To further corroborate our findings of differential gene expression between the BD patient CXCR4+ NPCs and neurons compared to their parental controls, and to overcome the limitation of having only one expandable CXCR4+ NPC line from each of the BD patient iPSCs, as an alternative differentiation method we turned to the generation of neutralized embryoid bodies (nEBs) formed from aggregating iPSCs under conditions that promote neuroectoderm formation (Sup. Fig. 17–19). Whole cell RNA was collected from multiple BD and unaffected nEBs at day one and day eleven. Using qRT-PCR, as expected, neuralization resulted in increased expression of SOX1 (SRY-box containing gene 1), a transcription factor that is an early maker of neuroectoderm formation, in both the unaffected and BD-patient iPSC lines. In contrast, two of the BD-patient nEBs failed to express PAX6, an evolutionarily conserved, homeobox-containing transcription factor downstream of SOX1 that plays an essential role in balancing cortical neural progenitor proliferation and differentiation59,60, whereas both unaffected parental lines upregulated PAX6 expression (Supp. Fig. 17). This downregulation of PAX6 expression in the BD-patient nEBs was in agreement with the reduced
