Stability and Pluripotency Status of Human Embryonic Stem Cell Lines Following Long-Term Cryopreservation

BACKGROUND: Human embryonic stem cell (hESC) lines preserved in biobanks represent valuable resources for developmental biology and regenerative medicine. However, the long-term effects of cryopreservation on genomic stability, differentiation capacity, and pluripotency state remain poorly characterized. The recent emergence of stem cell-based embryo models (SCBEMs) has further increased interest in identifying hESC lines exhibiting naïve pluripotency characteristics. METHODS: Four SNUhES lines (SNUhES35, SNUhES42, SNUhES43, and SNUhES49) cryopreserved for 2,443-4,242 days were recovered and evaluated for chromosomal integrity by G-banding karyotyping, pluripotency marker expression (Oct4, SSEA-4, and Tra-1-61), and tri-lineage differentiation capacity through embryoid body formation. Directed cardiomyocyte differentiation was assessed by cardiac lineage marker expression and multi-electrode array (MEA) analysis. Quantitative RT-PCR was performed to characterize pluripotency states using naïve-associated markers (TFCP2L1, KLF2, KLF4, and PRDM14) and DNMT3L. RESULT: All four hESC lines were successfully recovered following 6-13 years of cryopreservation and maintained normal karyotypes after thawing and expansion. Robust expression of pluripotency markers and preserved tri-lineage differentiation capacity were observed in all cell lines. Despite long-term storage under identical conditions, the lines exhibited distinct lineage-specific differentiation tendencies. Cardiomyocyte differentiation generated spontaneously beating clusters in all lines, with SNUhES43 showing the highest cardiac marker expression and electrophysiological activity as confirmed by MEA recordings. Pluripotency profiling revealed that SNUhES35 and SNUhES49 exhibited a naïve-like pluripotent state, whereas SNUhES42 and SNUhES43 displayed characteristics of primed pluripotency. CONCLUSION: Human ESC lines cryopreserved for up to 13 years retain genomic stability, pluripotency, and the capacity for functional differentiation, including the generation of electrophysiologically active cardiomyocytes. Importantly, archived hESC collections contain biologically distinct cell lines with different pluripotency states and lineage-specific differentiation propensities. The identification of naïve-like SNUhES lines highlights the value of systematic re-evaluation of long-term cryopreserved hESC biobanks and supports their application in stem cell-based embryo models, blastoid generation, and studies of early human development.