The protocol for transdifferentiating hSSCs into functional DA neurons included two steps. First, hSSCs were seeded at a density of 1 × 104 cells/cm2 on poly-lysine-coated plastic coverslips or 6-well plates with OECCM containing 15 ng/ml GDNF (R&D Systems), 5 μM RA, 250 ng/ml SHH (R&D System), 1 ng/ml TGFβ3 (R&D System), 100 ng/ml FGF8α (R&D Systems), 1 mM VPA (Sigma), 10 μM SB (Sigma), and 1 μM forskolin (Sigma). The induction medium was changed every 2 days. After 4 days of induction, this medium was supplemented with 250 ng/ml SHH (R&D System), 1 ng/ml TGFβ3 (R&D System), 100 ng/ml FGF8α (R&D Systems) and maintained for another 3–4 weeks. Notably, at least 5 duplicate wells or coverslips were set up in each experiment. Thereafter, the cultures were observed and imaged under an inverted contrast microscope.
Yang H., Hao D., Liu C., Huang D., Chen B., Fan H., Liu C., Zhang L., Zhang Q., An J, & Zhao J. (2019). Generation of functional dopaminergic neurons from human spermatogonial stem cells to rescue parkinsonian phenotypes. Stem Cell Research & Therapy, 10, 195.
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