All animal procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital (Affidavit of Approval of Animal Use Protocol No. 2019061902) and performed in accordance with the Guide for the Care and Use of Laboratory Animals.
Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC; Frederick, MD, USA)-approved animal facility in our hospital with controlled temperature and light cycles (24 oC and 12/12 light cycle).
Methodology of in vitro study of cell culturing for differentiation of human iPSC into mesenchymal stem cells (MSCs)
The procedure and protocol of human iPSC culture for differentiation into MSCs have been described in our previous study  and detailed in formation was illustrated as supplementary Figure 1. In details, at day 1, the human iPSCs [mTeSR™1; StemCell, #28315) were first washed by 5 mL PBS, followed by 2 mL Accutase (Gibco, #A1110501; Accutase: PBS = 1:1); the incubator reaction continued for 1 min. The 2 mL KO DMEM/F12 (Gibco, #12660012) was added and the cells were collected in 15 mL centrifuge tubes for 5-minute duration of centrifuge (x200 g). The cells were then cultured in a 10-cm dish for 24 h in mTeSR™1 culture medium.
By day 2, the cells (mTeSR™1) were collected and washed by 5 mL PBS. STEMdiffTM-ACF Mesenchymal Induction Medium (StemCell, #05241) was added to incubator culture and proceeded for 24 h. The STEMdiffTM-ACF Mesenchymal Induction Medium was exchanged once per day from days 1 to 3. This procedure was repeated on days 3 to 6. On days 7 to 21, the procedure was repeated but the culture medium was refreshed every 3 days.
miR-19a-3p and miR-20a-5p were candidates for double overexpression in iPS-MSCs (iPS-MSCdOex-mIRs) and treatment of DCM in rodent
The procedure and protocol were based on our recent report . We had identified that miR-19a-3p and miR-20a-5p were the two most suitable candidates among the five miRNAs (i.e., miR-374a-5p/ miR-19a-3p/ miR-106b-5p/miR-26b-5p/ miR-20a-5p) to be overexpressed (i.e., transfection) for the purpose of treatment of chronic kidney disease + ischemia-reperfusion animals . In detail, transfections of miR-19a-3p and miR-20a-5p mimics efficiently augmented the miRNA expressions and further decreased related gene expressions. Transfections of mimics (25 nM) were conducted with TransIT-X2 Dynamic Delivery System (Mirus), by following the manufacturer’s instruction. The iPS-MSCs were recognized >80% confluence on the day of transfection. TransIT-X2 reagent was mixed with miRNA mimics for 25 minutes at room temperature. The miRNA mimics-containing complexes were further distributed into cells. Two days later, relevant expressions of miRNAs and genes were validated by the real-time qPCR assay.
DCM induction in rodent by doxorubicin (Dox) and animal grouping
Pathogen-free, adult male Sprague-Dawley (SD) rats (n=32) weighing 320-350 g (Charles River Technology, BioLASCO Taiwan Co. Ltd., Taiwan) were utilized in this study. The procedure and protocol of Dox-induced rodent DCM model have been described in detail in our previous report . In detail, the accumulated dose of 12.5 mg per kg at 4 separated time points within 20 days (i.e., once every 5 days) in each rat by intraperitoneal (IP) administration was applied in the present study.
Animals were equally categorized into group 1 (sham-control, i.e., by IP administration of 1.0 cc saline four times within 20 days, followed by opening chest wall only at day 28 after DCM induction), group 2 (DCM only), group 3 [DCM + iPS-MSCs/1.2 x 106 cells/administered by day 28 after DCM induction) and group 4 (DCM + iPS-MSCdOex-mIRs/1.2 x 106 cells). The iPS-MSCs or iPS-MSCdOex-mIRs was implanted into the LV myocardium via opening the chest wall after intubation with animal ventilatory support by day 28 after DCM induction by Dox. Additionally, the dosage of MSCs in the present study was based on our previous studies [36, 39]. In the current study, the animals in each group were euthanized by day 60 and the heart specimen was harvested in each animal for individual study.
Determinant LVEF by utilizing the transthoracic echocardiography
Transthoracic echocardiography was performed in each group prior to and on days 28 and 60 after DCM induction. The procedure was performed by an animal cardiologist blinded to this experimental design using an ultrasound machine (Vevo 2100, Visualsonics). M-mode standard two-dimensional (2D) left parasternal-long axis echocardiographic examinations were conducted. Left ventricular (LV) internal dimensions [end-systolic diameter (ESD) and end-diastolic diameter (EDD)] were measured at the mitral valve level of the left ventricle, according to the leading-edge method of American Society of Echocardiography, by using at least three consecutive cardiac cycles. The left ventricular ejection fraction (LVEF) was calculated as follows: LVEF (%) = [(LVEDD3-LVEDS3)/LVEDD3] × 100%.
Western blot analysis of LV myocardium
The procedure and protocol have been described in detail in our previous reports [36-39].Briefly, primary antibodies against tumor necrosis factor (TNF)-α (1: 1000, Cell Signaling), nuclear factor (NF)-κB (1:1000, Abcam), tumor necrosis factor receptor-associated factor 6 (TRAF6) (1:2000, Abcam), toll-like receptor (TLR)-4 (1:1000, Novus), myeloid differentiation primary response 88 (MyD88) (1:1000, Abcam), myelin and lymphocyte protein (Mal) (1:1000, Abcam), TRIF (1:1000, Abcam), translocating chain-associated membrane protein (TRAM) (1:1000, Thermo Fisher Scientific), TNF receptor associated factor 6 (TRAF6) (1:2000, Abcam), IKK-α (1:5000, Abcam), IKK-ß (1:1000, Cell Signaling), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IKB-α) (1:1000, Cell Signaling), apoptosis signal-regulating kinase 1 (ASK1) (1:1000, Abcam), phosphorylated mitogen-activated protein kinase 4 (p-MMK4) (1:1000, Cell Signaling), p-MMK7 (1:1000, Thermo Fisher Scientific), p-JNK1/2 (1:1000, Abcam), p-cJUN (1:1000, Abcam), Atg5 (1:1000, Cell Signaling), Beclin1 (1:1000, Cell Signaling), interleukin (IL)-1ß (1:1000, Cell Signaling), matrix metalloproteinase (MMP)-9 (1:2000, Abcam), NOX-1 (1:1500, Sigma-Aldrich), NOX-2 (1:1000, Sigma-Aldrich), cytosolic cytochrome C (1:2000, BD), cyclophilin-D (1:3000, Abcam), dynamin-related protein 1 (DRP1) (1:1000, Cell Signaling), LC3B-II (1:2000, Abcam), LC3B-I (1:2000, Abcam), mitochondrial Bax (1:1000, Abcam), cleaved caspase 3 (1:1000, Cell Signaling), cleaved Poly (ADP-ribose) polymerase (c-PARP) (1:1000, Cell Signaling), Smad3 (1:1000, Cell Signaling) and transforming growth factor (TGF)-ß (1:1000, Abcam) were used. Signals were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse, goat anti-rat, or goat anti-rabbit IgG.
Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences), which was then exposed to Biomax L film (Kodak). For quantification, ECL signals were digitized using Labwork software (UVP).
Immunohistochemical (IHC) and immunofluorescent (IF) studies
The procedures and protocols for IHC and IF examinations were based on our previous reports [36-39]. Briefly, for IHC and IF staining, rehydrated paraffin sections were first treated with 3% H2O2 for 30 minutes and incubated with Immuno-Block reagent (BioSB, Santa Barbara, CA, USA) for 30 minutes at room temperature. Sections were then incubated with primary antibodies specifically against γ-H2AX (1:1000, Abcam) and CD14 (1:200, Thermo Fisher), while sections incubated with the use of irrelevant antibodies served as controls. Three sections of heart specimens from each rat were analyzed. For quantification, three randomly selected HPFs (400× for IF studies) were analyzed in each section.
