C6295

Immortalized cells were cultured according to standard practices. Briefly, cells were cryopreserved in low glucose DMEM (11885084, Thermo Fisher, Darmstadt, Germany) supplemented with 20% FBS (S0615-500ML, Sigma-Aldrich, Taufkirchen, Germany) and 10% DMSO (C6295, Sigma-Aldrich) in liquid N2. To bring them in culture, cryovials were thawed and resuspended in warmed complete medium made of DMEM supplemented with 10% FBS and 1% Anti/Anti (15240062, Thermo Fisher). The cell suspension was centrifuged at 0.5 rcf for 5′ to remove the DMSO, the cell pellet was resuspended in complete medium and transferred to a sterile T-75 flask. Cells were maintained in an incubator at 37 °C, 5% CO₂, 85–95% humidity and passaged once 90% confluent. For passaging, the medium was aspirated, the cells washed once with DPBS-CaCl₂ and then detached from the flask using 0.05% Trypsin-EDTA (5′ incubation at 37 °C, 5% CO₂). Cells were counted using 0.4% Trypan Blue Stain (T10282, Thermo Fisher) and the Countess3 FL Automated Cell Counter (Thermo Fisher) before seeding in a new recipient.

EZ spheres were prepared from iPSCs and then dissociated and sorted as described above. Cells in the CD271⁺ fraction and CD15^– fraction were transferred into expansion medium to reform spheres. A parallelly prepared group of cells that did not go through MACS was used as control, referred to as “unsorted cells.” After 6 weekly passages by mechanical chopping, spheres were transferred into the cell freezing medium (C6295, Sigma-Aldrich) and stored in liquid nitrogen for 2 years. Cryopreserved spheres were then thawed and transferred into the expansion medium. After spheres were passaged once a week and maintained for 6 weeks, 8 spheres per group were transferred into the wells of a low-attachment 96-well plate on day 0. Each well contained one sphere with a similar size (~20 μm diameter) in the expansion medium. On day 0, day 3, day 6, and day 9, diameters of the spheres were measured with the eyepiece reticle of the Nikon Eclipse TS 100 inverted microscope and used to calculate the sphere volume. Growth rates were calculated by normalizing each sphere's changes in volume relative to the initial volume on day 0. To verify the reliability of our findings, we repeated 3 independent experiments.