Induced pluripotent stem cell‐derived conditional medium promotes Leydig cell anti‐apoptosis and proliferation via autophagy and Wnt/β‐catenin pathway

Abstract Leydig cell transplantation is a better alternative in the treatment of androgen‐deficient males. The main purpose of this study was to investigate the effects of induced pluripotent stem cell‐derived conditioned medium (iPS‐CM) on the anti‐apoptosis, proliferation and function of immature Leydig cells (ILCs), and illuminate the underlying mechanisms. ILCs were exposed to 200 μmol/L hydrogen peroxide (H2O2) for 24 hours with or without iPS‐CM treatments. Cell apoptosis was detected by flow cytometric analysis. Cell proliferation was assessed using cell cycle assays and EdU staining. The steroidogenic enzyme expressions were quantified with Western blotting. The results showed that iPS‐CM significantly reduced H2O2‐induced ILC apoptosis through down‐regulation of autophagic and apoptotic proteins LC3‐I/II, Beclin‐1, P62, P53 and BAX as well as up‐regulation of BCL‐2, which could be inhibited by LY294002 (25 μmol/L). iPS‐CM could also promote ILC proliferation through up‐regulation of β‐catenin and its target proteins cyclin D1, c‐Myc and survivin, but was inhibited by XAV939 (10 μmol/L). The level of bFGF in iPS‐CM was higher than that of DMEM‐LG. Exogenous bFGF (20 ng/mL) or Wnt signalling agonist lithium chloride (LiCl) (20 mmol/L) added into DMEM‐LG could achieve the similar effects of iPS‐CM. Meanwhile, iPS‐CM could improve the medium testosterone levels and up‐regulation of LHCGR, SCARB1, STAR, CYP11A1, HSD3B1, CYP17A1, HSD17B3 and SF‐1 in H2O2‐induced ILCs. In conclusion, iPS‐CM could reduce H2O2‐induced ILC apoptosis through the activation of autophagy, promote proliferation through up‐regulation of Wnt/β‐catenin pathway and enhance testosterone production through increasing steroidogenic enzyme expressions, which might be used in regenerative medicine for future.


| INTRODUCTION
Leydig cells, distributing in clusters between the seminiferous tubules in the testis, are responsible for androgen production in the male. 1,2 Testosterone, referred to as the male hormone, plays a critical role in maintaining sexual function, muscle bulk and bone health. 3 Testosterone synthesis in Leydig cells depends on the luteinizing hormone (LH) secreted by the pituitary grand. 4 LH binds LH receptors (LHCGR, encoded by Lhcgr) on the Leydig cell plasma membrane and then leads to intracellular cyclic adenosine monophosphate (cAMP) cascade, 5 which further results in the rapid transport of cholesterol from the outer to the inner mitochondrial membrane, mediated by lipoprotein receptor (SCARB1, encoded Scarb1) and steroidogenic acute regulatory protein (STAR, encoded by Star). Subsequently, testosterone is synthesized through a series of steroidogenic enzymes: the cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1, encoded by Cyp11a1), 3b-hydroxysteroid dehydrogenase (HSD3B1, encoded by Hsd3b1), cytochrome P450 17a-hydroxylase (CYP17A1, encoded by Cyp17a1) and 17b-hydroxysteroid dehydrogenase 3 (HSD17B3, encoded by Hsd17b3), 6 while another critical factor for Leydig cells is the steroidogenic factor 1 (SF-1, or named NR5A1), which is essential for the commitment of stem Leydig cells to testosterone-producing Leydig cells. 7 It is well known that SF-1 is an essential factor for Leydig cell development and survival. 8 Overexpression of SF-1 has been shown to be capable of promoting ESCs into Leydig-like cells. 9 Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells that can be generated directly from adult cells. [10][11][12] share the features of ESCs that are capable of self-renewal and differentiation into three germ layers. 13 iPSCs can address immune rejection and ethical issues of the autologous cell transplantation.
They also offer an attractive approach to disease model, pharmaceutical screening, toxicology research and so on. 14,15 Conditioned medium can affect cell functions via biologically active components. It was reported that cytokines such as basic fibroblast growth factor (bFGF), nerve growth factor (NGF), stem cell factor (SCF), hepatocyte growth factors (HGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1) and brain derived neurotrophic factor (BDNF), which were secreted in the medium by cultured stem cells, could encourage the growth of cells. [16][17][18] Li et al 11 reported that induced pluripotent stem cell-derived conditioned medium (iPS-CM) potentially restored the bronchial microstructure in acute lung injury (ALI). Zhang et al 19 mentioned that iPS-CM contributed to recovery from the effect of endotoxin-induced ALI in mice. The stimulating proliferation and anti-apoptosis from iPS-CM were the result of cytokines secretion, which were generally safe and would not produce tumours. [19][20][21] The Wnt/b-catenin signalling pathway is the classic pathway involved in cell proliferation. 22 Basic fibroblast growth factor (bFGF) could promote the proliferation and migration of fibroblasts through the activation of Wnt/b-catenin signalling pathway. 23 Autophagy has been known as a conserved catabolism to sustain cellular homeostasis. 24,25 It plays critical roles in eliminating dysfunctional or surplus proteins and damaged intracellular organelles. 26 A major event in autophagosome formation is to generate LC3-I, which is then conjugated to phosphatidylethanolamine, generating membrane-bound LC3-II. 27,28 Beclin-1 is a key autophagic protein regulating autophagosome formation. 29,30 Meanwhile, P62 is a specific autophagic substrate protein and the hallmark representing autophagic flu. 30 It is known that the activation of autophagy can protect cells against apoptosis and inflammation. 31,32 In this research, we would investigate the effects of iPS-CM on the proliferation and the H 2 O 2 -induced apoptosis of immature Leydig cells (ILCs), and evaluate the influences of iPS-CM on testosterone production of ILCs. Moreover, the potential mechanisms underlying the effects of iPS-CM would be explored. This study was to develop a new way to improve the activities and function of ILCs used for future clinical cell transplantation.

| Animals
The Sprague-Dawley rats were provided by Laboratory Animal Center, Wenzhou Medical University, Wenzhou, China. They were raised in a 12-hour dark/light cycle temperature at 23 AE 2°C, and relative humidity of 45% to 55%. Water and food were accessed ad libitum.
This study was approved by the Wenzhou Medical University's Animal Care and Use Committee, and was performed in accordance with the Guide for the Care and Use of Laboratory Animals.

| Immature Leydig cells isolation
Eighteen 35-day-old male Sprague-Dawley rats were killed in CO 2 tank for the isolation of immature Leydig cells (ILCs). ILCs express all androgen biosynthetic enzymes 33 and are capable of proliferation. 34 The isolation of rat ILCs was performed as previously described. 33 Briefly, the testes were removed, perfused with collagenase via the testicular artery and digested with M-199 buffer containing collagenase (0.25 mg/mL) and DNase (0.25 mg/mL) for 15 minutes. Then, the cell suspension was filtered through 100-lm nylon mesh (BD, GUO ET AL. | 3615 CA, USA) and the cells were separated by the Percoll gradient. The cells with the density of 1.07-1.088 g/mL were collected. The purity of ILCs was evaluated by immunohistochemical staining HSD3B1, the biomarker of ILCs, as previously described. 35 The HSD3B1 staining solution contained with 0.4 mmol/L etiocholanolone as the steroid substrate and NAD + as a cofactor. 35 The purity of ILCs was more than 95%.

| Culture of ILCs
The isolated ILCs were directly seeded into wells in the 24-well culture plates with the density of 2 9 10 4 cells/well and incubated at a 37°C, 5% CO 2 incubator. The culture medium (DMEM-LG) contains low-glucose Dulbecco's modified Eagle's medium, 10% fatal bovine serum (FBS), and 1% penicillin and streptomycin as control.
2.5 | The culture of iPSCs and preparation of iPSC supernatant iPSCs were cultured as previously described. 36 In brief, culture plates were coated with 1% (v/v) Matrigel for 0.5 hours in advance, and then, iPSCs were cultured in mTeSR1 medium (StemCell Technologies Inc., Canada) at a 37°C, 5% CO 2 incubator. These cells would be passaged every 6 days using 0.05% trypsin-EDTA. Y-27632 (10 mmol/L) as ROCK inhibitor was added into the plates on the first day after passage. The supernatant of iPSC was derived from the mTeSR1 medium cultured with iPSCs for 24 hours. The supernatant was filtered (0.22 lm) to remove dead cells and then stored at À80°C for at least 2 weeks. iPS-CM was DMEM-LG mixed with iPSC supernatant at the ratio of 2:1.     Exogenous bFGF (20 ng/mL) was added into DMEM-LG+H 2 O 2 to obtain the control+H 2 O 2 +bFGF group. LY294002 (LY, 25 lmol/L) were added into iPS-CM+H 2 O 2 to get iPS-CM+H 2 O 2 +LY group.

