iPSC colonies were detached with accutase (Millipore) and grown as embryoid bodies (EBs) in suspension for 5–6 days in iPSC medium without basic fibroblast growth factor. EBs were allowed to attach and form rosettes. Ten-day-old rosettes were collected and grown in suspension as neurospheres. Neurospheres were dissociated after 3–4 weeks, and the cells were plated on glass coverslips (BD Biosciences) or plates coated with poly-D-lysine (0.1 mg/ml) and laminin (10 μg/m). Neurons were used after 2-4 weeks in culture.
iPSC colonies
grown as embryoid bodies (EBs) in suspension
5–6 days
days
rosettes
old
Ten-day
day
Neurospheres
dissociated
after 3–4 weeks
weeks
poly-D-lysine
0.1 mg/ml
mg/ml
laminin
10 μg/m
μg/m
Neurons
used
after 2-4 weeks
weeks