UC San Diego

Human embryonic stem cells offer unprecedented potential for the study of human development and carry a significant promise for advancing the future of medical practice. HESCs are particularly useful for the study of tissues and organ systems that exhibit poor endogenous regenerative capabilities, like the adult CNS. The corticospinal tract is part of CNS, and is required for skilled voluntary movement of the hands, fingers, and toes. Fezf2 has been identified as an important marker and regulator of the corticospinal motor neuron (CSMN) fate in mouse models, and is evolutionarily preserved from drosophila to humans. Nevertheless, the role of Fezf2 in human cortical and CST development requires further elucidation. Here, using a Fezf2:EYFP knock-in hESC reporter line, we sought to clarify the role of Fezf2 in human cortical development, and attempted to generate human CSMNs. Through directed differentiation, we were able to guide our cells through the process of cortical development. We found that Fezf2 is broadly expressed in early human cortical stem/ progenitor cells, and verified that these Fezf2 positive cells are mitotic, demonstrating that human Fezf2 expression is not limited to deep-layer post mitotic neurons. Further, we were able to verify that as differentiation progresses, so does the molecular identity of the progeny generated by these Fezf2 positive neural precursors. Gene expression analysis indicates that Fezf2 positive cells give rise to deep and superficial layer projection neurons, as well as glia, while Fezf2 negative cells give rise to other neural subtypes and cells expressing eye-associated markers. Through timing and FACS purification we were able to generate a population of post -mitotic neurons that were 94% Fezf2 positive, many of which co-expressed additional markers of deep-layer projection neurons. Finally, Fezf2 positive cells transplanted into neonatal mice were able to maintain Fezf2 expression, adopted pyramidal morphologies, and were able to correctly project down appropriate subcerebral motor pathways, even reaching the pyramidal decussation and the spinal cord.Additionally presented here is the development and implementation of two BAC constructs for use in permanently labeling populations of hESCs and their progeny