Membrane-derived vesicles (MV) are released from the surface of activated eucaryotic cells and exert pleiotropic effects on surrounding cells. Since the maintenance of pluripotency and undifferentiated propagation of embryonic stem (ES) cells in vitro requires tight cell to cell contacts and effective intercellular signaling, we hypothesize that MV derived from ES cells (ES-MV) express stem cell-specific molecules that may also support self-renewal and expansion of adult stem cells. To address this hypothesis, we employed expansion of hematopoietic progenitor cells (HPC) as a model. We found that ES-MV (10 μg/ml) isolated from murine ES cells (ES-D3) in serum-free cultures significantly (i) enhanced survival and improved expansion of murine HPC, (ii) upregulated the expression of early pluripotent (Oct-4, Nanog and Rex-1) and early hematopoietic stem cells (Scl, HoxB4 and GATA 2) markers in these cells, and (iii) induced phosphorylation of MAPK p42/44 and serine-threonine kinase AKT. Furthermore, molecular analysis revealed that ES-MV express Wnt-3 protein and are selectively highly enriched in mRNA for several pluripotent transcription factors as compared to parental ES cells. More important, this mRNA could be delivered by ES-MV to target cells and translated into the corresponding proteins. The biological effects of ES-MV were inhibited after heat inactivation or pretreatment with RNAse, indicating a major involvement of protein and mRNA components of ES-MV in the observed phenomena. We postulate that ES-MV may efficiently expand HPC by stimulating them with ES-MV expressed ligands (e.g., Wnt-3) as well as increase their pluripotency after horizontal transfer of ES-derived mRNA.
Cells communicate by secreted growth factors, cytokines, adhesion molecules and small molecular mediators such as nucleotides or bioactive lipids.1, 2, 3, 4 Recently, we and others have postulated that circular membrane fragments called microvesicles (MV) play an important but underappreciated role in cell to cell communication.5, 6, 7, 8 MV are shed from the surface of activated cells or are derived from the endosomal membrane compartment after fusion of secretory granules with the plasma membrane, where they exist as intraluminal membrane-bound vesicles.9, 10, 11 MV released from surface membranes are generated in a calcium flux–calpain-dependent manner and are relatively large (100 nm−1 μm), in contrast to smaller membrane fragments derived from the endosomal compartment (30–100 nm). These MV contain numerous proteins and lipids similar to those present in the membranes of the cells from which they originate.
We and others reported that MV may affect target cells (i) by stimulating them directly as ‘signaling devices’ by surface-expressed ligands,5, 6, 7 or (ii) by transferring surface receptors between cells.12, 13 This MV-mediated communication between cells may perhaps have developed very early in the course of eucaryocytic evolution, before soluble mediators emerged. Recently, we and others postulated that MV may play a role in the spread of certain infections (e.g., HIV or prions) after they have fused with the target cells and delivered infectious particles inside the MV to the cytoplasm, by a so-called ‘Trojan horse mechanism’ of infection.12, 14, 15, 16
Embryonic stem (ES) cells are a rich source of MV. The propagation of ES cells in the undifferentiated state as well as induction of differentiation in developing embryoid bodies is coregulated by intercellular communications within ES cell colonies or embryoid bodies through membrane-bound molecules. Hence, we hypothesize that ES-derived MV express various stem cell-specific molecules that may affect the growth of target cells and contribute to the cell-fate decision. Owing to these characteristics, undifferentiated ES-derived MV may represent one of the critical components that support self-renewal and expansion of pluripotent or multipotent stem cells in vitro.
Furthermore, it had been demonstrated that mature somatic cells cocultured with intact ES cells or extracts from these cells undergo epigenetic changes.17, 18 We hypothesize that some of these effects could be explained by a biological modification of the target cells via ES-MV.17, 18, 19, 20 If this is true, a reverse mechanism by which pluripotent ES cells influence growth of hematopoietic progenitor cells (HPC) could occur. To test this hypothesis we employed ES-MV obtained either from murine ES cells (D3) or human ES cells (CCTL14) in a model of expansion of murine HPC.
