Figure 1.

Flow diagram of the neurodifferentiation procedure from h/iPSCs and the characterization and stability of the neurospheres using defined culture conditions. (A) The five major stages and the corresponding cell types generated from these cultures. Neuronal initiation with SKSRM and 10% CO₂ at 37°C takes 3 days. Neuronal induction took one week in NMM with SB431542 and dorsomorphin in 5% CO₂ at 37°C. The AdSTEP (*) procedure was introduced to generate the neurosphederm from the neurosphere. After generating the neurosphederm and plating the cells, distinct neuronal rosettes appeared at 3–5 days in culture, and mature neurons and other sub type specific neurons appeared at 15–22 days in culture. At day 27, the neurons have fully functional synapses, as shown in the functional assays. (B, D) Generation of neurospheres from h/iPSCs with 10% and 5% CO₂, respectively. (C, E) Generation of the neurosphederm from h/iPSCs with 10% and 5% CO₂, respectively. (F). Graph of the relative expression levels of the SOX2, NESTIN, PAX6 and FOXG1 genes from neurospheres with 10% and 5% CO₂, respectively. The data are presented as the means ± SD. (G, H & I, J) Flow cytometry histogram of the time-dependent expression of SOX2 and NESTIN in the neurosphere and neuroectoderm cultures, respectively. Scale bars, 50 μm.