Fig. 3

Generation of neurospheres from the induced pluripotent stem cells (iPSCs). a Schema of neural induction and neurosphere generation. Neural induction using dual SMAD inhibition and subsequent neurosphere cultures were conducted to obtain the neurospheres. After 2 weeks of neural induction, the embryoid bodies (EBs) were dissociated and cultured in a medium containing bFGF, EGF, and LIF (scale bar = 50 μm). Representative photographs of EBs and neurospheres were derived from Patient A. b Quantitative RT-PCR for each TUBA1 isoform. Through neural induction, only TUBA1A expression was greatly increased. Changes in the expression of each TUBA1 isoform were different (relative to the expression in the undifferentiated iPSCs (201B7), fold change ± SD, n = 3, technical duplicates). c Immunostaining of each neurosphere (within 12 h after plating in neurosphere medium). Ubiquitous staining for the NPC markers NESTIN and SOX1 (scale bar = 50 μm) was observed in each cell. d Quantitative RT-PCR showed that the neurospheres expressed the neural marker genes N-cadherin (CDH2) and α tubulin 1A (TUBA1A) and the progenitor cell marker SOX1, all of which are expressed in the human neural stem cell (hNSC) line. The radial glial marker brain lipid-binding protein (BLBP) was also expressed (relative to the expression in the undifferentiated iPS, fold change ± SD, n = 3, technical duplicates). e Quantitative RT-PCR showed that the neurospheres had a caudalized regional identity characterized by high gastrulation brain homeobox 2 (GBX2) expression and low Forkhead box protein G1 (FOXG1) expression (relative to the expression of SFEB used as human forebrain control, fold change ± SD, n = 3, technical duplicates). f–k Spontaneous differentiation of the neurospheres. In 12 days, the βIII tubulin-positive neurites and few BLBP-positive radial glial-like fibers had migrated radially from the neurospheres (scale bar = 200 μm). Poorly ordered neurites and BLBP-positive radial glial-like fibers were observed in the TUBA1A-iPS-A-derived neurospheres (f–g). Radially extending neurites and BLBP-positive radial glial-like fibers were observed in the TUBA1A-iPS-B-derived neurospheres (H–I) and control iPSC-derived neurospheres (j-k)