Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues

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de Figueiredo Pessoa, Lais Vicari ; Lisboa Pires, Pedro Ratto ; del Collado, Maite ; Godoy Pieri, Naira Caroline ; Recchia, Kaiana ; Souza, Aline Fernanda ; Perecin, Felipe ; da Silveira, Juliano Coelho ; Cesar de Andrade, Andre Furugen ; Ambrosio, Carlos Eduardo ; Bressan, Fabiana Fernandes ; Meirelles, Flavio Vieira

• Tipo de documento: Artigo Científico
• Fonte: STEM CELLS INTERNATIONAL; v. 2019, p. 15-pg., 2019-01-01.

Resumo

Introduction. Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. Objectives. We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. Methods. Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. Results. Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. Conclusions. We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process. (AU)

• Processo FAPESP: 14/22887-0 - Exossomos e microvesículas contendo miRNAs modulam mudanças epigenéticas durante o cultivo in vitro de gametas e embriões em bovinos
• Beneficiário: Juliano Coelho da Silveira
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores
• Processo FAPESP: 08/57877-3 - Instituto Nacional de Ciência e Tecnologia em Células-Tronco e Terapia Celular - INCTC
• Beneficiário: Roberto Passetto Falcão
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 12/50533-2 - GIFT: melhoramento genômico de características relacionadas com a fertilização em gado bovino dinamarquês e brasileiro
• Beneficiário: Marcelo Fábio Gouveia Nogueira
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 15/26818-5 - Investigação de mecanismos celulares e moleculares da aquisição da toti- e pluripotência induzida in vitro - modelo translacional
• Beneficiário: Fabiana Fernandes Bressan
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores
• Processo FAPESP: 13/17955-3 - Estudo do potencial de pluripotência de células-tronco equinas derivadas de tecido adulto e cordão umbilical, submetidas à reprogramação induzida geneticamente (células IPS)
• Beneficiário: Laís Vicari de Figueiredo Pessôa
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 13/08135-2 - CTC - Centro de Terapia Celular
• Beneficiário: Dimas Tadeu Covas
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs