Enhanced Cardioprotection by Human Endometrium Mesenchymal Stem Cells Driven by Exosomal MicroRNA-21

1The present report describes the functional and mechanistic comparisons of mesenchymal stem cells (MSCs) from three different sources (bone marrow, adipose, and endometrium) as therapeutic agents for the treatment of cardiovascular disease. MSCs from the endometrium have markedly superior therapeutic potential relative to the other cell types, properties that can be explained by the paracrine actions mediated by exosomal microRNAs, in particular microRNA‐21, with downstream effects on cell survival and angiogenesis. The findings are important from a clinical perspective because the endometrium could be a preferential source of MSCs for cardiovascular cell therapy.

2The present report describes the functional and mechanistic comparisons of mesenchymal stem cells (MSCs) from three different sources (bone marrow, adipose, and endometrium) as therapeutic agents for the treatment of cardiovascular disease. MSCs from the endometrium have markedly superior therapeutic potential relative to the other cell types, properties that can be explained by the paracrine actions mediated by exosomal microRNAs, in particular microRNA‐21, with downstream effects on cell survival and angiogenesis. The findings are important from a clinical perspective because the endometrium could be a preferential source of MSCs for cardiovascular cell therapy.

3Stem cell infusion and intramyocardial injection remain promising approaches for the treatment of coronary artery disease, myocardial infarction (MI), and heart failure 1. Mesenchymal stem cells (MSCs) are potential off‐the‐shelf reagents for such therapy, although their full potential has not yet been achieved, and clinical studies have suggested that bone marrow MSCs (BMMSCs) confer only mild and, in some cases, negligible effects on heart function and infarct size 2 3 4 5. Endometrium‐derived MSCs (EnMSCs) can be conveniently isolated from menstrual blood and can be expanded to create almost infinite supplies 6. Our group recently reported the enhanced therapeutic properties of transplanted EnMSCs, including preservation of cardiac function, decreased infarct size, and ameliorated cardiac fibrosis, mediated primarily by paracrine effects in a rat model of MI 7, 8. Because the tissue origin of MSCs can critically determine the paracrine activity and therapeutic potential 5, 9, it is important to implement comparative studies in parallel to identify the optimal sources for clinical application.

4Exosomes are cell‐derived microvesicles that are released from the plasma membrane into the extracellular space, where they play key roles in intercellular signaling 10. Exosome communications can involve the transfer of proteins, cytokines, lipids, mRNAs, ribosomal RNAs, and microRNAs (miRs) 9, 11. Recently, exosomes have been shown to play key roles in the paracrine actions of MSCs and can be central mediators of MSC cytoprotection in the contexts of both myocardial infarction and ischemia/reperfusion injury 12.

5In the present study, we compared paracrine functions, in vivo therapy, and exosomal profiles of human EnMSCs, BMMSCs, and adipose‐derived MSCs (AdMSCs). We report the superior actions of EnMSCs in a rat MI model relative to BMMSCs and AdMSCs that correlated with enhanced paracrine actions and higher expression of exosomal miR‐21. The results are consistent with a role for miR‐21 by downregulation of phosphatase and tensin homolog (PTEN) and enhancement of Akt survival kinase activity in the superior actions of EnMSCs in this model.

6Human BMMSCs and EnMSCs were obtained from S‐Evans Biosciences (Hangzhou, China, http://www.sebio.net.cn). Human AdMSCs were purchased from Cyagen Bioscience (Guangzhou, China, http://www.cyagen.com). Human umbilical vein endothelial cells (HUVECs) were provided by AllCells (Shanghai, China, http://www.allcells.com). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Waltham, MA, http://www.thermofisher.com) with low glucose, supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) at 37°C in a humidified atmosphere with 5% CO2.

7MSCs at passages 6–8 were characterized for surface markers using a FACS Canto II Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, http://www/bdbiosciences.com). In brief, 1 × 106 cells suspended in 100 μl of phosphate‐buffered saline (PBS) with 1% FBS were stained with a panel of antibodies: fluorescein isothiocyanate (FITC)‐CD45, phycoerythrin (PE)‐CD29, allophycocyanin‐CD90, PE‐CD34, PE‐CD105, PE‐CD166, PE‐CD117, and isotype‐matched control for 1 hour. The cells were washed twice with PBS, and at least 10,000 cells per tube were acquired for each test. Surface marker expression was quantified using FACSCount II software (BD Biosciences).

8MSCs were cultured to 90% confluence in complete medium. Then, the media were replaced with DMEM containing 2% FBS for an additional 48 hours of incubation. The medium was collected, centrifuged at 2000g for 30 minutes, and stored at −80°C.

9Neonatal rat cardiomyocytes (CMs) were isolated and cultured as described previously 13. In brief, hearts from 1‐day‐old Sprague‐Dawley pups were minced and digested with 0.1% trypsin (Thermo Fisher Scientific). The dispersed cells were cultured with 10% FBS‐supplemented high glucose DMEM (glucose concentration, 4 g/l) containing 100 μmol/l 5′‐bromo‐2′‐deoxyuridine (Sigma‐Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) for 90 minutes at 37°C with 5% CO2. Nonadherent cells were collected and seeded onto 24‐well plates (2.5 × 105 cells per well) or 6‐well plates (1 × 106 cells per well; Corning, Tewksbury, MA, http://www/corning.com). The culture medium was replaced 48 hours after seeding. CMs were ready for experimentation after 72 hours of incubation.

10Terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) assays were performed in accordance with the manufacturer's protocol (Roche Applied Science, Indianapolis, IN, http://www.roche_applied_science.com). In brief, the cells or samples were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X‐100 for 10 minutes. Nucleotide and TdT enzyme mixture was added to the cells or samples for 1 hour of incubation at 37°C. The nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI). The percentage of apoptotic nuclei per total CM nuclei was calculated as the index of apoptosis. Images were taken at five randomly selected fields in the border zone of the ischemic myocardium in each sample or per well.

11Cells and heart samples were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.2% Triton. After 1 hour of blocking by 10% bovine serum albumin (BSA), the cells or tissue samples were incubated with primary antibodies as follows: anti‐troponin I (Abcam, Cambridge, MA, http://www.abcam.com). Sections or cells were washed with PBS‐0.05% Tween (PBST) followed by incubation of secondary antibody (fluorescein Isothiocyanate [FITC]‐conjugated; Abcam). After 1 hour of incubation at 37°C, cell nuclei were stained with DAPI. Images were obtained by fluorescence microscope (Leica Microsystems, Wetzlar, Germany, http://www.leica-microsystems.com) at five randomly selected fields for each sample or well and the mean positive/negative numbers calculated.

12For tube formation assays, 96‐well plates were precoated with 50 μl of growth factor‐reduced Matrigel (BD Biosciences) for 30 minutes at 37°C. HUVECs were seeded at a density of 20,000 cells per well and incubated with conditioned medium at 37°C. After 4–6 hours, capillary network structures were imaged by phase‐contrast microscopy (Leica Microsystems) and the total number of interconnecting branches in each group counted using Image‐Pro software (Media Cybernetics, Rockville, MD, http://www.mediacy.com).

13Conditioned medium was collected from each MSC type after culture in DMEM with 10% exosome‐depleted FBS for 48 hours. Media were passed through a 0.22‐μm filter to remove cell debris and incubated for 24 hours on ice with 0.5 volume of total exosome isolation reagent (Thermo Fisher Scientific). The mixture was centrifuged at 10,000g for 1 hour and the supernatant was discarded. Exosome pellets were resuspended in serum‐free medium and prepared for protein or RNA analysis.

14To investigate exosome morphology, 3 μl of exosome pellets were layered onto 200‐mesh copper electron microscopy grids for 5 minutes at room temperature and stained with uranyl acetate 14. After three washes with PBS, exosomes were observed under a transmission electron microscope (H7500 TEM, Hitachi, Tokyo, Japan, http://www.hitachi.com), and micrographs were taken to quantify the size of the exosomes.

15To label the exosomes, 4 µl of PKH‐26, (Sigma‐Aldrich) was added to 1 ml of diluent containing 30 μg of MSC‐derived exosomes and incubated at room temperature for 15 minutes. Then, 1 ml of 5% BSA was added to stop the reaction, and the suspension was centrifuged at 400g for 10 minutes. The supernatant was discarded, and the cell pellets were resuspended in 10 ml of PBS and centrifuged at 400g for 5 minutes. The cell pellets were washed twice with 10 ml of complete medium to remove any unbound dye.

