Objectives: To establish a straightforward single-cell passaging cultivation method that enables high-quality maintenance of human induced pluripotent stem cells 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 calculation and cell confluence. Standard procedures for human induced pluripotent stem cells monitoring as embryonic body formation, karyotyping and pluripotency markers expression were evaluated in order to assess the cellular state in long-term culture. Cells that underwent these tests were then subjected to differentiation into keratinocytes, cardiomyocytes and definitive endoderm to evaluate its differentiation capacity. Results: Human induced pluripotent stem cells 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, cardiomyocytes and definitive endoderm. Conclusions: Our findings support the routine of human induced pluripotent stem cells single-cell passaging as a reliable procedure even after long-term cultivation, providing healthy human induced pluripotent stem cells to be used in drug discovery, toxicity, and disease modeling as well as for therapeutic approaches.

In spite of the great advance in human induced pluripotent stem cells knowledge, several nonconsensual protocols to cultivate this peculiar cell type can be found in the literature. Laboratories and companies worldwide have been trying to provide equivalent results regarding long-term cultivation of hiPSCs and their derivatives, so it is mandatory the establishment of reproducible pipelines for cell generation, cultivation, differentiation, etc. Here, we validated a straightforward single-cell passaging cultivation method that enabled high-quality maintenance of human induced pluripotent stem cells (hiPSCs) over 50 passages without the appearance of karyotypic abnormalities or loss of pluripotency. Further, the hiPSC clones were able to generate derivatives from the three germ layers at high passages by embryoid body formation and high-efficient direct differentiation into keratinocytes, cardiomyocytes and definitive endoderm (DE). Thus, our findings support the routine of hiPSCs single-cell passaging as a reliable procedure even after long-term cultivation, providing healthy PSCs to be used in high-standard cellular modeling research and therapeutic approaches.