We found that both murine D3 cell-derived ES-MV and human CCTL14 cell-derived ES-MV are highly enriched in Wnt-3 protein and mRNA for several early pluripotent transcription factors as compared to the ES cells from which they originated. This selective increase in mRNA content in ES-MV compared to parental ES cells suggests the presence of a mechanism that enriches ES-MV in mRNA molecules before their shedding from ES cells. Intrigued by these observations, we investigated whether ES-MV could enter HPC as a kind of physiological ‘liposome’ and increase their pluripotency after delivering ES-derived mRNA.
We found that ES-MV may induce changes in HPC not only by stimulating them with surface-expressed ligands but also by delivery of ES-derived mRNA. This latter observation provides novel evidence for horizontal transfer of genetic information between cells. We also suggest that ES-MV could be employed as a new tool to expand adult stem cells. As we revealed that MV derived from human ES cells also express molecules that are critical for growth of stem cells, human ES cell-derived MV hold promise for expansion of human peripheral blood stem cells suitable in transplantation. Studies to identify other biologically active components of ES-MV in addition to mRNAs coding several stem cell-specific transcription factors and Wnt-3 protein are in progress.
Materials and methods
ES cell culture and isolation of ES-MV
Murine ES-D3 cells were purchased from ATCC (Rockville, MD, USA) and cultured in Dulbecco's Modified Eagle's Medium with 4 mM L-glutamine, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate supplemented with: 0.1 mM 2-mercaptoethanol, 15% heat-inactivated FBS and 5 ng/ml mLIF.
The human ES cell line CCTL14 was developed in the Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic. Complete information on derivation and characterization of this hESC line is available at http://www.isscr.org/science/sclines.htm. For this study, undifferentiated hESCs were maintained on mitomycin C-treated mouse embryonic fibroblasts in DMEM:F12 supplemented with 15% knockout serum replacement, 1 mM L-glutamine, 0.1 mM MEM nonessential amino acids, penicillin/streptomycin, 0.1 mM β-2 mercaptoethanol and 4 ng/ml FGF-2.
ES-MV were obtained from cultured ES-D3 cells and human CCTL-14 cells as previously described. As a control, MVs were also obtained from mitotically inactivated mouse embryonic fibroblasts alone. MV enriched in exosomes were washed once and resuspended in HEPES buffer, pH 7.4. The concentration of ES-MV was estimated by Bradford assay as described.21
Isolation of murine Sca-1+/kit+/lin− (SKL) cells
Sca-1+/kit+/lin− cells were isolated from a suspension of murine bone marrow (BM) mononuclear cells (MNC) by multiparameter, live sterile-cell sorting (FACSVantage SE; Becton Dickinson, Mountainview, CA, USA). Briefly, BMMNC (100 × 106 cells/ml) were resuspended in cell sort medium (CSM), containing 1 × Hank's balanced salt solution without phenol red (GIBCO, Grand Island, NY, USA), 2% heat-inactivated fetal calf serum (FCS; GIBCO), 10 mM HEPES buffer (GIBCO), and 30 U/ml Gentamicin (GIBCO). The following mAbs from B-D Biosciences – Pharmingen (San Diego, CA, USA) were employed to stain these cells: biotin-conjugated rat anti-mouse Ly-6A/E (Sca-1) (clone E13-161.7) FITC, anti-c-Kit allophycocyanin (clone 2B8), anti-CD45-APC-Cy7 (clone 30-F11), anti-CD45R/B220-PE (clone RA3-6B2), anti-Gr-1-PE (clone RB6-8C5), anti-TCRαβ PE (clone H57-597), anti-TCRγζ PE (clone GL3), anti-CD11b PE (clone M1/70) and anti-Ter-119 PE (clone TER-119 – which reacts with 52-kDA molecule associated with glycophorin-A). All mAbs were added at saturating concentrations and the cells were incubated for 30 min on ice and washed twice, then resuspended for sort in CSM at a concentration of 5 × 106 cells/ml. All mAbs were added at saturating concentrations and the cells were incubated for 30 min on ice and washed twice, then resuspended in CSM at a concentration of 5 × 106 cells/ml. In some experiments Sca-1+ positive cells were isolated by employing immunomagnetic beads as described previously.22
Effect of ES-MV on survival of murine SKL cells
Sca-1+ MNC were resuspended in Iscove's DMEM (Gibco BRL, Grand Island, NJ, USA) (104/ml) supplemented with 5% artificial serum containing 1% delipidated, deionized, and charcoal-treated BSA, 270 μg/ml iron-saturated transferrin, insulin (20 μg/ml), and 2 mmol/l L-glutamine (all from Sigma, St. Louis, MO, USA) and cultured for 5 days in the presence or absence of ES-MV (0–50 μg/ml). At day 5, cells were harvested and replated in secondary serum-free methylocellulose cultures to grow CFU-Mix, BFU-E, CFU-GM and CFU-Meg colonies as described.22, 23 Cultures were incubated at 37°C in a fully humidified atmosphere supplemented with 5% C02. Colonies were scored at days 7–10 using an inverted microscope.
Ex vivo expansion of murine HPC cells
Sca-1+ MNC were resuspended in Iscove's DMEM (Gibco BRL, Grand Island, NJ, USA) (104/ml) supplemented with 20 % artificial serum containing 1% delipidated, deionized, and charcoal-treated BSA, 270 mg/ml iron-saturated transferrin, insulin (20 mg/ml), and 2 mmol/l L-glutamine (all from Sigma, St Louis, MO, USA) and cultured for 5 days in the presence or absence of ES-MV (10 μg/ml) or in the presence of murine recombinant GFs (R&D, Minneapolis, MN, USA): mKL (25 ng/ml)+mFLT3 ligand (mFLT3L) (25 ng/ml)+mTPO (20 ng/ml)+mIL-6 (10 ng/ml). At day 5 cells were harvested, the number of MNC counted and the cell samples were evaluated for the number of CFU-S cells.
For the colony forming units-spleen (CFU-S) assays, BALB/c mice were irradiated with a lethal dose of γ-irradiation (950 cGy) and transplanted with 5 × 104 freshly isolated Sca-1+ marrow cells (control) or the same number of cells from expansion experiments (cells expanded with GFs or ESMV as described above). At day 12, spleens were removed and fixed in Tellysyniczky's fixative and CFU-S colonies were counted on the surface of the spleen using a magnifying glass as previously described.21
For analysis of mRNA levels, total mRNA was isolated from cells with the RNeasy Mini Kit (Quiagen, Inc.). mRNA was reverse-transcribed with TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA). Quantitative assessment of selected gene (Table 1) mRNA levels was performed by real-time RT–PCR using an ABI PRISM® 7000 Sequence Detection System (ABI, Foster City, CA, USA). All of the primer sequences are provided in Table 1. Primers were designed with Primer Express software. A 25 μl reaction mixture containing 12.5 μl SYBR Green PCR Master Mix and 10 ng of cDNA template (primers) were used. The threshold cycle (Ct), that is, the cycle number at which the amount of amplified gene of interest reaches a fixed threshold, was subsequently determined. Relative quantitization of the selected gene (Table 1) mRNA expression was performed with the comparative Ct method. The relative quantitization value of the target, normalized to an endogenous control β-actin (house-keeping) gene and relative to a calibrator, is expressed as (-fold difference), where ΔCt=(Ct of target genes listed in Table 1)–(Ct of endogenous control gene (β-actin), and ΔΔCt=(ΔCt of samples for target gene)–(ΔCt of calibrator for the target gene).
To avoid the possibility of amplifying contaminating DNA (i) all of the primers for real-time RT–PCR were designed containing a DNA intron sequence for specific cDNA amplification; (ii) reactions were performed with appropriate negative controls (template-free controls); (iii) a uniform amplification of the products was rechecked by analyzing the melting curves of the amplified products (dissociation graphs); (iv) gel electrophoresis was performed to confirm the correct size of the amplification and the absence of nonspecific bands.
Activation of cell growth-related kinases
Western blot analysis was performed on protein extracts from the cells as described.21 After the SKL cells were stimulated with ES-MV (10 μg/ml) for 5 or 10 min at 37°C Phosphorylation of serine/threonine kinase AKT and p44/42 mitogen-activated protein kinase (MAPK) was detected by protein immunoblotting using mouse monoclonal p44/42 phospho-specific MAPK antibody and rabbit phospho-specific polyclonal antibodies (all from New England Biolabs, Beverley, MA, USA) for each of the remainder, with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig) G or goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as a secondary antibody, as described. Equal loading in the lanes was evaluated by stripping the blots and reprobing them with the appropriate monoclonal or polyclonal antibodies: p42/44 anti-MAPK antibody clone 9102 and anti-AKT antibody clone 9272 (Santa Cruz Biotechnology). The membranes were developed with an ECL reagent (Amersham Life Sciences, Little Chalfont, UK), dried and exposed to film (HyperFilm, Amersham).
