About the Project
Parkinson’s disease (PD) is an incurable neurodegenerative disorder that affects between 8-10 million people worldwide. Grafting of human fetal midbrain tissue into the striatum of PD patients has demonstrated that a transplantation therapy can restore dopamine transmission to normal levels and significantly reduce morbidity for at least 15 years. However, fetal tissue has considerable ethical and logistical challenges precluding its widespread therapeutic application. An alternate cell source is clinical-grade midbrain dopaminergic (mDA) cells differentiated from human pluripotent stem cells (hPSCs)1,2. We have established a new mDA cell production paradigm that is precise, robust, and cost-effective through development of tools and methodologies to scale up hPSC-based therapies for PD. However, the midbrain region is very complex with three major dopaminergic systems; A8 (retrorubral area), A9 (substantia nigra), and A10 (ventral tegmental area), with the A9 neurons most severely affected in PD. The A9 nigral neurons can also be subdivided into regions, such as dorsal and ventral tier of the SN, and it is unclear whether they have differing efficacy upon transplantation. Recent single-cell molecular profiling has described at least 6 distinct mDA subtypes in rodents3. In order to characterize and quantify the different mDA subtypes present in human ESC/iPSC differentiated cultures, we will take advantage of emerging single-cell transcriptomic technologies. We expect to find a similar level of complexity, if not more, in our human hPSC-derived mDA populations. Combinatorial expression of known (PITX3, NURR1, EN1) and novel mDA-specific transcription factors and cell surface proteins will be used to dissect the heterogeneity of cell types in hPSC differentiated cultures, and determine which combinations of cells are most effective at restoring dopaminergic function in rodent models of PD.
Techniques
Human pluripotent stem cell culture
Differentiation of midbrain dopaminergic neurons
Immunofluorescence, confocal microscopy
Quantitative RT-PCR, RNA-seq, single cell RNA-seq
Fluorescence activated cell sorting (FACS)
Funding Notes
This project will be based at the Centre for Regenerative Medicine for instructions on how to apply please view http://www.crm.ed.ac.uk/join-us/phd-training/how-apply-phd
This project is part of a funding competition. Due to fund source regulations, this studentship is only available to UK and EU nationals. For information on eligibility and stipend amounts, please view http://www.mrc.ac.uk/skills-careers/studentships/studentship-guidance/
References
Devine MJ, Ryten M, Vodicka P, Thomson AJ, Burdon T, Houlden H, Cavaleri F, Nagano M, Drummond NJ, Taanman JW, Schapira AH, Gwinn K, Hardy J, Lewis PA, Kunath T. (2011) Parkinson's disease induced pluripotent stem cells with triplication of the α-synuclein locus. Nat Commun. 2:440
Canham MA, Van Deusen A, Brison DR, De Sousa PA, Downie J, Devito L, Hewitt ZA, Ilic D, Kimber SJ, Moore HD, Murray H, Kunath T. (2015) The Molecular Karyotype of 25 Clinical-Grade Human Embryonic Stem Cell Lines. Sci Rep. 5:17258
Poulin JF, Zou J, Drouin-Ouellet J, Kim KY, Cicchetti F, Awatramani RB. (2014) Defining midbrain dopaminergic neuron diversity by single-cell gene expression profiling. Cell Rep. 9:930-43
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