Nuclear transfer embryonic stem cells provide an in vitro culture model for Parkinson's disease

Somatic cell nuclear transfer enables the generation of embryonic stem cells (ESCs) that genetically match the donor and can be used to treat disease through cell replacement therapies or to recapitulate patient-specific disease via in vitro differentiation. We performed a "proof-of-principle" study using tail tip fibroblasts from a mouse model of Parkinson's disease (Aphakia) as the donor cell nuclei for nuclear transfer and derived "customized" ESCs for in vitro analysis. Aphakia mice contain deletions in the pitx3 gene and show selective loss of dopamine neurons of the substantia nigra, specifically the neuron population susceptible to degeneration in Parkinson's disease. Using electrofusion nuclear transfer, we produced cloned Aphakia oocytes at rates similar to those for control, cloned oocytes. Aphakia ESCs were isolated and live mice were generated using tetraploid embryo complementation. In vitro differentiation of Aphakia ESCs to dopaminergic neurons revealed significantly fewer TH+ neurons that expressed MAP2, DAT, synaptophysin, VMAT2, and AHD2 compared to control nuclear transfer ESC cultures, supporting a role for Pitx3 in mesodiencephalic dopamine neuron maturation. Taken together, our studies define a customized in vitro ESC culture system used to analyze gene-specific contribution to dopamine neuron generation, maturation, and susceptibility to degeneration.