Collagenase type 4 solution

Intermediate primary colonies with compact morphology were manually picked, digested in 1 mg/mL collagenase type IV solution (Worthington Biochemical Corporation, Lakewood, NJ, USA), and disaggregated by pipetting to single cells or small clumps. The cell suspension was seeded in 6 cm Geltrex dishes (30–40 × 10⁵ cells/dish) in marmoset iPSC medium (iPS-Brew supplemented with 3 µM IWR1 (Sigma-Aldrich, Munich, Germany), 0.5 µM CHIR99021, 0.7 µM CGP77675 (Selleckchem, Houston, TX, USA), 10 ng/mL human recombinant leukemia inhibitory factor (hrLIF) (PeproTech, Hamburg, Germany), and 7 µM Forskolin (Selleckchem, Houston, TX, USA)) and cultured for 2–3 passages in hypoxic conditions (5% O₂, 5% CO₂, and 90% N₂) until the appearance of colonies with iPSC-like morphology. These colonies were manually picked, disaggregated in collagenase solution to small clumps, and passed to fresh Geltrex dishes in iPSC medium with CGP77675 concentration reduced to 0.3 µM. The iPSCs were further maintained by splitting with collagenase at ratio 1:3–1:4 every 3–4 days. To increase survival, 5 µM Pro-survival Compound (MerckMillipore, Darmstadt, Germany) was added only on the day of splitting. Hypoxic conditions were maintained during the entire duration of iPSC culture.