Here, we investigated the expression of genes associated with the differentiation of NPCs during three weeks in dopaminergic differentiation media. Known early neuron markers, MAP2 and Tuj1 genes reached peak expression at two weeks while expression of dopaminergic neuronal genes (TH, Nurr1, VMAT2, AADC) was significantly increased in a time-dependent manner (p < 0.05) in two types of normal NPCs (Figure 1). Furthermore, expression of genes associated with glutamatergic (vGLUT1, vGLUT2, GLS2; Figure 2) and GABAergic (GABRB3; Figure 3) neurons was also induced and peaked at the end of three weeks. This shows that ATCC NPCs and dopaminergic differentiation media are capable of producing GABAergic and glutamatergic neurons, in addition to dopaminergic neurons. Noticeably, there was significant induction of motor neuron gene (EN1, LIM3, Hb9) expression in ACS-5003™ NPCs while there was significant induction of cholinergic (ChAT) neuron gene expression in ACS-5007™ NPCs, but not in ACS-5003™ NPCs during dopaminergic differentiation (Figure 3). Moreover, expression of TH, Tuj1, Nurr1, GLS2, and vGLUT2 neuronal markers in both ACS-5003™- and ACS-5007™-derived neurons has been confirmed by immunocytochemistry (Figure 4). To validate that ATCC NPCs and dopaminergic neuron differentiation media are suitable for drug screening, we conducted neurotoxicity screenings in the two types of NPCs and NPC-derived neurons by using a resazurin viability assay and high-content imaging analysis. We found that paclitaxel, a microtubule-stabilizing chemotherapeutic agent, significantly induced neurotoxicity (p < 0.001) in the two types of NPCs evaluated at a IC50 of ~ 1 µM, but not in NPC-derived neurons (Figures 5 and 6). Vincristine, amiodarone, and chlorhexidine significantly decreased viability of both NPCs and neurons, whereas piperine, cisplatin, and hydroxyurea did not induce any significant neurotoxicity in neurons (Figures 5 and 7). This study demonstrates that ATCC iPSC-derived NPCs and dopaminergic differentiation media are suitable for studying neurological development and neurotoxicity screening.
Figure 1. Induction of neuronal gene expression during 3 weeks of dopaminergic neuron differentiation. Expression of early neuronal genes (MAP2 and Tuj1) increased significantly in both ACS-5003™ and ACS-5007™ within a week (wk) of dopaminergic media treatment. In contrast, TH and dopaminergic neuronal transcription factor Nurr1 mRNA increased significantly and reached maximum level by the end of three weeks of differentiation. Related genes like VMAT2 and DAT with opposite functions also increased significantly. AADC also significantly increased in a time dependent manner in ACS-5003™ and ACS-5007™ during neuronal differentiation (n=1, *p < 0.05, **p < 0.01, ***p < 0.001 vs. day 0, Two-Way ANOVA).
Figure 2. Induction of glutamatergic neuronal gene expression during 3 weeks of dopaminergic neuron differentiation. Expression of GLS2, vGLUT1, and vGLUT2 mRNA increased significantly in a time dependent manner during 3 weeks (wk) of dopaminergic media treatment in ACS-5003™ and ACS-5007™ NPCs (n=1, *p < 0.05, **p < 0.01, ***p < 0.001 vs. day 0, Two-Way ANOVA).
Figure 3. Induction of GABAergic, motor, and cholinergic neuronal gene expression during 3 weeks (wk) of dopaminergic neuron differentiation. GABA and EN1 mRNA increased significantly in both ACS-5003™ and ACS-5007™ NPCs in a time- dependent manner during neuronal differentiation. There was a significant increase in LIM3, and Hb9 mRNA in ACS-5003™ while ChAT mRNA increased significantly in ACS-5007™ NPCs during dopaminergic differentiation (n=1, *p < 0.05, **p < 0.01, ***p < 0.001 vs. day 0, Two-Way ANOVA).
Figure 4. Immunocytochemistry of ACS-5003™- and ACS-5007™-derived neurons after 3 weeks of dopaminergic neuron differentiation. Consistent with qRT-PCR data, after three weeks incubation in dopaminergic differentiation media there was a marked increase in expression of TH, Nurr1, GLS2, vGLUT2, and Tuj1 neuronal markers. Original magnification, x20.
Figure 5. Dose-response curves for cell viability of ACS-5007™ NPCs and ACS-5007™ NPC-derived neurons treated with paclitaxel, cisplatin, piperine, vincristine, chlorhexidine, amiodarone, and hydroxyurea for two days. Paclitaxel, cisplatin, piperine, and hydroxyurea significantly induced neurotoxicity (p < 0.05) in NPCs, but not in NPC-derived neurons. Vincristine, chlorhexidine, and amiodarone significantly decreased viability (p < 0.01) of both NPCs and neurons (n=3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. DMSO control, Student’s T-test). The only concentration tested for amiodarone and chlorhexidine was 10 µM. Similar neurotoxicity was observed in ACS-5003™ cells (data not shown).
Figure 6. Dose-response curves for cell viability of ACS-5003™ and ACS-5007™ NPCs treated with paclitaxel for two days. The neurotoxic effect of paclitaxel was similar in both types of NPCs. IC50 of paclitaxel on ACS-5003™ and ACS-5007™ NPCs was 0.9 µM and 1.6 µM, respectively (n=2).
Figure 7. High-content imaging of chemotherapeutic-treated NPCs supports cell viability experiments. ACS-5003™ and ACS-5007™ NPCs-derived neurons were incubated with 10 µM paclitaxel, cisplatin, or chlorhexidine for two days, stained with Calcein Green AM, and Hoechst 33342, and then subjected to high content imaging. Consistent with the viability data, chlorhexidine significantly
inhibited the neurite outgrowth (n=2, **p < 0.01 vs. DMSO, Student’s T-test). Original magnification, x20.