Histopathological finding of myocardial fibrosis
The procedure and protocol were based on our previous studies . In details, hematoxylin and eosin and Masson's trichrome staining were utilized for identification of LV fibrotic area. Three serial sections of LV myocardium in each animal were prepared at 4 µm thickness by Cryostat (Leica CM3050S). The integrated area (µm2) of fibrosis on each section were calculated using the Image Tool 3 (IT3) image analysis software (University of Texas, Health Science Center, San Antonio, UTHSCSA; Image Tool for Windows, Version 3.0, USA). Three randomly selected high-power fields (HPFs) (100 x) were analyzed in each section. After determining the number of pixels in each fibrotic area per HPF, the numbers of pixels obtained from three HPFs were calculated. The procedure was repeated in two other sections of each animal. The mean pixel number per HPF for each animal was then analyzed by calculating all pixel numbers and dividing by 9. The mean integrated area (µm2) of fibrosis in LV myocardium per HPF was obtained using a conversion factor of 19.24 (since 1 µm2 represents 19.24 pixels).
MTT assay, qPCR analysisand flow cytometric analysis for identification of total cellular and mitochondrial oxidative stress and membrane potential of mitochondria in iPS-MSCs
For the purposes of in vitro study, the culturing cells were categorized into G1 (iPS-MSC), G2 (iPS-MSCdOex-mIRs), G3 (iPS-MSC + H2O2/100uM), and G4 (iPS-MSCdOex-mIRs + H2O2/100uM), respectively. The cells were finally collected for the flow cytometric analysis for assessment of total cellular (i.e., by H2DCFDA test) and mitochondrial (i.e., by Mito-SOX assay) oxidative stress and membrane potential of mitochondria [i.e., Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE assay)].
Additionally, the MTT assay was utilized in the present study to determine the cellular metabolic activity as an indicator of cell viability, proliferation, and cytotoxicity.
Furthermore, the cells were also collected after culturing for Western blot analysis. Finally, qPCR analysis was utilized to assess the success of overexpression of iPS-MSCOex-mIRs.
Procedure and protocol for measurement of reactive oxygen species (ROS)
The procedure and protocol have been described in our previous report . In detail, the 2′,7′-dichlorodihydrofluorescin diacetate (H2DCFDA, Molecular Probes) was dissolved in DMSO at a concentration of 25 mg/ml. After dilution with 50% ethanol to a final concentration of 2.5 mg/ml, 6 μg per gram for rat body weight was administered to each rat from the penis vein immediately following echocardiographic study. All animals were euthanized 30 minutes later following H2DCFDA administration.
The fluorescence and grayscale photos were captured by utilizing the DP controller 126.96.36.199 (Olympus). Grayscale photos for measuring the fluorescence intensity were processed by using Image J 1.37v (National Institutes of Health, USA). Nine grayscale photos from each slide were randomly acquired. As compared with the area of increased fluorescence intensity (IFI), the baseline fluorescence intensity (BFI) (arbitrary unit/400 x high-power field) was defined as the area in myocardium loaded without H2DCFDA. Six BFI areas were measured from each grayscale photo, from which 3 BFI areas were randomly chosen. The mean IFI and mean BFI were then calculated. The ratio of IFI to the BFI was determined as the relative fluorescence intensity.
The LV specimen were obtained, frozen rapidly in liquid nitrogen and then stored at -800 C.
Quantitative data are expressed as mean ± SD. Statistical analyses were performed using SAS statistical software for Windows Version 8.2 (SAS Institute, Cary, NC, USA). One-way ANOVA was conducted followed by Bonferroni multiple comparison post hoc test for comparing variables among groups. A probability value <0.05 was considered statistically significant.