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Within the scope of the FANTOM6 consortium, we perform a large-scale knockdown of 200 long non-coding RNAs (lncRNAs) in human induced pluripotent stem cells (iPSCs) and systematically characterize their roles in self-renewal and pluripotency. We find 36 lncRNAs (18%) exhibiting cell growth inhibition. From the knockdown of 123 lncRNAs with transcriptome profiling, 36 lncRNAs (29.3%) show molecular phenotypes. Integrating the molecular phenotypes with chromatin-interaction assays further reveals cis- and trans-interacting partners as potential primary targets. Additionally, cell-type enrichment analysis identifies lncRNAs associated with pluripotency, while the knockdown of LINC02595, CATG00000090305.1, and RP11-148B6.2 modulates colony formation of iPSCs. We compare our results with previously published fibroblasts phenotyping data and find that 2.9% of the lncRNAs exhibit a consistent cell growth phenotype, whereas we observe 58.3% agreement in molecular phenotypes. This highlights that molecular phenotyping is more comprehensive in revealing affected pathways.

Profiling of cis-regulatory elements (CREs, mostly promoters and enhancers) in single cells allows the interrogation of the cell-type and cell-state-specific contexts of gene regulation and genetic predisposition to diseases. Here we demonstrate single-cell RNA-5′end-sequencing (sc-end5-seq) methods can detect transcribed CREs (tCREs), enabling simultaneous quantification of gene expression and enhancer activities in a single assay at no extra cost. We showed enhancer RNAs can be detected using sc-end5-seq methods with either random or oligo(dT) priming. To analyze tCREs in single cells, we developed SCAFE (Single Cell Analysis of Five-prime Ends) to identify genuine tCREs and analyze their activities (https://github.com/chung-lab/scafe). As compared to accessible CRE (aCRE, based on chromatin accessibility), tCREs are more accurate in predicting CRE interactions by co-activity, more sensitive in detecting shifts in alternative promoter usage and more enriched in diseases heritability. Our results highlight additional dimensions within sc-end5-seq data which can be used for interrogating gene regulation and disease heritability.

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

Author(s): Yang, Pei-Chi; Perissinotti, Laura L; Lopez-Redondo, Fernando; Wang, Yibo; DeMarco, Kevin R; Jeng, Mao-Tsuen; Vorobyov, Igor; Harvey, Robert D; Kurokawa, Junko; Noskov, Sergei Y; Clancy, Colleen E | Abstract: KEY POINTS:This study represents a first step toward predicting mechanisms of sex-based arrhythmias that may lead to important developments in risk stratification and may inform future drug design and screening. We undertook simulations to reveal the conditions (i.e. pacing, drugs, sympathetic stimulation) required for triggering and sustaining reentrant arrhythmias. Using the recently solved cryo-EM structure for the Eag-family channel as a template, we revealed potential interactions of oestrogen with the pore loop hERG mutation (G604S). Molecular models suggest that oestrogen and dofetilide blockade can concur simultaneously in the hERG channel pore. ABSTRACT:Female sex is a risk factor for inherited and acquired long-QT associated torsade de pointes (TdP) arrhythmias, and sympathetic discharge is a major factor in triggering TdP in female long-QT syndrome patients. We used a combined experimental and computational approach to predict 'the perfect storm' of hormone concentration, IKr block and sympathetic stimulation that induces arrhythmia in females with inherited and acquired long-QT. More specifically, we developed mathematical models of acquired and inherited long-QT syndrome in male and female ventricular human myocytes by combining effects of a hormone and a hERG blocker, dofetilide, or hERG mutations. These 'male' and 'female' model myocytes and tissues then were used to predict how various sex-based differences underlie arrhythmia risk in the setting of acute sympathetic nervous system discharge. The model predicted increased risk for arrhythmia in females when acute sympathetic nervous system discharge was applied in the settings of both inherited and acquired long-QT syndrome. Females were predicted to have protection from arrhythmia induction when progesterone is high. Males were protected by the presence of testosterone. Structural modelling points towards two plausible and distinct mechanisms of oestrogen action enhancing torsadogenic effects: oestradiol interaction with hERG mutations in the pore loop containing G604 or with common TdP-related blockers in the intra-cavity binding site. Our study presents findings that constitute the first evidence linking structure to function mechanisms underlying female dominance of arousal-induced arrhythmias.

