Project Summary
Parkinson's disease (PD) is a movement disorder that involves the selective loss of midbrain dopamine (mDA)
neurons in the substantia nigra. Human stem cells, such as embryonic (hESCs) and induced pluripotent
(hiPSCs), represent a powerful technology to study and potentially treat PD. Methods to generate mDA neurons
from human stem cells have been pioneered by our group. Such work enabled applications of mDA neurons for
modeling PD in a dish and for the development of cell-based therapies. In fact, based on our work, the
transplantation of human mDA neurons is at the verge of clinical testing in PD. Despite such progress, current
strategies for generating mDA neurons are suboptimal and the resulting cells do not match all the molecular
features of mDA neurons in the brain. In addition, there are no reliable purification methods to specifically
enrich for mDA neurons. The lack of such methods is a problem, particularly in disease modeling, where mDA
neurons are compared across cell lines from many PD patients and where variability in yield can be a major
confounding factor. Furthermore, the use of purified mDA neurons will allow more precise transplantation
studies to define optimal graft composition. Another important challenge is the limited survival of mDA neurons
after transplantation (~10% of grafted cells), a problem that remains unresolved, and that can cause variability
in cell dosing and complicate the routine application of this technology. A final challenge is the lack of
knowledge how to preferentially generate mDA neurons of either A9 (substantia nigra) or A10 (ventral tegmental
area) identity. Both A9 and A10 are mDA neurons, but they represent subtypes with different molecular and
functional properties, and with A9 being the desired subtype for disease modeling and cell therapy in PD.
Here, we propose three specific aims to address these outstanding questions. In Aim1, based on exciting
preliminary data, we will refine our mDA neuron differentiation strategy to obtain mDA neurons with improved
molecular and functional properties and a sorting method that will enable routine purification of mDA neurons.
We propose the use of single cell gene expression analysis to assess whether mDA neurons under such
improved conditions more fully match mDA neurons in the developing or adult brain. In Aim 2, we will define the
factors that limit survival of mDA neurons upon cell transplantation. We have developed a very promising,
CRISPR-based screening technology to define survival factors, and already identified candidates acting either
directly within mDA neurons or via the host environment. Finally, in Aim 3, we will use single cell gene expression
and chromatin accessibility studies to map A9/A10 subtype diversity of mDA neurons from human stem cells.
The results from those in-depth single cell profiling studies will be used to identify and test factors that are
functionally important in subtype specification. Each of the three aims addresses a critical and complementary
challenge in the mDA field towards unlocking the full potential of human stem cell-derived mDA neurons for cell
therapy and human disease modeling.
