Search

Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells; however, current methods limit the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables ~4.2x expansion of cells within individual wells, across multiple wells, and between plates. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms to greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, on human cardiomyocytes (CMs) as measured by Hoechst signal across the nucleus. We show a dose dependent effect on nuclear DNA that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications.

Background: Articular cartilage degeneration can result from injury, age, or arthritis, causing significant joint pain and disability without surgical intervention. Currently, the only FDA cell-based therapy for articular cartilage injury is Autologous Chondrocyte Implantation (ACI); however, this procedure is costly, time-intensive, and requires multiple treatments. Mesenchymal stromal cells (MSCs) are an attractive alternative autologous therapy due to their availability and ability to robustly differentiate into chondrocytes for transplantation with good safety profiles. However, treatment outcomes are variable due to donor-to-donor variability as well as intrapopulation heterogeneity and unstandardized MSC manufacturing protocols. Process improvements that reduce cell heterogeneity while increasing donor cell numbers with improved chondrogenic potential during expansion culture are needed to realize the full potential of MSC therapy., Methods: In this study, we investigated the potential of MSC metabolic modulation during expansion to enhance their chondrogenic commitment by varying the nutrient composition, including glucose, pyruvate, glutamine, and ascorbic acid in culture media. We tested the effect of metabolic modulation in short-term (one passage) and long-term (up to seven passages). We measured metabolic state, cell size, population doubling time, and senescence and employed novel tools including micro-magnetic resonance relaxometry (µMRR) relaxation time (T 2 ) to characterize the effects of AA on improved MSC expansion and chondrogenic potential., Results: Our data show that the addition of 1 mM L-ascorbic acid-2-phosphate (AA) to cultures for one passage during MSC expansion prior to initiation of differentiation improves chondrogenic differentiation. We further demonstrate that AA treatment reduced the proportion of senescent cells and cell heterogeneity also allowing for long-term expansion that led to a > 300-fold increase in yield of MSCs with enhanced chondrogenic potential compared to untreated cells. AA-treated MSCs with improved chondrogenic potential showed a robust shift in metabolic profile to OXPHOS and higher µMRR T 2 values, identifying critical quality attributes that could be implemented in MSC manufacturing for articular cartilage repair., Conclusions: Our results suggest an improved MSC manufacturing process that can enhance chondrogenic potential by targeting MSC metabolism and integrating process analytic tools during expansion., (© 2024. The Author(s).)

Background: The functional heterogeneity of culture-expanded mesenchymal stem cells (MSCs) has hindered the clinical application of MSCs. Previous studies have shown that MSC subpopulations with superior chondrogenic capacity can be isolated using a spiral microfluidic device based on the principle of inertial cell focusing., Hypothesis: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function will overcome the challenge of the functional heterogeneity of expanded MSCs and will significantly improve MSC-based cartilage repair., Study Design: Controlled laboratory study., Methods: A next-generation, fully automated multidimensional double spiral microfluidic device was designed to provide more refined and efficient isolation of MSC subpopulations based on size. Analysis of in vitro chondrogenic potential and RNA sequencing was performed on size-sorted MSC subpopulations. In vivo cartilage repair efficacy was demonstrated in an osteochondral injury model in 12-week-old rats. Defects were implanted with MSC subpopulations (n = 6 per group) and compared with those implanted with unsegregated MSCs (n = 6). Osteochondral repair was assessed at 6 and 12 weeks after surgery by histological, micro-computed tomography, and mechanical analysis., Results: A chondrogenic MSC subpopulation was efficiently isolated using the multidimensional double spiral device. RNA sequencing revealed distinct transcriptomic profiles and identified differential gene expression between subpopulations. The delivery of a chondrogenic MSC subpopulation resulted in improved cartilage repair, as indicated by histological scoring, the compression modulus, and micro-computed tomography of the subchondral bone., Conclusion: We have established a rapid, label-free, and reliable microfluidic protocol for more efficient size-based enrichment of a chondrogenic MSC subpopulation. Our proof-of-concept in vivo study demonstrates the enhanced cartilage repair efficacy of these enriched chondrogenic MSCs., Clinical Relevance: The delivery of microfluidic-enriched chondrogenic MSCs that are consistent in size and function can overcome the challenge of the functional heterogeneity of expanded MSCs, resulting in significant improvement in MSC-based cartilage repair. The availability of such rapid, label-free enriched chondrogenic MSCs can enable better cell therapy products for cartilage repair with improved treatment outcomes., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: This work was supported by the National Research Foundation of Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) program through the Critical Analytics for Manufacturing Personalized–Medicine (CAMP) Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology (SMART). H.J. and J.H. have a US patent on microfluidic devices (“Multi-Dimensional Double Spiral Device and Methods of Use Thereof”; patent pending No. US20220088588A1 [2019]). J.H. has a US patent on microfluidic devices (“Micro-Fluidic Devices and Uses Thereof”; patent No. 9,789,485 [2017]). AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

Background: Mesenchymal stromal cells (MSCs) have broad potential as a cell therapy including for the treatment of drug-resistant inflammatory conditions with abnormal T cell proliferation such as graft-versus-host disease (GVHD). Clinical success, however, has been complicated by the heterogeneity of culture-expanded MSCs as well as donor variability. Here, we devise culture conditions that promote expansion of MSCs with enhanced immunomodulatory functions both in vitro and in animal models of GVHD., Methods: Human bone marrow-derived MSCs were expanded at high-confluency (MSC HC ) and low-confluency state (MSC LC ). Their immunomodulatory properties were evaluated with in vitro co-culture assays based on suppression of activated T cell proliferation and secretion of pro-inflammatory cytokines from activated T cells. Metabolic state of these cells was determined, while RNA sequencing was performed to explore transcriptome of these MSCs. Ex vivo expanded MSC HC or MSC LC was injected into human peripheral blood mononuclear cells (PBMC)-induced GVHD mouse model to determine their in vivo therapeutic efficacy based on clinical grade scoring, human CD45 +  blood count and histopathological examination., Results: As compared to MSC LC , MSC HC significantly reduced both the proliferation of anti-CD3/CD28-activated T cells and secretion of pro-inflammatory cytokines upon MSC HC co-culture across several donors even in the absence of cytokine priming. Mechanistically, metabolic analysis of MSC HC prior to co-culture with activated T cells showed increased glycolytic metabolism and lactate secretion compared to MSC LC , consistent with their ability to inhibit T cell proliferation. Transcriptome analysis further revealed differential expression of immunomodulatory genes including TRIM29, BPIFB4, MMP3 and SPP1 in MSC HC as well as enriched pathways including cytokine-cytokine receptor interactions, cell adhesion and PI3K-AKT signalling . Lastly, we demonstrate in a human PBMC-induced GVHD mouse model that delivery of MSC HC showed greater suppression of inflammation and improved outcomes compared to MSC LC and saline controls., Conclusion: Our study provides evidence that ex vivo expansion of MSCs at high confluency alters the metabolic and transcriptomic states of these cells. Importantly, this approach maximizes the production of MSCs with enhanced immunomodulatory functions without priming, thus providing a non-invasive and generalizable strategy for improving the use of MSCs for the treatment of inflammatory diseases., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Vascularization is driven by morphogen signals and mechanical cues that coordinately regulate cellular force generation, migration, and shape change to sculpt the developing vascular network. However, it remains unclear whether developing vasculature actively regulates its own mechanical properties to achieve effective vascularization. We engineered tissue constructs containing endothelial cells and fibroblasts to investigate the mechanics of vascularization. Tissue stiffness increases during vascular morphogenesis resulting from emergent interactions between endothelial cells, fibroblasts, and ECM and correlates with enhanced vascular function. Contractile cellular forces are key to emergent tissue stiffening and synergize with ECM mechanical properties to modulate the mechanics of vascularization. Emergent tissue stiffening and vascular function rely on mechanotransduction signaling within fibroblasts, mediated by YAP1. Mouse embryos lacking YAP1 in fibroblasts exhibit both reduced tissue stiffness and develop lethal vascular defects. Translating our findings through biology-inspired vascular tissue engineering approaches will have substantial implications in regenerative medicine.

Detection of cellular senescence is important quality analytics of cell therapy products, including mesenchymal stromal cells (MSCs). However, its detection is critically limited by the lack of specific markers and the destructive assays used to read out these markers. Here, we establish a rapid, live-cell assay for detecting senescent cells in heterogeneous mesenchymal stromal cell (MSC) cultures. We report that the T2 relaxation time measured by microscale Magnetic Resonance Relaxometry, which is related to intracellular iron accumulation, correlates strongly with senescence markers in MSC cultures under diverse conditions, including different passages and donors, size-sorted MSCs by inertial spiral microfluidic device, and drug-induced senescence. In addition, the live-cell and non-destructive method presented here has general applicability to other cells and tissues and can critically advance our understanding of cellular senescence., (© The Author(s) 2023. Published by Oxford University Press.)

Stem cells are remarkably small. Whether small size is important for stem cell function is unknown. We find that hematopoietic stem cells (HSCs) enlarge under conditions known to decrease stem cell function. This decreased fitness of large HSCs is due to reduced proliferation and was accompanied by altered metabolism. Preventing HSC enlargement or reducing large HSCs in size averts the loss of stem cell potential under conditions causing stem cell exhaustion. Last, we show that murine and human HSCs enlarge during aging. Preventing this age-dependent enlargement improves HSC function. We conclude that small cell size is important for stem cell function in vivo and propose that stem cell enlargement contributes to their functional decline during aging.

RNA polymerase II (RNAPII) pausing immediately downstream of the transcription start site is a critical rate-limiting step for the expression of most metazoan genes. During pause release, RNAPII encounters a highly conserved +1 H2A.Z nucleosome, yet how this histone variant contributes to transcription is poorly understood. Here, using an inducible protein degron system combined with genomic approaches and live cell super-resolution microscopy, we show that H2A.Z.1 modulates RNAPII dynamics across most genes in murine embryonic stem cells. Our quantitative analysis shows that H2A.Z.1 slows the rate of RNAPII pause release and consequently impacts negative elongation factor dynamics as well as nascent transcription. Consequently, H2A.Z.1 also impacts re-loading of the pre-initiation complex components TFIIB and TBP. Altogether, this work provides a critical mechanistic link between H2A.Z.1 and the proper induction of mammalian gene expression programs through the regulation of RNAPII dynamics and pause release.

Ferroptosis-an iron-dependent, non-apoptotic cell death process-is involved in various degenerative diseases and represents a targetable susceptibility in certain cancers 1 . The ferroptosis-susceptible cell state can either pre-exist in cells that arise from certain lineages or be acquired during cell-state transitions 2-5 . However, precisely how susceptibility to ferroptosis is dynamically regulated remains poorly understood. Here we use genome-wide CRISPR-Cas9 suppressor screens to identify the oxidative organelles peroxisomes as critical contributors to ferroptosis sensitivity in human renal and ovarian carcinoma cells. Using lipidomic profiling we show that peroxisomes contribute to ferroptosis by synthesizing polyunsaturated ether phospholipids (PUFA-ePLs), which act as substrates for lipid peroxidation that, in turn, results in the induction of ferroptosis. Carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo in mice, which is associated with extensive downregulation of PUFA-ePLs. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including neurons and cardiomyocytes. Together, our work reveals roles for the peroxisome-ether-phospholipid axis in driving susceptibility to and evasion from ferroptosis, highlights PUFA-ePL as a distinct functional lipid class that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases that involve ferroptosis.

