Generation of Naivetropic Induced Pluripotent Stem Cells from Parkinson's Disease Patients for High-Efficiency Genetic Manipulation and Disease Modeling.

Hu Z, Pu J, Jiang H, Zhong P, Qiu J, Li F, Wang X, Zhang B, Yan Z, Feng J1.
Source: Stem Cells Dev
Publication Date: (2015)
Issue: 24(21): 2591-25604
Research Area:
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Nucleofector® I/II/2b

A pair of TALENs were designed according to previously published sequences [29] and assembled with the Golden Gate method (Addgene TALEN Kit #1000000024) [35]. The gene-targeting vector PITX3-2A-eGFP-PGK-Puro [29] was provided by Dr. Rudolf Jaenisch at the Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology. One million naivetropic iPSCs in Human Nucleofector Stem Cell Kit 1 (Lonza) buffer were nucleofected with 5 µg of linearized targeting vector and 4?µg of each TALEN using Nucleofection Program A23. Cells were subsequently plated on DR4 MEF feeders for puromycin selection in naivetropic iPSC medium supplemented with ROCK-inhibitor for the first 24h. Puromycin selection (0.2µg/mL) started 48h later, after nucleofection. Individual colonies were picked up and expanded 7–10 days after puromycin selection. PCR was used to screen for homologous recombinants, which were confirmed by Southern blotting.


The lack of robust Parkinson's disease (PD) phenotype in parkin knockout rodents and the identification of defective dopaminergic (DA) neurotransmission in midbrain DA neurons derived from induced pluripotent stem cells (iPSC) of PD patients with parkin mutations demonstrate the utility of patient-specific iPSCs as an effective system to model the unique vulnerabilities of midbrain DA neurons in PD. Significant efforts have been directed at developing efficient genomic engineering technologies in human iPSCs to study diseases such as PD. In the present study, we converted patient-specific iPSCs from the primed state to a naivetropic state by DOX-induced expression of transgenes (Oct4, Sox2, Klf4, c-Myc, and Nanog) and the use of 2iL (MEK inhibitor PD0325901, GSK3 inhibitor CHIR99021, and human LIF). These patient-specific naivetropic iPSCs were pluripotent in terms of marker expression, spontaneous differentiation in vitro, and teratoma formation in vivo. They exhibited morphological, proliferative, and clonogenic characteristics very similar to naive mouse embryonic stem cells (ESC). The high clonal efficiency and proliferation rate of naivetropic iPSCs enabled very efficient gene targeting of GFP to the PITX3 locus by transcription activator-like effector nuclease. The naivetropic iPSCs could be readily reverted to the primed state upon the withdrawal of DOX, 2iL, and the switch to primed-state hESC culture conditions. Midbrain DA neurons differentiated from the reverted iPSCs retained the original phenotypes caused by parkin mutations, attesting to the robustness of these phenotypes and the usefulness of genomic engineering in patient-specific naivetropic iPSCs for studying PD.