Fig. 1

Anatomic protocol: differentiation and maturation (Ctrl1). A Schematic diagram outlining the steps for differentiation and maturation with the Anatomic compared to the Chambers protocol. Anatomic protocol requires 7 days of differentiation as compared to 10 days with Chambers protocol to achieve immature neurons. With Chambers protocol neurons were MACS sorted on DIV10 of differentiation. Neurons were then matured until DIV 35–40. DIV-Days in vitro, LDN-193189, SB431542 and SU5402, CHIR99021 and DAPT. DM-Differentiation medium, Chrono™ Senso-MM-Maturation medium. B Differentiation of Ctrl1 iPSCs with Anatomic protocol. Phase contrast images display single cell and clump seeding on DIV0 of differentiation. Differentiation involves formation of ectoderm within 24 h, spinal neural culture (DIV2), neural crest formation (DIV4) and generation of immature neurons by DIV7. Scale Bar—200 µm. C Maturation of Ctrl1 neurons with growth factors. Both seeding protocols resulted in formation of dense homogenous neuronal networks during the maturation period. No morphological differences could be observed during maturation from both protocols. Scale Bar—200 µm. D Immunostaining of neurons for Peripherin and Tuj1 on DIV8, 14, 28 and 35 with clump seeding. E Single cell seeded neurons staining for Peripherin and Tuj1 on DIV8, 14, 28 and 35. Scale Bar—200 µm. Peripherin-green, Tuj1-red and DAPI-blue fluorescence. DIV-Days in vitro