Midbrain dopaminergic (mDA) neurons residing in the ventral midbrain critically control voluntary movement, reward, and mood-related behaviors, and their degeneration/dysfunction is associated with major brain disorders such as Parkinson's disease (PD) and schizophrenia. In order to dissect the molecular and cellular mechanisms of mDA-related diseases and advance the development of novel therapeutics, it is critical to develop reliable and efficient disease model systems. A promising model system is the recently established induced pluripotent stem cell technology that potentially can provide unlimited cell sources with normal vs. disease phenotypes. Pluripotent stem cells, though able to be induced to differentiate to the mDA phenotype, tend to generate stochastic and heterogeneous differentiated progenies, which can obscure assay results and comparisons. Thus, it is essential to purify mDA cells prior to usage in order to guarantee a reliable and specific cell source for further application. During early brain development, regulatory cascades by key signals and transcription factors orchestrate intricate differentiation pathways in which specific neural precursors (NPs) are generated in different areas, leading to consequential differentiation to final subtype of neurons such as mDA neurons. Identification and isolation of such mDA-specified NPs will provide expandable cell sources that can readily generate mature mDA neurons. Even though no known single marker is available to purify mDA NPs, we hypothesize that mDA NPs can be identified and purified based on the knowledge gained from developmental studies of mDA neurons. mDA neurons were shown to be derived from the floor plate, which is specifically identified by the expression of the cell surface marker Corin. In addition, Frizzled-5 (Fzd5), the receptor for Wnt5a, is expressed in the forebrain and the ventricular zone of the midbrain in the developing CNS. Thus, the expression of these two genes overlaps only in the mDA domains in developing embryo. Based on these findings, we propose to purify human mDA NPs by the co-expression of these two surface markers from human ESC-derived and human iPSC-derived NPs. Purified human mDA NPs will be further characterized in vitro and in vivo for their proliferative and developmental potentials as well as their functionality following transplantation into a rodent model of PD. Our proposed experiments will identify and characterize human mDA NPs that will allow unprecedented dissection of mDA biology as well as serve as a platform to develop novel therapeutic approaches.

Public Health Relevance

Midbrain dopamine (mDA) neurons play critical roles in the regulation of voluntary movement, emotion, and reward-related behavior;their degeneration and/or dysfunction is associated with major brain disorders such as Parkinson's disease (PD), schizophrenia, and drug addiction. The proposed studies are aimed at isolating a pure population of mDA neuronal precursors (NPs) after in vitro differentiation of human pluripotent stem cells, and characterize their in vivo and in vitro proliferative, developmental and functional potential. These studies will not only facilitate further understanding of mDA NP biology but also open the door to novel therapeutic approaches for PD and other DA-related disorders.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Exploratory/Developmental Grants (R21)
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Clinical Neuroplasticity and Neurotransmitters Study Section (CNNT)
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Sutherland, Margaret L
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Mclean Hospital
United States
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