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

PLoS One. 2016 May 18;11(5):e0154890. doi: 10.1371/journal.pone.0154890. eCollection 2016.

Abstract

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.

MeSH terms

  • Cells, Cultured
  • Dopaminergic Neurons / cytology*
  • Dopaminergic Neurons / metabolism
  • Genome, Human
  • Glucosylceramidase / genetics
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism
  • Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 / genetics
  • Mitochondria / metabolism
  • Mitophagy
  • Mutation*
  • Neural Stem Cells / cytology*
  • Neural Stem Cells / metabolism
  • Neurogenesis*
  • Parkinson Disease / genetics*
  • Parkinson Disease / pathology
  • Ubiquitin-Protein Ligases / genetics
  • alpha-Synuclein / genetics

Substances

  • SNCA protein, human
  • alpha-Synuclein
  • Ubiquitin-Protein Ligases
  • parkin protein
  • LRRK2 protein, human
  • Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
  • Glucosylceramidase

Grant support

This work was supported by California Institute for Regenerative Medicine Grants TR-01856 and TG2-01155 to XZ (https://www.cirm.ca.gov/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. NxCell Science and Xcell Science provided support in the form of salaries for authors XZ and MSR, but did not have any additional role the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.