Culture of human iPSCs

The commercial human iPSCs used in this study were purchased from Saibei Biotechnology (Beijing, China). iPSCs (HiPSC-U1) were reprogrammed from human urine-derived cells of a 37-year-old male by the integration-free CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, MA, USA). iPSCs were cultured in 1% Matrigel-coated (BD Biosciences Co., Ltd., NM, USA) Petri dishes with E8 medium (Saibei Biotechnology) at 37C and 5% CO2. The medium was refreshed daily. iPSCs were passaged once every 6 days with 0.25% ethylenediaminetetraacetic acid (EDTA, Saibei Biotechnology). 1mL of 0.25% EDTA was added and the cells were placed at 37C and 5% CO2 for 5min. When iPSC colonies appeared white, the solution was gently removed, and the iPSCs were washed with Mg2+ and Ca2+-free Dulbeccos PBS (Sigma, MO, USA). iPSCs were then harvested by gently pipetting 710 times with 1mL E8 medium and seeded onto fresh six-well culture plates that were coated with 1% Matrigel at a ratio of 1:6. 10m Y-27632 (Sigma) was supplemented in the medium on the first day of passage.

Human LCs were obtained from nine male donors with a mean age of 45 years old through testes excision within 20h. Informed consent was obtained from each donor, and this study was approved by Human Research and Ethical Committee of Wenzhou Medical University. The testes were used to isolate ILCs. ILCs express all androgen biosynthetic enzymes56, and are capable of proliferation and differentiation57. The isolation of ILCs was performed as previously described56. In brief, the testes were perfused with collagenase (Sigma) via the testicular artery, and digested with M-199 buffer (Gibco, NY, USA) containing collagenase (0.25mg/ml) and DNase (0.25mg/mL, Sigma) for 15min. Then, the cell suspension was filtered through 100m nylon mesh and the cells were separated by a Percoll gradient (Sigma). The cells with the density of 1.071.088g/ml were collected. The purity of ILCs was evaluated by immunohistochemical staining HSD3B1, the biomarker of ILCs, as previously described58. The HSD3B1 staining solution contained with 0.4mm etiocholanolone (Sigma) as the steroid substrate and NAD+ as a cofactor58. The purity of ILCs was >95%.

The isolated ILCs were directly seeded into wells in the 24-well culture plates with the density of 2104 cells/well and incubated at a 37C, 5% CO2 incubator. The culture medium (LC-Medium) contains DMEM/F12 (Gibco), 5% fatal bovine serum (FBS, Gibco), 2.5% horse serum (HS, Gibco), and 1% penicillin/streptomycin (P/S, Gibco). In order to get ALCs, the culture medium were changed into differentiation-induced medium (DIM) contains DMEM/F12, 5mm ITS (insulin, transferrin, and selenium, Sigma), 5ng/ml luteinizing hormone (LH, PeproTech, NJ, USA), and 5mm lithium chloride (Li, Sigma) as our team previous report35.

The Sprague-Dawley rats (at 5 weeks of age) and immune deficiency (SCID) mice (at 5 weeks of age) were obtained from the laboratory animal center of Wenzhou Medical University, Wenzhou, China. All animals were kept under conditions with controlled temperature (232C), a 12h dark/light cycle, and relative humidity of 4555%. The standard drinking water and rodent diet were accessed ad libitum. All surgical procedures and postoperative care were approved by the Wenzhou Medical Universitys Animal Care and Use Committee, and were performed in accordance with the Guide for the Care and Use of Laboratory Animals.

