Neural Differentiation of CD117+ TSCs

Neural differentiation assay was performed as previously described [36 (link)] with modifications. CD117⁺ TSCs were cultured in differentiation conditions using DMEM/F12 medium supplemented with 10 ng/ml bFGF and B27 (1:50) for the next 2 days. To promote photoreceptor differentiation, cells were cultured in the differentiation medium plus N-2 supplement (1:100), 50 nM docosahexaenoic acid (Sigma), 2 μM retinoic acid (Sigma), 10 μM γ-secretase inhibitor (Sigma) for 2 days, and then changed to medium containing DMEM/F12 with B27 (1:50), 10 ng/ml nerve growth factor, 10 ng/ml insulin-like growth factor 1, and 10 ng/ml brain-derived neurotrophic factor (Sigma) for another 4–6 days. Cells were harvested for assessments of immunofluorescence staining (see “Immunocytochemistry”), flow cytometry, and qRT-PCR.