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Figure 1. Schematic for the STEMdiff™ Sensory Neuron Culture System Protocol

Sensory neurons can be generated in 7 days from sensory neuron precursors after 6 days in STEMdiff™ Sensory Neuron Differentiation Medium. For the differentiation of sensory neuron precursors from hPSC-derived neural crest cells, see the PIS.

Figure 2. STEMdiff™ Sensory Neuron Kits Promote Differentiation Across Multiple Embryonic Stem and Induced Pluripotent Stem Cell Lines

NCCs generated from hPSCs in mTeSR™ Plus using the STEMdiff™ Neural Crest Differentiation Kit were differentiated and matured to sensory neurons using the STEMdiff™ Sensory Neuron Differentiation and Maturation Kits. (A) Sensory neurons were generated after hPSC-derived NCCs were cultured with the STEMdiff™ Sensory Neuron Differentiation Kit for 6 days and then the STEMdiff™ Sensory Neuron Maturation Kit for 6 days. The resulting cultures contain a population of cells expressing sensory neuron markers peripherin (green) and BRN3A (red) along with (B) neuronal marker class III β-tubulin (TUJ1, red). (C) Midbrain neuron controls generated with STEMdiff™ Midbrain Neuron Differentiation and Maturation Kits do not have detectable peripherin (green) or BRN3A (red) expression, although they express (D) neuronal marker class III β-tubulin (TUJ1, red). Nuclei are labeled with DAPI (blue). Human ES and iPS cell lines were maintained in either mTeSR™1, TeSR™-E8™, or mTeSR™ Plus and differentiated with STEMdiff™ Neural Crest Differentiation Kit, followed by STEMdiff™ Sensory Neuron Differentiation and Maturation Kits. The percentage expression of (E) BRN3A+ and (F) TUJ1+ cells in the resulting cultures was quantified. This differentiation generated BRN3A+ sensory neurons (25.3% ± 6.9%, mean ± SEM; n=7 cell lines, 3 - 23 replicates per condition) that expressed neuronal marker class III β-tubulin (TUJ1; 90.3% ± 4.1%, mean ± SEM; n=4 cell lines, 3 - 12 replicates per condition). Numbers are % positive over total DAPI in a tiled image. NCCs = neural crest cells; hPSCs = human pluripotent stem cells; ES = embryonic stem; iPS = induced pluripotent stem

Figure 3. Temporal Gene Expression Profile of Sensory Neurons Generated Using STEMdiff™ Sensory Neuron Kits Reveals Loss of Pluripotency and Progressive Acquisition of Sensory Neuron Identity and Maturity

PCA of whole-transcriptome bulk RNA-seq data collected across four time points show distinct transcriptomic shifts from pluripotency to mature sensory neuron identity. Tight clustering of hPSCs, distinct from sensory neurons, indicates significant transcriptional divergence. Day 6 neural crest cells are positioned closer to hPSCs along the first principal component than the Day 12 and Day 18 sensory neurons, suggesting progressive differentiation over time. The number of DEGs increases with maturation: 3,321 DEGs between hPSCs and Day 6 neural crest cells, 3,399 DEGs between hPSCs and Day 12 sensory neurons, and 3,689 DEGs between hPSCs and Day 18 sensory neurons, reflecting dynamic transcriptomic shifts during neuronal development. PCA = principal component analysis; hPSCs = human pluripotent stem cells; DEGs = differentially expressed genes

Figure 4. hPSC-Derived Sensory Neurons Generated Using STEMdiff™ Kits Show Lineage-Specific Upregulation of Pain and Itch Genes Compared to hPSC-Derived Forebrain Neurons

hPSC-derived forebrain neuron precursor cells and sensory neurons were generated from hPSC lines (SCTi003-A, H1, and H9) using the STEMdiff™ Forebrain Neuron or STEMdiff™ Sensory Neuron Differentiation Kits, respectively. The neuron precursors were then matured with corresponding STEMdiff™ maturation kits for an additional 14 days (forebrain neurons) or 6 days (sensory neurons) according to recommended protocols. RNA from mature forebrain and sensory neurons along with parent hPSC controls was subsequently sequenced using bulk RNA-seq. The heatmap shows expression of selected pain- and itch-related genes, including sensory neuron ion channels and receptors involved in nociception and thermosensation (e.g. TRPV1/2, TRPM8, SCN9A/SCN10A, P2RX3, and the itch-associated neuropeptide GRP), along with markers of sensory neuron identity (POU4F1/BRN3A) and neuronal and regional controls (TUBB3, FOXG1). Over- (orange) and under-expression (grey) compared to the gene expression average is computed for each gene. Each column represents a replicate from a specific cell line. Three hPSC lines were used across all samples, with a greater number of replicates in the hPSC controls. hPSC = human pluripotent stem cell; iPSC = induced pluripotent stem cell