Induced pluripotent stem cell‐conditional medium inhibits H9C2 cardiomyocytes apoptosis via autophagy flux and Wnt/β‐catenin pathway

Abstract Induced pluripotent stem cell‐derived conditioned medium (iPS‐CM) could improve cell viability in many types of cells and may be a better alternative for the treatment of myocardial infarction. This study aimed to examine the influence of iPS‐CM on anti‐apoptosis and the proliferation of H9C2 cardiomyocytes and investigate the underlying mechanisms. H9C2 cardiomyocytes were exposed to 200 μmol/L hydrogen peroxide (H2O2) for 24 hours with or without pre‐treatment with iPS‐CM. The ratio of apoptotic cells, the loss of mitochondrial membrane potential (△Ψm) and the levels of intracellular reactive oxygen species were analysed by flow cytometric analysis. The expression levels of BCL‐2 and BAX proteins were analysed by Western blot. Cell proliferation was assessed using cell cycle and EdU staining assays. To study cell senescence, senescence‐associated β‐galactosidase (SA‐β‐gal) staining was conducted. The levels of malondialdehyde, superoxide dismutase and glutathione were also quantified using commercially available enzymatic kits. The results showed that iPS‐CM containing basic fibroblast growth factor significantly reduced H2O2‐induced H9C2 cardiomyocyte apoptosis by activating the autophagy flux pathway, promoted cardiomyocyte proliferation by up‐regulating the Wnt/β‐catenin pathway and inhibited oxidative stress and cell senescence. In conclusion, iPS‐CM effectively enhanced the cell viability of H9C2 cardiomyocytes and could potentially be used to inhibit cardiomyocytes apoptosis to treat myocardial infarction in the future.


| INTRODUC TI ON
In recent years, myocardial infarction has become one of the leading causes of mortality worldwide. 1 Myocardial infarction is defined as necrosis of cardiomyocytes as a result of prolonged ischaemia, which is responsible for heart failure, cardiac fibrosis and sudden death. 2,3 Oxidative stress plays an important role in cardiomyocyte proliferation, apoptosis and differentiation. 4 Currently, stem cell therapies for myocardial infarction have become promising alternatives to repair and regenerate injured tissues, and their applications are being extensively studied in various diseases. [5][6][7][8][9][10][11] Induced pluripotent stem cells (iPSCs), which can be directly obtained from adult cells using reprogramming factors (Oct 3/4, Sox2, Klf4 and c-Myc), 12 resemble embryonic stem cells with the abilities of self-renewal and differentiation into the three germ layers. 13 However, iPSCs have no immune rejection and ethical issues, which offer an attractive platform for disease model, pharmaceutical screening, and so on. 14,15 So far, iPSCs have been successfully differentiated into cardiomyocytes. 16,17 and can achieve in situ regeneration in infracted adult mouse hearts. 18 However, there are risks of tumorigenesis in rat hearts after cell transplantation. 19,20 Conditioned medium could affect cell functions and viability via biologically active components. It has been reported that basic fibroblast growth factor (bFGF), nerve growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF), insulinlike growth factor (IGF-1) and brain-derived neurotrophic factor could be secreted in the supernatant of cultured stem cells, which could improve cell viability. [21][22][23] Neel and Singla reported that iPSderived conditioned medium (iPS-CM) could reduce the occurrence of cardiac apoptotic nuclei in a diabetic cardiomyopathy rat model. 8 Lian et al showed that iPS-CM improved the proliferation and anti-apoptotic abilities of human adipose-derived stem cells. 24 Zhang et al concluded that paracrine factors of iPSCs could inhibit stress-induced senescence of H9C2 cardiomyocytes by inhibiting the p53-p21 and p16-pRb pathways. 25 The stimulation of cell proliferation and anti-apoptosis caused by iPS-CM is mainly because of the secreted cytokines that do not cause tumour formation. 8,[24][25][26][27] There is the report that showed iPSCs and iPS-CM have similar therapeutic effects on lung injury through the same signalling pathway. 26 Therefore, iPS-CM could be used to treat myocardial infarction.
However, it is imperative to understand how and which factors of iPS-CM affect cardiomyocyte viability and functions prior to of myocardial infarction therapy.
In this study, we investigated the effects of iPS-CM on the pro-

| Cell culture
H9C2 cardiomyocytes were cultured in medium (DMEM-HG) containing High-Glucose Dulbecco's Modified Eagle's Medium, 10% FBS, and 1% penicillin and streptomycin. The culture conditions were maintained at 37°C in a 5% CO 2 incubator. The cells were subcultured at confluence.

| Preparation of iPS-CM
Human iPSCs were acquired from the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences. iPSCs were produced from the umbilical cord matrix and amniotic mesenchymal cells through the transduction of the retroviral factors Oct4, Sox2, c-Myc and Klf4. 28 Previously, established protocols were followed to culture the iPSCs. 29 Briefly, culture dishes were coated with 1% Matrigel at least 30 minutes before cell seeding. Then the iPSCs were cultured in mTeSR1 medium in a 37°C incubator with 5% CO 2 .
Every 6 days, the iPSCs were subcultured using 0.05% trypsin-EDTA at 37°C for 5 minutes, and then seeded onto 1% Matrigel-coated culture plates. The supernatant of iPSCs was derived from the mTeSR1 medium cultured with iPSCs for 1 day. The supernatant was filtered (using a 0.22 μm filter) to remove dead cells and cell debris. The supernatant was then stored at −80°C for at least 2 weeks. The iPSC supernatant was mixed with DMEM-HG at a ratio of 1:2 to obtain iPS-CM.

| CCK-8 assay
Cell counting kit-8 was used to measure cell viability under different conditions. Briefly, H9C2 cardiomyocytes (1 × 10 3 cells/well) were seeded into 96-well plates. The cells were then treated under different conditions and incubated for 24 hours at 37°C in a 5% CO 2 incubator. Subsequently, 10 μL of CCK-8 solution was added to each well and incubated for 4 hours, after which the absorbance at 490 nm was measured using a microplate reader (Thermo, MA, USA).

| TUNEL staining
The TUNEL assay was conducted to detect cell apoptosis using an in situ Cell Death Detection kit according to the manufacturer's instructions. Briefly, H9C2 cardiomyocytes with different concentrations of H 2 O 2 treatments were fixed with 4% paraformaldehyde for 1 hour at room temperature and washed with PBS three times for 5 minutes each wash. Then, cells were incubated with blocking solution (3% H 2 O 2 in methanol) for 10 minutes at room temperature and washed with PBS three times for 5 minutes each wash. Then, cells were incubated in permeabilisation solution (0.1% Triton X-100) for 2 minutes on ice and treated with TdT/dUTP FITC labelling reaction mixture for 1 hour at 37°C in the dark, followed by three rinses with PBS. Cell nuclei were positive if they were labelled with FITC (green), whereas DAPI staining indicated the cell nucleus under the florescence microscope. The positive staining cells were counted using Image-Pro Plus 6.0 software.

| Annexin V and PI assay
Annexin V and PI assays were conducted to measure the apoptosis of H9C2 cardiomyocytes treated with H 2 O 2 . H9C2 cardiomyocytes were seeded into 6-well plates (1 × 10 6 cells/well).
The cells were then allowed to grow in DMEM-HG and iPS-CM for 24 hours at 37°C in a 5% CO 2 incubator. Then, 200 μmol/L of

| Measurement of H 2 O 2 -induced reactive oxygen species
The levels of reactive oxygen species (ROS) in the H9C2 cardiomyocytes were measured using a 2′7′-dichlorofluorescin diacetate   Table 1) overnight at 4°C. The following day, the membranes were washed five times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000; Bioword, MN, USA) for 2 hours at room temperature. The protein bands were detected by enhanced chemiluminescence (Pierce Chemical Co., IL, USA) and quantitated using ImageJ. The ratio of the expression of target proteins was determined after normalising to the β-Actin level.
The cells were incubated for 24 hours at 37°C in a 5% CO 2 incubator. The cells were then harvested and fixed in 75% ethanol at 4°C overnight. The following day, the cells were washed with PBS, and stained with PI in the dark for 30 minutes at room temperature. The stained cells were analysed using flow cytometry.

| EdU staining
A Click-iT ® EdU kit (Invitrogen, CA, USA) was used according to the manufacturer's instructions. H9C2 cardiomyocytes at a density of 1 × 10 5 cells/well were seeded in 6-well plates with DMEM-HG, iPS-CM and DMEM-HG+bFGF (30 ng/mL), and were then incubated at 37°C in a 5% CO 2 incubator for 24 hours. Then, 20 μmol/L EdU solutions were added to the cells, and incubated at 37°C for another 2 hours. The H9C2 cardiomyocytes were collected through digestion and centrifugation, and was fixed with 4% paraformaldehyde for 15 minutes and permeabilised using Triton X-100 solution for 30 minutes at room temperature in the dark. The fixed cells were then stained with Click-iT™ reaction mixture, and the cell nuclei were stained with 1 μg/mL DAPI in the dark, at room temperature for 30 minutes. The stained H9C2 cardiomyocytes were observed under an inverted fluorescence microscope and the cell fluorescence was quantified using flow cytometry. Quantification of the results was carried out by ImageJ software.

| Measurement of MDA, SOD and GSH
H9C2 cardiomyocytes (1 × 10 6 cells/well) were plated into 6-well plates and cultured with DMEM-HG and iPS-CM at 37°C in a 5%

| Enzyme-linked immunosorbent assay
An enzyme-linked immunosorbent assay (ELISA) kit was used to determine the levels of bFGF in DMEM-HG, iPS-CM and mTeSR1.
Briefly, 200 μL samples and 50 μL assay diluent were added to precoated wells of 96-well plates, and incubated at room temperature for 2 hours. The plates were then washed five times with washing buffer. Each well was treated with 100 μL of peroxidase-conjugated IgG anti-bFGF solution for 2 hours at room temperature. The plates were washed again five times with washing buffer. Then, 100 μL of substrate buffers were added into each well and incubated for 30 minutes at room temperature in the dark. Finally, the enzyme reaction was quenched with 50 μL of stop solution. The assays were analysed using a microplate reader at a wavelength of 550 nm with a correction wavelength of 450 nm.