Confocal fluorescence staining analysis
The study was performed on the rhabdomyosarcoma cell line RH30 (gift from Dr Barr, University of Pennsylvania, Philadelphia, PA, USA) and embryonal cell line ESD3 (Rockville, MD, USA). Cells were seeded on coverslips and nuclei were stained with Hoechst 33342 (2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride) (Sigma) by adding dye to culture medium for 1 h. MV were stained with PKH26 Red Fluorescent Cell Linker Mini Kit for General Cell Membrane Labeling (Sigma), washed with PBS and added to the culture medium. Cells incubated with MV were incubated overnight. After washing with PBS the coverslips were mounted.
Confocal laser scanning microscopy
Photographs were taken using a LSM510 Zeiss confocal laser scanning microscope (Carl Zeiss Microscopy, Germany; version 3.2 SP2) and a × 63 water immersion objective (Zeiss, C-Apochromat W Corr) with a numeric aperture of 1.2, by using separate channels for the analysis of phase-contrast images, red fluorescence and blue fluorescence. Filters LP 385 and LP560 for excitation at 364 nm (enterprise) for Hoechst 33342 and 543 nm (helium–neon laser) for PKH26, respectively, were employed. Images with 512 × 512 points were acquired in 1.6 s and had a size of 146.2 × 146.2 μm. To observe the location of the MV in the cell we took pictures of different sections. The size of the slices was 1.53 μm for 12 slices, 1.6 μm for 15 slices and 0.5 μm for 41, respectively. Representative photographs from three independent experiments are shown.
Detection of Wnt3 and Oct-4 by Western blot
Cell lysates from SKL cells that had been stimulated (or not) for 24 h with ES-MV (10 ng/ml) pretreated (or not pretreated) with RNAse 1 U/ml/1 h (Ambion Inc. Austin, TX, USA) were prepared as described above. Oct-4 protein was detected using anti-Oct-4 mouse monoclonal antibodies (Chemicon International Inc. Temecula, CA, USA) and goat-anti-mouse immunoglobulin IgG-conjugated with horseradish peroxidase (HRP) according to the manufacturer's protocol. Wnt-3 protein was detected by protein immunobloting using anti Wnt-3 goat-polyclonal IgG antibody (Santa-Cruz Biotechnology Inc., Santa Cruz California, CA, USA) and anti-goat IgG-conjugated with horseradish peroxidase HRP (Santa-Cruz Biotechnology Inc., Santa Cruz California, CA, USA) as a secondary antibody. At the same time the cell lysates from ES-D3 cells and from ESMV were prepared. The membranes were developed with an ECL reagent (Amersham Life Sciences, Little Chalfont, UK), dried and exposed to film (HyperFilm, Amersham).
The expression of Oct-4 protein in Sca-1+/kit+/lin− cells was verified by immunocytochemistry. Murine BM-derived Sca-1+/lin−/CD45+ BM-derived cells sorted by FACS were coincubated with ES-MV (10 mg/ml) for 24 h at 37°C in a presence or absence of RNAse (1 U/ml, Ambion Inc., Austin, TX, USA). After incubation cells were fixed in 1% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100, and incubated overnight at 4°C with mouse monoclonal anti-Oct-4 (Chemicon International Inc.) primary antibody. Anti mouse IgG – Alexa 594 labeled secondary antibody (Molecular Probes, Eugene, OR, USA) was used for the detection of Oct-4. Cells positive for Oct-4 were identified with the TE-FM Epi-Fluorescence system attached to a Nikon Inverted Microscope Eclipse TE300 (Carl Zeiss, Thornwood, NY, USA). All of the fluorescence images were captured by a cool snap HQ digital B/W CCD (Roper Scientific) camera. Nuclei were stained with DAPI (Molecular Probes, Eugene, Oregon).
