CXCR4-SF1 bifunctional adipose-derived stem cells benefit for the treatment of Leydig cell dysfunction-related diseases.

Abstract Stem cell transplantation is a candidate method for the treatment of Leydig cell dysfunction‐related diseases. However, there are still many problems that limit its clinical application. Here, we report the establishment of CXCR4‐SF1 bifunctional adipose‐derived stem cells (CXCR4‐SF1‐ADSCs) and their reparative effect on Leydig cell dysfunction. CD29+ CD44+ CD34− CD45− ADSCs were isolated from adipose tissue and purified by fluorescence‐activated cell sorting (FACS). Infection with lentiviruses carrying the CXCR4 and SF1 genes was applied to construct CXCR4‐SF1‐ADSCs. The CXCR4‐SF1‐ADSCs exhibited enhanced migration and had the ability to differentiate into Leydig‐like cells in vitro. Furthermore, the bifunctional ADSCs were injected into BPA‐mediated Leydig cell damage model mice via the tail vein. We found that the CXCR4‐SF1‐ADSCs were capable of homing to the injured testes, differentiating into Leydig‐like cells and repairing the deficiency in reproductive function caused by Leydig cell dysfunction. Moreover, we investigated the mechanism underlying SF1‐mediated differentiation and testosterone synthesis in Leydig cells, and the B‐box and SPRY Domain Containing Protein (BSPRY) gene was proposed to be involved in this process. This study provides insight into the treatment of Leydig cell dysfunction‐related diseases.

hypothalamus-pituitary axis and its side effects. [5][6][7] In addition, stem cell techniques show great promise in the treatment of Leydig cell dysfunction, and some preclinical trials have been conducted. It may offer a non-hormonal, potentially permanent option that would be within a working hormonal axis. Some studies have indicated that Leydig stem cells isolated from the testes can be implanted into Leydig cell-injured testes to increase the testosterone level. 8,9 Besides, this method could not only provide therapeutic benefits for men who would otherwise not have treatment options (such as Klinefelters), but also avoid the issues associated with excessive or insufficient hormone supplementation. However, because of difficulties in sourcing Leydig stem cells and ethical concerns, the popularization and application of this approach in the clinic are limited. Therefore, methods to effectively reduce the occurrence of androgen deficiency diseases, improve reproductive system development and dysfunction caused by hypoandrogenism and treat patients effectively have become urgently needed in society.

Mesenchymal stem cells (MSCs) have multilineage differentiation
potential and greater potential for application than Leydig stem cells.
Compared with other MSCs, adipose-derived stem cells (ADSCs) have the advantages of easy acquisition, reduced immunogenicity and an enhanced differentiation capacity. [10][11][12] Many preclinical studies have studied the roles of ADSCs in promoting chronic diabetic wound healing and skin regeneration as well as the regeneration of cartilage tissue and liver cells. [13][14][15][16] ADSC therapy for Leydig cell dysfunction-related diseases has also been evaluated. Yang et al 17 injected ADSCs into an ageing rat model and found that these ADSCs migrated to damaged testis areas and the testosterone level increased. However, they noted that the effect of the ADSCs was more likely to be the result of reduced apoptosis than direct differentiation. Furthermore, we successfully induced ADSCs to differentiate into Leydig-like cells, and the expression of 3β-HSD and testosterone was previously detected in the induced cells. 18 All these findings indicate that ADSCs have great potential and value in the treatment of Leydig cell dysfunction-related diseases. It is known that the self-renewal and differentiation of stem cells depend on the functional ecological pool formed by the cells and the cytokines they secrete. 19,20 The differentiation of Leydig cells in vivo depends on the testicular microenvironment. Therefore, there are two problems in repairing Leydig cell dysfunction with ADSCs: one is how to effectively induce ADSCs to migrate to the sites of Leydig cell dysfunction in the testes so that these cells can be regulated by the testicular microenvironment, and the other is how to improve the ability of ADSCs to differentiate into Leydig-like cells after colonization of the testes. Zhiyv et al 21 reported that the stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor-4 (CXCR4) pathway is the most important biological axis for promoting MSC homing to injured tissues and that MSCs positive for CXCR4 expression can migrate to injured tissues with the help of SDF-1. However, in vitro MSC culture reduces CXCR4 expression and weakens this homing ability. Steroidogenic factor-1 (SF1), also known as Ad4BP or NR5A1, is vital to the differentiation and development of the adrenal gland, gonads and pituitary gland and the synthesis of steroid hormones. Yazava et al 22 found that bone marrow-derived mesenchymal stem cells (BMSCs) transfected with SF1 could differentiate into Leydig-like cells in vitro and that the expression of P450scc, 3β-HSD and 17β-HSD was detectable.
In this study, we constructed CXCR4-SF1 bifunctional ADSCs (CXCR4-SF1-ADSCs) and injected them into model mice with BPAmediated Leydig cell injury via the tail vein. We found that these cells had the ability to home to the testes with Leydig cell dysfunction and differentiate into Leydig-like cells in the testicular microenvironment. Furthermore, the mechanism of ADSC differentiation into Leydig-like cells was preliminarily studied.

