Establishment and longitudinal characterisation of a feeder-free embryonic stem-like cell line (ATES1) derived from blastomeres of the climbing perch, Anabas testudineus.

Embryonic stem (ES) cell-based platforms in non-model teleosts remain scarce, which may constrain in vitro studies in functional genomics and developmental biology in aquaculture-relevant species. Here, we report the derivation and characterisation of ATES1, an ES-like cell line derived from Anabas testudineus, a commercially important freshwater fish. ATES1 has been maintained for over 250 passages (> 1500 days) under feeder-free conditions in Leibovitz-15 medium supplemented with foetal bovine serum, fish serum, embryo extract, and human basic fibroblast growth factor (hbFGF). Proliferation was optimal at 28 °C, with both fish serum and hbFGF significantly enhancing cell growth. The cell line exhibited multiple characteristics associated with ES-like cells in vitro during early passages (approximately up to passages 25-35), including ES cell-like morphology, high self-renewal capacity (5-bromo-2'-deoxyuridine incorporation > 90% and a doubling time of 39.9 h), alkaline phosphatase activity, responsiveness to differentiation induction conditions, and expression of stemness-associated genes (sox2, klf4, sall4, nanog, myc). Immunocytochemistry confirmed Sox2 protein expression. However, real-time PCR revealed a significant decline in klf4 and sall4 expression, along with a lack of responsiveness to differentiation cues, increased heterogeneity, and the emergence of chromosomal abnormalities, suggesting compromised maintenance of ES-like properties during extended passaging. Despite these limitations, ATES1 remained continuously proliferative under feeder-free conditions and supported moderate to high (~ 45.8 ± 4.26%) non-viral transgene delivery efficiency via lipofection, suggesting its potential utility as a genetically manipulable in vitro system for future cellular and biotechnological applications in Anabas testudineus.