16EnMSCs were cultured to 90% confluence in complete medium. The media were replaced with DMEM containing 2% exosome‐depleted FBS, and the cells were conditioned with or without 20 μM GW4869 (Sigma‐Aldrich) for an additional 48 hours. Conditioned media were collected for in vitro studies, and EnMSCs were prepared for in vivo studies.

17Total RNA was extracted from exosomes using a total exosome RNA isolation kit (Thermo Fisher Scientific), according to the manufacturer's instructions. RNA pellets were dissolved in RNase‐free water, and RNA content was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, http://www.genomics.agilent.com). RNA samples of 100 ng were used for miR microarrays. RNA was labeled using a miRCURY Hy3/Hy5 Power labeling kit (Exiqon, Vedbaek, Denmark, http://www.exiqon.com), hybridized on a miRCURY LNA Array, version 18.0 (Exiqon), and scanned using an Axon GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA, http://www.moleculardevices.com). Expression data were analyzed using GenePix Pro 6.0 software (Axon, Jakarta, Indonesia, http://www.axoninstruments.biz).

18Recombinant lentivirus was constructed by Genechem (Shanghai, China, http://www.genechem.com). For viral infection of EnMSCs, CMs, and HUVECs, the cells were seeded at a density of 1 × 105 cells per well in a six‐well plate and infected with lentiviral vectors in the presence of 10 μg/ml polybrene (EMD Millipore, Merck KGaA, Darmstadt, Germany, http://www.emdmillipore.com). After 12 hours of infection, the medium was replaced with growth medium. At 72 hours after infection, the efficiency of the infection was assessed as green fluorescent protein expression by fluorescence microcopy and precisely quantified by real‐time polymerase chain reaction (RT‐PCR).

19Total RNAs and miRs were extracted, miRs were reverse transcribed using a miDETECT A Track quantitative RT (qRT)‐PCR Kit (RiboBio, Guangzhou, China, http://www.ribobio.com), and mRNAs were reverse transcribed using a PrimeScript Stand cDNA Synthesis Kit (Takara Bio Inc., Kusatsu, Japan, http://www.takara-bio.com). The mRNA primers were selected using Primer3 Input online software (available at: http://primer3.ut.ee/) as shown in supplemental online Table 1. The primers of miRs were designed using miRBase online software (available at: http://www.mirbase.org/) as shown in supplemental online Table 2. RT‐PCRs were conducted using SYBR green reaction mixture in the Step One Plus Real‐Time PCR System (ABI PRISM 7900HT; Thermo Fisher Scientific). The PCR conditions were 95°C for 10 minutes and 40 cycles at 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. We used β‐actin as the internal control for normalization and calculated the relative expression of mRNA using a (ΔCt) value method. Relative expression of miRs were also evaluated by a ΔCt method and normalized to U6.

20In brief, cells and exosomes were rinsed with PBS and lysed in RIPA buffer (50 mM TrisHCl, pH 8, 150 mM NaCl, 1% NP‐40, 0.5% sodium deoxycholate, and 0.1% SDS; Beyotime Institute of Biotechnology, Haimen, China, http://www.beyotime.com) containing Protease Inhibitor Cocktail Set III, EDTA‐Free (1:200 dilution; Calbiochem; EMD Millipore) on ice for 30 minutes. Then, cell lysates were collected and centrifuged at 12,000 rpm for 30 minutes at 4°C to acquire extracts. Protein concentrations were assessed using a BCA Protein Assay Kit (Thermo Fisher Scientific). Next, 30 μg of protein was analyzed by SDS‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (EMD Millipore). After 1 hour of blocking with PBST containing 5% BSA at room temperature, primary antibodies, including anti‐Bcl‐2, Bcl‐2‐associated X protein (Bax), caspase‐3, phosphorylated‐Akt/total‐Akt, PTEN, vascular endothelial growth factor (VEGF), CD63, and β‐actin (Abcam) were diluted to 1:1,000 and incubated with the membrane overnight at 4°C. The membranes were washed with PBST and incubated with 1:3,000 secondary conjuncted with horseradish peroxidase (HRP) antibody for 1 hour at room temperature. The membranes were immersed in enhanced chemiluminescence solution and images acquired by digital development with β‐actin as control for normalization.

