Modeling chemotherapeutic neurotoxicity with human induced pluripotent stem cell-derived neuronal cells

Abstract

There are no effective agents to prevent or treat chemotherapy-induced peripheral neuropathy (CIPN), the most common non-hematologic toxicity of chemotherapy. Therefore, we sought to evaluate the utility of human neuron-like cells derived from induced pluripotent stem cells (iPSCs) as a means to study CIPN. We used high content imaging measurements of neurite outgrowth phenotypes to compare the changes that occur to iPSC-derived neuronal cells among drugs and among individuals in response to several classes of chemotherapeutics. Upon treatment of these neuronal cells with the neurotoxic drug paclitaxel, vincristine or cisplatin, we identified significant differences in five morphological phenotypes among drugs, including total outgrowth, mean/median/maximum process length, and mean outgrowth intensity (P < 0.05). The differences in damage among drugs reflect differences in their mechanisms of action and clinical CIPN manifestations. We show the potential of the model for gene perturbation studies by demonstrating decreased expression of TUBB2A results in significantly increased sensitivity of neurons to paclitaxel (0.23 ± 0.06 decrease in total neurite outgrowth, P = 0.011). The variance in several neurite outgrowth and apoptotic phenotypes upon treatment with one of the neurotoxic drugs is significantly greater between than within neurons derived from four different individuals (P < 0.05), demonstrating the potential of iPSC-derived neurons as a genetically diverse model for CIPN. The human neuron model will allow both for mechanistic studies of specific genes and genetic variants discovered in clinical studies and for screening of new drugs to prevent or treat CIPN.

Fig 1. Representative images from iCell Neuron chemotherapeutic treatment.

Representative micrographs comparing neurite outgrowth of iCell Neurons upon 72 h treatment with the neuropathy-inducing drugs paclitaxel, vincristine, and cisplatin and the non-neuropathy-inducing negative control hydroxyurea. Images taken at 10x magnification, scale bar = 100 μm, green = Calcein AM (cell-permeant stain, live cells), blue = Hoechst 33342 (cell-permeant, nucleic acid stain, live cell nuclei), red = Ethidium Homodimer-2 (cell-impermeant, nucleic acid stain, dead cells).

Fig 2. Neurite outgrowth response upon chemotherapeutic treatment of iCell Neurons.

Nine phenotypes measured after 72 h of drug exposure were tested for differences across drugs by one-way ANOVA of the area under the curve (AUC) calculated from 0.001–10 μM including relative (A) total outgrowth (P = 0.014), (B) number of processes (P = 0.623), (C) mean process length (P = 0.005), (D) median process length (P = 0.002), (E) maximum process length (P = 0.009), (F) number of branches (P = 0.065), (G) straightness (P = 0.081), (H) cell body area (P = 0.176), and (I) mean outgrowth intensity (P = 0.005). Error bars represent the standard error of the mean from three independent experiments of >500 cells per dose.

Fig 3. Neurite outgrowth, viability, and apoptotic response upon chemotherapeutic treatment of and gene perturbation of TUBB2A in iCell Neurons.

Relative viability measured by CellTiter-Glo (black lines, right Y-axes) was significantly higher than relative total outgrowth (colored lines, left Y-axes) upon treatment of iCell Neurons for 72 h with (A) paclitaxel (Welch's t-test of AUC P = 0.0005) and (B) vincristine (P = 0.027), but not (C) cisplatin (P = 0.71). Three replicates of the total outgrowth assay and four replicates of the viability assay were performed. (D) Relative caspase 3/7 activity measured by Caspase-Glo 3/7 after 48 h treatment significantly differed among drugs (one-way ANOVA of AUC calculated from 0.001–10 μM P = 0.002). Five replicates of the apoptosis assay were performed. (E) Paclitaxel-induced decrease in neurite outgrowth of iCell Neurons is influenced by TUBB2A expression. Left, relative TUBB2A expression 24 to 48 h post-transfection. Right, decreased expression of TUBB2A by siRNA transfection causes a greater decrease in relative total neurite outgrowth of iCell Neurons (Welch's t-test P = 0.011) 24 h post-treatment with 0.1 μM paclitaxel (48 h post-transfection). Error bars represent the standard error of the mean from three independent transfection experiments. NTC = non-targeting control.

Fig 4. Neurite outgrowth and apoptotic response upon chemotherapeutic treatment of 4 genetically distinct LCL-iPSC-derived Neuron lines.

Phenotypes measured after 72 h of drug exposure that significantly differed across individuals (ANOVA of AUC calculated from 0.001–100 μM) include relative paclitaxel-induced (A) total outgrowth (P = 0.005), (B) mean process length (P = 0.019), (C) median process length (P = 0.022), (D) maximum process length (P = 0.009), (E) number of branches (P = 0.018), and vincristine-induced (F) number of processes (P = 0.022). Relative caspase 3/7 activity measured by Caspase-Glo 3/7 after 48 h treatment of (G) paclitaxel, (H) vincristine, and (I) cisplatin. Doses of drug that caused a mean greater than 2-fold increase in caspase activity relative to control were tested for significant differences among individual cell lines by one-way ANOVA, *P < 0.05 and **P < 0.001. Error bars represent the standard error of the mean from three independent experiments of >500 cells per dose.