confirming the relevance of surface expressed molecules in the process of MV internalization by target cells. Similar results were obtained using rat hepatocytes (data not shown).
In vitro proliferative and anti-apoptotic effects of MVs
Incubation of human hepatocytes with MVs derived from HLSC promoted a significant proliferation in respect to control cells incubated with vehicle alone (Fig. 2A). In addition, incubation of human hepatocytes with increasing doses of MVs significantly inhibited apoptosis induced by GalN (Fig. 2B). However, when MVs were incubated with RNase, that we previously showed to induce a complete degradation of the RNA shuttled by MVs without affecting their structure , proliferation and apoptosis resistance elicited by MVs were significantly reduced (Fig. 2A, B). As previously shown , RNase treatment did not modify the expression of surface molecules or the internalization of MVs (not shown). These results suggest that the MV biological effects were mediated by the transfer of mRNA following MV internalization, as previously described [21, 23]. DNase treatment was ineffective (not shown).
HLSC-derived MVs enhanced hepatic regeneration in rat with 70% hepatectomy
In the rat model of 70% hepatectomy, we observed a significant rise in AST and ALT and a significant reduction in serum albumin at 24, 48 and 72 hrs (Fig. 3A–C). In MV-treated rats the rise of AST and ALT was significantly lower after 72 hrs (Fig. 3A, B) in respect to the untreated. Moreover, in MV-treated rats an enhanced production of albumin was detected at 24 hrs (Fig. 3C). The liver/body weight ratio gradually increased after 70% hepatectomy in rats treated or not with MVs. However, after 72 hrs a significant enhancement of liver/body ratio was observed in MV-treated in respect to untreated rats (Fig. 3D). The histological examination of 70% hepatectomized rats showed a marked hepatocyte swelling and vacuolization (Fig. 4A). The staining for triglyceride with Oil red O indicated a diffuse hepatic steatosis (Fig. 4B). When animals were treated with MVs, the lesions were less severe at all the time points in respect to animals with 70% hepatectomy treated with vehicle alone (Fig. 4A) and the steatosis was minimal or absent (Fig. 4B). As shown in Figs 5 and 6, MV-treatment of rats with 70% hepatectomy significantly enhanced in vivo hepatocyte proliferation as detected by BrdU+ and PCNA+ cells in respect to hepatectomized rats injected with vehicle alone (Figs 5A, B and 6A, B). The enhanced proliferation was detected at all the different time points after MV treatment. RNase treatment of MVs abrogated MV-induced recovery of liver lesions and significantly inhibited hepatic proliferation induced by MVs (Figs 5A and 6A). The activation of a proliferative program in hepatocytes was also confirmed by Q-PCR showing a significantly enhanced expression of cyclin A1 in rats treated with MVs in respect to the untreated (Fig. 6C).
After hepatectomy a significant enhancement of apoptosis of hepatocytes was seen only after 72 hrs (Fig. 7A, B). When hepatectomized rats were treated with MVs, a significant reduction of apoptosis was observed.
In vivo evidence of de novo human protein expression in the liver of hepatectomized rats treated with human MVs
Q-PCR and RT-PCR for human AGO2, used as reporter gene, confirmed the accumulation of injected MVs within the liver of hepatectomized rats treated with MVs but not in those treated with vehicle alone (Fig. 8A, B). Using anti-human AGO2 antibody, the protein was detected both by Western blot analysis (Fig. 8C) of liver extracts and by immunofluorescence (Fig. 8D) in hepatectomized rats treated with MVs but not in those treated with vehicle alone. By confocal microscopy AGO2 protein was detected with a perinuclear localization in liver parenchyma. HLSC-derived MVs contained AGO2 mRNA (Table 1) but not the protein (not shown). These observations suggest that human specific mRNA shuttled by MVs could be translated in protein in liver cells during regeneration.
In the present study, we demonstrated that MVs derived from human HLSC promoted hepatocyte proliferation and suppressed hepatocyte cell death. MV administration accelerated in vivo liver regeneration in 70% hepatectomized rats. The RNase treatment of MVs abrogated both the in vitro and in vivo effects of MVs, suggesting that the RNA shuttled by MVs was the final effector of their biological effect.
