iPSK3 Cell Differentiation Protocol

Undifferentiated human iPSK3 cells were seeded into ultra-low attachment (ULA) 24-well plates (Corning Inc., Corning, NY) at 3 × 10⁵ cells/well in differentiation medium composed of DMEM/F-12 plus 2% B27 serum-free supplement (Life Technologies, Carlsbad, CA). iPSK3 cells were seeded in the presence of Y27632 (10 μM). After 24 h, Y27632 was removed and the formed embryoid bodies (EB) were treated with dual SMAD signaling inhibitors of 10 μM SB431542 (Sigma-Aldrich, St. Louis, MO) and 100 nM LDN193189 (Sigma) over 7 days. Then on day 8, the spheroids were treated with fibroblast growth factor (FGF)-2 (10 ng/mL, Life Technologies) and cyclopamine (an Shh inhibitor, 1 μM, Sigma) for cortical differentiation for 21 days.^{22 (link),33 (link),34 (link)} The cells were replated onto growth factor reduced Matrigel-coated surfaces and treated with different iron oxide nanoparticles for another 2–4 days prior to further downstream experiments (Figure 1B, C). On the basis of our previous studies,^{35 (link),36 (link)} the labeling efficiency for microsized particles of iron oxides (MPIO) can reach 50–80%. It was estimated that the labeling efficiency for nanoscale iron oxides should be similar or higher than MPIO.