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TISSUE-SPECIFIC STEM CELLS |
aDepartment of Cardiology, Keio University School of Medicine, Tokyo, Japan;
bDepartment of Reproductive Biology and Pathology, National Research Institute for Child Health and Development, Tokyo, Japan;
cDepartment of Pathology, Keio University School of Medicine, Tokyo, Japan;
dInstitute for Advanced Cardiac Therapeutics, Keio University School of Medicine, Tokyo, Japan;
eDepartment of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan;
fVirology Division, National Cancer Center Research Institute, Tokyo, Japan;
gDepartment of Obstetrics and Gynecology, Kanazawa University, School of Medicine, Kanazawa, Japan;
hInstitute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan
Key Words. Cardiomyogenesis human mesenchymal stem cell • Menstrual blood endometrial gland • Cell sheet technology cardiac precursors
Correspondence: Correspondence: Shunichiro Miyoshi M.D., Ph.D., Keio University School of Medicine, 35-Shinanomachi, Shinjuku-ku, Tokyo, 160-8582 Japan. Telephone: +81-3-3353-1211 (ext 62310); Fax: +81-3-3353-2502; e-mail: smiyoshi{at}cpnet.med.keio.ac.jp
Received on October 2, 2007;
accepted for publication on April 6, 2008.
First published online in STEM CELLS EXPRESS April 17, 2008.
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ABSTRACT |
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 TopFootnotes Abstract IntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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Our previous study showed that umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) [11] and placental chorionic plate cells (PCPCs) [12] have a phenotype of mesenchymal cells and have higher cardiomyogenic differentiation ability in vitro. Since these materials are deemed medical waste and can be obtained without any ethical problems, they may be a suitable stem cell source for cardiac regenerative therapy. But the population of UCBMSCs in umbilical cord blood is scant [13] and there is also a problem in establishing PCPCs, since placental tissue contains a lot of maternal decidua-derived mesenchymal cells that could contaminate PCPCs. Therefore, it is difficult to obtain enough of these cells without using a limiting dilution method and/or massive ex vivo propagation, which may cause instability of the genome [14]. Consequently, material that contains a large amount of mesenchymal cells during the first few passages should be a highly suitable source of stem cells.
A previous paper suggests that endometrium contains an MSC-like population [15] and menstrual blood-derived mesenchymal (MMCs) cells have a pluripotent differentiation ability in vitro [16]. The data presented here demonstrate that human menstrual blood-derived mesenchymal cells and uterine endometrial gland-derived mesenchymal cells (EMCs) have a strong potential for cardiomyogenic transdifferentiation in vitro and in vivo. Moreover, large amounts of MMCs could be obtained from the first passage of menstrual blood culture, and MMCs have been shown to restore impaired cardiac function through marked cardiomyogenesis in vivo.
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MATERIALS AND METHODS |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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Isolation of Marrow-Derived Mesenchymal Stem Cells
Bone marrow-derived mesenchymal stem cells (BMMSCs) were obtained from a 41-year-old male as described previously [2].
Coculture with Murine Fetal Cardiomyocytes
MMCs, EMCs, and BMMSCs were infected with enhanced green fluorescent protein (EGFP) expressing adenovirus [2]. Fetal cardiomyocytes were obtained from hearts of day-17 mouse fetuses, as previously described [2]. The isolated cardiomyocytes were replated at 5 x 104/cm2 on top of a floating athelocollagen membrane (CM-6, 40-µm thickness; Koken, Tokyo, http://www.kokenmpc.co.jp/english/products/collagen/cell_culture/cm-6_24/index.html) that is permeable for only small molecules (less than 5,000 MW). The next day, the athelocollagen membrane was plated upside down on the culture dish. Harvested EGFP-labeled MMCs and EMCs were then seeded upon the athelocollagen surface (bottom surface) at 7 x 103/cm2 (Fig. 1M). In several experiments (Figs. 1G–1L, 2, 3E, 3H, 3K–3M, 4, supplemental online Fig. 2, examination of chromosome chimeras), we did not use the athelocollagen membrane for the coculture system.
Immunocytochemistry and Immunohistochemistry
A laser confocal microscope (FV1000; Olympus, Tokyo, http://www.olympus-global.com) was used for immunocytochemical analysis. Samples were stained with mouse monoclonal anti-cardiac troponin-I antibody (4T21 Lot 98/10-T21-C2; HyTest, Euro, Finland, http://www.hytest.fi/) or with mouse monoclonal anti-sarcomeric 
-actinin antibody (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), or anti-connexin 43 antibody (Sigma-Aldrich) diluted 1:300 overnight at 4°C, then stained with TRITC-conjugated anti-mouse antibody (Sigma-Aldrich), TRITC-conjugated anti-rabbit antibody (Sigma-Aldrich), and Cy5-conjugated anti-mouse IgG (Chemicon, Temecula, CA, http://www.chemicon.com) diluted 1:100, containing 4'-6-diamidino-2-phenylindole (DAPI; Wako Chemical, Osaka, Japan, http://www.wako-chem.co.jp/english) at 1:300 for 30 minutes at 25°C–28°C. See also supplemental online data 1 for detail of method.
Functional Analysis
The method of action potential (AP) recording was as previously described [2] but with slight modification. A fluorescence inverted microscope (IX-70; Olympus) was used for AP recording. The microscope was equipped with a recording chamber and a noiseless heating plate (Microwarm Plate; Kitazato Supply, Fujinomiya, Shizuoka, Japan, http://www.kitazato-supply.com). A 10-mM volume of HEPES (Sigma-Aldrich) was added to the culture medium to stabilize the pH of the perfusate at 7.5. Standard glass microelectrodes having a direct current resistance of 15–25 M
when filled with pipette solution were used. Alexa 568 compound was dissolved to a concentration of 0.5 mM in 2 M of KCl solution in order to completely dissolve the Alexa 568 in the pipette solution. The electrodes were positioned with a motor-driven micromanipulator (PCS-5000; Burleigh Instrument, Inc., New York) under optical control. Spontaneously beating EGFP-positive cells were selected as targets, and after the APs of the target cells had been recorded, the dye was injected by iontophoresis (–7 nA for 10–20 seconds). The extent of dye transfer was monitored under a fluorescence microscope, and digital images were recorded with a digital photo camera (EOS-digital; Canon, Tokyo, http://www.canon.com) mounted on the microscope. The recording pipette was connected to a patch-clamp amplifier (MEZ-8300; Nihon Kohden, Tokyo, http://www.nihonkohden.com). The amplified signal was filtered with a 4-pole Bessel filter (NF-3625; NF electronic instrument; NF Corp., Tokyo, http://www.nfcorp.co.jp/english/index.html) set at 2 kHz, then digitized with an A/D converter with a sampling frequency of 10 kHz (Digidata 1,322A; Molecular Devices Corp., Union City, CA, http://www.moleculardevices.com). Pacemaker potential was defined by the slowly depolarizing membrane potential at phase IV of the AP.
Alexa 568 was injected into cells via recording microelectrodes to stain the cells and confirm that the AP was generated by EGFP-positive cells (Fig. 1G–1I, 3E, 3H). Since the dye did not diffuse into the EGFP-negative murine cardiomyocytes, there were no tight cell-to-cell heterologous connections (i.e., gap junctions), at least in the in vitro condition. In some experiments, Alexa 568 diffused into the EGFP-positive satellite EMCs and MMCs, suggesting that a homologous cell-to-cell connection had been established at least 1 week after cocultivation. The measured parameters of the APs were averaged and are shown in Figure 1K.
The fluorescent image of the beating MMCs and EMCs was monitored using a CCD camera (Ikegami Tsushin Co., Ltd, http://www.ikegami.co.jp) and was stored using digital video. The video images (National Television Standards Committee format, 29.97 frame/second) of contraction of EMCs and MMCs were stored in a personal computer as MPEG-2 format files, then analyzed later. Both edges of the EGFP-positive EMCs and MMCs along the line (Figs. 1L, 3K) were automatically detected, and the distance between both edges was measured from each video frame using an image edge-detection program using Igor Pro 4 (Wavemetrics Inc., Lake Oswego, OR) [11].
Calculation of Induction Rate
The MMCs and EMCs were exposed to 3 µM 5-azacytidine (5-azaC; Sigma-Aldrich) for 24 hours to induce cell differentiation, or were left untreated. The 5-azaC-treated and nontreated MMCs or EMCs, cultivated with or without murine fetal cardiomyocytes, were enzymatically dissociated and stained, then observed by confocal laser microscope (supplemental online data 2 for detail of method). The cardiomyogenic induction rate (average of 10 separate experiments) was calculated as the fraction of cardiac troponin-I-positive cells in the EGFP-positive cells.
Examination of Chromosomes of MMCs or EMCs and Murine Cell Chimeras
To rule out cell fusion-dependent cardiomyogenesis, chromosomes from MMCs or EMCs cocultivated without separation by the athelocollagen membrane from murine cardiomyocytes for 1 week were stained using a human chromosome-specific probe and a mouse chromosome-specific probe (Chromosome Science Labo, Hokkaido, Japan, http://www.chromoscience.jp/en/probe/page01/page01e.html) and spectral karyotyping with fluorescent in situ hybridization chromosome painting technique (Applied Spectral Imaging, Vista, CA, http://www.spectral-imaging.com), according to the manufacturer's protocol.
RNA Extraction and RT-PCR
Reverse transcriptase polymerase chain reaction (RT-PCR) was done as described previously [2]. Primers for the following genes were used: cardiac transcription factors—Csx/Nkx-2.5 and GATA4; cardiac hormones—atrial natriuretic peptide and brain natriuretic peptide; cardiac structural proteins—cardiac troponin I, cardiac troponin T, myosin light chain-2a, myosin light chain-2v, and cardiac-actin; and ion channel—cyclic nucleotide-gated potassium channel 2 (supplemental online Table 1). The internal control was 18S rRNA. PCR primers were prepared such that they would amplify the human but not the mouse genes.
Flow Cytometric Analysis
The cells were analyzed using an EPICS ALTRA analyzer (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). Antibodies (anti-human CD10, CD13, CD14, CD24, CD29, CD31, CD34, CD44, CD45, CD54, CD55, CD59, CD71, CD73, CDw90, CD105, CD106, CD117, CD133, CD140a, CD166, CD309, HLA-ABC, and HLA-DR) [12] were purchased from Beckman Coulter, Immunotech (Luminy, France, http://www.beckmancoulter.com/products/pr_immunology.asp), Cytotech (Hellebaek, Denmark, http://www.cytotech.dk/index.html), Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com), RDI (Research Diagnostics, Inc., Concord, MA, http://www.researchd.com), and Pharmingen Pharmaceutical, Inc. (San Diego, http://www.bdbiosciences.com/index_us.shtml).
In Vivo Cardiomyogenic Differentiation of EMCs
EGFP-labeled EMC tissue graft, made by a novel 3-dimensional cell sheet manipulation, was transplanted into male F344 nude rats (Clea, Tokyo, http://www.clea-japan.com/) (8 weeks of age). EMC100s and EMC214s (2 x 105/cm2) were plated onto fibrin polymer-coated culture dishes. Four days after plating, EMCs were detached as previously described [18], and transplanted onto the surface of the recipient heart (Fig. 5A) [19]. At 2 weeks after transplantation, immunohistochemical analysis was performed. EGFP-labeled EMC tissue graft on the fibrin polymer-coated culture dish did not show cardiomyogenic differentiation in vitro.
MMC Transplantation in Myocardial Infarction Model In Vivo
Recipient male F344 nude rats (Clea) (6 weeks of age) were anesthetized with 2% isoflurane gas. After left thoracotomy, the left ventricle was exposed and left anterior coronary artery was ligated by 6–0 silk suture. The complete occlusion of the coronary artery was confirmed by the cyanotic color and dyskinetic motion of the left ventricular anterior wall. In some rats, we did not ligate the coronary artery (Sham). The chest was closed and animals survived for 2 weeks to create complete myocardial infarction.
Two weeks after the first operation, rats with myocardial infarction were randomized for the control myocardial infarction (MI) group, the MI+BMMSC group, and the MI+MMC group, and were blinded immediately before the cell injection. Echocardiograms were performed on the anesthetized (2% isoflurane) rats. Data were collected three times and averaged. Immediately before transplantation, 
1–2 x 106 of EGFP-positive MMC or BMMSC suspension was drawn up into a 50-µl Hamilton syringe (Hamilton Co., Reno, NV, http://www.hamiltoncompany.com/main_usa.asp) with a 31-gauge needle. A 10-µl portion of the cell suspension was injected into the center and margin of the infarcted myocardium (MI+MMC, Fig. 7A). In the control MI group, culture medium or 1–2 x 106 of murine cardiac fibroblast was injected. Immediately before cell transplantation, 2-dimensional and M-mode echocardiographic (8.5 MHz linear transducer, EnVisor C; Phillips Medical System, Andover, MA, http://www.medical.philips.com/index.html) images were obtained to assess left ventricular (LV) end-diastolic dimension and LV end-systolic dimension at the mid-papillary muscle level.
Two weeks after the transplantation, a similar echocardiogram was performed again; then after opening the abdomen, a blood sample was drawn from the abdominal great vein; then the left diaphragm was dissected to insert a 22-gauge manometer line into the left ventricle, which was connected to the transducer (model TP-400T; Nihon Kohden) to monitor left ventricular pressure. The electrocardiogram and measured pressure were digitized by PowerLabo (ADInstruments, Milford, MA, http://www.adinstruments.com) at the sample frequency of 10 KHz and stored in a personal computer (Macintosh iBook G4; Apple, Cupertino, CA, http://www.apple.com).
Tissue samples were obtained by fixing and slicing along the short axis of the left ventricle, for every 1-mm depth of the ventricle. After Masson's trichrome staining, digital images of samples were collected using a light microscope (IX-70; Olympus). The images were digitized and analyzed using an Igor Pro 4 (Wavemetrics Inc.). The pixel area of blue color (fibrosis area) was defined as the infarcted area, and the pixel area of red color was defined as "survived" myocardium. The data on each pixel area from each slice were collated and the percentage fibrosis area was calculated as follows: % Fibrosis = 100 x (Pixel area of blue color)/(Pixel area of blue color and red color).
Statistical Analysis
All data are shown as the mean value ± SE. The difference among mean values was determined with analysis of variance. The posthoc test (Bonferroni) was used when three or more groups were compared. Student's t test was used when two values were compared. Statistical significance was set at p < .05.
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RESULTS |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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-actinin in the differentiated MMCs (Fig. 2B) and diffuse dot-like staining pattern of connexin 43 around the margin of each EGFP-positive cardiomyocyte (Fig. 2C–2F), suggesting that these human transdifferentiated cardiomyocytes have tight electrical coupling with each other. APs were recorded from spontaneously beating MMCs. The APs obtained from MMCs showed clear cardiomyocyte-specific sustained plateaus and slowly depolarizing resting membrane potentials—so-called "pacemaker potentials" (Fig. 1J, 1K)—and were, therefore, determined to be APs of cardiomyocytes, not of smooth muscle cells, nerve cells, or skeletal muscle cells. The fractional shortening (% FS) of the MMCs was analyzed (Fig. 1L) using a cell edge detection program. The EGFP-positive cells contracted simultaneously within the whole visual field. The % FS was 5.9 ± 0.5% (n = 19).
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Cardiomyogenic Transdifferentiation of EMCs
We hypothesized that the origin of cardiomyogenic cells in the MMCs was the endometrial gland, since MMCs have a high content of detached endometrial glands, whereas circulating blood-derived endothelial progenitor cells [21] or marrow-derived MSCs [2] do not have such high cardiomyogenic differentiation ability. We consequently established a line of EMCs (Fig. 3B, 3D) with a lifespan prolonged by a cell cycle-mediated gene to ensure a supply of cells for analysis. Almost all EMCs beat strongly in a synchronized manner (supplemental online Video 1), and 76.4%–96.5% became positive for cardiac troponin-I antibody as a result of cocultivation (Figs. 3A, 3C, 4B, 4C, supplemental online Fig. 2E–2L). EMCs were also positive for sarcomeric -actinin and connexin 43 (Fig. 2G–2L). APs were recorded from EMCs. The APs obtained from EMCs showed clear cardiomyocyte-specific sustained plateaus and, in some cells, pacemaker potentials (Fig. 3E–3J). The EGFP-positive EMCs contracted simultaneously within the whole visual field (Fig. 3L, 3M). Nuclear fusion between the cocultivated EMC100s or EMC214s and murine cardiomyocytes without separation of the athelocollagen membrane was observed in only 0.57% (6/1058) or 0.28% (5/1758), respectively.
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There is no difference between surface markers of the MMCs and EMCs. Both cells were positive for CD29 (integrin β1), CD59, and negative for CD14, CD34, CD45, CD309 (Flk-1), etc. (Fig. 4E, supplemental online Fig. 3A–3C).
Cardiomyogenic Effects In Vivo
An EGFP-labeled EMC tissue graft made by a novel 3-dimensional cell sheet manipulation [18] was transplanted into male F344 nude rats to ensure in vivo cardiomyogenic transdifferentiation ability. The EGFP-positive cell layer (green) was observed at the epicardial surface of the host heart (Fig. 5B–5D). Whole EMCs throughout the layer expressed a clear striation staining pattern of sarcomeric -actinin (Fig. 5B–5G), suggesting extremely high cardiomyogenic transdifferentiation ability of EMCs in situ.
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-actinin (supplemental online Fig. 5), suggesting an extremely high in situ cardiomyogenic transdifferentiation ability of MMCs, which contributed to improvement in cardiac function.
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DISCUSSION |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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Origin of the MMCs and EMCs
Cell surface marker analysis revealed that MMCs are neither encirculating endothelial progenitor cells [22] nor macrophages, but are mesenchymal phenotype cells. We speculated that MMCs may originate in uterine endometrial glands since a lot of detached endometrial glands were observed in menstrual blood and EMCs have the same surface marker as the MMCs, as well as an extremely high cardiomyogenic potential (76.4%–96.5% and 33.2%, respectively). As has been reported, MSCs cannot be detected in circulating blood and all tissues have MSC reservoirs localized in the perivascular niche [23], so EMCs and MMCs do not seem to originate from BMMSCs.
Clinical Contribution
In the present study, MMC transplantation improved impaired cardiac function in vivo. Since MMCs were transplanted at 2 weeks after coronary occlusion, when myocardial necrosis had been completed, the improvement of cardiac function is not due only to transplanted MMC-induced neovascularization [7, 8] or an antiapoptotic [9] effect on infarcted cardiomyocytes. Since they display high cardiomyogenic transdifferentiation ability in vitro and massive cardiomyogenic transdifferentiation in vivo, MMC-derived cardiomyocytes may play a role in the improvement of cardiac function in the present study. Myocardial infarction is known to suppress contraction ability of cardiomyocytes even at normal zone by left ventricular remodeling. Therefore MMC-derived paracrine factors may also play an important role in recovery of % LVFS by prevention of development of LV remodeling.
Neovascularization and the antiapoptotic effect are important for improving cardiac function to some extent. However, the feasible effect is dependent on the number of residual host cardiomyocytes in the infarcted myocardium. To achieve further improvement of cardiac function, a stem cell source that can be expected to exhibit powerful cardiomyogenic transdifferentiation in situ is required. MMCs can be transdifferentiated into cardiomyocytes in situ on the recipient heart, suggesting that they are a promising source for cardiac stem cell-based therapy material, significantly more efficient for cardiomyogenesis than BMMSCs.
MMCs can be readily obtained in a noninvasive manner from young female volunteers, and stored. It should therefore be possible to obtain MMCs of all the HLA types, possibly enabling the establishment of an MMC bank system to facilitate cardiac stem cell-based therapy.
Role of Established Cardiomyogenic EMC Cell Line for Determining Cardiomyogenic Factors
Several stem cell types are used for clinical patients. Of these, MSCs are reported to show cardiomyogenesis in vitro. Thus, the analysis of key mechanisms for cardiomyogenic differentiation in the human mesenchymal cell is extremely important in order to expand the efficacy of current cardiac stem cell therapy. However, it is very difficult to specify the key factor of cardiomyogenesis by in vivo experiment only. Establishment of EMCs and an in vitro cardiomyogenic differentiation assay system are essential. Stable and high cardiomyogenic transdifferentiation ability in our established system enables us to observe, with wide dynamic range, the effects of treatment for cardiomyogenesis. Moreover, the primary culture condition of murine cardiomyocytes usually fluctuates due to variations in environments, the skill of individual researchers, and institutional differences in isolation protocols. Our established EMCs may provide a good positive control for a cardiomyogenic assay system in vitro to check whether the feeder cell condition is suitable for cardiomyogenic assay. When feeder conditions are suitable, we can survey for possible cardiomyogenic assistant factors or appropriate culture conditions for human BMMSCs by applying various agents or modifying culture conditions systematically. Thus, by using our EMCs and cocultivation system, we may be able to expand the cardiomyogenic differentiation potential of marrow-derived MSCs. Consequently, we may be able to increase the efficacy of cardiac stem cell-based therapy dramatically.
Neither passive stretching of EMCs nor an application of the supernatant of murine cardiomyocyte culture medium to the EMCs alone caused cardiomyocyte differentiation. Taking these findings into account, the multiple environmental factors, including mechanical stretching and/or feeder cardiomyocyte-derived humoral factors, seem to contribute to cardiomyogenic transdifferentiation in human mesenchymal cells. Further experiments should be done.
Study Limitations
Cell fusion between the human cells (MMCs or EMCs) might be a major cause of EGFP-positive cardiomyocytes in the present study. However, EGFP-positive cardiomyocytes could be observed, even when human cells and murine cardiomyocytes were cocultured separately by the athelocollagen membrane that is permeable for only small molecules (less than 5,000 MW)—thus allowing no possible penetration of cells or organelles through the membrane (supplemental online Fig. 6). Furthermore, even if the cells were cocultured without the athelocollagen membrane, nuclear fusion between EMC100s, EMC214s, or MMCs and fetal murine cardiomyocytes was less than 1% in the present study. Moreover, transdifferentiated EMCs at the external layer of the cell sheet graft on the epicardial surface did not directly contact the host cardiomyocytes (Fig. 5). Taking these results into account, we concluded that the cell fusion did not play a major role in the observed significant cardiomyogenic potential of MMCs and EMCs in the present study.
Infarcted heart tissue may increase auto-fluorescence in some fixative conditions and such auto-fluorescence of host cardiomyocytes might be confused as EGFP-positive like cells. However, autofluorescence of the host myocardium adjacent to the infarcted area was not significant in our present condition (Figs. 5B, 6B, supplemental online Fig. 5B, 5F). Therefore, EGFP-positive tissue in the present study can be defined as of human cell origin and easily distinguished from the host heart by the EGFP fluorescent intensity.
The transfection of the cell cycle-mediated gene may increase cardiomyogenic differentiation to some extent. However, our previous study in human BMMSCs, [2] with the same combination of cell cycle-mediated gene transfection, did not show any increase in efficiency. Furthermore, non-gene-transfected MMCs have an extremely high cardiomyogenic efficiency compared to gene-transfected BMMSCs. Taking these results into account, we concluded that transfection of those genes does not play an essential role in causing such high cardiomyogenic differentiation efficiency in EMCs.
In comparison to previous papers, there was no observable effect of BMMSC transplantation on cardiac function in the present study. This discrepancy may be caused by different experimental conditions, that is, species difference between BMMSCs and the host animal [24], transplantation at acute myocardial infarction [25–27], and usage of immunosuppressive agents, etc [24–27].
In the present study, we did not use a pressure-tipped catheter, therefore the LV dp/dt value may be underestimated.
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SUMMARY |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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ACKNOWLEDGMENTS |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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FOOTNOTES |
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REFERENCES |
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TopFootnotesAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosure of Potential...AcknowledgmentsReferences |
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