Episomal Reprogramming of Human iPSCs

The human iPSC lines used in this study were first generated from a healthy male patient, WTC11^{45 (link),46 (link)} using the episomal reprogramming method.^{47 (link)} Informed consent was obtained for this procedure. iPSCs were maintained on Matrigel (Corning) in mTeSR Plus media (StemCell Technologies), which was exchanged every other day. For passaging, at ~70% confluency, iPSCs were passaged using Versene (Gibco) and replated in mTeSR Plus + 10 μM Y-27632, a Rho-associated kinase (ROCK) inhibitor (StemCell Technologies), to promote cell survival, and media was changed the next day to mTeSR Plus. For harvesting, iPSCs at ~70% confluency were washed once with 1x PBS with no Mg²⁺ or Ca²⁺(DPBS, Thermo Fisher Scientific), passaged with Versene, washed two times with 1x DPBS, pelleted, and frozen at −80°C. To ensure equal numbers of cells during cell pelleting, cells were counted using NucleoCounter NC-200 and Via-1 Cassettes (Chemometec). During harvesting, an aliquot of iPSCs was taken to verify pluripotency status of iPSCs. The presence of pluripotency markers Oct3/4 and SSEA-4 were validated using flow cytometry. Each cell pellet contained approximately 10 million harvested cells and was frozen at −80°C.