We made a number of important observations with the current conditions. First, single-cell analysis over the entire 125 day period of culture revealed that cell-type production proceeded as expected based on in vivo studies, with the production of unventralized telencephalic progenitors early, followed by mitotic NKX2-1+ MGE progenitors, ASCL1+NKX2-1+ intermediate progenitors, and glia. Interneurons to immature to be classified as cortical interneurons appeared earliest, followed by ERBB4+SST+ cortical interneurons, and finally immature oligodendrocyte precursors. We mapped expression data from GAD1+ migrating interneurons from D100 dpc fetal human cortex onto the PCA space generated from the in vitro data and found that they most closely resembled neurons obtained from D54 in vitro. Between D100 and D125 in vitro, the rate of transcriptomic changes appeared to slow. A slow-down in gene expression dynamics has been observed in previous studies of human cortical development (Bakken et al., 2016; Colantuoni et al., 2011). It is possible that our in vitro observations reflect similar changes occurring at equivalent ages in vivo, or that some extrinsic element necessary for continued development of these cells is missing from our protocol, as previous work has suggested that development is comparatively slow in vitro (Sadegh and Macklis, 2014).
