Versene

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.

In the present study, pluripotent H9 hESC (WA09, WiCell Research Institute, Madison, WI, USA) and hiPSC (IMR90 clone #4, WiCell) were used. All work with H9 was reviewed and approved by the Stem Cell Oversight Committee, University of Wisconsin.
H9 and IMR90 cells were maintained in the presence of 5% CO₂ at 37°C on 6-well tissue culture plates (Greiner Bio-One Cellstar, Monroe, NC, USA) coated with matrigel (WiCell) in mTeSR1 media (Stem Cell Technologies). The culture medium was changed daily, and cells were passaged with versene (Fisher Scientific, Hampton, NH, USA) every 4 to 6 days.