Stem Cell-Derived Immature Human Dorsal Root Ganglia Neurons to Identify Peripheral Neurotoxicants

Abstract

Safety sciences and the identification of chemical hazards have been seen as one of the most immediate practical applications of human pluripotent stem cell technology. Protocols for the generation of many desirable human cell types have been developed, but optimization of neuronal models for toxicological use has been astonishingly slow, and the wide, clinically important field of peripheral neurotoxicity is still largely unexplored. A two-step protocol to generate large lots of identical peripheral human neuronal precursors was characterized and adapted to the measurement of peripheral neurotoxicity. High content imaging allowed an unbiased assessment of cell morphology and viability. The computational quantification of neurite growth as a functional parameter highly sensitive to disturbances by toxicants was used as an endpoint reflecting specific neurotoxicity. The differentiation of cells toward dorsal root ganglia neurons was tracked in relation to a large background data set based on gene expression microarrays. On this basis, a peripheral neurotoxicity (PeriTox) test was developed as a first toxicological assay that harnesses the potential of human pluripotent stem cells to generate cell types/tissues that are not otherwise available for the prediction of human systemic organ toxicity. Testing of more than 30 chemicals showed that human neurotoxicants and neurite growth enhancers were correctly identified. Various classes of chemotherapeutic agents causing human peripheral neuropathies were identified, and they were missed when tested on human central neurons. The PeriTox test we established shows the potential of human stem cells for clinically relevant safety testing of drugs in use and of new emerging candidates.

Significance: The generation of human cells from pluripotent stem cells has aroused great hopes in biomedical research and safety sciences. Neurotoxicity testing is a particularly important application for stem cell-derived somatic cells, as human neurons are hardly available otherwise. Also, peripheral neurotoxicity has become of major concern in drug development for chemotherapy. The first neurotoxicity test method was established based on human pluripotent stem cell-derived peripheral neurons. The strategies exemplified in the present study of reproducible cell generation, cell function-based test system establishment, and assay validation provide the basis for a drug safety assessment on cells not available otherwise.

Keywords: Human pluripotent stem cells; In vitro drug and toxicity testing; Neural differentiation; Peripheral neuropathies.

Figure 1.

Generation of human immature dorsal root ganglia neuron (iDRG) cells for toxicity testing. Pluripotent stem cells were differentiated in a two-step procedure, as indicated. (A–D): Immunocytochemical characterization of hESC-derived cells. Labels are color keyed to images. (E): Proliferating cells (EdU⁺) were quantified (mean ± SEM; n = 3). (F–I): Immunocytochemical characterization of iDRG cells after thawing. Labels are color keyed to images. Scale bars = 100 µm (A) and 50 µm (B–D, F–I). Abbreviations: BDNF, brain-derived neurotrophic factor; DoD, day of differentiation; GDNF, glia-derived neurotrophic factor; hESC, human embryonic stem cell; NGF, nerve growth factor.

Figure 2.

Quantification of calcium signaling of immature dorsal root ganglia neuron cells. (A): Intracellular calcium levels measured on DoD7 after depolarization. Each cell is depicted by a circle. ***, p < .001. (B): Before depolarization by KCl, cells were pretreated with Verap (100 µM) or Nifed (100 µM; n = 3; ***, p < .001). Abbreviations: DMSO, dimethyl sulfoxide; HBSS, Hanks' buffered salt solution; Nifed, nifedipine; rel., relative; Verap, verapamil; Verat, veratridine.

Figure 3.

Differentiation tracking by transcriptome analysis of iDRG cells. (A): Samples were obtained at different developmental stages for whole transcriptome analysis; data are displayed as a principal component analysis (PCA) map, together with legacy data from cell cultures or from human dorsal root ganglion, brain, and liver tissue. The red arrow indicates the cell differentiation track of the two-step iDRG cell differentiation protocol. Note: DoD7 of the two-step protocol is 16 days older than hESCs (i.e., roughly corresponding to DoD16* of the one-step protocol in differentiation time). (B): Number of regulated genes over time. (C): Top 20 significantly upregulated (red) and downregulated (blue) genes. (D): Hierarchical clustering analysis of the top 100 genes with the highest variance during peripheral neuronal differentiation. (E): Relative gene expression during differentiation (n = 4–6). Abbreviations: DoD, day of differentiation; FC, fold change; hESC, human embryonic stem cell; iDRG, immature dorsal root ganglia neuron; PC1, principal component 1; PC2, principal component 2; rel., relative.

Figure 4.

Characterization and quantification of neurite growth of early iDRG cells. (A): Immunocytochemical characterization of early neurites; labels are color keyed to images. (B): Quantification of neurite area over time (mean ± SEM; n = 5). (C): Effects of vincristine, colchicine, and cytochalasin D on neurite area and viability under PeriTox test conditions (mean ± SEM; n = 3–4) and exemplary sample images (**, p < .01; ***, p < .001). Scale bars = 20 µm (A, left) and 50 µm (B, right; C). Abbreviations: DoD, day of differentiation; untr., untreated.

Figure 5.

Profiling and quantification of negative and positive control toxicant effects on immature dorsal root ganglia neuron neurites. Tool compounds were used as in (4C). (A): Negative control. (B): Unspecific toxicants. (C): Guanylyl cyclase inhibitor as pathway-specific control. *, p < .05; **, p < .01; ***, p < .001 versus untr. cells. (D): Test of reversibility. After 24 hours of exposure, ODQ was washed out, and neurites were measured again 24 hours later (n = 3–4). (E): ROCK inhibitor as pathway-specific control for accelerated growth. (F): Positive controls (drugs causing peripheral neuropathy). Scale bars = 50 µm. Abbreviations: SDS, sodium dodecyl sulfate; untr., untreated.

Figure 6.

Classification of toxicant effect on immature dorsal root ganglia neuron neurites. (A): Overview of specificity of all compounds tested. EC₅₀ values for effects on neurite area were plotted against EC₅₀ for effects on viability. The solid line indicates an EC₅₀ ratio of 1. The dashed line indicates an EC₅₀ ratio of 3 (n ≥3). (B): Comparison of positive hits in the PeriTox test and published data on the LUHMES test (based on central neurons). For both tests, the EC₅₀ ratio (EC₅₀ viability to EC₅₀ neurite area) of various compounds is shown. Abbreviation: HDAC, histone deacetylase.

Figure 7.

Effect of environmental toxicants on immature dorsal root ganglia neuron neurites. Neurite area and viability were measured under PeriTox test conditions. Effect of acrylamide (A) or acrylic acid (B) (mean ± SEM; n = 3–5). (C): Immunostaining of cells treated with acrylamide as in (A). (D): Test of reversibility. After 24 hours of exposure, acrylamide was washed out, and neurites were measured again 24 hours later (n = 3–4). (E–G): Toxicant testing as in (A). (H): Reversibility of rotenone effects was tested as in D. Scale bars = 50 µm (A, C).