| Establishment of BPA-induced Leydig cell damage model
Six-week-old Balb/c male mice were purchased from the DaShuo Experimental Animal Centre (Chengdu, China). Mice were divided into two groups randomly and then injected intraperitoneally with vehicle (corn oil, Sigma) or BPA (100 mg/kg, Sigma) for 5 days. The bodyweight and food intake of the mice were weighed every day.
All mice were housed in a controlled condition of 12-hour light/dark cycle at 22°C. All animal studies were performed in accordance with the relevant guidelines and regulations and approved by the Ethics Committee of West China Hospital, Sichuan University.

| Isolation and characterization of mouse ADSCs
The groin adipose tissue was removed from the male mouse (6-weekold) and minced into pieces aseptically. Then, the minced tissue was digested with 0.25% trypsin (Gibco) at 37°C for 1 hour and followed by 1 mg/mL type I collagenase (Invitrogen) in Dulbecco's modified Eagle's medium (DMEM)/F12 (Invitrogen) at 37°C for 1 hour. The digest was treated with the red blood cell lysis buffer for 5 minutes and centrifuged at 240 g for 10 minutes at room temperature. The sample was washed with phosphate-buffered saline (PBS) twice, filtered through a cell strainer at the size of 40-µm pore (BD Falcon), resuspended with Balb/c mouse adipose-derived mesenchymal stem cell complete medium (Cyagen) and cultured at 37°C under an atmosphere of 95% humidified air with 5% CO 2 .
The isolated ADSCs were characterized and sorted by flow cytometry with antibodies against the surface marker CD29, CD44, CD34 and CD45 (CD29-APC, CD34-FITC, CD44-PE-Cyanine7, CD45-PE, eBioscience™). The ADSCs we got were positive for CD29 and CD44, while negative for CD34 and CD45. Sorted ADSCs (2nd passage) in the logarithmic growth phase were placed in a 6-well plate and incubated at 37°C under an atmosphere of 95% humidified air with 5% CO 2 until the cell density reached 50% or 60%. Control and target gene lentiviruses (LV-Vector, LV-CXCR4, LV-SF1 and LV-CXCR4-SF1) were placed on ice to melt, and the lentiviruses (MOI: 50) were diluted with 1 mL culture medium containing 10% foetal bovine serum and polybrene (5 µg/ mL). Then, the mixture was added to the corresponding well after gentle mixing. The next day, the original medium was replaced with

| Quantitative real-time polymerase chain reaction (qRT-PCR)
The total RNA was extracted from cells using RNAiso Plus

| Western blotting analysis
The testes or collected cells were lysed with appropriate amount of lysis buffer containing RIPA buffer and protease inhibitor, and lysed on ice for 30 minutes. Then, the lysates were centrifuged (4°C, 24 000 g) and the supernatant was harvested. The protein content was detected in accordance with the Bio-Rad Protein Assay (BIO-RAD). Equivalent proteins extracted were loaded, separated via 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore).
Densitometric ratio analyses were performed using ImageJ software.

| In vitro differentiation of Lentiviralinfected ADSCs
The Vector-ADSCs, CXCR4-ADSCs, SF1-ADSCs and CXCR4-SF1- were purchased from Cyagen, and the induced procedure was conducted according to the instructions. After the inducement, Oil red O was used for adipogenesis identification, and Alizarin Red S was used for osteogenesis identification.

| ELISA
The blood of the mouse was placed at 37°C for 3 hours and followed by 4°C for 1 hour. Next, the blood sample was centrifuged at 1500 g for 10 minutes at 4°C to get the serum. For testosterone measurement, the cell culture suspensions or the serum was collected and measured using a Testosterone ELISA Kit (ENZO, ADI-900-065) as the manufacturer's instructions.

| Tissue preparation
The mouse was anaesthetized by intraperitoneal injection of chloral hydrate (10%) and killed by cervical dislocation. Immediately, the testes, epididymides, lung, kidney and liver were collected. Then, one side of the testes and epididymides was frozen in liquid nitrogen, while the other side was fixed mDF for 72 hours as reference. 23,24 The lung, kidney and liver were fixed in 4% paraformaldehyde for 48 hours.
To get the testis homogenates, the testis tissue frozen in liquid nitrogen was weighed, placed in normal saline (NS) containing protease inhibitor (a ratio of 0.1 g:1 mL) and homogenized on ice. After homogenization, the homogenate was centrifugation at 2800 g at 4°C for 15 minutes. The supernatant was collected and stored at −80°C.

| Haematoxylin-eosin (HE) staining, immunohistochemistry (IHC) and immunofluorescence (IF)
The tissues were paraffin embedded and then sliced into 4-μm sections. Then, the sections were got dewaxed and hydrated, and stained by haematoxylin-eosin (Beyotime Biotechnology) as the instructions. The Photo Imaging System (Olympus DP20) was applied to take the photomicrographs of these sections.
For IHC assay, 3% H 2 O 2 was used to block the endogenous peroxidase for 15 minutes; then, high-pressure antigen repair method was required to get the antigen retrieved. The primary antibodies were incubated overnight at 4°C. After that, these sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibodies (Sangon Biotech) (1:1000 dilution) for 1 hour at 37°C. Subsequently, the sections were counterstained with haematoxylin. Photomicrographs were also taken by a Photo Imaging System (Olympus DP20).
For IF assay, the work procedures were the same as the IHC assay until the application of the primary antibodies. Next, these sections were incubated with an Alexa Fluor 594-conjugated secondary antibody (1:100 dilution; Boaosen) for 1 hour at 37°C. And DAPI (Invitrogen) was used for nuclei staining. The photomicrographs were taken by a fluorescence microscope (Olympus Optical Co).

| Mating assay
The mating experiment was conducted 1 week, 3 weeks and 5 weeks after the cell injection. One male mouse was mated with two randomly selected normal female mice, and every group contained 5 male mice.
Thus, these female mice got grouped the same with their male counterparts. The female mouse was separated to another cage after pregnancy. The numbers of pups were counted soon after the birth. At last, the average offspring number of every female mouse was counted.

| Cell proliferation assay
Cell suspension (100 μL/well) was seeded into a 96-well plate at a density of 3000 cells/well. The cell proliferation ability was detected by CCK8 kits (4A Biotech Co. Ltd) according to the instructions. All the experiments were repeated 3 times.

| Analysis of sperm concentration, sperm motility and abnormal sperm rate
One side of the epididymides was put in a plate containing normal saline (preheated at 37°C) and cut into small pieces. 25 The plate was gently mixed and placed in a 37°C water bath for 15 minutes. After that, the suspension was filtered with a cell filter. Then, the suspen-

| Statistical analysis
Data were presented as the means ± standard error of the mean (SEM). And SPSS software (version 20.0; SPSS, Inc, Chicago, IL) was used to perform statistical tests. The significance between groups was performed using Student's t test or unpaired t test with Welch's correction. One-way analysis of variance (ANOVA), followed by Tukey's post hoc test was used among three or more groups. P < .05 was considered to indicate statistically significant.

