Digestion and culture of testicular cellsES-like cells were generated from 7-week-old C57BL/6 mouse testis. The neonatal mouse was killed, and both testes were removed via a small insecure in the lower part of the abdomen. Mice were sacrificed by carbon dioxide after being used in the experiment. After decapsulation of the testis, tissue was mechanically dissected and dissociated via a two-step mechanical and enzymatic digestion solution that contained 0.05 mg/ml DNase (Sigma Aldrich, USA), 0.5 mg/ml collagenase (Sigma Aldrich, USA), and 0.5 mg/ml dispase (Sigma Aldrich, USA) in an HBSS buffer (PAA, USA) (with shaking and pipetting) at 37˚C for 8 min. Digestion enzymes were stopped with 10% ES cell-qualified FBS and pipetted up and down to obtain a single-cell suspension. After centrifugation, specimens were washed with DMEM/F12, filtered through a 70 μm cell strainer, and centrifuged for 10 min at 1500 rpm. The supernatant was removed, and the suspension of testicular cells was plated onto 0.2% gelatine-coated culture dishes. Cells cultured in the mouse GSC (mGSC) medium consisted of StemPro-34 medium, 1% L-glutamine (PAA, USA), 1% N2-supplement (Invitrogen, USA), 6 mg/ml D + glucose (Sigma Aldrich, USA), 1% penicillin/streptomycin (PAA, USA), 5 µg/ml bovine serum albumin (Sigma Aldrich, USA), 0.1% s-mercaptoethanol (Invitrogen, USA), 30 ng/ml oestradiol (Sigma Aldrich, USA), 60 ng/ml progesterone (Sigma Aldrich, USA), 1% non-essential amino acids (PAA, USA), 10 ng/ml FGF (Sigma Aldrich, USA), 100 U/ml human LIF (Millipore), 1% MEM vitamins (PAA, USA), 8 ng/ml GDNF (Sigma Aldrich, USA), 20 ng/ml epidermal growth factor (EGF, Sigma Aldrich, USA), 30 µg/ml pyruvic acid (Sigma Aldrich, USA), 1% ES cell qualified FBS, 100 µg/ml ascorbic acid (Sigma Aldrich, USA) and 1 µl/ml DL-lactic acid (Sigma Aldrich, USA) with 37 °C and 5% CO2 in air35,36.Generation of ES-like cellsNeonate mouse testicular cells were cultivated in GS medium. Undifferentiated spermatogonia colonies were manually picked in the primary culture or after the subculture of primary supernatants into the MEF feeder layer. ES-like colonies were generated from undifferentiated spermatogonia cells in a time window between 40 and 125 days after culture initiation. Single cells of ES-like colonies were obtained after trypsinization under mouse ES medium condition consisting of KO-DMEM (or DMEM high-glucose medium), 15% Fetal Bovine Serum (FBS), 1% MEM NEAA solution, 1% L-glutamine, 1% Pen-Strep, 0.1% ß-mercaptoethanol (Sigma Aldrich) and 1000 U/ml Leukemia Inhibitory Factor (LIF; Sigma Aldrich) under MEF feeder layer. ES-like colonies were grown in mESCs media and were passaged every 3–4 days. At the beginning of culture, some undifferentiated spermatogonia cells expressed a low level of Oct4-GFP, but this signal was down-regulated after long-term culture. During the cultivation of undifferentiated spermatogonia cells, we found colonies (rate of 25about 33%) that were similar to mESCs (ES-like cells) or cultured epiblast cells that expressed a high level of Oct4-GFP about 41–125 days after initiation of culture. Generated ES-like cells and mESCs were subcultured in mESCs medium. These cells reached confluence about 4–5 days after initiation of culture. Cells were passaged to a new MEF feeder after washing with PBS and treatment with Trypsin-EDTA for 3 min. Trypsin-EDTA was inactivated with 15% of FBS37.Immunocytochemical (ICC) stainingCells were cultured in 24 well plates and fixed with 4% paraformaldehyde. After rinsing with PBS, samples were permeabilized with 0.1% Triton/PBS and blocked with 1% BSA/PBS. After removing the blocking solution, the cells were incubated overnight with primary antibodies. After 29 rinsing, the process was followed by incubation with species-specific secondary antibodies conjugated with different fluorochrome. Labeled cells were counterstained with 0.2 µg/ml DAPI (4’, 6-diamidino 2-phenylindole) for 3 min at room temperature and fixed with Mowiol 4–88 reagent. Each primary antibody in the sample was omitted as a negative control for all markers. Labeled cells were examined with a confocal Zeiss LSM 700 microscope, and images were acquired with a Zeiss LSM-TPMT camera38.Gene expression analyses on the Fluidigm Biomark systemThe expression level of the NANOG gene in SSCs, ESCs, ES-like cells, and Epiblast cells was examined by the Fluidigm Biomark system. Glyceraldehyde-3-phosphate dehydrogenase was utilized as a housekeeping gene for standardization. SSCs, ESCs, ES-like, and Epiblast cells were picked up with a micromanipulator technique, lysed with a solution of lysis buffer that contained 9 µl RT-PreAmp Master Mix (5.0 µl Cells Direct 2× Reaction Mix, Invitrogen, USA), 2.5 µl 0.2× assay pool and 1.3 µl TE buffer, 0.2 µl RT/Taq Superscript III (Invitrogen, USA). Then, the amount of the amplified product of RNA-targeted copies was examined with TaqMan real-time PCR on a BioMark Real-Time Quantitative PCR (qPCR) system. Samples were analyzed in two technical repeats. The Ct values were calculated using Excel and GenEx software5,38.PPI network construction and analysisTo construct the PPI network, we used the Search Tool for the STRING version 11.5 (Retrieval of Interacting Genes/Proteins database, https://string-db.org/). STRING is a web database that intends to integrate all known and predicted interactions between proteins, including physical interactions and functional associations. STRING app in Cytoscape Software (v 3.8.2) was used to construct the PPI network. The STRING: PubMed query was used as the data source to generate a network among the first 100 proteins in the “Stem cell” in Mus musculus, and the minimum confidence score cut-off was set at 0.40. Subsequently, we identified the first neighbors’ nodes with NANOG and created a sub-network for them.Gene enrichment analysisTo investigate the functions of some genes involved in the sub-network and their localization in some tissues, we have performed the STRING Enrichment analysis in the Cytoscape Software (https://cytoscape.org/). We selected 14 genes that are connected to NANOG, including POU5F1, SOX2, SOX9, Pax7, Notch1, Kit, Stat3, Tet2, Stra8, Klf4, Ctnnb1, Cd44, and Myc. We studied several functional enrichments related to our laboratory data without considering a specific FDR value.Statistical analysisThe trials were replicated at least 3 times. All groups’ average gene expression was calculated, and the groups were evaluated using one-way analysis of variance (ANOVA), covered by Tukey’s post-hoc tests. The expression of genes was compared with non-parametric Mann-Whitney’s test. The variation between groups was considered statistically reliable if a value of P < 0.05 had been acquired. PPI networks were analyzed based on relevant databases or online data analysis tools.
