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

Autor(es):Mostrar menos -

Cruvinel, Estela ^[1] ; Ogusuku, Isabella ^[1] ; Cerioni, Rosanna ^[1] ; Rodrigues, Sirlene ^[1] ; Goncalves, Jessica ^[1] ; Goes, Maria Elisa ^[2] ; Alvim, Juliana Morais ^[1] ; Silva, Anderson Carlos ^[3] ; Lino, Vanesca de Souza ^[4] ; Boccardo, Enrique ^[4] ; Goulart, Ernesto ^[5] ; Pereira, Alexandre ^[3] ; Dariolli, Rafael ^{[6, 1]} ; Valadares, Marcos ^[1] ; Biagi, Diogo ^[1]

Afiliação do(s) autor(es)

^[1] PluriCell Biotech, 2242 Prof Lineu Prestes Ave, BR-05508000 Sao Paulo, SP - Brazil

^[2] Univ Sao Paulo, Inst Chem, Dept Biochem, Sao Paulo - Brazil

^[3] Univ Sao Paulo, Med Sch, Heart Inst InCor, Sao Paulo - Brazil

^[4] Univ Sao Paulo, Inst Biomed Sci, Dept Microbiol, Sao Paulo - Brazil

^[5] Univ Sao Paulo, Human Genome & Stem Cell Res Ctr, Inst Biosci, Dept Genet & Evolutionary Biol, Sao Paulo - Brazil

^[6] Icahn Sch Med Mt Sinai, Dept Pharmacol Sci, New York, NY 10029 - USA

• Tipo de documento: Artigo Científico
• Fonte: SAGE OPEN MEDICINE; v. 8, OCT 2020.
• Citações Web of Science: 0

Resumo

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. (AU)

• Processo FAPESP: 15/50224-8 - Caracterização de cardiomiócitos derivados de células tronco de pluripotência induzida e padronização de ensaios celulares
• Beneficiário: Diogo Gonçalves Biagi dos Santos
• Modalidade de apoio: Auxílio à Pesquisa - Pesquisa Inovativa em Pequenas Empresas - PIPE
• Processo FAPESP: 16/50082-1 - Viabilização comercial de queratinócitos derivados de células-tronco pluripotentes induzidas e desenvolvimento de equivalente de pele
• Beneficiário: Estela Mitie Cruvinel
• Modalidade de apoio: Auxílio à Pesquisa - Pesquisa Inovativa em Pequenas Empresas - PIPE