
Long-term single-cell passaging of human iPSC fully supports pluripotency and high-efficient trilineage differentiation capacity
Author(s) -
Estela Cruvinel,
Isabella Ogusuku,
Rosanna Cerioni,
Sirlene Rodrigues,
Jéssica Gonçalves,
Maria Elisa Góes,
Juliana Morais Alvim,
Anderson Carlos Silva,
Vanesca de Souza Lino,
Enrique Boccardo,
Ernesto Goulart,
Alexandre da Costa Pereira,
Rafael Dariolli,
Marcos Valadares,
Diogo Biagi
Publication year - 2020
Publication title -
sage open medicine
Language(s) - English
Resource type - Journals
ISSN - 2050-3121
DOI - 10.1177/2050312120966456
Subject(s) - induced pluripotent stem cell , embryoid body , embryonic stem cell , germ layer , microbiology and biotechnology , biology , stem cell , kosr , cellular differentiation , endoderm , population , cell potency , genetics , medicine , gene , environmental health
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.