Self-renewal potential of thawed hPSCs. A. IHC of pluripotency-related molecules (POU5F1, SSEA-4, TRA 1-60, NANOG and TRA 1-81) in hPSCs (KhES-1 and PFX#9) at five passages after thawing. These molecules were detected by specific antibodies and visualized with secondary Alexa Fluor 488 (green) or 594 (red) -labeled antibodies. Phase contrast image of KhES-1 and PFX#9 cells is shown in upper left. Scale bar = 100 μm. B. Pluripotency-related genes (POU5F1, NANOG, SOX2, KLF4, cMYC, hTERT, NODAL, LEFTY, and REX1) in KhES-1 and PFX#9 cells before and six passages after thawing were examined by qRT-PCR. Gene expression was compared by the ΔΔCt method. C. Flow cytometric analysis of pluripotency-related surface markers (SSEA-3, SSEA-4, and TRA 1-60) in KhES-1 and PFX#9 cells at five passages after thawing. D. Growth of KhES-1 and PFX#9 cells after thawing and passaging without cryopreservation. X axis represents cell numbers, Y axis represents passage number.