Abstracts

Rationale: One hundred million Americans are affected by one or more neurological diseases. Developing drugs that target these pathologies relies heavily on animal studies, which require significant time, labor, and financial investments over the course of 10 years on average. Furthermore, less than 12% of compounds that pass animal studies and undergo human clinical trials gain FDA market approval. Since in vitro studies circumvent the price tag and ethical restrictions of animal work, in vitro experiments done in human cells could reduce the number of compounds tested in animals, expediting drug discovery and reducing the number of animals in research. Pluripotent stem cells are a potential source of differentiated human neuron/glia populations; however, the time necessary for differentiation limits their usefulness in drug discovery assays. Here, we optimized and characterized a protocol that consistently and rapidly differentiates neurons and astrocytes from human NTera2/cl.D1 (NT2) cells.

Methods: NT2 cells treated for six days with the nucleoside analogue cytosine-B-D-arabinofuranoside (AraC) produce a population of irreversibly post-mitotic cells (Stem Cell Res 2016 vol 16, pp 541-551). We used immunocytochemical techniques and anti-NeuN and anti-S100β to determine neuronal/astrocyte ratios after six days of treatment and up to 12 days post-treatment. We also used anti-VGlut1 and anti-GAD67 to determine neuronal excitatory/inhibitory ratios after six days of AraC treatment. Cells were cultured onto multielectrode arrays for functional and pharmacologic characterization.

Results: A six-day treatment with AraC produces a population of neurons and astrocytes that are viable for at least 12 days post-treatment. NT2-derived neurons and astrocytes were present in a 2:1 ratio. Additionally, glutamatergic and GABAergic neuronal phenotypes were present in a 4.8:1 ratio, which mimics the ratio found in vivo in the human brain. Furthermore, preliminary functional studies indicate that this cell population forms synapses and that NT2 neurons produce spontaneous action potentials.

Conclusions: This study tested the hypothesis that a six-day treatment with AraC would produce a differentiated population of neurons and astrocytes capable of forming functional networks. Our results reveal that this novel NT2-cell differentiation protocol yields an electrically-active and heterogeneous population of mostly excitatory neurons and astrocytes, all of which are stable in fresh media for at least 12 days post-differentiation. Cumulatively, these findings provide a basis against which future quantifications and characterizations may be compared.

Funding: Please list any funding that was received in support of this abstract.: NS085389, Creighton University Internal Grant.