Deciphering the genetic and epigenetic regulation of cardiomyocyte proliferation in organisms that are capable of robust cardiac renewal, such as zebrafish, represents an attractive inroad towards regenerating the human heart. Using integrated high-throughput transcriptional and chromatin analyses, we have identified a strong association between H3K27me3 deposition and reduced sarcomere and cytoskeletal gene expression in proliferative cardiomyocytes following cardiac injury in zebrafish. To move beyond an association, we generated an inducible transgenic strain expressing a mutant version of histone 3, H3.3 K27M , that inhibits H3K27me3 catalysis in cardiomyocytes during the regenerative window. Hearts comprising H3.3 K27M -expressing cardiomyocytes fail to regenerate, with wound edge cells showing heightened expression of structural genes and prominent sarcomeres. Although cell cycle re-entry was unperturbed, cytokinesis and wound invasion were significantly compromised. Collectively, our study identifies H3K27me3-mediated silencing of structural genes as requisite for zebrafish heart regeneration and suggests that repression of similar structural components in the border zone of an infarcted human heart might improve its regenerative capacity., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (βOHB), distinguishes self-renewing Lgr5 + stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes βOHB levels in Lgr5 + ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous βOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, βOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through βOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of βOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Microdeletions involving TBX1 result in variable congenital malformations known collectively as 22q11.2 deletion syndrome (22q11.2DS). Tbx1-deficient mice and zebrafish recapitulate several disease phenotypes, including pharyngeal arch artery (PAA), head muscle (HM), and cardiac outflow tract (OFT) deficiencies. In zebrafish, these structures arise from nkx2.5 + progenitors in pharyngeal arches 2-6. Because pharyngeal arch morphogenesis is compromised in Tbx1-deficient animals, the malformations were considered secondary. Here, we report that the PAA, HM, and OFT phenotypes in tbx1 mutant zebrafish are primary and arise prior to pharyngeal arch morphogenesis from failed specification of the nkx2.5 + pharyngeal lineage. Through in situ analysis and lineage tracing, we reveal that nkx2.5 and tbx1 are co-expressed in this progenitor population. Furthermore, we present evidence suggesting that gdf3-ALK4 signaling is a downstream mediator of nkx2.5 + pharyngeal lineage specification. Collectively, these studies support a cellular mechanism potentially underlying the cardiovascular and craniofacial defects observed in the 22q11.2DS population., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs - "4-way", "7-way", and "10-way" - each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS "physiome-on-a-chip" approaches in drug discovery.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising platform for cardiac studies in vitro, and possibly for tissue repair in humans. However, hiPSC-CM cells tend to retain morphology, metabolism, patterns of gene expression, and electrophysiology similar to that of embryonic cardiomyocytes. We grew hiPSC-CM in patterned islands of different sizes and shapes, and measured the effect of island geometry on action potential waveform and calcium dynamics using optical recordings of voltage and calcium from 970 islands of different sizes. hiPSC-CM in larger islands showed electrical and calcium dynamics indicative of greater functional maturity. We then compared transcriptional signatures of the small and large islands against a developmental time course of cardiac differentiation. Although island size had little effect on expression of most genes whose levels differed between hiPSC-CM and adult primary CM, we identified a subset of genes for which island size drove the majority (58%) of the changes associated with functional maturation. Finally, we patterned hiPSC-CM on islands with a variety of shapes to probe the relative contributions of soluble factors, electrical coupling, and direct cell-cell contacts to the functional maturation. Collectively, our data show that optical electrophysiology is a powerful tool for assaying hiPSC-CM maturation, and that island size powerfully drives activation of a subset of genes involved in cardiac maturation.

Long non-coding RNAs (lncRNAs) are an emerging class of transcripts that can modulate gene expression; however, their mechanisms of action remain poorly understood. Here, we experimentally determine the secondary structure of Braveheart (Bvht) using chemical probing methods and show that this ∼590 nt transcript has a modular fold. Using CRISPR/Cas9-mediated editing of mouse embryonic stem cells, we find that deletion of 11 nt in a 5' asymmetric G-rich internal loop (AGIL) of Bvht (bvht dAGIL ) dramatically impairs cardiomyocyte differentiation. We demonstrate a specific interaction between AGIL and cellular nucleic acid binding protein (CNBP/ZNF9), a zinc-finger protein known to bind single-stranded G-rich sequences. We further show that CNBP deletion partially rescues the bvht dAGIL mutant phenotype by restoring differentiation capacity. Together, our work shows that Bvht functions with CNBP through a well-defined RNA motif to regulate cardiovascular lineage commitment, opening the door for exploring broader roles of RNA structure in development and disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Myocardial mass is a key determinant of cardiac muscle function and hypertrophy. Myocardial depolarization leading to cardiac muscle contraction is reflected by the amplitude and duration of the QRS complex on the electrocardiogram (ECG). Abnormal QRS amplitude or duration reflect changes in myocardial mass and conduction, and are associated with increased risk of heart failure and death., Objectives: This meta-analysis sought to gain insights into the genetic determinants of myocardial mass., Methods: We carried out a genome-wide association meta-analysis of 4 QRS traits in up to 73,518 individuals of European ancestry, followed by extensive biological and functional assessment., Results: We identified 52 genomic loci, of which 32 are novel, that are reliably associated with 1 or more QRS phenotypes at p < 1 × 10(-8). These loci are enriched in regions of open chromatin, histone modifications, and transcription factor binding, suggesting that they represent regions of the genome that are actively transcribed in the human heart. Pathway analyses provided evidence that these loci play a role in cardiac hypertrophy. We further highlighted 67 candidate genes at the identified loci that are preferentially expressed in cardiac tissue and associated with cardiac abnormalities in Drosophila melanogaster and Mus musculus. We validated the regulatory function of a novel variant in the SCN5A/SCN10A locus in vitro and in vivo., Conclusions: Taken together, our findings provide new insights into genes and biological pathways controlling myocardial mass and may help identify novel therapeutic targets., (Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Motor units are the fundamental elements responsible for muscle movement. They are formed by lower motor neurons and their muscle targets, synapsed via neuromuscular junctions (NMJs). The loss of NMJs in neurodegenerative disorders (such as amyotrophic lateral sclerosis or spinal muscle atrophy) or as a result of traumatic injuries affects millions of lives each year. Developing in vitro assays that closely recapitulate the physiology of neuromuscular tissues is crucial to understand the formation and maturation of NMJs, as well as to help unravel the mechanisms leading to their degeneration and repair. We present a microfluidic platform designed to coculture myoblast-derived muscle strips and motor neurons differentiated from mouse embryonic stem cells (ESCs) within a three-dimensional (3D) hydrogel. The device geometry mimics the spinal cord-limb physical separation by compartmentalizing the two cell types, which also facilitates the observation of 3D neurite outgrowth and remote muscle innervation. Moreover, the use of compliant pillars as anchors for muscle strips provides a quantitative functional readout of force generation. Finally, photosensitizing the ESC provides a pool of source cells that can be differentiated into optically excitable motor neurons, allowing for spatiodynamic, versatile, and noninvasive in vitro control of the motor units.

The ST-segment and adjacent T-wave (ST-T wave) amplitudes of the electrocardiogram are quantitative characteristics of cardiac repolarization. Repolarization abnormalities have been linked to ventricular arrhythmias and sudden cardiac death. We performed the first genome-wide association meta-analysis of ST-T-wave amplitudes in up to 37 977 individuals identifying 71 robust genotype-phenotype associations clustered within 28 independent loci. Fifty-four genes were prioritized as candidates underlying the phenotypes, including genes with established roles in the cardiac repolarization phase (SCN5A/SCN10A, KCND3, KCNB1, NOS1AP and HEY2) and others with as yet undefined cardiac function. These associations may provide insights in the spatiotemporal contribution of genetic variation influencing cardiac repolarization and provide novel leads for future functional follow-up., (© The Author 2016. Published by Oxford University Press.)

Genetic variants identified by genome-wide association studies explain only a modest proportion of heritability, suggesting that meaningful associations lie 'hidden' below current thresholds. Here, we integrate information from association studies with epigenomic maps to demonstrate that enhancers significantly overlap known loci associated with the cardiac QT interval and QRS duration. We apply functional criteria to identify loci associated with QT interval that do not meet genome-wide significance and are missed by existing studies. We demonstrate that these 'sub-threshold' signals represent novel loci, and that epigenomic maps are effective at discriminating true biological signals from noise. We experimentally validate the molecular, gene-regulatory, cellular and organismal phenotypes of these sub-threshold loci, demonstrating that most sub-threshold loci have regulatory consequences and that genetic perturbation of nearby genes causes cardiac phenotypes in mouse. Our work provides a general approach for improving the detection of novel loci associated with complex human traits.

Failure of the human heart to maintain sufficient output of blood for the demands of the body, heart failure, is a common condition with high mortality even with modern therapeutic alternatives. To identify molecular determinants of mortality in patients with new-onset heart failure, we performed a meta-analysis of genome-wide association studies and follow-up genotyping in independent populations. We identified and replicated an association for a genetic variant on chromosome 5q22 with 36% increased risk of death in subjects with heart failure (rs9885413, P = 2.7x10-9). We provide evidence from reporter gene assays, computational predictions and epigenomic marks that this polymorphism increases activity of an enhancer region active in multiple human tissues. The polymorphism was further reproducibly associated with a DNA methylation signature in whole blood (P = 4.5x10-40) that also associated with allergic sensitization and expression in blood of the cytokine TSLP (P = 1.1x10-4). Knockdown of the transcription factor predicted to bind the enhancer region (NHLH1) in a human cell line (HEK293) expressing NHLH1 resulted in lower TSLP expression. In addition, we observed evidence of recent positive selection acting on the risk allele in populations of African descent. Our findings provide novel genetic leads to factors that influence mortality in patients with heart failure.