Data are reported as mean±s.d. All statistical analyses were performed on a Macintosh computer (PowerBase 180) using the Instat 1.14 software (GraphPad, San Diego, CA, USA). Data were analyzed using the Student's t-test for unpaired samples. Statistical significance was defined as P<0.01.
ES-MV enhance survival of murine SKL progenitor cells
First we tested whether ES-MV enhance survival of early murine hematopoietic SKL cells cultured under serum-free conditions. To address this issue purified murine SKL cells were seeded on 24-well plates in IMDM in the absence or presence of 0, 1, 10 or 50 μg of ES-MV. At time 0 the aliquot of control SKL cells (unexposed to ES-MV) was plated in methylcellulose cultures and stimulated with a combination of the appropriate growth factors to grow CFU-Mix, BFU-E, CFU-GM and CFU-Meg colonies. The rest of the cells were cultured for 5 days in liquid culture. At day 5, the cells were removed from the wells and plated as described above for control cells in secondary methylcellulose cultures to grow CFU-Mix, BFU-E, CFU-GM and CFU-Meg-derived colonies.
As shown in Table 2 the clonogenecity of SKL cells not exposed to ES-MV decreased after 5 days of culture in medium alone to 34, 21, 15 and 19% for CFU-Mix, CFU-GM, BFU-E and CFU-Meg, respectively. Addition of ES-MV (1 μg/ml) to medium maintained and even slightly increased the number of clonogeneic progenitors in the cultures. Increased concentration of ES-MV (10 μg/ml) enhanced their number (∼ × 3) compared with the initial number of these cells present in freshly purified SKL cells at time 0. Thus, these data indicate that ES-MV not only protected clonogeneic SKL cells from undergoing apoptosis but also significantly expanded their input number (Table 2).
ES-MV enhance expansion of murine Sca-1+ cell-derived CFU-S
To better address the effect of ES-MV on the expansion of HPC, we seeded aliquots of Sca-1+ cells on 24-well plates in IMDEM supplemented with 20% of artificial serum in medium alone, or in the presence of optimal doses of KL+mFLT3L+TPO+IL-6 or ES-MV (50 μg/ml). The combination of growth factors/cytokines employed in this study was selected as the most potent combination of factors to expand SKL cells based on our previous studies. At time 0 aliquots of purified Sca-1+ cells were tested for the presence of day-12 CFU-S able to form spleen colonies in irradiated syngeneic recipients and mRNA samples were isolated for real-time RT–PCR analysis of gene expression. The rest of the purified Sca-1+ cells were expanded for 5 days, and subsequently we evaluated (i) the total number of MNC present in the expansion cultures, (ii) number of day-12 CFU-S able to form spleen colonies in irradiated syngeneic recipients and (iii) using real-time RT–PCR, expression of mRNA for early stem cell-related transcription factors in expanded cells.
Figure 1 shows that although ES-MV were less efficient than the cocktail of GFs in expanding the total number of MNC cells from Sca-1+ cells (Figure 1A), they expanded better than the cocktail of GFs with respect to total number of day-12 CFU-S (∼6 times and ∼4 times, respectively) (Figure 1B). Furthermore, the expansion of CFU-S in the presence of ES-MV correlated with an increase of mRNA for early transcription factors such as Oct-4, Rex-1, Nanog and HoxB4 in expanded cells (Figure 1C). This effect was abrogated if ES-MV were heat-inactivated before expansion, suggesting a major involvement of thermo-labile components of ES-MV in the observed phenomena (not shown).
Interestingly, when we compared freshly isolated SKL cells and cells expanded in the presence of a cocktail of GFs using real-time RT–PCR expression of pluripotent transcription factors, we observed a slight decrease in expression of Oct-4, Nanog and Rex-1 (∼x2, x4 and x3, respectively) in cells expanded with GFs (not shown). This suggests that the primitive potential of HPC decreases after exposure to these factors.
