Objectives To validate a straightforward single-cell passaging cultivation method that enables high-quality maintenance of hiPSC without the appearance of karyotypic abnormalities or loss of pluripotency.Methods Cells were kept in culture for over 50 passages, following a structured chronogram of passage and monitoring cell growth by population doubling time (PDT) calculation and cell confluence. Standard procedures for iPSC monitoring as embryonic body (EB) formation, karyotyping and pluripotency markers expression were evaluated in order to monitor the cellular state in the long-term culture. Cells that underwent these tests were then subjected to differentiation into keratinocytes and cardiomyocytes to evaluate its differentiation capacity.Results hiPSC clones maintained its pluripotent capability as well as chromosomal integrity and were able to generate derivatives from the three germ layers at high passages by embryoid body formation and high-efficient direct differentiation into keratinocytes and cardiomyocytes.Conclusion Our findings support the routine of hiPSC single-cell passaging as a reliable procedure even after long-term cultivation, providing healthy PSCs to be used in drugs discovery, toxicity and disease modeling as well as for therapeutic approaches.

Long-term single-cell passaging of human iPSC fully supports pluripotency and high-efficient trilineage differentiation capacity