| Construction of CXCR4-SF1-ADSCs
Adipose Next, we used lentiviral infection to construct different types of ADSCs, which were named Vector-ADSCs, CXCR4-ADSCs, SF1-ADSCs and CXCR4-SF1-ADSCs; the Vector-ADSCs were used as a negative control. All lentiviruses carried a GFP gene, and green fluorescence could be detected under a fluorescence microscope for these four types of ADSCs ( Figure 1C). Subsequently, Western blotting and qPCR were used to detect the expression of CXCR4 and SF1 at the protein and mRNA levels ( Figure 1D). These results indicated that the lentivirus-infected ADSCs were successfully established.
Whether a lentivirus can affect the pluripotency of ADSCs has not been reported thus far. In our study, we showed that the induction  Figure S1).

| The migration and Leydig-like cell lineage differentiation ability of lentivirus-infected ADSCs in vitro
After construction, we verified the migration capacities of the four cell types by a Transwell migration assay (Figure 2A) showed no differences among the four groups ( Figure 2F). For further verification, we also detected 3β-HSD, the most representative Leydig lineage-specific marker, 30,31 and StAR, the protein involved in the rate-limiting step of testosterone synthesis, 31 at the protein level, and the results were consistent with those for the mRNA levels ( Figure 2G). These results indicated that overexpression of the SF1 gene could promote the differentiation of ADSCs into Leydig-like cells automatically.

| CXCR4-SF1-ADSCs restored the damage to the testes caused by BPA
After Considering that the ADSCs used were treated with a lentivirus, we evaluated morphological changes in the liver, lungs and kidneys as well as changes in liver and kidney function indexes in mice from every group after inoculation, and the results showed that the cells

F I G U R E 2
The migratory and Leydig-like cell lineage differentiation abilities of lentivirus-infected ADSCs. (A) A Transwell assay was used to detect the migratory ability of lentivirus-infected ADSCs. Scale bar, 50 µm. The data from the Transwell assay are represented as the mean ± SD. The significance of differences was determined using one-way ANOVA; ****P < .0001; NS, non-significant difference; compared with the negative control. qPCR to detect the mRNA levels of 3β-HSD (B), 17β-HSD (C), CYP17a1 (D), P450scc (E) and StAR (F) and WB to detect the protein levels of 3β-HSD and StAR after differentiation induction (G). *P < .05; **P < .01; ****P < .001; ****P < .0001; NS, nonsignificant difference treated with the lentivirus had no toxic effects on the mice ( Figure   S3, Tables S2 and S3).

| CXCR4-SF1-ADSCs restored the reproductive capacity of mice treated with BPA
The ultimate purpose of this study was to rescue the reproductive dysfunction caused by Leydig cell dysfunction. The previous results proved that CXCR4-SF1-ADSCs could repair the testicular structure Furthermore, Figure 4D shows the morphologies of normal and abnormal sperm (acrosome absence deformity, head deformity, tail rotation, double tail deformity, etc). These results demonstrated that CXCR4-SF1-ADSCs had a direct reparative effect on the reproductive capability of mice with Leydig cell dysfunction.