The point at which iPSCs were expanded to ~70% confluency in the E8 medium was defined as day 2, and at this point when iPSCs were changed into E7 medium (no FGF2) for 2 days to prepare differentiation. From day 5 to 0, the medium was refreshed daily. Prior to the beginning of differentiation, iPSCs were cultured in a differentiation-inducing medium composed of DMEM/F12, 1% bovine serum albumin (BSA) (Sigma), 5mm ITS, 5ng/mL LH. From 07 days, 0.2m SAG (DHH agonist, Sigma), 5m 22R-OHC (Steraloids, RI, USA), and 5mm Li were added into iPSC-DIM. From 710 days, 5ng/mL PDGF-AA (Sigma) and 5ng/mL FGF2 (Sigma) were added into iPSC-DIM. From 1017 days, 5ng/mL PDGF-AA, 5nM IGF1 (Sigma), and 10m Androgen (Sigma) were added into iPSC-DIM. From 1720 days, 10ng/mL PDGF-AA and 10ng/mL FGF2 were added into iPSC-DIM. From 2025 days, 5ng/mL LH, 0.5mm retinoic acid (RA, Sigma) and 1mm 8-Br-cAMP (Sigma) were added into iPSC-DIM. From day 0 to 25, the medium was changed every 2 days by fresh iPSC-DIM. From 2530 days, the cells were mechanically enriched by scraping away clonal iPSC-like cells. The remaining Leydig-like cells were kept in Enrichment Medium contained DMEM/F12, 5% FBS, 2.5% HS, 1sodiumpyruvate (Invitrogen), 1GlutaMAX (Invitrogen), and 1% P/S for the subsequent assays. The medium was changed every 2 days by fresh Enrichment Medium.

For TEM, the cells in different groups were prefixed with 2.5% glutaraldehyde in 0.1m PBS for 24h at 4C. Then, they were washed with PBS, and post-fixed with 1% osmium tetroxide. After gradient dehydration of acetone, they were embedded in Araldite M (Sigma Aldrich). Ultrathin sections (1m) were subsequently cut with an ultramicrotome, mounted on nickel grids, and stained with uranyl acetate and lead citrate. At last, the samples were sent to the electron microscope room at Wenzhou Medical University for subsequent processing and testing using a transmission electron microscope (H-600A-2; Hitachi, Tokyo, Japan).

The cell culture supernatants and serum were collected at each experimental time point for the quantitative measurement of testosterone. For the cell culture supernatants, 10ng/mL LH was in advance at least 3h added into the medium (just having DMEM/F12) to stimulate the testosterone production of LCs or iPSC-LCs. Testosterone levels were measured with a tritium-based radioimmunoassay using anti-testosterone antibody as previously described59. Standards ranging between 10 and 2000pg/mL testosterone were prepared in triplicate. Standards and samples were incubated with tracer and antibody at 4C overnight 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 scintillation counter (PE, CA, USA). The minimum detectable concentration for testosterone was 5pg/mL. Quality control samples contain 100pg/mL testosterone. The intra-assay and inter-assay coefficients of variation were within 10%.

Immunofluorescence was used to identify iPSC-LCs as a previous report60. In brief, after fixation with 4% paraformaldehyde (Sigma) for 15min, cells were washed three times with PBS. Then cells were permeabilized with 0.1% TritonX-100 in PBS for 15min at room temperature, and incubated with 3% (w/v) BSA in PBS for 1h at room temperature. The cells were then incubated with primary antibodies as TableS1 overnight at 4C, and then with fluorescein isothiocyanate (FITC)-conjugated anti-mouse, FITC-conjugated anti-rabbit, Cy3-conjugated anti-mouse, and Cy3-conjugated anti-rabbit IgG secondary antibodies (1:1000, Bioword, USA) for 60min at room temperature. Then the cells were rinsed three times with PBS thrice for 5min each and then incubated for 15min with DAPI (Sigma) for nuclear staining and washed three times with PBS before examination by an inverted fluorescence microscope (OLYMPUS, Japan).

Total RNA from the cells was extracted using Trizol reagent (Invitrogen, CA, USA) according to the manufacturers instruction. The RNA was reversely transcribed into cDNA using the Superscript II kit (Invitrogen). The cDNAs templates were diluted 1:10, which were used to perform RT-PCR and qPCR to analyze the gene expressions. RT-PCR was performed using an authorized thermal cycler (Eppendorf, Hamburg, GER). After amplification, 1L of 6Loading buffer and 5L of each PCR product were mixed and electrophoresed on a 2% agarose containing 0.5g/mL ethidium bromide. Gels were scanned for further analysis. qPCR was performed using the Thunderbird SYBR qPCR Mix (Takara, Tokyo, Japan) according to the manufacturers instructions. Signals were detected using a Light Cycler 480 Detection System (Roche, Basel, Switzerland). The relative expression of genes was normalized to GAPDH. The melting curve was examined for the quality of PCR amplification for each sample, and quantification was performed using the comparative 2-Ct method. The primer sequences were shown in TableS2.

