Directing fate, subtype identity and survival in human pluripotent-derived midbrain dopamine neurons

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.