TY - JOUR
T1 - High-content screen in human pluripotent cells identifies miRNA-regulated pathways controlling pluripotency and differentiation
AU - De Souza Lima, Ildercílio Mota
AU - Schiavinato, Josiane Lilian Dos Santos
AU - Paulino Leite, Sarah Blima
AU - Sastre, Danuta
AU - Bezerra, Hudson Lenormando De Oliveira
AU - Sangiorgi, Bruno
AU - Corveloni, Amanda Cristina
AU - Thomé, Carolina Hassibe
AU - Faça, Vitor Marcel
AU - Covas, Dimas Tadeu
AU - Zago, Marco Antônio
AU - Giacca, Mauro
AU - Mano, Miguel
AU - Panepucci, Rodrigo Alexandre
N1 - Publisher Copyright:
© 2019 The Author(s).
PY - 2019/7/8
Y1 - 2019/7/8
N2 - 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.
AB - 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.
KW - Cell differentiation
KW - Human embryonic stem cells
KW - MicroRNA
KW - Microscopy, fluorescence
KW - Pluripotent stem cells
KW - Receptors, Notch
UR - http://www.scopus.com/inward/record.url?scp=85068892176&partnerID=8YFLogxK
U2 - 10.1186/s13287-019-1318-6
DO - 10.1186/s13287-019-1318-6
M3 - Article
C2 - 31287022
AN - SCOPUS:85068892176
SN - 1757-6512
VL - 10
JO - Stem Cell Research and Therapy
JF - Stem Cell Research and Therapy
IS - 1
M1 - 202
ER -