| Statistical analysis
The collected data were statistically analysed using GraphPad Prism software (version 6; GraphPad Software Inc., San Diego, CA, USA).
Comparisons of different groups were performed using one-way Mean ± SE, n = 5. *P < 0.05, **P < 0.01 designate significant differences when compared to control (1:0 or 0:1). The n.s designates no significant difference. Scale bars 100 μm ANOVA followed by Tukey's test. The values were expressed as the mean ± SD, and P < 0.05 was considered to be statistically significant.  Figure 1A). To establish the optimal apoptosis model, H9C2 cardiomyocytes were exposed to different concentra- was selected as the optimal concentration for the following apoptosis model ( Figure 1C).

| iPS-CM inhibited H 2 O 2 -induced apoptosis of H9C2 cardiomyocytes
Annexin V and PI assays were used to evaluate apoptosis in dif-

| The effects of iPS-CM on BAX and BCL-2 expression in H 2 O 2 -treated H9C2 cardiomyocytes
To  were also less than those in the iPS-CM+H 2 O 2 group ( Figure 6E).

| iPS-CM promoted the proliferation of H9C2 cardiomyocytes
These results suggested that iPS-CM could promote the proliferation of H9C2 cardiomyocytes even under the H 2 O 2 administration.

| iPS-CM inhibited H 2 O 2 -induced cell senescence and oxidative stress in H9C2 cardiomyocytes
Cell senescence is defined as the irreversible cell cycle arrest of mitotic cells, leading to a change in the cellular phenotype. Cell senescence can be induced by external stimuli, such as oxidative stress. H9C2 cardiomyocytes were exposed to a low dose of

| The potential mechanism underlying the promotion of H9C2 cardiomyocyte anti-apoptosis and proliferation by iPS-CM
To elucidate the underlying mechanism of the anti-apoptotic and proliferative properties of iPS-CM on H9C2 cardiomyocytes, we first analysed the levels of different growth factors, such as bFGF, IGF-1 and VEGF in DMEM-HG, iPS-CM and mTeSR1 using ELISA.
Only bFGF, but not IGF-1 and VEGF (data not shown), showed significant differences among these groups, and the level of bFGF in iPS-CM was significantly greater than that in DMEM-HG or mTeSR1 ( Figure 8A). Based on this result, exogenous bFGF (30 ng/mL) was added to the DMEM-HG to establish a new group that mimicked the iPS-CM group, and the underlying signalling pathways were further investigated.
To  Figure 9C.
The potential mechanisms underlying the promotion of H9C2 cardiomyocyte proliferation by iPS-CM. A, Assessment of protein expression levels of p-β-catenin (ser 675), β-catenin (nuclear protein), β-catenin (cytosolic protein), Cyclin D1, c-Myc and Survivin in different groups using Western blot assays. B, Quantification of Western blot assays. C, Diagram of the potential mechanisms underlying the promotion of H9C2 cardiomyocytes anti-apoptosis and proliferation by iPS-CM. Mean ± SE, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, designate significant differences. The n.s designates no significant difference Previous studies have shown that the proliferative ability of iPS-CM is attributed to the products secreted by iPSCs in culture medium. 29 Guo et al showed that the levels of bFGF and Activin A in iPS-CM were greater than in fresh mTeSR1 medium. 29 Villageois et al found that Activin A, which was secreted by hASCs isolated from various fat deposits of donors with different ages, promoted human multipotent adipose-derived stem cell proliferation and adipocyte differentiation. 45 In our study, bFGF levels in iPS-CM were also significantly greater than those in DMEM-HG and mTeSR1, which were determined using an ELISA assay. Based on this result, we added exogenous bFGF to DMEM-HG to mimic the function of iPS-CM.

| D ISCUSS I ON
Apoptosis is defined as highly regulated, programmed cell death that requires much energy and plays a key role in a variety of biological systems. Saraste et al showed that in myocardial samples obtained from acute myocardial infarction patients, a subset of cardiomyocytes underwent apoptosis. 46 Apoptosis was more prominent in the border zones of recent infarctions. 47 Recently, increasing attention has been focused on autophagy flux, which is the progression of autophagosome formation to cargo delivery and degradation in lysosomes caused by lysosomal proteases. 59,60 Autophagy flux can be activated by cells to protect against apoptosis and inflammation under stress. 32,61 It is well known that LC3 and Beclin-1 are involved in the progression of autophagosome formation, while P62 is an endogenous substrate of the autophagic process, which is considered a marker of autophagic flux, and the accumulation of P62 indicates disrupted autophagy flux. 62

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.