Collagenase

H1(WA01), H9 (WA09) and DF19 (iPS DF19-9-7T) were all purchased from the National Stem Cell Bank at www.wicell.org. H1 and H9 were initially expanded in hESCs media composed of 80% DMEM/F12 (cat#11330057, Life Technologies), 20% knockout serum replacer (cat#10828028, Life Technologies), 1% NEAA (cat# 11140050, Life Technologies), 4 ng/ml bFGF (cat# 100-18B, PeproTech), 1 uM L-Glutamine (G8540, Sigma) and 0.07 µM beta-mercaptoethanol (ES-007-E, Millipore) on MEF feeder cells (GSC-6001M, mitomycin inactivated CF-1 cells, GlobalStem), whereas DF19 was expanded in TeSR1 media (5850, Stem Cell Technologies) on matrigel (354234, BD). For culturing on inactivated hMSCs, all cell lines were grown in hESCs media supplemented with different concentrations of bFGF. Cells were long-term passaged using 1 mg/ml collagenase (cat# 17104-019, Life Technologies) and colonies were detached by physical scraping using the tip of a glass pipet after 7 minutes of collagenase incubation. Single cell dissociation was carried out using accutase (cat# A1110501, Life Technologies) by following the provided manufacturer's protocol. Cells were passed through a sterile cell strainer (cat# 08-771-01, Thermofisher Scientific) to achieve single cell homogeneity and counted using the Countess automated cell counter (cat# C10227, Life Technologies), average of 3 chambers/sample.

Mandibular slice cultures were performed as previously described (Wells et al., 2013 (link); Li et al., 2016 (link)). For the bead experiment, two types of beads were used to help distinguish between the control and treated conditions. For the Fgf10-treated explants, heparin beads (Sigma, 100-200 mesh) were incubated overnight at 4°C with 100 μg/ml Fgf10 (R&D Systems). For the control, Affi-Gel blue beads (Bio-Rad,153-7302) were treated with 0.5% BSA. For inhibiting Fgf receptor signalling or the Erk pathway, explant cultures were treated with 2.5 μM SU5402 (Merck) or 5 μM U0126 (Cell Signaling Technology), respectively, made up in DMSO. Control cultures were treated with equivalent concentrations of DMSO (0.25% DMSO for the SU5402 and 0.5% DMSO for the U0126 experiment). For the collagenase treatment, whole E12.5 submandibular glands were dissected and treated for 2 days with 1 μg/ml collagenase, Type II (Thermo Fisher Scientific) and HBSS-treated glands were used as a control. Spooner ratios were calculated as the number of buds at the end of culture divided by the number of buds at the start of culture.

All experiments were approved by the Animal Ethics Committee of Gansu Agricultural University. Clinically healthy adult yaks were selected from the grasslands of Xining, China, at an altitude of 3,800 m; the yaks were euthanized by administering pentobarbital sodium (200 mg/kg) via intravenous injection. One oviduct was collected from each yak and two adult female yaks were selected for single-cell experiments. The oviduct tissue was washed with sterile saline, placed in a tissue-protection fluid, and transported to the laboratory for subsequent RNA sequencing. The oviduct tissue was cut into 1 mm³ cubes and digested with collagenase (GIBCO) and trypsin (GIBCO) for 25 min (containing 2 mmol EDTA). Digestion was then stopped and the oviduct tissue was filtered. Cells were washed twice with 10% BSA to obtain a single-cell suspension and RNA sequencing was performed on a Chromium.

Fresh tissue specimens were extensively washed with serum-free RPMI 1640, minced, and enzymatically dissociated for 2 h at 37 °C with agitation in serum-free RPMI 1640 containing 0.4 mg/mL collagenase (Gibco, Grand Island, NY, USA), 0.5 mg/mL dispase (Gibco), and 0.2 mg/mL DNase I (Roche, Basel, Switzerland), as described previously⁵³ . The cells were cultured in RPMI 1640 supplemented with 10% foetal bovine serum. Experiments were conducted within four passages after PDC derivation.

For preparing Leydig cells, sperm cell, and Sertoli cells, Percoll density gradient centrifugation was performed as previously described by Umehara and colleagues [52 (link)]. Briefly, 10 decapsulated testes from male mice were incubated for 10 min at 37 °C with gentle stirring in 50 ml DMEM (Nacalai Tesque) supplemented with 0.1% BSA (Sigma) and 0.5 mg/ml collagenase (GIBCO). Centrifugation was performed using a discontinuous density gradient of Percoll (20%, 30%, 50%, and 60%; GE Healthcare UK Ltd., Amersham Place, UK). The gradient was centrifuged at 2,500 × g for 60 min at 4 °C, as low temperature significantly prevents cell aggregation. Following centrifugation, the gradient was fractionated into plastic tubes and each fraction was centrifuged at 200 × g for 10 min at 4 °C, then washed twice with cold DMEM and finally centrifuged again at 200 × g for 10 min at 4 °C. Germ cells at different developmental stages including spermatozoa were fractioned at the interphase between 20% and 30% of Percoll. Sertoli cells were between 30% and 50% of Percoll, and Leydig cells were present through the 50% Percoll zone.

Tumor, spleen, and lung tissues were extracted at the end of the study. Tumor and spleen tissues were harvested, minced with a scalpel blade, and incubated with 100 U/mL collagenase (Gibco, Grand Island, NY, USA) and 0.2 mg/mL DNase (Roche, Basel, Switzerland) in Hank’s Balanced Salt Solution for 30 min at 37 °C. Gross images of the extracted tumor and lung tissues were collected prior to any additional processing.