Retinoic Acid-FGF Antagonism during Motor Neuron Differentiation of Human ES Cells
Retinoic acid is a metabolic derivative of vitamin A that has recently been shown to stimulate differentiation of human embryonic stem cells into motor neurons. However, almost nothing is known about how retinoic acid may perform this function. The recent discovery that retinoic acid antagonizes the action of fibroblast growth factor suggests a possible mechanism for retinoic acid function during motor neuron differentiation. We plan to use our knowledge of retinoic acid-fibroblast growth factor interaction to understand how retinoic acid stimulates human embryonic stem cells to go down the motor neuron lineage. Such knowledge will allow us to devise rational strategies for optimal use of retinoic with other reagents to reliably differentiate human embryonic stem cells into motor neurons. Our studies will contribute to the development of cell-replacement therapies for motor neuron loss in patients with amyotrophic lateral sclerosis or spinal cord injury. We plan to study the effect of retinoic acid on differentiation of human embryonic stem cell lines that are ineligible for federal funding. Because all of the human embryonic stem cell lines approved for federal funding were generated using methods that involved mixing human cells with mouse feeder cells, they are poorly suited for clinical use and are likely to have undergone modifications with unpredictable consequences. Given the scarcity of federally-approved human embryonic stem cell lines, and recognizing that cells passaged for long periods of time in culture tend to become genetically unstable, it is necessary to have the means of characterizing new human embryonic stem cell lines. The studies proposed here on human embryonic stem cell lines that are ineligible for federal funding will provide new insight into how retinoic acid and fibroblast growth factor can best be used to generate motor neurons for therapeutic purposes.
The studies we perform should benefit the state of California in several ways:(1) We hope to increase the ability to generate motor neurons that can be used in cell-replacement therapies for motor neuron loss in patients with amyotrophic lateral sclerosis or spinal cord injury. This will directly benefit patients in California and elsewhere.(2) The human embryonic stem cell research we perform may bring new biotechnology jobs to California, thus increasing the state’s visibility as a leader in stem cell technology.(3) New therapeutic methods developed using our discoveries could bring revenues to California due to the ability of the state to obtain licensing fees on technology generated using CIRM funds.
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.
- PLoS Biol (2011) Retinoic Acid functions as a key GABAergic differentiation signal in the Basal Ganglia. (PubMed: 21532733)
- Cell (2008) Retinoic acid synthesis and signaling during early organogenesis. (PubMed: 18805086)