ES-MV induce phosphorylation of MAPKp42/44 and AKT in murine Sca-1+ cells
Since an effect on proliferation/survival of early murine hematopoietic cells was observed, we assessed whether ES-MV can affect signaling pathways in murine BM-derived Sca-1+ cells that are crucial for cell proliferation/survival. As shown in Figure 2, we noticed that ES-MV enhanced the phosphorylation of stress-induced kinases MAPK p42/44 and serine/threonine kinase AKT (Figure 2). Again, activation of MAPK p42/44 was not visible and activation of AKT was less pronounced when ES-MV were heat inactivated before adding them to the cultures, again suggesting a major involvement of thermo-labile molecules in the observed phenomena (Figure 2, lane 3).
ES-MV are enriched in Wnt-3 and Oct-4 proteins and mRNA for several pluripotent transcription regulatory factors
Based on these observations we attempted to identify potential temperature-labile components that are crucial to these phenomena. First, based on the observation that Wnt-3 plays a pivotal role in stem cell expansion,24, 25, 26 we tested whether Wnt-3 protein is detectable in ES-MV. As shown in Figure 3, we determined by Western blotting that Wnt-3 isoforms A and B are a normal constituent of ES-MV. Thus, it is likely that Wnt-3 present on ES-MV could contribute to the expansion of HPC. This, however, requires further investigations. Interestingly, we also found that ES-MV are enriched in the pluripotent stem cell transcription factor, Oct-4 protein (Figure 3).
Next, based on the report that apoptotic bodies are selectively enriched in mRNA,27, 28 we evaluated whether ES-MV are similarly enriched in ES-derived mRNA. First, using spectroscopic analysis and direct Pyronin Y fluorescence staining we confirmed that ES-MV are highly enriched in mRNA (not shown). Second, we evaluated by real-time RT–PCR expression of selected early pluripotent stem cells the transcription factors Oct-4, Rex-1, Nanog, SCL and GATA-2 in ES-MV. Figure 4A shows that murine ES-MV are highly enriched in messages for these early pluripotent transcription factors compared to parental ES-D3 cells. We obtained similar results by analyzing human ES-MV derived from the CCTL14 cell line (Figure 4B).
Interestingly, when we compared the expression of several other mRNAs between ES-MV and parental ES-D3 cells (Table 3) we found that ES-MV are to different degrees enriched in mRNA for early transcription factors, some cytokines and receptors. This indicates ES-MV are selectively enriched in mRNA during their formation.
ES-MV act as ‘physiological liposomes’, attach to target cells, fuse with them and deliver their content into the cytoplasm
As (i) apoptotic bodies have been described as being involved in horizontal transfer of DNA29, 30, 31, 32 between cells and (ii) cell extracts have been demonstrated to induce epigenetic changes in permeabilized target cells,18, 19, 33 we hypothesized that ES-MV, acting as a kind of ‘physiological liposome,’ could also potentially deliver their content into normal cells (physiological lipofection).
To address this notion we exposed parental ES cells (Figure 5A), sarcoma RH30 cells (Figure 5B) as well as murine SKL cells (Figure 5C) to ES-MV that were labeled with lipid-fluorochrome PKH26. We observed that ES-MV attach to target cells and after this interaction/fusion with the target cells their labeled lipid components could be detected inside the cells (Figure 5). We obtained similar results with human CD34+ cells (not shown).
Oct-4 mRNA is delivered by ES-MV to SKL cells and subsequently becomes translated into Oct-4 protein
We tested the hypothesis that mRNA in ES-MV (Figure 3C) could be delivered to target cells and translated to the appropriate proteins using SKL cells as a model and evaluating expression of Oct-4 by Western blot and immunofluoresence before and after incubation of these cells with ES-MV (Figure 6).
As reported above, ES-MV were found both to be highly enriched both in mRNA for Oct-4 (Figure 1C) and to express Oct-4 protein (Figure 3). Further, to examine the contribution of Oct-4 mRNA versus Oct-4 protein as delivered by the ES-MV to the SKL cells; control experiments were carried out using ES-MV pretreated with RNAse before addition to the target cells. Figure 6A shows that Oct-4 is not detectable by Western blot in freshly isolated SKL cells (lane 1), but these cells acquire expression of Oct-4 protein after incubation with ES-MV (lane 2), and that this expression was significantly diminished when ES-MV were exposed to RNAse before incubation with SKL cells (lane 3). Of note, no Oct-4 mRNA was found by RT–PCR in ES-MV that were pretreated with RNAse (not shown). However, as expected, RNAse did not affect expression of Oct-4 protein in ES-MV (lanes 4 and 5). As pretreatment with RNAse significantly decreased expression of Oct-4 in SKL cells, this suggests that ES-MV-derived mRNA makes a significant contribution to Oct-4 expression. However, at this point we cannot estimate how much in addition Oct-4 expression resulted from stimulation of KSL by ES-MV expressed ligands and/or from intracellular delivery of Oct-4 protein by ES-MV (Figure 3).