21Rats (Sprague‐Dawley, male, 6–8 weeks old, weight, 200–250 g) were fed a standard laboratory diet. All animal research protocols met the Guide for the Care and Use of Laboratory Animals (NIH Publication no. 85‐23, revised 1996) and were approved by the Animal Care and Use Committee of Zhejiang Province Medical Institute. The MI model was established as described previously 15. In brief, MI was induced in anesthetized rats by permanent ligation of the left anterior descending coronary artery with 8‐0 silk suture. Successful coronary occlusion was confirmed by blanching of the myocardium distal to the coronary ligation. The rats were randomized into seven groups: control group (PBS treatment), BM group (BMMSC treatment), Ad group (AdMSC treatment), En group (EnMSC treatment), En‐Null group (treatment with EnMSCs infected with null vector), En‐miR‐21‐KD group (treatment with EnMSCs infected with miR‐21 knockdown vector), and En‐GW4869 group (EnMSCs preconditioned with 20 μM GW4869). At 30 minutes after ligation, 30 μl of cell suspension containing 1 × 106 cells were injected around the MI border zone at five sites (2 × 105 cells per site). The control groups received the same volume of PBS.

22Echocardiography was implemented at baseline and 3, 28, and 56 days after surgery. The rats were anesthetized with 2% isoflurane in 100% O2 and placed in the supine position. Two‐dimensional and M‐model echocardiographic images (17.5 MHz transducer; Vevo 2100; VisualSonics, Toronto, ON, Canada, http://www.visualsonics.com) were obtained at the level of the papillary muscles in an investigator‐blinded manner. The left ventricular end‐systolic diameter (LVESD) and left ventricular end‐diastolic diameter (LVEDD) were measured for at least three separate cardiac cycles. The left ventricular end‐systolic volume (LVESV) and left ventricular end‐diastolic volume (LVEDV) were calculated as 7.0 × LVESD3/(2.4 + LVESD) and 7.0 × LVEDD3/(2.4 + LVEDD), respectively. The ejection fraction (EF) and fractional shortening (FS) were calculated as [(LVEDV − LVESV)/LVEDV] × 100 and [(LVEDD − LVESD)/LVEDD] × 100, respectively.

23The rats were sacrificed at 3, 28, and 56 days after MI. The hearts were excised and cut into transverse slices at the ventricle midline between the atrioventricular groove and the apex. The hearts were dehydrated in 30% sucrose solution for 12–24 hours at 4°C, embedded in Tissue‐Tek O.C.T. compound (Sakura Finetek, Tokyo, Japan, http://www.sakura-finetek.com) and snap frozen on dry ice. Samples were cut at 7 μm thickness and preserved at −80°C.

24For immunohistochemistry, the heart tissue sections were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X‐100, and blocked with PBST containing 5% BSA. The samples were incubated with anti‐α‐SMA antibody (Abcam) and anti‐CD31 antibody (Abcam) at 4°C overnight. After several washes with PBST, the samples were reacted with HRP‐conjugated secondary antibody for 1 hour at room temperature. Immunopositive reactions were developed using a polymer 3,3′‐diaminobenzidine and sections mounted on gelatin‐coated slides. Images were taken at four to five randomly selected high‐magnification fields in the border zone of the ischemic myocardium, and the average number of microvessels per visual field was calculated.

25At 28 and 56 days after MI, Masson trichrome staining was performed for infarct zone evaluation. In brief, sections from different groups were fixed with 4% paraformaldehyde, washed with PBS, and stained using a Masson trichrome kit (Maixin‐Bio, Fuzhou, China, http://www.maxim.com.cn). The infarct area was calculated by the sum of the endocardial and epicardial length of the infarct zone divided by the total length of the endocardial and epicardial left ventricle using Image‐Pro software (Media Cybernetics) and expressed as a percentage of infarct size.

26Statistical analyses were calculated using SPSS, version 16.0, software (IBM Corp., Armonk, NY, http://www.ibm.com). One‐way analysis of variance tests were used to compare the differences between groups. Data are presented as the mean ± SD. Differences were considered statistically significant at p <.05.

27After adherent culture, BMMSCs, AdMSCs, and EnMSCs all appeared as spindle‐shaped fibroblast‐like cells ( supplemental online Fig. 1A). Cell surface marker expression of MSCs from each source was determined by flow cytometry. All samples coexpressed equivalent mesenchymal surface markers, including CD29, CD90, CD105, CD166, and were negative for hematopoietic surface markers CD34 and CD45. The expression of CD117 was very low in all MSCs ( supplemental online Fig. 1B).

28Paracrine actions can contribute significantly to the cardioprotective effects of MSC‐based therapy 16. To analyze paracrine activities, cardiac myocytes or HUVECs were exposed to conditioned medium from each of the three types of MSCs, and the effects on apoptosis or angiogenesis, respectively, were determined. Apoptosis was monitored by TUNEL assay of CMs during exposure to 48 hours of simulated ischemia with or without conditioned medium. As shown in Figure 1A, 1C, CMs cocultured with conditioned medium from EnMSCs displayed significantly fewer apoptotic cells relative to all other groups (TUNEL‐positive ratio, EnMSCs, 12.44% ± 3.84%; BMMSCs, 23.85% ± 5.85%; AdMSCs, 20.68% ± 4.60%; and control, 30.86% ± 4.42%). The HUVEC tube length served as an in vitro surrogate for angiogenesis. The mean tube length from the EnMSC group (28,578 ± 5,770 μm) was markedly increased relative to that of all other groups (control, 15,628 ± 2,271 μm; BMMSCs, 20,840 ± 2,576 μm; AdMSCs, 21,805 ± 5,672 μm; Fig. 1B, 1D). These findings suggest that EnMSC‐derived conditioned medium confers enhanced cardioprotection and proangiogenic stimuli relative to either of the other MSC types.

29The role of paracrine action as a mechanism for MSC‐mediated therapy for myocardial infarction is well recognized. Therefore, the paracrine effects of each MSC treatment group were compared by quantifying apoptosis, neovascularization, infarct size, and cardiac function in parallel in a rat model of MI.

30TUNEL staining of cardiac cells in the border zones after MI revealed that hearts in the EnMSC group had the lowest apoptotic index compared with that of the other groups (17.06% ± 6.30% vs. control, 44.26% ± 4.88%; BMMSCs, 28.24% ± 6.40%; and AdMSCs, 27.78% ± 6.32%; Fig. 2A, 2D). Neovascularization, determined by α‐SMA/CD31 immunohistochemical staining, revealed that myocardial tissues in the EnMSC group had the highest density of microvessels relative to the other groups (Fig. 2B, 2E, 2F). Similarly, the infarct size as determined by Masson trichrome staining at 28 and 56 days after MI (Fig. 2C, 2G) was most reduced by EnMSC transplantation relative to all other groups (28 days after MI: EnMSCs, 27.34% ± 9.77%; control, 50.35% ± 3.23%; BMMSCs, 41.98% ± 9.32%; and AdMSCs, 39.55% ± 12.76%; and 56 days after MI: EnMSCs, 33.39% ± 9.86%; control, 59.90% ± 6.23%; BMMSCs, 49.93% ± 8.70%; and AdMSCs, 47.18% ± 8.63%). Similar trends were observed for cardiac function. Echocardiography revealed that transplantation of all MSCs reduced left ventricular dilation and preserved systolic function compared with the control group. However, the cardiac performance in the EnMSC treatment groups was significantly better than that of the BMMSC or AdMSC groups at both 28 and 56 days postoperatively (Fig. 2H–2K, supplemental online Fig. 2A). These results indicate the superior paracrine properties and therapeutic actions of EnMSCs relative to adipose‐ or BM‐derived cells.

31If exosomes regulate the therapeutic effects of EnMSCs, inhibition of exosome release would be expected to abolish the cardioprotection. To verify this hypothesis, we treated EnMSCs with GW4869, a reversible blocker of neutral sphingomyelinase that inhibits exosome secretion 17, 18. Exosome production was inhibited by GW4869 in a dose‐dependent manner, with complete blockage at a concentration of 20 μM ( supplemental online Fig. 3A). We found that suppression of exosome secretion by GW4869 abrogated the antiapoptosis and proangiogenesis actions of EnMSCs in vitro as evidenced in either the apoptotic indexes of CMs under hypoxia or capillary structure formations by HUVECs in Matrigel (BD Biosciences; Fig. 3A–3D). In vivo, EnMSCs pretreated with GW4869 conferred no infarct size reduction or improvement of cardiac performance as indicated by echocardiography (Fig. 3E–3H, supplemental online Fig. 3B). These results confirm that secreted exosomes are essential for EnMSC‐mediated cardiac repair.

32To investigate the possible molecular mediators of MSC‐derived paracrine effects, we isolated exosomes from conditioned medium as described in Materials and Methods. Transmission electron microscopy, implemented as described previously 19, revealed the predicted shape and size of exosomes 20 (Fig. 4A). Immunoblotting also confirmed enrichment of the exosomal surface marker CD63 in purified exosomal fractions (Fig. 4B). To confirm cellular exosome uptake, exosome fractions from each conditioned medium were labeled with PKH26 and cocultured with CMs and HUVECs. After 6 hours, the cells were immunostained and analyzed by confocal laser microscopy to detect PKH26 fluorescence. As shown in Figure 4C, both CMs and HUVECs stained positively for PKH26, confirming exosomal uptake.

33We compared miR profiles in exosomes derived from each MSC group by microarray analyses as described in Materials and Methods. As shown in Figure 4D, exosomal miR profiles from EnMSCs were different from those of the other groups, in particular, miR‐1275, miR‐21‐5p, miR‐23‐3p, miR‐3940‐5p, miR‐4708‐3p, miR‐548ap‐5p, and miR‐642b‐5. qRT‐PCR validation confirmed these results and further revealed that the expression of miR‐21 was selectively increased more significantly in EnMSC exosomes relative to all other groups (Fig. 4E). Enhanced expression of miR‐21 selectively in the EnMSC group was also seen in CMs and HUVECs after 24 hours of coculture with each exosomal fraction (Fig. 4F, 4G).

34Target genes were predicted by gen ontology analysis, and genes were classified into three groups: molecular function, biological process, and cellular component. Apoptosis‐ and angiogenesis‐related miR‐21 targets were selected for further identification. Putative miR‐21 apoptotic target genes included HS1‐associated protein X‐1 (Hax‐1), programmed cell death 4 (PDCD‐4), S‐phase kinase‐associated protein 2 (Skp‐2), Yes‐associated protein 1 (Yap‐1), POC1 centriolar protein A (Pix‐2), YOD1 deubiquitinase (YOD‐1), signal transducer and activator of transcription 3 (Stat3), phosphatase and tensin homolog (PTEN), toll‐like receptor 4 (TLR‐4), sprouty homolog 1 (SPRY‐1), and Bcl‐2 interacting mitochondrial ribosomal protein 2 (BMRP‐2). Angiogenesis target genes included polycystin 2, transient receptor potential cation channel (PKD‐2), reversion‐inducing cysteine‐rich protein with Kazal motif (RECK), Kruppel‐like factor 6 (KLF‐6), fibroblast growth factor 18 (FGF‐18), Stat3, PTEN, TLR‐4, SPRY‐1, and BMRP‐2. Target gene primers were selected using Primer3 Input online software (available at: http://primer3.ut.ee/), as listed in supplemental online Table 1. To identify possible apoptotic target genes, CMs were cocultured with exosome fractions under hypoxia (1% O2; 5% CO2), and RNA and protein were extracted after 48 hours. For angiogenesis, HUVECs were incubated with exosomes and RNA and protein extracted after 24 hours. Putative target genes were identified by qRT‐PCR. Both assays revealed that PTEN mRNA was uniquely decreased by treatment with EnMSC exosomes (Fig. 5A–5C). Western blots further confirmed that PTEN protein was most attenuated in recipient cells incubated with EnMSC exosomes, and this coincided with enhanced Akt phosphorylation and Bcl‐2 expression in CMs and VEGF expression in HUVECs (Fig. 5D, 5E).

35To validate a role for miR‐21 in EnMSC‐specific paracrine and cardioprotective effects, EnMSCs were transfected with a miR‐21 antagonist miR or negative control as described in Materials and Methods. For in vitro study, exosomes derived from EnMSCmiR‐21‐KD, EnMSCNull, EnMSCs, BMMSCs, and AdMSCs were incubated with CMs or HUVECs under hypoxic or normoxic conditions, followed by apoptosis or angiogenesis assays, respectively. For in vivo validation, EnMSCmiR‐21‐KD and EnMSCNull, respectively, were delivered into infarcted rat hearts. PTEN and its downstream targets were simultaneously evaluated by Western blotting. In parallel experiments, CMs and HUVECs were transfected directly with miR‐21 or a negative control and subjected to hypoxic or normoxic incubations. As shown in Figure 6, significantly higher apoptotic indexes were observed in CMs incubated with exosomes from EnMSCmiR‐21‐KD than CMs cocultured with EnMSCNull and EnMSC‐derived exosomes, and HUVECs incubated with exosomes of EnMSCmiR‐21‐KD had a lower capillary density (Fig. 6A, 6B, supplemental online Fig. 4C, 4D). The efficiencies of miR‐21 knockdown in both EnMSCs and exosomes were quantified ( supplemental online Fig. 4A, 4B). Western blots confirmed that suppression of miR‐21 in exosomes conferred upregulation of PTEN, inactivation of Akt phosphorylation, and reduced expression of Bcl‐2 and VEGF in recipient cells (Fig. 6C, 6D). Moreover, the results shown in Figure 6 confirmed the absence of therapeutic enhancement in the EnMSCmiR‐21‐KD treatment group, including the loss of antiapoptosis and cardioprotective properties (Fig. 6E, 6I), loss of proangiogenic actions (Fig. 6F, 6G, 6J, 6K), absence of a reduction in infarct size (Fig. 6H, 6L), and no improvement in cardiac function (Fig. 6M–6P, supplemental online Fig. 5D). We attributed these effects to downregulation of miR‐21 and upregulation of PTEN in heart tissues of these treatment groups ( supplemental online Fig. 5A–5C). Compared with the null groups, CMs transfected with miR‐21 inhibitor displayed higher apoptotic indexes after hypoxia incubation, but miR‐21 KD HUVECs had lower microvessel densities ( supplemental online Fig. 6A, 6B, 6D, 6E). High efficiencies of miR‐21 knockdown by our procedures were confirmed in both CMs and HUVECs ( supplemental online Fig. 6C, 6F). Western blotting confirmed that blockage of miR‐21 conferred upregulation of PTEN, inactivation of Akt phosphorylation, and reduced expression of Bcl‐2 and VEGF in recipient cells ( supplemental online Fig. 6G, 6H).

36Our results suggest that MSCs derived from the endometrium have greater therapeutic potential compared with cells derived from bone marrow or adipose tissue, the two most common sources of therapeutic MSCs 21, 22. We report stronger paracrine effects of EnMSC on cellular apoptosis and angiogenesis parameters in vitro and in vivo and greater myocardial salvage and enhanced cardiac function after MI in the EnMSC‐treatment groups. The enhanced paracrine actions and therapy with EnMSCs might be mediated by higher exosomal expression and cellular delivery of miR‐21 with downstream effects on the target PTEN‐Akt survival pathway (Fig. 7).

37Clinical trials of MSCs derived from bone marrow or adipose tissue have demonstrated improved cardiac function that is probably mediated primarily by paracrine actions of secreted cytokines rather than by cardiac regeneration or myogenic differentiation 23, 24. However, bone marrow and adipose tissue might not be optimal cell sources for practical and functional reasons 20, 25. EnMSCs have several advantages, including simple, noninvasive isolation procedures, embryonic‐like properties, consistent quality, and improved function 7. Our present findings are the first to directly demonstrate the possible superiority of EnMSCs over MSCs from other sources for cardiovascular anti‐MI therapy. The improved therapy is driven by enhanced paracrine effects of EnMSCs that confer both improved cardioprotection and angiogenesis, as demonstrated by both in vitro and in vivo analyses.

