hiPSC-Derived Neurons Provide a Robust and Physiologically Relevant In Vitro Platform to Test Botulinum Neurotoxins

Abstract

Botulinum neurotoxins (BoNTs) are zinc metalloproteases that block neurotransmitter release at the neuromuscular junction (NMJ). Their high affinity for motor neurons combined with a high potency have made them extremely effective drugs for the treatment of a variety of neurological diseases as well as for aesthetic applications. Current in vitro assays used for testing and developing BoNT therapeutics include primary rodent cells and immortalized cell lines. Both models have limitations concerning accuracy and physiological relevance. In order to improve the translational value of preclinical data there is a clear need to use more accurate models such as human induced Pluripotent Stem Cells (hiPSC)-derived neuronal models. In this study we have assessed the potential of four different human iPSC-derived neuronal models including Motor Neurons for BoNT testing. We have characterized these models in detail and found that all models express all proteins needed for BoNT intoxication and showed that all four hiPSC-derived neuronal models are sensitive to both serotype A and E BoNT with Motor Neurons being the most sensitive. We showed that hiPSC-derived Motor Neurons expressed authentic markers after only 7 days of culture, are functional and able to form active synapses. When cultivated with myotubes, we demonstrated that they can innervate myotubes and induce contraction, generating an in vitro model of NMJ showing dose-responsive sensitivity BoNT intoxication. Together, these data demonstrate the promise of hiPSC-derived neurons, especially Motor Neurons, for pharmaceutical BoNT testing and development.

Keywords: botulinum neurotoxins; human induced pluripotent stem cells; in vitro translational models; motor neurons; neuromuscular junction.

FIGURE 1

Characterization of multiple neuronal models derived from hiPSC. (A) Phase contrast images taken with a 20X objective at day 15 after thawing. Scale bar: 25 μm. (B–E) Time-course gene expression analysis of neuronal markers (B) and phenotypic markers (C,E) for the four models. Expression is normalized to GAPDH and to a control cDNA (human total brain: TB). For every model, each shade bar represents a different time point: 1 day, 7 days, 14 days and 28 days after thawing; 1 day being the lighter shade. (F) Immunolabelings at day 15 after thawing of markers for neurons (TUJ1 or MAP2), synapses (SYN), GABAergic neurons (GABA), peripheric neurons (PRPH), glutamatergic neurons (vGlut2) and motor neurons (Islet1). Nuclei are stained in blue (DAPI). Scale bar: 25 μm. (G) % of islet1+ cells amongst total cells and amongst neurons (TUJ1+).

FIGURE 2

Expression of the different BoNT SNARE substrates, BoNT receptors and gangliosides. (A–C) Time-course gene expression analysis of various SNARE proteins (A), BoNT proteic receptors (B) and enzymes involved in the biosynthesis of gangliosides (C) for the 4 models. For every model, each shade bar represents a different time point: 1 day, 7 days, 14 days and 28 days after thawing; 1 day being the lighter shade. Expression is normalized to GAPDH and to a control cDNA (human total brain: TB).

FIGURE 3

Immunolabelings of hiPSC-derived Neurons at day 15 after thawing: (A–E) TUJ1 (green) and (A) SNAP25 (red); (B) SV2A (red); (C) VAMP2 (red); (D) GM1 (red) and (E) GT1b (red). Nuclei are stained in blue (DAPI). Scale bar: 25 μm.

FIGURE 4

Sensitivity of the 4 models to rBoNT/A and rBoNT/E. (A,B): Western Blots showing the cleavage of SNAP25 in the 4 different cellular models after treatment of BoNT/A (A) or BoNT/E (B) at different doses. (C,D) Dose response curves of SNAP25 cleavage for BoNT/A (C) and BoNT/E (D). (E) Table of the potencies (pEC⁵⁰) for BoNT/A and BoNT/E in each model; n = 3 for all models.

FIGURE 5

RNASeq analysis of hiPSC-derived Motor Neurons. (A) Principal Component Analysis showing distinct signatures at different time points during maturation of hiPSC-derived Motor neurons and Spinal cord RNA sample. (B) Heat map illustrating the time-course of Motor Neurons progenitors and mature Motor Neurons expression (C) Enrichment analysis showing classes of genes that are over-represented in Motor Neurons after 2 weeks of maturation compared to a human tissue sample (Spinal Cord) and classes of genes that are over-represented over-represented in human spinal cord compared to Motor Neurons after 2 weeks of maturation. Expressions are in ‐logp value. Only the top gene sets of the Gene Ontology collection are represented.

FIGURE 6

Functional characterization of Motor neurons. (A) Representatives traces of Ca2+ oscillations obtained by whole-well recording followed by FDSS. Waveformanalysis of Cal520 dye loaded 28 days old-iCell MN cultures. Cells were stained with Cal520 the day of the recording. Ca2+ oscillations were recorded before (i.e. baseline condition) and after addition of culture medium, 0.1% DMSO or 1 μM TTX. (B) Ca2+ oscillations frequency of iCell Motor Neurons after addition of culture medium, 0.1% DMSO or 1 μM TTX, compared to baseline condition without treatment. Data are represented as mean ± SEM. ANOVA with Sidak correction (****p < 0.0001, n.s. not significant). (C) Left: Patch-clamped iPSC-derived MN (arrowhead) filled with lucifer yellow (LY) and identified after recording by labelling for Islet1 (inset) and Tuj1. Scale bar 20 μm. Right: Voltage-clamp whole-cell current-voltage relationship. Top: representative trace and corresponding voltage steps; bottom: current densities plotted against voltage pulses. (D) Left, Top: Current-clamp whole-cell current-voltage relationship with current pulses applied. Bottom: table summarizing action potentials properties and resting membrane potential (RMP). Right: Synaptic inputs and zoom in on one event. Bottom: spontaneous firing of action potentials.

FIGURE 7

Coculture of human myotubes and human motor neurons. (A) Phase images of co-cultures maintained in P96 during 7 or 13 days. Scale bar: 25 µm. (B) Immunocytochemical characterization of NMJ formation. Muscular fibers are identified with Myosin/MF20 or Desmin, Motor Neurons neurites are identified with TUJ1 or SMI-32 and MN nuclei are identified with Islet 1 and Acetylcholine receptor with alpha-bungarotoxin. Nuclei are represented in blue (DAPI). Top: 20X images; scale bar: 50 µm. Bottom: 40X images; scale bar: 25 µm. (C) Effect of TTX and Tubocurarine on muscle cells contractions after 6 h of exposure. (D) Dose-response effects of BoNT/A on contraction of myotubes after 6 h of exposure. *p < 0.05; **p > 0.01; ***p < 0.001; ****p < 0.0001.