| BSPRY might participate in the process of SF1mediated ADSC differentiation into Leydig-like cells
In previous experiments, we verified that CXCR4-SF1-ADSCs had the ability to home to injured testes and differentiate into Leydig-like cells. We knew that SDF-1/CXCR4 acted in the role of promoting the homing of the bifunctional ADSCs to the testes.  Figure 5C), but the level of testosterone produced by the TM3 cells decreased significantly ( Figure 5D). Moreover, the mRNA levels of 3β-HSD, 17β-HSD and StAR ( Figure 5F) were significantly decreased, but those of CYP17a1 and P450scc ( Figure 5G) showed no significant changes in the TM3 cells with knocked down BSPRY expression.
Interestingly, SF1 mRNA levels were slightly increased in these cells ( Figure 5E). In addition, the changes in the 3 β-HSD, StAR and SF1 protein levels ( Figure 5H) agreed with their mRNA level changes. These results indicated that BSPRY might be involved in the SF1-mediated testosterone synthesis process. These results were also confirmed in ADSCs. We transfected siRNAs (shBSPRY #1 and #2) into ADSCs, and the proliferation of the ADSCs was not influenced ( Figure 5I). In addition, we transfected these siR-NAs into Vector-ADSCs or SF1-ADSCs and cocultured these cells with 8-Br-cAMP. We found that interference with the BSPRY gene could offset the enhancement of Leydig-like cell differentiation mediated by SF1 in the ADSCs ( Figure 5J). This finding implies that BSPRY may assist SF-1 in promoting the differentiation of ADSCs into Leydig-like cells.

| D ISCUSS I ON
Currently, the clinical application of stem cell therapy has been con- However, 5 weeks later, the GFP fluorescence expressed by the CXCR4-SF1-ADSCs was too weak to be detected, so further work is needed to prove the function of the CXCR4-SF1-ADSCs. In addition, the structural damage in the testes caused by BPA also recovered following inoculation. We also evaluated testosterone levels in the supernatant of 8-Br-cAMP-treated cells in vitro, but the expression of testosterone was not detected. This might be because during the inducement process, testosterone was diluted during culture medium changes and was therefore lower than the detection limit of the kit. Leydig cell dysfunction and worked well. Therefore, this intravenous F I G U R E 5 BSPRY might participate in the process of SF1-mediated ADSC differentiation into Leydig-like cells. (A) An immunoprecipitation assay was utilized to verify the relationship between SF1 and BSPRY. qPCR and Western blotting (B) were applied to detect the expression of BSPRY in TM3 cells transfected with shNC or shBSPRY #1/#2/#3. (C) The CCK8 method was used to detect the proliferation rates of TM3 cells transfected with shNC or shBSPRY #1/#2/#3. (D) An ELISA method was used to detect the testosterone concentration in culture supernatants of these cells. **P < .01, ****P < .001. (E, F, G) qPCR was used to determine SF1, 3β-HSD, 17β HSD, StAR, P450scc and Cyp17a1 mRNA expression in TM3 cells treated with shNC or shBSPRY #1/#2/#3. (H) Western blotting was used to verify the SF1, 3β-HSD and Star protein expression of the cells mentioned above. H3 is presented as a control. (I) The CCK8 method was used to detect the proliferation rates of ADSCs transfected with shNC or shBSPRY #1/#2. (J) Western blotting was used to detect the expression of SF1, BSPRY, 3β-HSD and Star in ADSCs transfected with LV-SF1 alone, shBSPRY #1/#2 alone or both vectors and then treated with 8-Br-cAmp inoculation method has relatively great clinical application potential.
Moreover, considering that all the ADSCs studied were treated with a lentivirus, we evaluated morphological changes in the liver, lungs and kidneys as well as changes in liver and kidney function indexes in mice in different groups after cell inoculation, and the results showed that the lentivirus-treated cells had no toxic effects on the mice.
Various factors, such as dosage-sensitive sex reversal gene 1 However, how BSPRY cooperates with SF1 to affect the function of Leydig cells needs to be further explored.
In conclusion, we successfully constructed CXCR4-SF1-ADSCs and inoculated them into BPA-treated mice via the caudal vein.
Additionally, we confirmed that the CXCR4-SF1-ADSCs could markedly increase testosterone levels in mice with Leydig cell dysfunction and restore the reproductive function of these mice. Thus, CXCR4-SF1-ADSCs have the potential to become a new therapeutic for treating Leydig cell dysfunction-related diseases and are expected to be applied in clinical practice.

ACK N OWLED G EM ENTS
This study was supported by Sichuan Province foundation (2016SZ0060) from Science and Technology Department of Sichuan Province.

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.