The nucleofection procedure was performed based on the manufacturer’s instruction [Human Stem Cell Nucleofector Kit 1 (Lonza, cat. no. VPH-5012)]. According to the manufacturer manual: the efficiency of nucleofection is drastically influenced by the cell type, the cell source, the cell viability, confluence and number of cell passage. Thus, providing a unique optimized protocol with predefined conditions i.e. one program (electrical parameter) which is guaranteed to work with any hiPSCs clone is not possible. For our PMSC-hiPSCs clones, it is recommended to first determine the optimal program. In brief, on the day of nucleofection, PMSC-hiPSCs were pre-treated with 10 µmol/L Rho kinase inhibitor (Y-27632) (R&D Systems, EMD Millipore) for 2-3 hours before nucleofection. To prepare a single-cell suspension, the culture was treated with collagenase IV (Sigma). Detached PMSC-hiPSCs were collected and centrifuged at 100 x g for 3 min at RT. The hiPSCs pellet was suspended and dispensed (8 x 105 cells) into the seven separated tubes, and spin down in low-speed centrifugation. Then, ready-to-use nucleofection solutions and 7 µg of fluorescent protein-expressing plasmid DNA were added into the Nucleocuvette containing the cell suspension and mixed by tapping gently. Finally, to find the best nucleofection program,  six different programs of A-012, A-13, A-023, A-027, A-033, and B-016 were applied. Nucleofected PMSChiPSCs were recovered and transferred onto the feeder layers. On the following day, nucleofection efficiency was monitored using Flow cytometry analysis. Also, the expression of GFP was examined to find and mark the nucleofected PMSC-hiPSCs by fluorescence microscope (Figure 4). Then, PMSC-hiPSCs were cultured at 37°C until distinct colonies appear large enough for colony picking. About 70 clear single colonies were picked under sterile conditions. The picked up single colonies were cultured and propagated, separately.