A more detailed analysis of the process of differentiation was performed by examining NSC gene expression comparisons and comparisons after cells had been differentiated to dopaminergic neurons. An unsupervised two-way hierarchical cluster analysis of differentially expressed genes across ESC/iPSC, NSCs, and dopaminergic neuron samples is also shown in Figure 4B. As expected, the heat map shows a clear separation of ESC/iPSC, NSC, and dopaminergic cell types, and a separation of six differentially expressed gene clusters in different colors. The hESCs and iPSCs types were strongly associated with cluster six (yellow), which was upregulated in hESCs and iPSCs, whereas downregulated in the other cell types. The red genes (cluster 1 and cluster 2) appear to be dopaminergic-specific, which were upregulated in the dopaminergic cells, whereas downregulated or expressed at low levels in undifferentiated and NSC samples. More specifically, Table 2 shows the expression levels of four markers representative of NSCs in iPSC- and hESC-derived NSC populations. No significant difference was observed among the lines. Of the 20 genes associated with dopaminergic/neuronal specification, differentiation, and maturation, or PD listed in Table 2,
