To better understand cell type specification during organoid development, we performed single cell RNA-seq (scRNA-seq) in organoids (Figure 5A). After quality control, data from a large number of single cells (59,235 cells in total) from two biological replicates of hMGEOs and hCOs at two developmental stages (early: day 30; late: day 72∼79) were obtained (Figure 5A and Figure S3A). Analysis by t-distributed Stochastic Neighbor Embedding (t-SNE) revealed production of both unique and overlapping cell types between hMGEOs and hCOs (Figure 5B). Compared with those at day 30, hMGEO and hCO cells at day 72 were more distinct, indicating progression of further lineage commitment during the organoid development. Batch effect between two replicates was minimized by regressing out cell-cell variation in gene expression driven by batch (p>0.308, Figure S3B) (Macosko et al., 2015). Almost exclusive expression of TBR1, a marker unique for cortical excitatory neurons (referred to as cortical neuron, CN) or GFAP for astrocytes confirmed a minimal doublet rate in our scRNA-seq (Figure S3C).