| Cell treatments
Then, cells were cultured for another 48 hours and harvested for Western blotting. For exploration of the proliferation mechanism, the mediums were changed into DMEM-LG (control) or iPS-CM to establish control group or iPS-CM group. XAV939 (XAV, 10 lmol/L) was added into iPS-CM to get iPS-CM+XAV group. Exogenous bFGF (20 ng/mL) was added into DMEM-LG to get control+bFGF group.
LiCl (20 mmol/L) was added into DMEM-LG to get control+LiCl group. Then, cells were cultured for another 48 hours and harvested for Western blotting.

| Western blotting
Cells were washed twice with cold PBS and then were lysed in the radio immunoprecipitation assay buffer (Bocai Biotechnology, Shanghai, China) supplemented with a protease inhibitor (Amyjet Scientific Inc, Wuhan, China). Lysate was centrifuged at 12 000 g for 15 minutes at 4°C. The protein concentrations in the supernatants were measured using the BCA assay kit (Takara, Japan) as the manufacturer's instructions. Sample proteins (50 lg) were subjected to 10% polyacrylamide gel containing sodium dodecyl and then transferred into the polyvinylidene fluoride membrane. After being blocked with 5% free-fat milk in Tween 20-containing Tris-buffered saline for 2 hours at 4°C, the membranes were incubated with primary antibodies over night at 4°C (listed in Table 1). Then, membranes were washed with Tween 20-containing Tris-buffered saline for five times and incubated with horseradish peroxidase-conjugated secondary antibody (1:5000, Bioword, MN, USA) for 1 hour at room temperature and then were washed with the buffer for three times again.
The protein bands were visualized with enhanced chemiluminescence (Pierce Chemical Co, IL, USA). The intensities of proteins were quantified using Image J software.

| Testosterone measurement by radioimmunoassay
Medium testosterone concentrations were measured with a tritiumbased radioimmunoassay using antitestosterone antibody as previously described. 37 Standards ranging between 10 and 2000 pg/mL testosterone were prepared in triplicate. Standards and samples were incubated with tracer, and charcoal-dextran suspension was used to separate the bound and free steroids. The bound steroids were mixed with a scintillation buffer and counted in a b scintillation counter (PE, CA, USA). The minimum detectable concentration for testosterone was 5 pg/mL. Quality control samples contain 100 pg/mL testosterone. The intra-assay and interassay coefficients of variation were within 10%.  Then, plates were washed five times with washing buffer. Then 100 lL substrate buffers were added to each well and incubated in

