Derivation, Characterization, and Neural Differentiation of Integration-Free Induced Pluripotent Stem Cell Lines from Parkinson's Disease Patients Carrying SNCA, LRRK2, PARK2, and GBA Mutations

We report generation of induced pluripotent stem cell (iPSC) lines from ten Parkinson's disease (PD) patients carrying SNCA, PARK2, LRRK2, and GBA mutations, and one age-matched control. After validation of pluripotency, long-term genome stability, and integration-free reprogramming, eight of these lines (one of each SNCA, LRRK2 and GBA, four PARK2 lines, and the control) were differentiated into neural stem cells (NSC) and subsequently to dopaminergic cultures. We did not observe significant differences in the timeline of neural induction and NSC derivation between the patient and control line, nor amongst the patient lines, although we report considerable variability in the efficiency of dopaminergic differentiation among patient lines. We performed whole genome expression analyses of the lines at each stage of differentiation (fibroblast, iPSC, NSC, and dopaminergic culture) in an attempt to identify alterations by large-scale evaluation. While gene expression profiling clearly distinguished cells at different stages of differentiation, no mutation-specific clustering or difference was observed, though consistent changes in patient lines were detected in genes associated mitochondrial biology. We further examined gene expression in a stress model (MPTP-induced dopaminergic neuronal death) using two clones from the SNCA triplication line, and detected changes in genes associated with mitophagy. Our data suggested that even a well-characterized line of a monogenic disease may not be sufficient to determine the cause or mechanism of the disease, and highlights the need to use more focused strategies for large-scale data analysis

Mitochondrial Alterations by PARKIN in Dopaminergic Neurons Using PARK2 Patient-Specific and PARK2 Knockout Isogenic iPSC Lines

In this study, we used patient-specific and isogenic PARK2-induced pluripotent stem cells (iPSCs) to show that mutations in PARK2 alter neuronal proliferation. The percentage of TH+ neurons was decreased in Parkinson’s disease (PD) patient-derived neurons carrying various mutations in PARK2 compared with an age-matched control subject. This reduction was accompanied by alterations in mitochondrial:cell volume fraction (mitochondrial volume fraction). The same phenotype was confirmed in isogenic PARK2 null lines. The mitochondrial phenotype was also seen in non-midbrain neurons differentiated from the PARK2 null line, as was the functional phenotype of reduced proliferation in culture. Whole genome expression profiling at various stages of differentiation confirmed the mitochondrial phenotype and identified pathways altered by PARK2 dysfunction that include PD-related genes. Our results are consistent with current model of PARK2 function where damaged mitochondria are targeted for degradation via a PARK2/PINK1-mediated mechanism

Pluripotency verification of PD patient iPSC.

<p>A: Pluripotency score for iPSC lines A6, B119, I3, K20, P1, S110, T101,L1 and Y09. ESC H9 line served as a positive control, whereas fibroblasts from the healthy subject (Y line) were used as a negative control. Pluripotency range is depicted in red, whereas non-pluripotent range is shown in blue. B: Novelty score for tested samples. Low novelty score (green bars) is characteristic for pluripotent cell lines, whereas high novelty sore (red) highlights sample that deviated from the pluripotent transcriptional signature. C: Hierarchical clustering of all samples. D: Combination of pluripotency and novelty scores illustrates that iPSC and ESC samples are grouped together (red background—high pluripotency and low novelty scores). Y fibroblast line had the opposite result (blue background—low pluripotency and high novelty scores).</p

Integration-free iPSC lines generated from PD patient and control fibroblasts.

<p>Integration-free iPSC lines generated from PD patient and control fibroblasts.</p

Gene up or down regulated in <i>SNCA</i> triplication dopaminergic cultures relative to healthy control.

<p>Gene up or down regulated in <i>SNCA</i> triplication dopaminergic cultures relative to healthy control.</p

Microarray gene expression data quality control.

<p>A: the number of genes detected at p-value < 0.05 (red line) and p-value < 0.01 (blue line). Detection p-value is a measurement of confidence that a given transcript is expressed above the background level. B: Sample quality assessment by comparison of 95^th signal intensity values (red line) and signal-to-noise ratio (blue line) across samples. Signal-to-noise ratio is calculated as a ration of 95^th and 5^th percentile (p95/p05) in non-normalized data. C: Hierarchical clustering of samples after normalization and averaging of biological replicates.</p

Neural and dopaminergic differentiation.

<p>A-B: Immunocytochemistry in <i>SNCA</i> triplication (A6) NSCs with antibodies against NSC markers SOX1, NESTIN, and PAX6. C-D: Immunocytochemistry for dopaminergic (TH and LMX1A), and midbrain (FOXA2) markers. Scale bar as marked.</p

Genes up or down regulated 3 fold in control and <i>SNCA</i> triplication dopaminergic cultures after MPP treatment.

<p>Genes up or down regulated 3 fold in control and <i>SNCA</i> triplication dopaminergic cultures after MPP treatment.</p

Genes up or down regulated in all PD-patient specific dopaminergic cultures relative to control.

<p>Genes up or down regulated in all PD-patient specific dopaminergic cultures relative to control.</p