iPSC laboratory

Induced pluripotent stem cells (iPSCs) have gained increasing importance for stem cell-based disease modeling whereby tissue from a patient is reprogrammed to pluripotency (iPSC) and then differentiated into a cell type closely resembling the tissue bearing the brunt of disease. We use iPSC technology to functionally examine disease pathology in patient-derived, biologically relevant cells. The tasks of our “iPSC platform” are to undertake the reprogramming, biobanking, and differentiation required in the context of various projects. To reprogram patient skin fibroblasts into iPSCs, we employ Sendai virus transduction to deliver the four reprogramming factors OCT4, SOX2, KLF4, and cMYC by using a commercially available kit (CytoTune-iPS 2.0 Sendai Reprogramming Kit, Thermo Fisher Scientific Inc). Sendai virus is a non-integrating RNA virus that produces iPSC clones with high efficiency and of best quality, as has been shown by us and others. Stringent quality control steps established in our laboratory are applied to ensure that the iPSC lines generated are free of karyotypic abnormalities and demonstrate robust differentiation potential. All iPSC lines are cultured under feeder-free conditions and a basic stock of cryovials of each iPSC line is generated for dissemination. For the neurological movement disorders Parkinson disease and dystonia, we focus on dopaminergic neurons. Based on our previous work1-5, we differentiate iPSCs into midbrain dopaminergic neurons. The underlying protocol6 ventralizes the iPSCs into a midbrain floor plate phenotype first (the floor plate being a key organizer of the central nervous system, where the midbrain dopaminergic neurons originate). This is accomplished by using strong bone morphogenic protein inhibition coupled with Sonic Hedgehog and Wnt pathway activation to induce the floor plate-like phenotype. Neural induction is performed by adding numerous recombinant proteins and small molecules at various time points. After 75 days of differentiation, there is a cell culture consisting of more than 90% neurons, of which approximately 40% represent electrophysiologically active dopaminergic neurons based on whole-cell recordings and the expression of key markers tyrosine hydroxylase (marker for dopaminergic cells), TUJ1 (neuronal marker), and FOXA2 (midbrain marker).