Efficient in vitro differentiation into specific cell types is more important than ever after the breakthrough in nuclear reprogramming of somatic cells and its potential for disease modeling and drug screening. Key success factors for neuronal differentiation are the yield of desired neuronal marker expression, reproducibility, length, and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation, embryoid body (EB) differentiation, and direct neuronal differentiation. Here, we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC) lines from patients with Parkinson's disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage.

The protein α-synuclein is involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Its toxic potential appears to be enhanced by increased protein expression, providing a compelling rationale for therapeutic strategies aimed at reducing neuronal α-synuclein burden. Here, feasibility and safety of α-synuclein suppression were evaluated by treating monkeys with small interfering RNA (siRNA) directed against α-synuclein. The siRNA molecule was chemically modified to prevent degradation by exo- and endonucleases and directly infused into the left substantia nigra. Results compared levels of α-synuclein mRNA and protein in the infused (left) vs. untreated (right) hemisphere and revealed a significant 40–50% suppression of α-synuclein expression. These findings could not be attributable to non-specific effects of siRNA infusion since treatment of a separate set of animals with luciferase-targeting siRNA produced no changes in α-synuclein. Infusion with α-synuclein siRNA, while lowering α-synuclein expression, had no overt adverse consequences. In particular, it did not cause tissue inflammation and did not change (i) the number and phenotype of nigral dopaminergic neurons, and (ii) the concentrations of striatal dopamine and its metabolites. The data represent the first evidence of successful anti-α-synuclein intervention in the primate substantia nigra and support further development of RNA interference-based therapeutics.

α-Synuclein expression is increased in dopaminergic neurons challenged by toxic insults. Here, we assessed whether this upregulation is accompanied by pathological accumulation of α-synuclein and protein modifications (i.e. nitration, phosphorylation and aggregation) that are typically observed in Parkinson disease and in other synucleinopathies. A single injection of the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to squirrel monkeys caused a buildup of α-synuclein but not of β-synuclein or synaptophysin within nigral dopaminergic cell bodies. Immunohistochemistry and immunoelectron microscopy (IEM) also revealed large numbers of dystrophic axons labeled with α-synuclein. Antibodies that recognize nitrated and phosphorylated (at serine 129) α-synuclein stained neuronal cell bodies and dystrophic axons in the midbrain of MPTP-treated animals. Following toxicant exposure, α-synuclein deposition occurred at the level of neuronal axons in which amorphous protein aggregates were demonstrated by IEM. In a subset of these axons, immunoreactivity for α-synuclein was evident after tissue digestion with proteinase K, further indicating the accumulation of insoluble protein. These data indicate that toxic injury can induce the α-synuclein modifications that have been implicated in the pathogenesis of human synucleinopathies. The findings are also consistent with a pattern of evolution of α-synuclein pathology that may begin with the accumulation and aggregation of the protein within damaged axons.