Long non-coding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes and yet, their functions remain largely unknown. We systematically knockdown 285 lncRNAs expression in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNA exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest to-date lncRNA knockdown dataset with molecular phenotyping (over 1,000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

Key points This study represents a first step toward predicting mechanisms of sex‐based arrhythmias that may lead to important developments in risk stratification and may inform future drug design and screening.We undertook simulations to reveal the conditions (i.e. pacing, drugs, sympathetic stimulation) required for triggering and sustaining reentrant arrhythmias.Using the recently solved cryo‐EM structure for the Eag‐family channel as a template, we revealed potential interactions of oestrogen with the pore loop hERG mutation (G604S).Molecular models suggest that oestrogen and dofetilide blockade can concur simultaneously in the hERG channel pore. Abstract Female sex is a risk factor for inherited and acquired long‐QT associated torsade de pointes (TdP) arrhythmias, and sympathetic discharge is a major factor in triggering TdP in female long‐QT syndrome patients. We used a combined experimental and computational approach to predict ‘the perfect storm’ of hormone concentration, I Kr block and sympathetic stimulation that induces arrhythmia in females with inherited and acquired long‐QT. More specifically, we developed mathematical models of acquired and inherited long‐QT syndrome in male and female ventricular human myocytes by combining effects of a hormone and a hERG blocker, dofetilide, or hERG mutations. These ‘male’ and ‘female’ model myocytes and tissues then were used to predict how various sex‐based differences underlie arrhythmia risk in the setting of acute sympathetic nervous system discharge. The model predicted increased risk for arrhythmia in females when acute sympathetic nervous system discharge was applied in the settings of both inherited and acquired long‐QT syndrome. Females were predicted to have protection from arrhythmia induction when progesterone is high. Males were protected by the presence of testosterone. Structural modelling points towards two plausible and distinct mechanisms of oestrogen action enhancing torsadogenic effects: oestradiol interaction with hERG mutations in the pore loop containing G604 or with common TdP‐related blockers in the intra‐cavity binding site. Our study presents findings that constitute the first evidence linking structure to function mechanisms underlying female dominance of arousal‐induced arrhythmias., Key points This study represents a first step toward predicting mechanisms of sex‐based arrhythmias that may lead to important developments in risk stratification and may inform future drug design and screening.We undertook simulations to reveal the conditions (i.e. pacing, drugs, sympathetic stimulation) required for triggering and sustaining reentrant arrhythmias.Using the recently solved cryo‐EM structure for the Eag‐family channel as a template, we revealed potential interactions of oestrogen with the pore loop hERG mutation (G604S).Molecular models suggest that oestrogen and dofetilide blockade can concur simultaneously in the hERG channel pore.

Because spontaneous host regeneration of damaged tissues is limited, novel therapeutics utilizing cultured cells with the aid of tissue engineering methods are promising alternatives for tissue replacement. One critical shortcoming is current requirement for invasive cell harvest from culture to fabricate cell-based devices. Although microglia that secrete neurotrophic factors are attractive candidates for novel cell transplantation therapy for damaged central nervous system tissue, the intact harvest of cultured microglia is presently not achievable. Therefore, primary microglia were plated onto culture surfaces grafted with the temperature-responsive polymer, poly( N -isopropylacrylamide) (PIPAAm). This surface undergoes rapid, reversible temperature-dependent changes in its hydration state and surface hydrophilicity. Microglia attached and proliferated on PIPAAm-grafted dishes at 37°C. By reducing culture temperature, more than 90% of the cells spontaneously detached from the dishes within several minutes without trypsin or EDTA treatment. Recovered and replated microglia exhibited phenotypic properties comparable to those of primary microglia freshly isolated from brain. By contrast, less than 60% of the cells were harvested by trypsin digestion, and exhibited significant alteration of characteristic cellular properties as monitored by pathological states in vivo. This new technology exhibits utility for the preparation of cell sources required for cell transplantation as well as microglial function analysis.

