Figure 2.

Generation of hNPCs from Ctrl and A-T hiPSCs. To obtain stable and proliferating hNPCs, we followed the protocol depicted in a and a representative image of the cell culture obtained is shown. In b, the characterization of the hNPCs by immunofluorescence demonstrates the loss of the pluripotency markers Tra-1-81 and Oct3/4, the expression of neural markers (Sox2, Nestin and Vimentin) and the proliferation ability (Ki67). A comparative analysis between hiPSCs and proliferating (hNPCs) and differentiated (D15) hNPCs was performed by western blot. In c, proliferating hNPCs were exposed to IR (5 Gy), and the time-dependent DDR activation was evaluated by analyzing the phosphorylation of the indicated ATM substrates, and of cleaved PARP. β-Actin was used as a loading control. The real capacity of hNPCs to differentiate into neurons is shown in d, where a high number of MAP2+, β-tubulin III+ and GABA+ cells are detected at D15 and D50 (e). hNPCs and neurons at 15–30 or 50 days of differentiation were collected and analyzed by western blot for the expression of differentiation and maturation markers (f) or for the indicated proteins (g and h)