Cytotune ips reprogramming kit

In compliance with the Declaration of Helsinki of the World Medical Association, informed consent form was approved by the Institutional Review Board of Mackay Memorial Hospital and was obtained from the patient before any investigation of this study. Primary skin fibroblasts derived from patient with MELAS syndrome harboring mtDNA A3243G mutation were cultured in standard Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Life Technologies, Grand Island, NY, USA) and 1% penicillin G/streptomycin sulfate, in a humidified atmosphere of 5% (v/v) CO₂ at 37 °C. Reprogramming of fibroblasts was carried out with a modified, non-transmissible form of Sendai virus according to the manufacturer’s protocol (CytoTune-iPS Reprogramming Kit, Thermo Fisher Scientific, Waltham, MA, USA). After transduction, undifferentiated iPS colonies were isolated manually and propagated in Essential 8™ Medium (Thermo Fishers Scientific) on vitronectin-coated culture dishes. When indicated, the iPS cells were plated at a density of 400,000 cells per well of a six-well cell culture plate overnight. The next day, iPS cells were treated with or without 2 μM Carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 4 h in the absence or presence of Bafilomycin (BAF).

A 3 mm skin biopsy was obtained from the lower leg of a 45-year-old individual with normal ocular history and used for generation of patient-specific induced pluripotent stem cells (iPSCs) as described previously (Sharma et al., 2017 (link); Wiley et al., 2016b (link), 2017 (link)). Briefly, 250,000 patient-specific dermal fibroblasts were plated in one well of a six-well culture dish and transduced with non-integrating Sendai viral vectors driving expression of OCT4, SOX2, KLF4 and c-MYC at a multiplicity of infection of 3 (CytoTune-iPS Reprogramming Kit; Thermo Fisher Scientific). IPSC colonies were cultured under feeder-free conditions on recombinant human Laminin-521-coated plates (Thermo Fisher Scientific) and maintained in Human Essential 8™ media (Thermo Fisher Scientific) (Wiley et al., 2017 (link)). All patient-specific iPSCs are authentic and routinely tested for mycoplasma contamination using the MycoAlert PLUS Mycoplasma Detection Kit (Lonza Walkersville, Inc.,Walkersville, USA).

iPSCs were derived from donors who had given signed informed consent for derivation of iPSC lines from skin biopsies as part of the EU IMI-funded programme StemBANCC. Cyto Tune-iPS reprogramming kit (ThermoFisher) was used to reprogram fibroblasts through expression of OCT4, SOX2, KLF4 and c-MYC by four separate Sendai viral vectors. iPSCs were generated from a familial Parkinson’s disease patient carrying gene triplication of SNCA encoding α-synuclein (Patient 1) and a patient carrying a point mutation in SNCA (A53T, Patient 2). Control 1 was derived from Lonza fibroblasts (CC-2511); Control 2 was derived by StemBANCC from an unaffected volunteer. All iPSCs were from female donors except for Control 1. iPSCs were maintained on Matrigel in Essential 8 medium and passaged with collagenase IV. Fibroblasts were cultured in DMEM with l-glutamine and passaged with trypsin-EDTA.