Year 2

Retinoic acid is a metabolic derivative of vitamin A that stimulates differentiation of human embryonic stem cells into neurons in vitro. However, the types of neurons generated by retinoic acid treatment can vary greatly depending on the in vitro cell culture conditions, the amount of retinoic acid used, when it is added during cell culture, and for how long the cells are treated. This project was designed to examine in more detail the ability of retinoic acid to stimulate differentiation of neurons that control motor function. In the first year of this project we learned how to maintain human embryonic stem cells in an undifferentiated state for long-term culture and how to differentiate these stem cells into neurons. We learned how to differentiate human embryonic stem cells into spinal cord motor neurons using previously published techniques involving treatment with retinoic acid and other compounds. During our initial studies we also discovered a technique for differentiating human embryonic stem cells into forebrain GABAergic neurons which are known to control motor function through an inhibitory mechanism. Defects in GABAergic neurons are believed to be involved in Huntington’s disease, a severe neurologic disorder that arises during early to middle adulthood and ultimately leads to death caused by an inability to control motor functions. As methods to generate GABAergic neurons from human embryonic stem cells have not previously been described, we further investigated this significant discovery. We found that human embryonic stem cells allowed to detach from the culture dish and form embryoid bodies were much more efficient in generating GABAergic neurons than cells attached to the culture dish. We also found that retinoic acid worked best when used at 10 uM concentration with treatment limited to day 3 to day 6 after initiation of embryoid body formation, resulting in generation of GABAergic neurons within 28 days. Such knowledge will be helpful to devise rational strategies for optimal use of retinoic acid to reliably differentiate human embryonic stem cells into large numbers of GABAergic neurons. Our studies will contribute to the development of cell-replacement therapies for neuron loss in patients with Huntington’s disease.