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

Abstract

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. Electronic supplementary material The online version of this article (10.1186/s13287-019-1318-6) contains supplementary material, which is available to authorized users.

Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell‐Cardiomyocyte Differentiation

“…miRNA from the first cluster (miR‐378a, miR‐143, miR 125a), strongly expressed in the mammalian heart, and largely involved in muscle development and hypertrophy, [ 52 , 53 , 54 , 55 , 56 ] were downregulated in hiPSC‐EV and gradually upregulated along cardiomyocyte differentiation (with exception of miR‐378a, which is also expressed in hiPSC‐EV). The second miRNA cluster (miR‐200b/c, miR‐335, miR‐302b/c, miR‐363, miR‐183, miR‐182), contained pluripotency‐associated miRNAs, [ 31 , 32 , 33 , 57 ] which were progressively downregulated in EV secreted from committed and mature cells. The third cluster is composed solely of two miRNAs (miR‐483, miR‐375), involved in lineage specification, which explains their absence in hiPSC‐EV and CMm‐EV.…”

“…[ 19 ] Not all miRNAs are ubiquitously expressed, with tissue‐specific subsets, [ 20 ] such as myomiRs (miR‐1, miR‐133a, miR‐133b, miR‐206, miR‐208a, miR‐208b, miR‐486, and miR‐499), exclusively or preferentially expressed in striated muscle, [ 21 , 22 , 23 , 24 , 25 ] and involved in muscle cell proliferation and/or differentiation, or angiomiRs (miR‐126, miR‐130a, let‐7f, and the miR‐17‐92 cluster), expressed in endothelial cells and identified as key regulators in angiogenic processes. [ 26 , 27 , 28 , 29 , 30 ] Similarly, miRNA clusters were shown to be essential in stem cell maintenance (miR‐106a‐363, [ 31 ] miR‐302a‐367, [ 32 , 33 ] miR‐17‐92, [ 31 ] and C19MC clusters, [ 34 ] and miR‐200c/b) [ 35 , 36 ] and differentiation. [ 31 , 37 ] In fact, combinations of miRNAs were capable of inducing direct cellular reprogramming (reviewed in refs.…”

“…[ 26 , 27 , 28 , 29 , 30 ] Similarly, miRNA clusters were shown to be essential in stem cell maintenance (miR‐106a‐363, [ 31 ] miR‐302a‐367, [ 32 , 33 ] miR‐17‐92, [ 31 ] and C19MC clusters, [ 34 ] and miR‐200c/b) [ 35 , 36 ] and differentiation. [ 31 , 37 ] In fact, combinations of miRNAs were capable of inducing direct cellular reprogramming (reviewed in refs. [ 38 , 39 ]), either back to a pluripotent state, or producing phenotype switches relevant in a cardiac repair context.…”

Tracking telomere fusions through crisis reveals conflict between DNA transcription and the DNA damage response

“…Archetypes of pertinent signalling pathways that might prove pivotal for crisis transition were selected for expression analysis (Figure 6 ). Of these, only the expression patterns of WIF1 ( 62 ) and DTX1 ( 63 , 64 ) (regulators of Wnt and Notch signalling, respectively) were reproduced by RT-PCR assays, both demonstrating increased mRNA levels in the transformed fibroblasts at the late crisis stage that, importantly, were manifestly higher than those measured in the Early (proliferating) and Late (senescent) NEO control fibroblasts ( Supplementary Figure S1A ). By RT-PCR, WIF1 was 3.5-fold increased ( P <0.0001) between early- and late-crisis E6E7 cells (compared with 99.5-fold by RNA-seq; P = NS) and 5.24-fold ( P < 0.0001) upregulated in the late-stage E6E7 compared with the NEO cells.…”

TRAF3, a Target of MicroRNA-363-3p, Suppresses Senescence and Regulates the Balance Between Osteoblastic and Adipocytic Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells

“…Some studies have shown that miR-363-3p is associated with cell differentiation. For example, miR-363-3p promotes pluripotency maintenance through repression of NOTCH1 and PSEN1 and suppression of Notch-induced differentiation of human pluripotent cells [26]. Dendritic cells from peripheral blood of patients with rheumatoid arthritis promote Th17 cell differentiation through the miR-363-3p-integrin av-TGF-b axis [27].…”