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High-content screen in human pluripotent cells identifies miRNA-regulated pathways controlling pluripotency and differentiation

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de Souza Lima, Ildercilio Mota [1, 2] ; dos Santos Schiavinato, Josiane Lilian [1, 2] ; Paulino Leite, Sarah Blima [1, 2] ; Sastre, Danuta [1] ; de Oliveira Bezerra, Hudson Lenormando [1, 2] ; Sangiorgi, Bruno [1, 2] ; Corveloni, Amanda Cristina [1, 2] ; Thome, Carolina Hassibe [3] ; Faca, Vitor Marcel [3] ; Covas, Dimas Tadeu [1, 2] ; Zago, Marco Antonio [1, 2] ; Giacca, Mauro [4] ; Mano, Miguel [5, 4] ; Panepucci, Rodrigo Alexandre [1, 2]
Número total de Autores: 14
Afiliação do(s) autor(es):
[1] Reg Blood Ctr Ribeirao Preto, LFBio, Ctr Cell Based Therapy CTC, Rua Tenente Catao Roxo 2501, BR-14051140 Ribeirao Preto, SP - Brazil
[2] Univ Sao Paulo FMRP USP, Ribeirao Preto Med Sch, Dept Genet & Internal Med, Ribeirao Preto, SP - Brazil
[3] Univ Sao Paulo FMRP USP, Ribeirao Preto Med Sch, Dept Biochem & Immunol, Ribeirao Preto - Brazil
[4] ICGEB, Mol Med Lab, Trieste - Italy
[5] Univ Coimbra, Ctr Neurosci & Cell Biol CNC, Coimbra - Portugal
Número total de Afiliações: 5
Tipo de documento: Artigo Científico
Fonte: STEM CELL RESEARCH & THERAPY; v. 10, JUL 8 2019.
Citações Web of Science: 0

BackgroundBy 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.MethodsWe investigated the effects of 31 miRNAs on NTera-2 and H1 hESCs, by transfecting miRNA mimics. Following 3-4days 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.ResultsDespite 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 naive and primed pluripotent states.ConclusionsWe 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. (AU)

Processo FAPESP: 15/15864-6 - Avaliação funcional de microRNAs na proliferação e diferenciação celular da linhagem de carcinoma embrionário NTera-2 por high content screening
Beneficiário:Ildercílio Mota de Souza Lima
Linha de fomento: Bolsas no Exterior - Estágio de Pesquisa - Doutorado
Processo FAPESP: 13/27061-0 - O papel dos microRNAs na proliferação e diferenciação celular da linhagem pluripotente de carcinoma embrionário humano NTera-2
Beneficiário:Ildercílio Mota de Souza Lima
Linha de fomento: Bolsas no Brasil - Doutorado
Processo FAPESP: 15/08070-3 - Análise em larga-escala da função dos microRNAs no ciclo celular, pluripotência, auto-renovação e diferenciação de células-tronco utilizando High Content Screening
Beneficiário:Rodrigo Alexandre Panepucci
Linha de fomento: Bolsas no Exterior - Pesquisa
Processo FAPESP: 13/08135-2 - CTC - Centro de Terapia Celular
Beneficiário:Dimas Tadeu Covas
Linha de fomento: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs