Parkinson’s disease (PD) is a progressive neurodegenerative disorder that affects 1% of people over age 60, and more than 5 million people worldwide . PD results primarily from the selective loss of dopaminergic neurons, which first affects movement, then cognitive function, with late-stage disease often accompanied by dementia.
The absence of physiologically relevant cellular models for PD represents a major bottleneck for PD research. Novel models are urgently needed to accelerate the discovery of disease mechanisms and drug targets which could rapidly translate into a wide range of clinical and therapeutic applications.
Recently, Life Technologies has partnered with the Parkinson’s Institute in Sunnyvale, California, to develop PD model systems using donor fibroblasts that have been collected at the Institute.
Modify and grow: deriving iPSCs
The Ion PGM™ Sequencer was used to evaluate the donor fibroblasts for additional mutations prior to reprogramming. The fibroblasts were found to have the previously detected LRRK2 and/or GBA mutation in the expected lines, and no unexpected mutations. Induced pluripotent stem cells (iPSCs) were generated from the donor fibroblasts using the CytoTune®-iPS Sendai Reprogramming Kit, an integration-free reprogramming tool utilizing Sendai virus. The iPSCs were cultured and expanded using Gibco® KnockOut™ DMEM/F12 and KnockOut™ Serum Replacement.
Detect: iPSC characterization through cell health assessment
Cell health was routinely assessed during iPSC expansion using the Molecular Probes® Alkaline Phosphatase Live Stain to quickly confirm pluripotency on the EVOS® XL Imaging System. After cells were reprogrammed, detection of transgenes was determined utilizing the TaqMan® iPSC Sendai Detection Kit.
Analyze: iPSC characterization using gene expression profiles
The QuantStudio™ 12K Flex Real-Time PCR System was used for gene expression analysis of the iPSCs. This included a broad-based gene expression analysis of the iPSCs with 609 genes in the TaqMan® OpenArray® Human Stem Cell Panel. Pluripotency genes were shown to be upregulated in all iPSCs lines and fibroblasts were easily distinguished from iPSCs.