Year 3 + NCE

A major goal of stem cell research is to generate various functional human cell types from stem cells both for developing cell transplantation therapies and for better understanding human biology. Our lab studies the repair of central nervous system after injury and in particular spinal cord injury. To complement our studies of the molecular control of axon regeneration using animal models of spinal cord injury, we have been developing ways to derive human corticospinal motor neurons from human embryonic stem cells through this CIRM funded project. These neurons are of paramount importance to skilled voluntary movement in humans, loss or damage of which leads to paralysis and disability in patients of spinal cord injury and amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease). We took advantage of a reporter line we developed with a prior CIRM SEED grant to generate human corticospinal motor neurons. This reporter line carries a fluorescent reporter gene under the control of an endogenous gene encoding a molecular marker and determinant of corticospinal motor neurons, Fezf2. The idea was that whenever the cells carrying the reporter gene lights up – literally, we would know the cells are expressing Fezf2. Using this approach, we have learned quite a bit about human cells that express Fezf2. First, there are a large population of human neural stem cells that express Fezf2 early in neural differentiation, which is likely mirrored in human development. Fezf2 positive neural stem cells can become Fezf2 positive neurons, but they can also become Fezf2 negative neurons. On the contrary, Fezf2 negative stem/progenitor cells do not become Fezf2 positive neurons. During neural differentiation in culture starting from human embryonic stem cells, Fezf2 expression is dynamic. Earlier Fezf2-expressing neural stem cells have different properties from the late Fezf2-expressing neural stem cells in that they have different capabilities to turning into differential neuronal types. Particular in this last year of funding, we conducted in-depth characterization of the molecular signature of Fezf2 positive and negative neural stem/progenitor cells, as well as neurons that had been derived from these progenitors, at different times in differentiation. Hierarchical cluster analysis not only provided new insights on the different cell populations in the differentiation culture over time but also on the different molecular markers based on studies in mice. We have also extended our in vivo transplantation studies to determine how well these neural progenitor cells may survive and integrate into the mouse nervous system. The data indicate that neural progenitors that express the fluorescence reporter can survive, integrate into the host nervous system and send out extensive axonal trajectories. Some axons grew along the appropriate paths expected for corticospinal and related subcerebral projection neurons while others appear to wonder off the course. These data indicate that one challenge in future research will be to elucidate mechanisms of control the re-connection of transplanted stem cell derivatives with appropriate host targets when cell transplantation therapies are used to replace lost or damaged neurons in disease or injury.