Investigation of early neurogenic proceedings and neuronal subtypes' specification during in vitro neurogenesis using embryonic stem cell model

Abstract

Embryonic stem cells (ESC) derived dopaminergic (DA) neurons have been used in reversing the Parkinson Disease (PD) symptoms in rodents. One of the major limitations associated with ESCs, hindering their clinical applications is their tendency to generate asynchronous culture during differentiation. Therefore, their efficient and safer use would require isolating the cells of interest from this culture. Here, we have deduced a strategy to demarcate and isolate the ventral mesencephalic (VM) neural progenitors. Three types of ESC clones (MB1, MB2 & MB3) have been generated using different intron2 regions of nestin, regulating EGFP expression to demarcate VM progenitors during differentiation. Based on their EGFP intensity, they display two hierarchical populations of neural progenitors; early and late. MB2 progenitors are more specific for nigrostriatal DA neurons, while MB3 the least specific. These DA neurons display functionality as they release DA upon activation. Moreover, transplantation of all these progenitors demonstrates functional recovery in PD animal models with their efficiency of recovery and engraftment in the tissue highly dependent on their midbrain specificity. Thus, our study illustrates that specific elements within nestin intron2 can be used to track VM DA progenitors during ESCs differentiation. Further, Notch signalling has been studied to understand the developmental mechanisms involved during neural differentiation for augmenting a particular population in culture. ESCs during maintenance remain impervious to Notch inhibition. However, Notch activation promotes differentiation even in presence of LIF displaying ligand preference associated lineage discrimination; where Jagged-1 favours neural commitment and Delta-like-4, the mesoderm. This differential ligands action involves a combination of Notch receptors influencing specific downstream target genes expression. Though Notch activation during early neural differentiation specifically promotes neural stem cells or early neural progenitors and delays their maturation; its inhibition promotes late neural progenitors and expedites neurogenesis preferring neurons to glia. However, gliogenesis is promoted upon Notch activation only when executed in combination with ciliary neurotrphic factor. Thus, our investigation also underscores a multifaceted role of Notch demonstrating the interdependency of ligand usage and lineage specification and Notch acting as a master switch displaying stage specific influence on neurogenesis. Overall, this study emphasizes the importance of ESCs as an elegant developmental model in studying early neurogenesis in vitro, and also in the isolation of region specific neuronal progenitors possessing therapeutic potency.