Maintenance of pluripotency under xeno-free, feeder-free, and MEF growth conditions. (A): Colonies grown under xeno-free, feeder-free, and MEF conditions exhibited similar morphological features when viewed under bright-field microscopy. Magnification, ×4. (B–D): Uniform and homogeneous expression of pluripotency associated factors such as OCT-4, SOX2, and NANOG was observed in the undifferentiated colonies, irrespective of the system they were grown in. Magnification, ×20. (E–G): Similar expression of cell surface markers including TRA-1-60, TRA-1-81, and SSEA-4 was observed in the xeno-free cells when compared with feeder-free and MEF systems, further confirming the pluripotency of these cells. Magnification, ×20. (H): Reverse transcription-polymerase chain reaction analysis confirmed the expression of pluripotency genes in hiPSCs maintained in all conditions, as well as the relative absence of markers of differentiation. Abbreviations: F-F, feeder-free; MEF, mouse embryonic fibroblast; X-F, xeno-free.