Figure 1.

Directed differentiation produces neurons with a nociceptor phenotype. (a) Directed differentiation protocol for human pluripotent stem cells (hPSC)-sensory. LDN193189 (LDN), SB-431542 (SB), CHIR99021 (CHIR), and SU5402 (SU). Open arrows denote timing of microarray analysis, black arrowheads denote timing of single cell qPCR analysis, and the black arrow denotes timing of electrophysiological analysis. (b) Phase contrast images of hPSC-sensory through the differentiation protocol. Cellular morphology after 5i is seen in left panel with emerging islands of neuronal cell bodies (arrow head). After growth factor addition many neuronal islands can be seen (arrow heads) which stain positive for sensory neuronal markers peripherin, NeuN, and Brn3a (right panel), scale bar 400 µm. (c) Sample clustering based on a principal components analysis of genome-wide expression levels across all samples. Although measured in triplicate, for clarity each time point is represented by a single sample comprising the median expression level. Samples from the differentiating hES are colored red through blue according to the time of sampling. hDRG samples are colored brown. Key time points are labeled. The percentage of total variation accounted for by each principal component is given in parentheses. (d) Relative expression levels of key marker genes across the time course on log₂ scale. The median expression levels of each gene across replicates are mean centered and scaled to unit variance. BDNF, brain-derived neurotrophic factor; GDNF, glial derived neurotrophic factor; hES, human embryonic stem cell; NGF, neuronal growth factor.