Total RNA from each sample was extracted using Trizol reagent (Invitrogen, CA, USA). 12g total RNA was used to prepare the sequencing library. To sequence the libraries, the barcoded libraries were mixed, denatured to single stranded DNA, captured on Illumina flow cell, amplified in situ, and subsequently sequenced for 150 cycles for both ends on Illumina HiSeq 4000 instrument. Sequence quality was examined using the FastQC software. The transcript abundances for each sample were estimated with StringTie, and the FPKM value for gene and transcript level were calculated with R package Ballgown. The differentially expressed genes and transcripts were filtered using R package Ballgown. The correlation analysis was based on gene expression levels. Hierarchical Clustering, Gene Ontology, Pathway analysis, scatter plots and volcano plots were performed with the differentially expressed genes in R, Python for statistical computing and graphics61.

Cells were washed with cold PBS and were lysed in 1radioimmunoprecipitation assay lysis buffer in the presence of a protease inhibitor mixture/1% phosphatase inhibitor mixture (Roche). 50g of protein samples were applied to a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred into the polyvinylidene difluoride membranes (Sigma) by an electroblot apparatus. After being blocked with a blocking solution (5% fat-free milk) for 2h at 4C, the membranes were incubated with primary antibodies as TableS1 in the blocking solution at 4C overnight. The membranes were washed with tris-buffered saline with Tween 20 (TBST) five times (10min each), and incubated with horseradish peroxidase-conjugated secondary antibody (1:3000, Bioword) at room temperature for 2h. The membranes were then washed five times (10min each) with TBST. Bands were visualized with enhanced chemiluminescence (ECL, Pierce, USA). The protein expression was normalized to -actin.

The cell samples were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% TritonX-100 (Sigma). The samples were then labeled with primary or isotype control antibodies for 30min at 4C. Primary and isotype control antibodies that were not conjugated to fluorophores were labeled with fluorophore-conjugated secondary antibody for 30min at 4C. The labeled samples were detected by flow cytometry analyzer (BD, USA). Data analysis was performed on FCS Express 4 Flow Research Edition software.

The standard protocol was conducted as PKH26 Product Information Sheet (Sigma, MINI2). In brief, the suspension containing 2107 cells were centrifuged (400g, 5min) and were washed once using fresh medium without serum. After centrifuging, the supernatant was removed and no more than 25L of 2Cell Suspension was prepared by adding 1mL of Diluent C, the cells were resuspended with gentle pipetting to ensure complete dispersion. 2Dye Solution (4106m) was prepared by adding 4L of the PKH26 ethanolic dye solution to 1mL of Diluent C and mixed well. Then 1mL of 2Dye Solution was rapidly added into the 1mL of 2Cell Suspension. Final concentration after mixing was 2106m PKH26 with 1107 cells/well. The mixing suspension was incubated at room temperature for 5min with periodic mixing. The action of the staining was stopped by adding an equal volume (2mL) of serum. Then the suspension was centrifuged at 400g for 10min and washed three times. Finally, the cells tagged with PKH26 were seeded on fresh wells and used for injection.

For evaluating whether iPSC-LCs could facilitate the recovery of LC dysfunction of rats, iPSC-LC transplantation was performed as previously described with some modifications62. Sixty 49-days-old male Sprague-Dawley rats (n=5 for each group at each time point) were used in this study. Before transplantation, male rats were administered a single intraperitoneal injection of EDS (75mg/kg, Pterosaur Biotech Co., Ltd., Hangzhou, China), which was dissolved in dimethyl sulfoxide (Sigma): H2O (1: 3, v/v). This treatment resulted in the elimination of LCs in the adult testes of rats63. Then iPSC-LCs labeled with PKH26 (red) were resuspended manually, and harvested in a 15mL Falcon tube. Cells were rinsed twice with PBS following centrifugation at 200g for 5min. Finally each pellet was resuspended in PBS for transplantation. Cells were loaded into a 1mL syringe for injection into the testis of adult Sprague-Dawley male rats that had been treated with EDS. Approximately 2106 PKH26-labeled iPSC-LCs in 40mL of PBS were injected into the parenchyma of recipient testes 7 days after the rats received EDS. The control animals for the experimental group were EDS-treated rats that had received a testicular injection of the PBS vehicle. Testes from all animals were examined at 0, 7, 14, and 21 days after EDS treatment.