38Exosomal mRNA and miRs could be key components for biological information exchange and cell‐to‐cell communication 26. Recent work has suggested that MSC‐derived exosomes and exosomal miRs might contribute to the pathogenesis of stroke 27, multiple myeloma 28, breast cancer 29, and myocardial infarction 30. We have described, for the first time, high levels of miR‐21 in exosomes from EnMSCs that might be principal effectors of the superior paracrine effects of EnMSCs relative to bone marrow and adipose tissue. We found that exosomal miR‐21 released from EnMSCs can be shuttled to recipient CMs and endothelial cells to regulate apoptosis and angiogenesis in CMs and HUVECs, respectively, and ameliorate cardiac function after MI. Knockdown of miR‐21 in exosomes abolished these actions, further supporting an important role for miR‐21. Although our studies have confirmed essential contributions of miR‐21 to cardioprotection by EnMSC‐derived exosomes, it might not be the exclusive mediator of such protection. As shown in Figure 4, we have also confirmed the enhanced expression of miR‐23 in EnMSC‐derived exosomes and EnMSC‐exosome pretreated recipient cells. It has been reported that miR‐23 can promote angiogenesis in cardiac endothelial cells by targeting Sprouty2 and Sema6A and thereby activating proangiogenic signaling 31, 32. Therefore, it is possible that miR‐23 contributes in a similar way to the cardioprotective effects described in the present study for EnMSCs and their derived exosomes.

39It was recently reported that exosomal miR‐21 derived from induced pluripotent stem cells protected the ischemic myocardium from apoptosis 33 and stimulated angiogenesis in transfected bronchial epithelial cells 34. Furthermore, as a downstream target of miR‐21, PTEN has important roles in both apoptosis and angiogenesis by regulating Akt phosphorylation 35, 36. Regulation of PTEN by miR‐21 was established in previous work in carcinoma 37 and cardiovascular cells 38. Consistent with this, we found that stimulation of miR‐21 in EnMSC exosomes reduced expression of PTEN and increased phosphorylation of Akt in exosome‐treated target cells and that these effects were reversed by knockdown of miR‐21, providing a molecular mechanism for the enhanced paracrine and cardioprotective effects of EnMSCs.

40Our study has important clinical implications. MSCs are potential off‐the‐shelf reagents that are immune‐privileged, without autologous restrictions, and with wide application. The safety and feasibility of MSC therapy for ischemic myocardial disease has been well documented in both preclinical and early‐phase clinical trials, but improvements in myocardial function have been modest 23, 39 40 41 42. Thus, a medical need exists for new methods to improve therapy by MSCs. Our results demonstrate for the first time that EnMSCs confer significantly improved therapeutic benefit in the context of MI relative to BMMSCs and AdMSCs in parallel assays. The results support the use of EnMSCs as a preferential alternative to these other more commonly used cells, perhaps in combination with hypoxic preconditioning 43, for enhanced clinical treatment of heart disease.

41We have described the superior properties of EnMSCs relative to AdMSCs or BMMSCs for the treatment of myocardial infarction. EnMSCs conferred enhanced myocardium salvage and microvessel regeneration that was associated with significant improvement of cardiac function after MI. The possible mechanism for enhanced paracrine activity and therapy by EnMSC treatment might involve elevated expression and cellular delivery of exosomal miR‐21, with corresponding downstream effects on PTEN and Akt. Further investigations and clinical trials are essential to confirm EnMSCs as the preferred MSC source for myocardial regenerative medicine. Our study was limited to only three sources of MSCs. Thus, it is possible that MSCs from other early‐stage tissue sources, including placenta, fetus, and embryo, might have similar or superior properties to EnMSCs.

42K.W.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; Z.J.: collection and/or assembly of data, data analysis and interpretation; K.A.W.: conception and design, data analysis and interpretation; J.C.: data analysis and interpretation, manuscript writing; H.H., Y.Z., L.W., Y.W., C.N., and Q.L.: collection and/or assembly of data; J.Z.: collection and/or assembly of data, data analysis and interpretation; Z.Z.: data analysis and interpretation, manuscript writing; C.X: provision of study material or patients; L.Z.: data analysis and interpretation, manuscript writing; R.W., W.Z., and H.Y.: data analysis and interpretation, manuscript writing; X.H. and J.W.: conception and design, data analysis and interpretation, provision of study material or patients, financial support, final approval of manuscript.

43K.A.W. is founder, president, and CEO of Integene International LLC, a gene therapy company based in Miami, FL. The other authors indicated no potential conflicts of interest.