Glutamate transporters play an important role in the re-uptake of glutamate after its release from glutamatergic synapses. So far five of such transporters subtypes have been cloned from rodent and human brains. The densities of glutamate transporters are recognised to be developmentally regulated, but the role of glutamate transporters in the mechanisms underlying the occurrence of neuronal traumatic injury has not been widely studied. In the present study quantitative Western blotting and immunohistochemical technique were employed to study the expression of GLT-1/EAAT2 in the facial nuclei of adult rats following unilateral facial nerve axotomy. The total content of GLT-1 protein decreased in the ipsilateral axotomised rat facial nucleus. However, activated microglia surrounding motoneurons showed high expression of GLT-1 after facial nerve axotomy. Parallel studies revealed that primary cultured microglial cells also showed GLT-1-immunoreactivity. To our knowledge, this is the first direct demonstration of the expression of GLT-1 protein in activated microglial cells, suggesting a neuroprotective role of microglia against glutamate excitotoxicity following nerve axotomy.

Microglia represent a population of brain macrophage precursor cells which are intrinsic to the CNS parenchyma. Transection of the facial nerve in the newborn rat causes death of the affected motor neurons which is accompanied by massive activation of local microglia. Many of these cells develop into macrophages as can be shown by immunocytochemistry for OX-42 and ED1. Using the new polyclonal microglial marker ionized calcium binding adapter molecule 1, iba1, in combination with immunocytochemical double-labeling for the proliferating cell nuclear antigen (PCNA), or [3H]thymidine autoradiography, and confocal microscopy, qualitative as well as quantitative differences can be demonstrated between the newborn and the adult axotomized rat facial nucleus. While microglial cells are the only cell population which responds to axotomy by cell division in the adult facial nucleus, GFAP positive reactive astrocytes can be shown to undergo mitosis following axotomy in the newborn rat. Furthermore, ED1 immunoreactivity, early expression of MHC class II molecules and morphological transformation of microglia into macrophages can only be observed under conditions of neuronal degeneration, i.e., in the neonatal rat facial nucleus. Thus, the combination of cellular markers described here should be useful for studies employing the neonatal rat facial nucleus as an in vivo assay system to test the efficacy of neurotrophic factors.

Special attention is directed to the potential application of human induced pluripotent stem (iPS) cell-derived cardiomyocytes for cardiac safety pharmacology and toxicology with fewer legal and ethical issues. Supply of commercially available products enables many researchers to test the utility in their own systems. Although variations of electrophysiological properties have been reported among pluripotent cell lines by classifying cardiac subtypes into nodal, atrial and ventricular cells, most cells are spontaneously contracting, which makes difficult to be implicated in adult human hearts. Thus, we sought objective description on action potential (AP) parameters recorded from perforated patch-clamped iCell cardiomyocytes (iCell-CMs, AJ/CDI) by systematically comparing with those from human ES-derived cardiomyocytes (hES-CMs, Cellartis). Mean +/- SE values of APD50 in iCell-CMs (382 +/- 38 ms, n=36) were significantly longer than those in hES-CMs (278 +/- 28 ms n=64), while APD90 in iCell-CMs (1210 +/- 346 ms, n=36) tended to be longer than those in hES-CMs (776 +/- 136 ms n=64). As for APD90, despite of the large sample size, there was no statistical significance between iPS-CM and hES-CMs. Detailed analysis of the variation by Gaussian fitting revealed that marked differences in shapes of probability distribution between iPS-CMs and hES-CMs. Although further studies are necessary to know if the variation of AP parameters affect drug responses, our data provide important information for cardiac safety assessments.