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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

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]
Total Authors: 14
[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
Total Affiliations: 5
Document type: Journal article
Source: STEM CELL RESEARCH & THERAPY; v. 10, JUL 8 2019.
Web of Science Citations: 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)

FAPESP's process: 15/08070-3 - Large-scale analysis of microRNA function in cell cycle, pluripotency, self-renewal and differentiation of stem cells using high-content screening
Grantee:Rodrigo Alexandre Panepucci
Support Opportunities: Scholarships abroad - Research
FAPESP's process: 15/15864-6 - Functional evaluation of microRNAs in cell proliferation and differentiation of embryonal carcinoma cell line NTera-2 using high content screening
Grantee:Ildercílio Mota de Souza Lima
Support Opportunities: Scholarships abroad - Research Internship - Doctorate
FAPESP's process: 13/27061-0 - The role of microRNAs on the proliferation and cell differentiation of the embryonal carcinoma pluripotent cell line NTera-2.
Grantee:Ildercílio Mota de Souza Lima
Support Opportunities: Scholarships in Brazil - Doctorate
FAPESP's process: 13/08135-2 - CTC - Center for Cell-Based Therapy
Grantee:Dimas Tadeu Covas
Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC