Figure 6

TET1-deficient hESCs cannot be differentiated to neurons in the morphogen-free condition. (a) Protocol for the differentiation of neuroepithelial cells to neurons in morphogen-free condition. N2, N2 supplements. (b–f) At day 28 of differentiation, neuroectoderm cells generated from wild-type (b–e) or TET1-deficient (b’-e”) H9 hESCs were costained for SOX1 (b–b”), NESTIN (c–c”), and DAPI (d–d”). Merged images (e–e”) were used to quantify the percentage of SOX1 and NESTIN double positive cells among all DAPI⁺ cells (f). *p < 0.001, Student’s t-test, n = 3. (g–k) At day 50, cells differentiated from wild-type or TET1-deficient H9 hESCs were co-stained for the pan-neural marker TUJ1 (g–g”), mature neuronal marker MAP2 (h–h”), and DAPI (i–i”). Merged images (j–j”) were used to quantify the percentage of MAP2 and TUJ1 double positive neuron among all DAPI⁺ cells (k). *p < 0.001. Student’s t-test, n = 3. Bars, 100 µm.