Finally, to confirm that Oct-4 expression in SKL cells and its nuclear localization in SKL cells exposed to ES-MV occur, we performed immunofluorescence confocal analysis of Oct-4 expression in these cells exposed or not exposed to ES-MV. As shown in Figure 6B, Oct-4 protein which was not detectable in SKL cells unexposed to ES-MV became highly expressed in the nuclei of these cells after preincubation with ES-MV. As in Western blot analysis (Figure 6A), the expression of Oct-4 was significantly decreased in SKL cells when ES-MV were pretreated with RNAse.
MV are secreted by activated normal healthy cells and play an important role in cell to cell communication acting as signaling devices that stimulate target cells via the ligands expressed on their surface.7, 9, 10 MV were also reported to transfer between cells certain cell surface-expressed adhesion molecules or receptors, for example, CD41 integrin or CXCR4,5, 6, 12, 34 as well as infectious particles (e.g, HIV, prions).14, 15, 35, 36 Assuming that ES-MV express some unique molecules that regulate self-renewal of pluripotent cells in embryoid bodies, we investigated whether ES-MV could be used as a new tool to expand ex vivo adult murine HPC.
We found that murine BM-derived HPC cells cultured in the presence of ES-MV became highly enriched for clonogenic progenitors from all major hematopoietic lineages (CFU-Mix, CFU-GM, CFU-Meg and BFU-E) as well as in CFU-S. It is important to note that ES-MV could be obtained from expotentially growing ES cells. This suggests that the mechanism for release of MV from ES cells is inherent. At the molecular level, SKL cells expanded in the presence of ES-MV highly expressed mRNA for early transcription factors such as Oct-4, Rex-1, Nanog and HoxB4 operating at the level of pluripotent stem cells. We hypothesized that upregulation of these transcription factors is a result of stimulation of SKL cells with ES-MV-expressed ligands (e.g., Wnt-3) and/or a result of mRNA transfer/delivery by ES-MV from ES to HPC.
In support of this latter notion, we found that ES-MV are highly enriched in mRNA for various transcription factors, cytokines and receptors. Furthermore, the selective differences in expression of mRNA in ES-MV and parental ES suggest involvement of a regulatory mechanism in this process. In this context our data point to a novel mechanism that may operate in all embryonic cells starting at the inner cell mass stage of the developing blastocyst and extending through to the inner cell mass-derived ES cells. It is noteworthy that there are significant differences between mouse and human ES cells in self-renewal ability after dissociation to single cell suspension or small clumps with fewer than 10 cells. Human ES cells have a very limited capacity to self-renew when plated in single cell suspension or in small clumps. In addition, cell density is also critical for expansion of human ES cells. Hence, it is widely accepted that human ES cells require close cell–cell contact and intensive crosstalk via membrane-associated factors to maintain their undifferentiated state and to proliferate. Such a microenvironment could be, at least partially, created by ES-MV. Furthermore, since MV are secreted not only by ES but also by various normal and cancer cells that have been activated, MV may play an important role in the horizontal transfer of mRNA and communication between several types of eucaryotic cells. To our surprise, we found that Oct-4 mRNA that was delivered by ES-MV to HPC was actively translated into protein. Whether a similar mechanism operates for mRNA for other proteins requires further study.