The 70% hepatectomy in rats is a classical model to study liver regeneration. The remaining hepatic lobes of the rats can restore the liver mass two weeks after partial hepatectomy . It has been suggested that the regeneration process is due to the proliferation of residual mature hepatocytes that re-enter into the cell cycle and undergo proliferation . It is not clear whether resident or bone marrow derived stem cells may contribute to liver regeneration and whether resident stem cells may orchestrate the cell cycle entry of differentiated cells . Recently, Quesenberry and Aliotta  proposed a dynamic reconsideration of the stem cell niches suggesting that the lability of stem cell phenotype depends on complex interactions with differentiated cells based on MV mediated cell-to-cell transfer of genetic information. Moreover, these authors suggested that transfer of genetic information from injured cells may explain stem cell functional and phenotypic changes without the need of transdifferentiation into tissue cells . On the other hand, we showed that transfer of genetic information from stem cells may re-direct altered functions in target cells suggesting that stem cells may repair damaged tissues without directly replacing parenchymal cells [21, 23]. Indeed, the genetic information shuttled by MVs may be exploited to activate angiogenic and regenerative programs in target cells [21, 23].
Shedding of MVs is no longer considered an artefact [38, 39] and it has been shown to occur in vivo. It has been suggested that shedding vesicles could participate in important biological processes and their significant number at the surface of a Cajal-like cell of the heart  suggests that these structures may transport important information to other cells.
In the present study, we demonstrated that MVs derived from HLSC may induce in vitro proliferation and apoptosis resistance in human and rat hepatocytes. HLSC is a pluripotent stem cells population resident in the adult human liver . In vivo HLSC were shown to contribute to liver regeneration in a model of acute liver injury in SCID mice . In the present study we found that HLSC spontaneously released MVs in culture. MVs released from HLSC had a size similar to that described for MVs released from endothelial progenitors cells  or from bone marrow derived mesenchymal stem cells  and expressed several adhesion molecules known to be present on the surface of HLSC such as CD44, CD29 (b1-integrin), α4-integrin. It has been previously reported that the adhesion molecules expressed by MVs are instrumental in their internalization into target cells . We found that α4-integrin blockade prevented MV incorporation into hepatocytes whereas blockade of CD44 and CD29 had only minor effects.
The internalization of MVs in target cells was previously found to be essential for their biologic activity but not the final mechanism of action . Indeed, RNase treatment almost completely abrogated MV-induced angiogenic and regenerative effects suggesting a critical role of RNA transfer mediated by MVs [21, 23]. In the present study, we found that MV treatment with RNase abrogated the in vitro proliferation and resistance to apoptosis of hepatocytes.
The in vivo administration of MVs derived from HLSC was found to accelerate liver regeneration in 70% hepatectomized rats. This was possibly related to an in vivo stimulation of proliferation of hepatocytes, as indicated by the incorporation of BrdU, the expression of PCNA and of cyclin A1 by hepatocytes. Moreover, MV treatment significantly reduced liver apoptosis as seen by TUNEL. Pre-treatment of MVs with RNase abrogated also the protective effect of MVs in 70% hepatectomized rats. One can speculate that the delivery of exogenous mRNA by MVs to hepatocytes may stimulate hepatic regeneration. Indeed, MVs derived from HLSC contained a defined pattern of mRNA associated with several cell functions involved in the control of transcription, translation and proliferation. In vivo evidence for an effective horizontal transfer of mRNA was obtained by the presence of the human specific mRNA for AGO2, chosen as reporter gene, and by its de novo protein expression in the liver of MV-treated hepatectomized rats. AGO2 is a regulatory protein with a central role in RNA silencing processes . The results of present study are in line with the recent evidence that MVs may shuttle biologically active mRNA or microRNA able to regulate cell function of different target cells [20–23, 41–43]. Of interest, the pattern of genes present in HLSC-derived MVs was substantially different from that of endothelial precursors and mesenchymal stem cells [21, 23] indicating a specificity for the cell of origin. Moreover, MVs were shown to contain Staufen 2 suggesting a role of ribonucleoproteins in carrying and stabilizing RNAs  within MVs.
In conclusion, MV-mediated transfer of mRNA from HLSC to hepatocytes may represent a mechanism that contribute to liver regeneration and that could be exploited in regenerative medicine.
This work was supported by Fresenius Medical Care and by Regione Piemonte, progetto PISTEM.