Mouse gdnf

Testicular tissue was obtained from 6-day-old male mice. To obtain SSC suspension from the tissue, a two-step enzymatic digestion protocol was applied (Guan et al., 2009 (link)). Briefly, decapsulated testes were treated with collagenase type IV (1 mg/ml) for 15 min at 37°C, followed by digestion in 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA) for 10 min at 37°C. The singly dissociated cells were incubated overnight in dish coated with gelation to removed somatic cells. Non-adherent and weakly adherent cells were collected and then labeled with CD326 (EpCAM) MicroBeads by Mini MACS Starting Kit (Miltenyi Biotec, Stevenage, United Kingdom) according to the manufacturer’s protocol. Cells were rinsed with PBS containing 0.5% BSA (Sigma, Poole, Dorset, United Kingdom), and the CD326-positive cells were collected. The CD326-positive cells were plated onto DTM hydrogel, Matrigel (10 mg/ml) hydrogel, or laminin (2 mg/ml) coated plates with SSC culture medium. The SSC medium was composed of Dulbecco’s modified Eagle’s medium (DMEM), 15% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, United States), 50 μM-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA, United States), 1 × minimal essential medium (MEM) non-essential amino acids (Thermo Fisher Scientific, Waltham, MA, United States), and 10 ng/ml mouse GDNF (Peprotech, Rocky Hill, United States).

Spermatogonial stem cell derived from testes of six‐day‐old male mice. To obtain SSCs suspension from the testicular tissue, a two‐step enzymatic digestion protocol was applied.¹⁸ Briefly, decapsulated testes were treated with collagenase type IV (1 mg/mL) for 15 minutes at 37℃, followed by digestion in 0.25% trypsin and 1 mmol/L EDTA for 10 minutes at 37°C. The singly dissociated cells were incubated overnight in dished to removed somatic cells. Non‐adherent and weakly adherent cells were collected and then labelled these cells with CD326 (EpCAM) MicroBeads by Mini MACS Starting Kit (Miltenyi Biotec) according to the manufacturer's protocol. Cells were rinsed with PBS containing 0.5% BSA (Sigma), and the CD326‐positive cells were collected. Then, the CD326‐positive cells were plated onto laminin (2 mg/mL) coated plates with SSC culture medium. The SSC medium was composed of DMEM, 15% foetal bovine serum (FBS, Thermo Fisher Scientific), 50 µmol/L‐mercaptoethanol (Thermo Fisher Scientific), 1 × minimal essential medium (MEM) non‐essential amino acids (Thermo Fisher Scientific), and 10 ng/mL mouse GDNF (Peprotech).

We isolated and purified SSCs from neonatal mouse testes (6 days old, C57BL/6) following a previously described protocol [48 (link)]. We cultured SSCs on STO feeder cells in MEMα basal medium (Invitrogen) supplemented with 10% FBS (Life Technologies), 1 mM sodium pyruvate (Amresco), 2 mM L-glutamine (Amresco), 50 μM β-mercaptoethanol (Biotech), 1 mM NEAA (Invitrogen), 20 ng/ml mouse EGF (PeproTech), 10 ng/ml human bFGF (PeproTech), 10 ng/ml mouse GDNF (PeproTech), 10 ng/ml ESGRO (Santa Cruz Biotechnology), and 100 μg/ml transferrin (Sigma). Spermatogonial stem cells were identified by immunofluorescence staining and RT-PCR (Additional file 1: Figure S2).