The fact that mRNA molecules after translocation from the nucleus to the cytoplasm may bind to and are transported inside cells on membranous organelles or vesicles to specific subcellular sites explains why ES-MV are highly enriched for mRNA. A similar mechanism may also explain why vesicles derived from apoptotic cells are also highly enriched in mRNA.27, 28, 37, 38
In support of the concept of MV (e.g., ES-MV) playing a role in horizontal exchange of molecules between cells, it has been demonstrated that eucaryotic cells possess highly sophisticated membrane trafficking pathways that define specific membrane domains and provide a means for moving vesicles between them. Recently, it was demonstrated, for example, that cells cultured in vitro may exchange MV in so-called tunneling nanotubules.39 By performing three-dimensional live-cell microscopy it has been shown that eucaryotic cells in cultures can communicate by exchanging MV and some organelles in ultrafine intracellular structures that connect the cytoplasm of distantly located cells.39, 40 Based on this we postulate that MV, both those secreted as ‘free MV’ into the extracellular space or those trafficking inside nanotubules can play a significant role as messengers between eucaryotic cells. Furthermore, MV may also deliver to cells not only mRNA but also proteins, bioactive lipids and other molecules.7, 41 As MV are derived from the membranes of normal cells, they seem to be optimal fusion partners for target cells and thus, the MV-mediated transfer/delivery of molecules into cells can be likened to ‘physiological lipofection’. A similar transfer/fusion mechanism was recently described for apoptotic microvesicles that may be involved in horizontal transfer of genes encoding some oncogenes or viral DNA.14, 30, 31
Lending further support our hypothesis, it has been well documented that cell extracts may induce epigenetic changes in cocultured target cells; however, the mechanisms responsible for this phenomenon are not clear. This phenomenon has been demonstrated, for example, for permabilized fibroblasts exposed to extracts from lymphocytic cells.18, 33 These fibroblasts after exposure to extracts from lymphocytic cells began to express several genes typical for lymphocytes.33 Similarly, a phenomenon was described in the cultures of murine hematopoietic stem cells (HSC) exposed to the extracts from damaged liver cells. After this exposure HSC began to express some genes specific for hepatocytes,42 a finding that supports the notion that proteins and mRNA molecules exogenously delivered to the cells may induce changes in target cells. Based on this we envision that some of the previously published data, where the trans-dedifferentiation or plasticity of stem cells was demonstrated could be explained at least in part by changes in transplanted stem cells due to horizontal transfer of mRNA/proteins from the damaged tissues.
In this paper, we also clearly demonstrated that MV may contribute to the developmental program of target cells. Our heat inactivation and RNAse exposure data suggest a major contribution of (i) ES-MV expressed Oct-4 mRNA and/or protein transferred to cells or (ii) de novo synthesis of Oct-4 protein in SKL cells stimulated by ES-MV-expressed ligands (e.g., Wnt-3). These phenomena could be explained by changes in target cells by ES-MV-derived ES cell mRNA for pluripotent transcription factors as well as direct stimulation of target cells, for example, by ES-MV-expressed ligands (e.g., Wnt-3). We are aware, however, that involvement of other protein components of ES-MV as well as other mRNA molecules requires further investigation and careful analysis. It is also not clear whether the reprogramming capacity of ES-MV is involved in physiological modulation of cell biology and how important is MV-mediated crosstalk between other cell types. Furthermore, since several pluripotent stem cell transcription factors were upregulated in SKL cells expanded in the presence of ES-MV, but were downregulated in the presence of a cocktail of recombinant cytokines and growth factors, our data indicate that ES-MV may have some advantage over recombinant growth factors/cytokines by reversing the phenotype of HPC to a more primitive pluripotent stem cell stage. In particular, MV isolated from human ES cell-conditioned medium may represent a promising tool for the expansion of human HSC. However, further investigations of the possible transmission of histocompatibility antigens that may trigger the immune rejection process is needed.
We expect that proteomic analysis of ES-MV will reveal some molecules that may operate during formation of the inner cell mass in the blastocyst and in undifferentiated ES cells. Later during differentiation, such molecules can be downregulated and/or expressed only locally in restricted tissue locations in stem cell-specific niches. Thus, perhaps by studying protein/lipid composition of ES-MV, we will be able to identify molecules crucial for stem cell renewal/expansion without inducing their differentiation. Such molecules are not completely identified yet, but their development would be crucial for optimizing strategies for stem cell expansion. Studies to identify other biologically active components of ES-MV are in progress.
This work was supported by NIH Grant R01 CA106281-01 to MZR and a GA CR 301/03/1122 Grant to PD.