To generate suspended cellular aggregates of pluripotent cells, we used cultures of hiPSCs grown on feeders. We used seven hiPSC lines derived from five subjects and successfully generated hCSs from each clone. Rather than using single-cell suspensions, we enzymatically detached intact hiPSC colonies from inactivated feeders (Fig. 1a). Suspended colonies were subsequently transferred into low-attachment plates in a KnockOut Serum (Invitrogen)-based medium without fibroblast growth factor 2 (FGF2). Within a few hours, the floating hiPSC colonies folded into spherical structures. To achieve rapid and efficient neural induction, both the BMP and TGF-β signaling pathways were inhibited with small molecules: dorsomorphin (also known as compound C) and SB-431542 (ref. 10). On the sixth day in suspension, the floating spheroids were moved to serum-free Neurobasal with B-27 (Invitrogen) medium containing FGF2 and epidermal growth factor (EGF). We changed the medium daily in the first 10 d and every other day for the subsequent 9 d.
