Article Figures & Data
Figures
- Figure 1.
Experimental pipeline for the identification of factors essential for SNc development in vivo and the systematic study of their potential in stem cell therapy for Parkinson's disease. Novel SNc-specific factors can be identified by global expression analysis of mdDAn subsets. Since no specific reporter lines exist to purify developing SNc neurons, these factors can be identified based on transcriptome analysis after anatomical separation, by single-cell RNA-Seq of mdDAns, and/or by analysis of mutants that display SNc-specific degeneration. Novel putative markers can be used to generate reporter lines, which in turn can be used to explore the SNc-specific transcriptome in more detail. In the next phase, putative subset-specific factors, preferably secreted factors for their easy application and therapeutic potential, should be tested for their ability to protect against SNc-mdDAn degeneration in vitro (e.g., in ventral midbrain primary cultures of mutant mice), as well as their ability to increase SNc-mdDAn yield from PSCs in vitro. An appropriate control condition for such an experiment would consist of a standard protocol that generates bona fide mdDAns. It may be critical to test different dosages, combinations, and timing schemes, which will require a high-throughput experimental system. This can be achieved by the use of reporter lines carrying fluorescent proteins under the control of an SNc-specific marker, in combination with a pan-specific mdDAn marker (e.g., PITX3-GFP), which can be used in combination with FACS to assess both mdDAn differentiation efficiency and shifts in SNc/VTA-mdDAn ratio. Finally, behavioral recovery in animal models of PD upon grafting of PSC-derived SNc-mdDAns needs to be demonstrated, and PSC-derived SNc-mdDAns should be compared with their in vivo counterpart in terms of their physiology, epigenome, and transcriptome to assess that they are bona fide SNc-mdDAns.
