Differential expression of retinoic acid alpha and beta receptors in neuronal progenitors generated from human embryonic stem cells in response to TTNPB (a retinoic acid mimetic)

Retinoic acid (RA), an active metabolite of vitamin A, plays a critical role in the morphogenesis and differentiation of various tissues, especially in the central nervous system. RA is the most commonly used morphogen for the differentiation of human embryonic stem cells (hESCs) into neuronal progenitor cells (NPCs), an abundant source of healthy neuronal tissues for regenerative therapy. During the differentiation process, the activity of RA is governed by the involvement of RA receptor subtypes (RAR alpha, beta, and gamma) and their isoforms in the nucleus. However, little is known about the involvement of specific RAR subtypes during neuronal differentiation in humans. It is essential to elucidate the dynamic function of different RAR subtypes and their influence on the phenotypic outcome. Here in this study, we used TTNPB, an analog and stabilized form of retinoic acid that potently and selectively activates retinoic acid receptors. Here we determined the optimum concentration of TTNPBfor the efficient generation of early NPCs from hESCs. Using the optimized concentration of -TTNPB, we found that RAR alpha is the functionally dominant subtype and controls the RA-mediated neurogenesis of hESCs. Importantly, we also found that the RAR gamma subtype also played a role in neuronal differentiation. In contrast, the RAR beta subtype negatively correlates with neuronal differentiation. Therefore, pharmacological inhibition of RAR beta in the TTNPB-mediated differentiation process could be used as a strategy to generate a large number of NPCs in vitro. In summary, our results show that RAR alpha and RAR gamma play a vital role in the TTNPB-mediated neuronal differentiation of hESCs, -whereas RAR beta acts as a negative regulator.