Embryonic stem (ES) cells are pluripotent cells that can differentiate into all three main germ layers: endoderm, mesoderm, and ectoderm. Although a number of methods have been developed to differentiate ES cells into neuronal phenotypes such as sensory and motor neurons, the efficient generation of GABAergic interneurons from ES cells still presents an ongoing challenge. Because the main output of inhibitory GABAergic interneurons is the gamma-aminobutyric-acid (GABA), a neurotransmitter whose controlled homeostasis is required for normal brain function, the efficient generation in culture of functional interneurons may have future implications on the treatment of neurological disorders such as epilepsy, autism, and schizophrenia. The goal of this work was to examine the generation of GABAergic neurons from mouse ES cells by comparing an embryoid body-based methodology versus a hydrogel-based encapsulation protocol that involves the use of all-trans-retinoid acid (RA). We observed that 1) there was a 2-fold increase in neuronal differentiation in encapsulated versus non-encapsulated cells and 2) there was an increase in the specificity for interneuronal differentiation in encapsulated cells, as assessed by mRNA expression and electrophysiology approaches. Furthermore, our results indicate that most of the neurons obtained from encapsulated mouse ES cells are GABA-positive (~87%). Thus, these results suggest that combining encapsulation of ES cells and RA treatment provide a more efficient and scalable differentiation strategy for the generation in culture of functional GABAergic interneurons. This technology may have implications for future cell replacement therapies and the treatment of CNS disorders.

Embryonic Stem (ES) cells are pluripotent cells that can be induced to differentiate into cells of all three lineages: mesoderm, endoderm, and ectoderm. In culture, ES cells can be differentiated into mature neurons by treatment with Retinoic Acid (RA) and this effect is mediated mainly through the activation of the RA nuclear receptors (RAR α, β, and γ), and their isoforms. However, little is known about the role played by specific RAR types on ES cell differentiation. Here, we found that treatment of ES cells with AC55649, an RARβ2 agonist, increased endodermal marker gene expression. On the other hand, we found that the inhibition of RARβ with 5 μM LE135, together with RA treatment, increased the efficiency of mouse ES cell differentiation into neurons by more than 4-fold as compared to cells treated with RA only. Finally, we performed proteomic analyses on ES cells treated with RA vs RA plus AC55649 in order to identify the signaling pathways activated by the RARβ agonist. Our proteomic analyses using antibody microarrays indicated that proteins such as p38 and AKT were upregulated in cells treated with RA plus the agonist, as compared to cells treated with RA alone. Our results indicate that RARβ may function as a repressor of neuronal differentiation through the activation of major cell signaling pathways, and that the pharmacological inhibition of this nuclear receptor may constitute a novel method to increase the efficiency of ES to neuronal differentiation in culture.