Parkinson’s Disease Modeling
Modeling Parkinson’s Disease with Human iPS Cell-derived DA Neurons
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic (DA) neurons from the pars compacta of the substantia nigra, and the formation of Lewy bodies.
- DA neurons: Produce dopamine; found in different regions in the central nervous system (CNS) with the largest concentration in the midbrain.
- Dopamine: Play a role in voluntary movement and a broad array of behavioral processes, such as mood, reward, addiction, and stress.
- Midbrain DA neurons: Located in the substantia nigra compacta (SNc) and the ventral tegmental area (VTA); send fibers to tissues in both sides of the brain.
- PD: Caused by selective degeneration of the SNc DA neurons.
Numerous animal and cellular models of PD have been established to study the molecular mechanisms, with mitochondrial dysfunction, oxidative stress, autophagy, kinase signaling, neuro-inflammation, and calcium homeostasis all being implicated. However, due to species-specific differences between rodent and human, many discoveries garnered from these models may not accurately reflect the pathogenesis seen in PD. The ability to generate unlimited numbers of human DA neurons from iPS cells offers a promising new avenue for research into PD.
The prodrug MPTP is oxidized in glial cells by monoamine oxidase B (MAO-B) into the neurotoxic species, MPP+. Neurodegeneration induced by MPP+ is specific to DA neurons as it is taken up by the cells through the dopamine transporter. It concentrates in the mitochondria leading to ATP depletion and eventually death of dopaminergic neurons in the substantia nigra. To model PD with an induced phenotype, iCell® DopaNeurons were treated with MPP+ as detailed in Figure 3.
iCell DopaNeurons from CDI, which are terminally-differentiated, human iPS cell-derived midbrain floorplate dopaminergic neurons, were treated with increasing concentrations of MPP+ and a decrease in the channel bursting rate was observed. No changes were detected upon addition of MPTP (no astrocytes present) or DMSO (vehicle control; Ctrl). Experiments were performed using the Maestro MEA (Axion BioSystems).
Several detailed applications utilizing iCell DopaNeurons are available. In addition, for instructions on using iCell neural products with MEA technology, refer to the application protocol: Measuring Synchronous Neuronal Activity on the Maestro Multielectrode Array.
Mutations in several different genes have been clearly linked to PD. Despite its low occurrence, SNCA A53T is one of the most highly penetrant and widely studied mutations. SNCA encodes for the alpha-synuclein (α-syn) protein, which is predominantly expressed in the brain at presynaptic terminals. The A53T mutation renders α-syn more susceptible to aggregation and accumulation, which are hallmark indicators of PD pathology. The combination of cutting-edge genome-editing and iPS cell technologies offers the opportunity to study patient-specific risk factors or disease-specific mutations (such as A53T) in a physiologically-relevant cell type (dopaminergic neurons) and compare the function and phenotype in a series of assays to cells derived from healthy control iPS cell lines.
To generate an isogenic A53T allelic variant, nuclease-mediated SNP alteration of a healthy control iPS cell line was performed to introduce this site-specific mutation into the gene for SNCA. This genome-engineered iPS cell was then differentiated into human midbrain floorplate dopaminergic neurons according to protocols adapted from the Lorenz Studer lab (Memorial Sloan Kettering) and industrialized at CDI.
Isogenic iCell DopaNeurons (WT control) and MyCell DopaNeurons with genotype SNCA A53T were cultured in BrainPhys Neuronal Medium (STEMCELL Technologies) for ~2 weeks. Data from the Maestro MEA (Axion Biosystems) showed >3-fold decrease in burst frequency (BPM) and ~4-fold increase in burst intensity for A53T with an equal number of active electrodes versus WT.
For further characterization of the MyCell® DopaNeurons with genotype SNCA A53T, including gene expression, mitochondrial assay, and calcium assay, view our scientific poster presented at SfN.
For ordering information, visit the MyCell catalog.
In partnership with the Michael J. Fox Foundation (MJFF) and Indiana University, CDI is deriving iPS cells from 85 different donors (with and without PD) in the MJFF-sponsored Parkinson’s Progression Markers Initiative (PPMI). These iPS cells, as well as clinical and imaging data on the donors, will be made available to the scientific community. For more information on CDI’s stem cell banking initiatives, including the CIRM iPSC Repository, visit the MyCell searchable catalog.