Characterizing an AraC-Differentiated, Heterogeneous NT2-Derived Population

Abstract

Background & Problem: 100 million Americans are affected by one or more neurological diseases. The initial stages of new pharmacotherapy identification rely heavily on in vitro or in vivo studies (e.g., animals or animal tissues such as primary cultures of isolated neurons)—the latter of which entails significant financial, labor, and time investment. There exist in vitro culture systems that bypass the investments and restrictions inherent to animal research.

Aim: The current study aims to characterize one such novel in vitro culture system that reduces these time and financial costs.

Methods & Results: NTera2/cl.D1 (NT2) cells, a clonally-derived pluripotent human embryonal carcinoma cell line, are known to differentiate into functional neurons following treatment with retinoic acid (RA) for 4 weeks followed by another 4 weeks of treatment with MI (totaling 56 days of treatment). Here, based on work by González-Burguera et al. (2016), we found that treatment with 20μM of the nucleoside analogue cytosine-B-D-arabinofuranoside (AraC) for 6 days resulted in differentiated neurons and astrocytes that were viable for at least 12 days post-differentiation.

Conclusions: Using immunofluorescence (IFC), we determined that NT2 neurons (NT2N; anti-NeuN) and astrocytes (NT2A; anti-S100β) were present in a 2:1 ratio, respectively. Furthermore, we found that the NT2N cells consisted of glutamatergic (anti-VGluT1) and GABAergic (anti-GAD67) subtypes in a 4.8:1 ratio, respectively, which is similar to the ratio found in vivo. Preliminary functional studies performed via electrophysiology indicate that these cells possess the machinery for spontaneous action potentials. Development of this in vitro system of accelerated neuronal network establishment will improve costs associated with drug screening and potentially reduce the use of animals in preclinical studies.