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Título: | High-content screen in human pluripotent cells identifies miRNA-regulated pathways controlling pluripotency and differentiation | Autor: | de Souza Lima, Ildercílio Mota Schiavinato, Josiane Lilian Dos Santos Paulino Leite, Sarah Blima Sastre, Danuta Bezerra, Hudson Lenormando de Oliveira Sangiorgi, Bruno Corveloni, Amanda Cristina Thomé, Carolina Hassibe Faça, Vitor Marcel Covas, Dimas Tadeu Zago, Marco Antônio Giacca, Mauro Mano, Miguel Panepucci, Rodrigo Alexandre |
Palavras-chave: | Human embryonic stem cells; Pluripotent stem cells; MicroRNA; Cell differentiation; Receptors; Notch; Microscopy; fluorescence | Data: | 8-Jul-2019 | Editora: | Springer Nature | Projeto: | This work was supported by São Paulo Research Foundation (FAPESP), the National Council for Scientific and Technological Development (CNPq), and the International Centre for Genetic Engineering and Biotechnology (ICGEB). | Título da revista, periódico, livro ou evento: | Stem Cell Research and Therapy | Volume: | 10 | Número: | 1 | Resumo: | Background: By post-transcriptionally regulating multiple target transcripts, microRNAs (miRNAs or miR) play important biological functions. H1 embryonic stem cells (hESCs) and NTera-2 embryonal carcinoma cells (ECCs) are two of the most widely used human pluripotent model cell lines, sharing several characteristics, including the expression of miRNAs associated to the pluripotent state or with differentiation. However, how each of these miRNAs functionally impacts the biological properties of these cells has not been systematically evaluated. Methods: We investigated the effects of 31 miRNAs on NTera-2 and H1 hESCs, by transfecting miRNA mimics. Following 3–4 days of culture, cells were stained for the pluripotency marker OCT4 and the G2 cell-cycle marker Cyclin B1, and nuclei and cytoplasm were co-stained with Hoechst and Cell Mask Blue, respectively. By using automated quantitative fluorescence microscopy (i.e., high-content screening (HCS)), we obtained several morphological and marker intensity measurements, in both cell compartments, allowing the generation of a multiparametric miR-induced phenotypic profile describing changes related to proliferation, cell cycle, pluripotency, and differentiation. Results: Despite the overall similarities between both cell types, some miRNAs elicited cell-specific effects, while some related miRNAs induced contrasting effects in the same cell. By identifying transcripts predicted to be commonly targeted by miRNAs inducing similar effects (profiles grouped by hierarchical clustering), we were able to uncover potentially modulated signaling pathways and biological processes, likely mediating the effects of the microRNAs on the distinct groups identified. Specifically, we show that miR-363 contributes to pluripotency maintenance, at least in part, by targeting NOTCH1 and PSEN1 and inhibiting Notch-induced differentiation, a mechanism that could be implicated in naïve and primed pluripotent states. Conclusions: We present the first multiparametric high-content microRNA functional screening in human pluripotent cells. Integration of this type of data with similar data obtained from siRNA screenings (using the same HCS assay) could provide a large-scale functional approach to identify and validate microRNA-mediated regulatory mechanisms controlling pluripotency and differentiation. | URI: | https://hdl.handle.net/10316/107392 | ISSN: | 1757-6512 | DOI: | 10.1186/s13287-019-1318-6 | Direitos: | openAccess |
Aparece nas coleções: | I&D CNC - Artigos em Revistas Internacionais |
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High-content screen in human pluripotent cells identifies miRNA-regulated pathways controlling pluripotency and differentiation.pdf | 2.2 MB | Adobe PDF | Ver/Abrir |
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