One of the newest CDI additions, iCell Neurons, represents a population of cryopreserved human iPSC-derived neurons that, upon reanimation and culture, quickly display a typical neuronal morphology as shown through the presence of dense axonal and dendritic processes (Figure 1A). These human neurons, once plated onto tissue culture plates pre-coated with standard neuron substrates (i.e., a Poly-L-ornithine (PLO)/Laminin double coating), provide an adherent single-cell morphology.
Post-thaw, iCell Neurons develop branched networks within 24 hours and remain viable and adherent for an extended period in culture (≥14 days). The stable morphology of iCell Neurons is an important attribute for drug discovery, as it facilitates the use of these cells in various assay platforms.
Phenotypically, iCell Neurons represent a highly pure population as assayed through immunostaining for the presence of class III beta-tubulin (TuJ1) and the absence of the type VI intermediate filament nestin, a marker of neural stem/progenitor cells. As quantified through flow cytometry (Figure 1B), iCell Neurons display >90% positive staining for TuJ1 with very low levels of nestin immunopositivity.
This population of neurons is predominantly composed of a mix of GABAergic and Glutamatergic subtypes as tested through staining for the mature synaptic markers vGAT (vesicular GABA transporter) and vGLUT2 (vesicular glutamate transporter 2) (data not shown). In addition, double immunostaining of iCell Neurons for the microtubule-associated protein 2 (MAP2) and the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) reveals ~50% of the population displaying a GABAergic phenotype (Figure 1C).
To serve as a viable neuroscience discovery model, it is imperative that iCell Neurons provide a robust, consistent, and highly pure cell product amenable to common discovery applications. These applications may include high-content imaging, automated electrophysiology, and cell-based assays.
For example, iCell Neurons cultured for 7–14 days post-thaw on PLO/Laminin pre-coated 96-well plates exhibit an expected sensitivity to known compounds including the ATP-competitive kinase inhibitor staurosporine and the phenothiazine antipsychotic chlorpromazine as assayed using the Cell-Titer-Glo® Luminescent Cell Viability Assay (Promega), a measure of metabolically active cells (Figure 1D).
An analysis of electrophysiological characteristics reveals that iCell Neurons possess a functional phenotype. Evoked action potentials from these cells display an average resting membrane potential of -46 mV within 17 days post-thaw (n=12). All action potentials demonstrate an overshoot of the depolarization phase above 0 mV and an undershoot of the repolarization phase below baseline before correction to steady-state (Figure 2A). Also, iCell Neurons are capable of firing spontaneous action potentials with the same characteristics seen in the evoked action potentials (Figure 2B). Addition of the classic neuron ion-channel antagonists tetraethylammonium (TEA, 30 mM) and tetrodotoxin (TTX, 100 nM) effectively block outward potassium and inward sodium currents, respectively (Figure 2C, D).
In summary, CDI’s iCell Neurons represent a robust in vitro cell model, not only providing the expected phenotype associated with human neurons but also possessing functional electrophysiological attributes. These human iPSC-derived cells provide a more relevant alternative to the current cell models commonly employed in drug discovery workflows.