Recombinant human fgf9

THY‐1⁺ germ cell cultures were established as described previously.⁸ Briefly, male C57 LacZ pups (5‐8 days post‐partum) were sacrificed and testes were digested using Trypsin‐EDTA (Gibco, USA). Cells were magnetically sorted using CD90.2 beads (Miltenyi, USA) and plated onto Sandos inbred mouse (SIM)–derived 6‐thioguanine‐resistant and ouabain‐resistant (STO) feeders (SNL76/7, ATCC). Germ cell cultures were maintained in a humidified atmosphere at 37°C contains 5% CO₂ with Mouse serum‐free media (mSFM) with 20 ng/mL recombinant human GDNF (R&D, USA), 150 ng/mL recombinant rat GFRα1 (R&D) and 1 ng/mL recombinant human FGF2 (Corning, USA). Other reagents were used as indicated: recombinant human FGF9 (R&D), SB203580, GW788388 and Tofacitinib citrate (all from MedChemExpress, USA). Germ cell counts were determined with a hemocytometer. Feeder cells were identified and discounted from counts based on morphology.²² SSC number was calculated using the following formula, adapted from reference ⁴: $SSCgrowth=\frac{\mathit{Cellsharvested}}{\mathit{Cellsseeded}}\times \frac{\mathit{Colonies}}{{10}^{5}cellstransplanted}$
Here, ‘cells seeded’ indicates the number of cultured THY‐1⁺ germ cells seeded at the beginning of each time period and ‘cells harvested’ is the cell count at the end. ‘Colonies’ indicates the number of distinct colonies counted per testis and is divided by the number of cultured THY‐1⁺ germ cells transplanted per testis.

As adapted from Takasato et al. [16 (link)] and our earlier publication [14 (link)], iPSC were plated at a density of 20,000–25,000 cells/cm² and cultured in STEMdiff APEL2 medium (APEL2) (STEMCELL Technologies, Vancouver, Canada) supplemented with 8 µM CHIR99021 (R&D Systems, Minneapolis, United States), 5% Protein Free Hybridoma Medium II and 1% Antibiotic-Antimycotic (both Thermo Fisher Scientific) for 3-4 days. Hereafter, 200 ng/mL recombinant human FGF9 (R&D Systems) and 1 μg/mL heparin (Sigma Aldrich, Zwijndrecht, Netherlands) were added until day 7. 500,000 cells were transferred as a pellet onto a 0.4 μm pore polyester transwell membrane (Corning, New York, United States) at an air–liquid interphase and treated for 1 h with 5 μM CHIR99021. Organoids were stimulated with 200 ng/mL recombinant human FGF9 and 1 μg/mL heparin for 5 days, followed by 13 days culture without growth factors. Medium was refreshed every other day.