Neural stem cells (NSCs) are defined by their ability to self-renew and to differentiate into neurons, astrocytes and oligodendrocytes. However, during CNS development, the generation of neurons and glia is temporally regulated. In early embryonic development, NSCs are restricted and predominantly differentiate into neurons. Transitioning from early to late gestation, NSCs become competent towards glial differentiation. This suggests the presence of an epigenetic switch triggering the onset of glial production. Timing of the switch can be recapitulated in vitro;using primary or embryonic stem cell (ESC) derived NSCs. In human ESCs the switch towards efficient glial cell production occurs at 2-3 months after differentiation, a protracted time frame that represents a major practical hurdle for the application of glial cells in disease modeling and regenerative medicine. Here we propose to identify the mechanisms involved in the switch from neurogenic to gliogenic NSCs. Towards this goal we will establish a glial specific reporter human ESC lines targeting the glial fibrillary aciic protein (GFAP) and the aquaporin 4 (AQP4) to identify astrocytes and glial competent NSCs. Our preliminary results suggest that the several factors, including transcription factors as well a microRNA regulators, are differentially regulated in early versus late NSCs. Initially we will functionally test our candidate proteins for the ability to activate the gliogenic program in early NSCs. The reporter cell lines will serve as readout for a large-scale shRNA screen, aimed at identifying novel candidates that mediate the epigenetic switch in NSCs. Additionally, we found that the epidermal growth factor receptor (EGFR) is expressed in NSCs that correlate with glial competency. We will utilize EGFR to distinguish gliogenic NSCs (EGFR+ GFAP+) from glial cells (EGRF- GFAP+). Overall, the proposed studies are designed to yield novel insights into CNS fate choice and further our understanding of glial cell biology, ultimately identifying factors that may accelerate their differentiation from human pluripotent cells.
The overall goal of this multidisciplinary proposal is to build reporter lines and use them to identify factors that aid in the acceleration of glial differentiatin for modeling neurodegenerative diseases. Currently, a major hurdle in using human glial cells to model neurodegenerative disease is the prolonged timeframe it takes to derive these cells. Glial cells make up the majority of cells in the brain and have been recently implicated to be involved in neurodegenerative diseases, our studies will shed light on the mechanisms involved in acquiring glial cell fate in order to effectively use these cells in disease modeling studies.
