Research Project
9199485
SUMMARY/ABSTRACT Drug addiction is a complex disease. There are a variety of potential neural systems and circuits involved, some of which are physiologically or epigenetically altered through addiction. Drug abuse and addiction weigh heavily on the U.S. through health and crime?related costs, as well as broader societal issues related to safety, family, education and employment. In order to fully understand the physiological changes in the addicted brain on a cellular level we must have the proper tools for study. The first step toward creating these tools is the establishment of an in vitro system of human dopaminergic neurons. Such a system would not only enable cellular, molecular, and physiological studies into the mechanisms of addiction but also open the door to the screening and development of small molecules and drugs that can potentially block the onset of changes associated with addiction or even reverse these changes. Human pluripotent stem cells (hPSC) can be differentiated into populations of neurons enriched for dopaminergic neurons. However, current protocols yield low percentages of dopaminergic neurons and are not robust and reproducible. Additionally, most of the stem cell?to?dopaminergic neuron research is focused on Parkinson's Disease and very little effort has been dedicated to deriving the specific type of dopaminergic neurons implicated in addiction. Transfection of synthetic mRNA into cells is technology to fine?tune gene expression. This technology has been used to reprogram adult somatic cells to pluripotent stem cells and to transdifferentiate adult somatic cells to neurons and other cell types. We propose to develop novel reagents and protocols for mRNA?mediated directed differentiation of hPSC to dopaminergic neurons relevant for addiction research. This project will yield novel, cell?specific, high efficiency mRNA transfection reagents and protocols. It will result in a detailed work flow for the directed differentiation of A10 dopaminergic neurons from undifferentiated human iPSC. And finally, it will produce a platform system for the study of mechanisms of drug abuse at the cellular and molecular level, while allowing screening assays to be performed on this system. A realistic future application of this platform (Phase II) is the creation of iPSC from cells taken from addicted individuals carrying identified genetic variants, identified to be risk factors for addictive behavior. These patient?specific iPSC can then be differentiated to A10 DA neurons and studied for possible insights into how these genetic variations affect the development of addictive behavior, among other things.
