Neural stem cells (NSCs) can propagate in vitro while retaining ability to differentiate into neurons, astrocytes and oligodendrocytes. However, because of the nature of their developmental progression, NSCs isolated at different developmental stages or regions exhibit remarkable differences in their ability to yield specific neuron types. Specifically, NSCs grown in vitro rapidly lose access to the full neuronal spectrum. Limited NSC potential is one of the major impediments for applications in regenerative medicine, specifically for Parkinson’s and motoneuron-related diseases. Therefore, there is an enormous need to understand how NSCs self-renew.Human embryonic stem cells (hESCs) are known to provide access to early neural fates. We have recently isolated a novel type of early NSCs derived from hESCs termed rosette-NSCs (R-NSCs), which respond to such regional patterning cues. We proved the unique NSC stage of R-NSCs based on their cytoarchitecture, marker expression, stem cell properties and differentiation potential. Nevertheless, we only partially identified growth requirements and signaling pathways governing the R-NSCs stage.Here we would like to address these limitations by defining heterogeneity within R-NSCs and develop genetic strategies to prospectively isolate fully patternable R-NSCs. Generating transgenic hESC reporter lines will serve as reliable readout for defining R-NSC stage, identity and function, and will be used for establishing correlative genome-wide chromatin state and promoter methylation maps. Finally we will systematically probe function of extrinsic/intrinsic factors affecting R-NSC identity, neural patterning potential and epigenetic state. This should provide fundamental insights into the genetic/epigenetic mechanisms of neural patterning and ultimately result in novel conditions for the continued in vitro expansion of fully patternable R-NSC - a key step towards establishing a stable expandable universal NSC population.