Generating Human-Induced Pluripotent Stem Cells

Healthy fibroblast donor was recruited from Kuopio University Hospital (Kuopio, Finland; Approved by the committee on Research Ethics of Northern Savo Hospital district (license no 64/2014). Written informed consent was obtained from the donor. Skin biopsy derived fibroblasts were reprogrammed with CytoTune® iPS Sendai Reprogramming kit (Thermo Scientific, MA, USA) as previously described (Holmqvist et al., 2016 (link)), with slight modifications. Briefly, fibroblasts (1 × 10⁵) were transduced with 3 or 4 separate vectors including the four Yamanaka factors OCT-3/4, KLF-4, SOX-2 and c-MYC. One week after transduction, 0.75 × 10⁵ cells were seeded on the top of mitotically inactivated (10 μg/ml mitomycin-C for 2.5 h in 37°C) human foreskin fibroblast feeder cells (CRL-2429, ATCC, Manassas, VA) growing in 10 cm petri dish. First colonies started to appear a week later, and they were re-seeded by picking up individual colonies. The pluripotency of created hiPSC line was assessed as in our earlier studies (Qu et al., 2013 (link)).