To more specifically investigate the in vitro neural differentiation capacity of each of the 12 iPSCs lines from the Family-811 quartet, directed neural differentiation was used to generate NPCs. To initially derive heterogeneous populations of NPCs containing both central nervous system (CNS) and peripheral nervous system (PNS) progenitors, each of the 12 iPSC lines were cultured individually on MEF feeder cells and colonies were passaged manually to feeder-free cultures on Matrigel in the presence of bFGF. After 3 days, bFGF was withdrawn to induce differentiation and after 7 days the media was switched to neural induction media consisting of Neurobasal media supplemented with B27 (without retinoic acid), EGF and bFGF. All 12 of the iPSC lines, regardless of whether they were from the healthy control parents or BD sons, were found to be capable of forming neural rosettes with a morphology characteristic of early neuroepithelium, which upon picking and expansion on poly-ornithine/laminin coated plates gave rise to self-renewing NPCs that stained for a panel of markers, including SOX1, SOX2, NESTIN and MUSASHI (Fig. 2A and data not shown).