One testis from each rat was used for immunohistochemistry (Vector Laboratories, Inc., Burlingame, CA, USA) according to the manufacturers instructions. The rats were killed with an overdose of sodium pentobarbital (Sigma). Testes were removed and fixed in 4% paraformaldehyde overnight at 4C. Then testes were dehydrated with a graded series of ethanol and xylene and subsequently embedded in paraffin. Five micrometer-thick transverse sections (5m) were cut, de-waxed in water, and were mounted on glass slides. Antigen retrieval was performed by microwave irradiation for 10min in 10mm (pH 6.0) of citrate buffer, after which endogenous peroxidase was blocked with 0.5% of H2O2 in methanol for 30min. Some sections were fixed with 4% paraformaldehyde for 15min and washed 3 times with PBS. Then they were permeabilized with 0.1% TritonX-100 in PBS for 15min at room temperature, and incubated with 3% (w/v) BSA (Sigma) in PBS for 1h at room temperature. Then these sections were then incubated with an CYP11A1 polyclonal antibody diluted 1:1000 for 2h at room temperature, and then with FITC-conjugated IgG secondary antibodies (1:1000, Bioword) for 1h at room temperature. These sections were rinsed with PBS three times for 5min each time. Then the sections were incubated for 15min with DAPI (10g/mL, Sigma) for nuclear staining and washed three times with PBS. The sections were cover-slipped with resin (Thermo Fisher Scientific, Waltham, UK). At last, they were examined by an inverted fluorescence microscope (OLYMPUS). The cells with CYP11A1 staining in the interstitial area represent the LC64.

Other sections were directly incubated with CYP11A1 polyclonal antibody diluted 1:1000, for 2h at room temperature. Diaminobenzidine was used for visualizing the antibodyantigen complexes, positive labeling LCs by brown cytoplasmic staining. Mayer hematoxylin was applied in counterstaining. The sections were then dehydrated in graded concentrations of alcohol and cover-slipped with resin (Thermo Fisher Scientific, Waltham, UK). Lastly, they were examined by a fluorescence microscope (LEICA). The cells with CYP11A1 staining in the interstitial area represent the LCs.

For teratoma formation, iPSCs (5106 cells) were dissociated with 0.5mm EDTA, centrifuged, resuspended in 100L E8 with 1% Matrigel, and injected into the hind limbs of 6-week-old male SCID mice. Teratomas were collected after 6 weeks, and fixed in 4% paraformaldehyde for paraffin embedding and hematoxylin and eosin staining. Slides were imaged and analyzed by a qualified clinical pathologist.

The division of iPSCs was blocked with 50g/mL of colcemid solution (Invitrogen, USA). Cells were washed with PBS and harvested with trypsin at room temperature for 2min. Then cells were fixed in methanol/glacial acetic acid (3:1) for three times and dropped onto slides for chromosome spreads. At last, the slides were baked at 55C for overnight. Standard G-banded karyotypes were obtained using Giemsa solution staining (Giemsa, Japan).

To enumerate CYP11A1-positive Leydig cell numbers, sampling of the testis was performed according to a fractionator method as our previous report65. Identification of all Leydig cell lineages was done by the staining of CYP11A1. About 10 testis sections per rat were sampled from each testis. The total number of LCs was calculated by multiplying the number of LCs counted in a known fraction of the testis by the inverse of the sampling probability.

All experiments were performed at least thrice, and the data are presented as the meanstandard error of the mean. Statistical analyses were evaluated using an unpaired Students t test or one-way analysis of variance for more than two groups. P<0.05 was considered statistically significant.

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