Isolation of LMX1a Ventral Midbrain Progenitors Improves the Safety and Predictability of Human Pluripotent Stem Cell-Derived Neural Transplants in Parkinsonian Disease

Human pluripotent stem cells (hPSCs) are a promising resource for the replacement of degenerated ventral midbrain dopaminergic (vmDA) neurons in Parkinson's disease. Despite recent advances in protocols for the in vitro generation of vmDA neurons, the asynchronous and heterogeneous nature of the differentiations results in transplants of surprisingly low vmDA neuron purity. As the field advances toward the clinic, it will be optimal, if not essential, to remove poorly specified and potentially proliferative cells from donor preparations to ensure safety and predictable efficacy. Here, we use two novel hPSC knock-in reporter lines expressing GFP under the LMX1A and PITX3 promoters, to selectively isolate vm progenitors and DA precursors, respectively. For each cell line, unsorted, GFP(+), and GFP(-) cells were transplanted into male or female Parkinsonian rodents. Only rats receiving unsorted cells, LMX1A-eGFP(+), or PITX3-eGFP(-) cell grafts showed improved motor function over 6 months. Postmortem analysis revealed small grafts from PITX3-eGFP(+) cells, suggesting that these DA precursors were not compatible with cell survival and integration. In contrast, LMX1A-eGFP(+) grafts were highly enriched for vmDA neurons, and importantly excluded expansive proliferative populations and serotonergic neurons. These LMX1A-eGFP(+) progenitor grafts accelerated behavioral recovery and innervated developmentally appropriate forebrain targets, whereas LMX1A-eGFP(-) cell grafts failed to restore motor deficits, supported by increased fiber growth into nondopaminergic target nuclei. This is the first study to use an hPSC-derived reporter line to purify vm progenitors, resulting in improved safety, predictability of the graft composition, and enhanced motor function.