| Statistical analysis
All data are presented as the mean AE standard errors (SE). Statistical significance was analysed using one-way ANOVA followed by ad hoc Turkey multiple comparisons to the control. Statistical analyses were performed using GraphPad Prism (version 6, GraphPad Software Inc., San Diego, CA, USA). *P < .05, **P < .01 or ***P < .001 were considered statistically significant.  Figure 1A). Base on these data, we considered this proportion as iPS-CM to treat ILCs in following studies.
To establish the optimal apoptosis model, ILCs were exposed to LG) almost had no apoptotic cells, with only 1.98 AE 0.15%. The con-trol+H 2 O 2 group had 25.59 AE 0.37% apoptotic cells, which is much higher than that of control (**P < .01). The apoptotic ratio in iPS-CM+H 2 O 2 group was 7.97 AE 0.21%, which was higher than that of control group (*P < .05) but lower than that of control+H 2 O 2 group (**P < .01) ( Figure 2B). This result demonstrated that iPS-CM could inhibit H 2 O 2 -induced ILC apoptosis.
As the loss of MΨm was associated with early apoptosis, JC-1 probe was used to analyse the effects of iPS-CM on the loss of  | 3619 also more than that of control+H 2 O 2 group (40.55 AE 1.12%) (**P < .01) ( Figure 4B). In addition, EdU staining showed that the EdUpositive cells in control group was less than that of iPS-CM group, and iPS-CM+H 2 O 2 group was more than control+H 2 O 2 group ( Figure 4C).
Meanwhile, the flow cytometry assays also demonstrated that the fluorescent density of EdU-positive cells in control group was less than iPS-CM group (**P < .01), and iPS-CM+H 2 O 2 group was more than that of control+H 2 O 2 group (**P < .01) ( Figure 4D,E). These results suggested that iPS-CM could promote the proliferation of ILCs.  Figure 6F. Mean AE SE, n = 3. *P < .05, **P < .01, ***P < .01 designate significant differences apoptosis in the streptozotocin of diabetic cardiomyopathy (SIDC) rat model. 21 Zhang et al 20 reported that iPS-CM could not only suppress apoptosis by inhibiting p53/p21 and p16/pRb pathways, but also promote proliferation by attenuating G1 phase arrest of cell cycle in H9C2 cells. In our study, we also found that iPS-CM could increase the percentage of ILCs entering the S and G2 phases and EdU staining-positive ILCs with or without H 2 O 2 treatment, which indicated that iPS-CM could significantly promote the proliferation of ILCs.
iPS-CM could enhance alveolar epithelial regeneration in vivo partially due to containing hepatocyte growth factor. 53 iPS-CM could also promote the growth of other cells. 54,55 In addition, we also found that iPS-CM could significantly increase the medium testosterone levels of ILCs with or without up-regulation, suggesting that iPS-CM up-regulated these proteins via increasing SF-1 expression. Indeed, many studies had showed that CYP11A1, HSD3B1, CYP17A1 and HSD17B3 promoters had SF-1 binding sites. 56,57 Although the exact mechanism is still unclear, the significant increase in SF-1, a critical transcription factor for the expression of steroidogenic enzymes, 7 might be involved in possible inherent mechanism.
As is known to all that the activation of autophagy can protect cells against apoptosis and inflammation, 31,32 in this study, it was discovered that the level of bFGF in iPS-CM was higher than that of control (DMEM-LG) with or without H 2 O 2 treatment. iPS-CM could up-regulate the expression of autophagic proteins LC3 II/I, Beclin-1 and P62 but down-regulate the expression of apoptotic protein P53 in H 2 O 2 -induced ILCs. However, the up-regulated autophagic proteins and the down-regulated apoptotic protein could be inhibited by LY294002 (25 lmol/L), an inhibitor of autophagy. 38 Additionally, exogenous bFGF (20 ng/mL) added into DMEM-LG could achieve the similar effects of iPS-CM. These results suggested that iPS-CM might act as an agonist of autophagy pathway to inhibit apoptosis of ILCs, which was markedly contributed to the inherent bFGF. The Wnt/b-catenin signalling pathway is the classic pathway-mediated cell proliferation. 22 A closely related report demonstrated that XAV939 (an inhibitor of the Wnt/b-catenin signalling pathway) could counteract the proliferation of preterm umbilical cord mesenchymal stem cells (UC-MSCs) compared to term UC-MSCs. 58 In this study, it was showed that iPS-CM could significantly promote the expression levels of Wnt/b-catenin signalling proteins such as b-catenin, cyclin D1, c-Myc and survivin, but could be inhibited by XAV939 (10 lmol/L). Exogenous bFGF (20 ng/mL) or LiCl (20 mmol/L) added into DMEM-LG could obtain almost similar effects of iPS-CM. LiCl is a widely used GSK-3b inhibitor that results in the activation of Wnt/ b-catenin signalling pathway. 59 These data suggested that iPS-CM might mainly rely on inherent bFGF to promote the proliferation of ILCs through up-regulation of Wnt/b-catenin signalling pathway.
Taken as a whole, our study discovered that both the inhibition ILC apoptosis mediated by the activation of autophagy pathway and the promotion ILC proliferation mediated by the Wnt/b-catenin signalling pathway might be attributed to the inherent bFGF of iPS-CM. While this study just hints the potential mechanisms underlying the promotion anti-apoptosis and proliferation of ILCs by iPS-CM, the convincing molecular mechanisms should need further study in the future.
In conclusion, we reported that iPS-CM might mainly rely on inherent bFGF to dramatically inhibit H 2 O 2 -induced apoptosis, stimulate proliferation and enhance testosterone production of