Histone variant H2A.Z occupies the promoters of active and poised, bivalent genes in embryonic stem cells (ESCs) to regulate developmental programs, yet how it contributes to these contrasting states is poorly understood. Here, we investigate the function of H2A.Z.1 monoubiquitylation (H2A.Z.1ub) by mutation of the PRC1 target residues (H2A.Z.1(K3R3)). We show that H2A.Z.1(K3R3) is properly incorporated at target promoters in murine ESCs (mESCs), but loss of monoubiquitylation leads to de-repression of bivalent genes, loss of Polycomb binding, and faulty lineage commitment. Using quantitative proteomics, we find that tandem bromodomain proteins, including the BET family member BRD2, are enriched in H2A.Z.1 chromatin. We further show that BRD2 is gained at de-repressed promoters in H2A.Z.1(K3R3) mESCs, whereas BRD2 inhibition restores gene silencing at these sites. Together, our study reveals an antagonistic relationship between H2A.Z.1ub and BRD2 to regulate the transcriptional balance at bivalent genes to enable proper execution of developmental programs., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The histone variant H2A.Z is a hallmark of nucleosomes flanking promoters of protein-coding genes and is often found in nucleosomes that carry lysine 56-acetylated histone H3 (H3-K56Ac), a mark that promotes replication-independent nucleosome turnover. Here, we find that H3-K56Ac promotes RNA polymerase II occupancy at many protein-coding and noncoding loci, yet neither H3-K56Ac nor H2A.Z has a significant impact on steady-state mRNA levels in yeast. Instead, broad effects of H3-K56Ac or H2A.Z on RNA levels are revealed only in the absence of the nuclear RNA exosome. H2A.Z is also necessary for the expression of divergent, promoter-proximal noncoding RNAs (ncRNAs) in mouse embryonic stem cells. Finally, we show that H2A.Z functions with H3-K56Ac to facilitate formation of chromosome interaction domains (CIDs). Our study suggests that H2A.Z and H3-K56Ac work in concert with the RNA exosome to control mRNA and ncRNA expression, perhaps in part by regulating higher-order chromatin structures., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Transcriptional and epigenetic regulation is critical for proper heart development, cardiac homeostasis, and pathogenesis. Long noncoding RNAs have emerged as key components of the transcriptional regulatory pathways that govern cardiac development as well as stress response, signaling, and remodeling in cardiac pathologies. Within the past few years, studies have identified many long noncoding RNAs in the context of cardiovascular biology and have begun to reveal the key functions of these transcripts. In this review, we discuss the growing roles of long noncoding RNAs in different aspects of cardiovascular development as well as pathological responses during injury or disease. In addition, we discuss diverse mechanisms by which long noncoding RNAs orchestrate cardiac transcriptional programs. Finally, we explore the exciting potential of this novel class of transcripts as biomarkers and novel therapeutic targets for cardiovascular diseases., (© 2015 American Heart Association, Inc.)

Rationale: Neonatal mice have the capacity to regenerate their hearts in response to injury, but this potential is lost after the first week of life. The transcriptional changes that underpin mammalian cardiac regeneration have not been fully characterized at the molecular level., Objective: The objectives of our study were to determine whether myocytes revert the transcriptional phenotype to a less differentiated state during regeneration and to systematically interrogate the transcriptional data to identify and validate potential regulators of this process., Methods and Results: We derived a core transcriptional signature of injury-induced cardiac myocyte (CM) regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo CM differentiation, in vitro CM explant model, as well as a neonatal heart resection model. The regenerating mouse heart revealed a transcriptional reversion of CM differentiation processes, including reactivation of latent developmental programs similar to those observed during destabilization of a mature CM phenotype in the explant model. We identified potential upstream regulators of the core network, including interleukin 13, which induced CM cell cycle entry and STAT6/STAT3 signaling in vitro. We demonstrate that STAT3/periostin and STAT6 signaling are critical mediators of interleukin 13 signaling in CMs. These downstream signaling molecules are also modulated in the regenerating mouse heart., Conclusions: Our work reveals new insights into the transcriptional regulation of mammalian cardiac regeneration and provides the founding circuitry for identifying potential regulators for stimulating heart regeneration., (© 2014 American Heart Association, Inc.)

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

The replacement of nucleosomal H2A with the histone variant H2A.Z is critical for regulating DNA-mediated processes across eukaryotes and for early development of multicellular organisms. How this variant performs these seemingly diverse roles has remained largely enigmatic. Here, we discuss recent mechanistic insights that have begun to reveal how H2A.Z functions as a molecular rheostat for gene control. We focus on specific examples in metazoans as a model for understanding how H2A.Z integrates information from histone post-translational modifications, other histone variants, and transcription factors (TFs) to regulate proper induction of gene expression programs in response to cellular cues. Finally, we propose a general model of how H2A.Z incorporation regulates chromatin states in diverse processes.

Polycomb Repressive Complex 2 (PRC2) catalyzes histone H3 lysine 27 tri-methylation (H3K27me3), an epigenetic modification associated with gene repression. H3K27me3 is enriched at the promoters of a large cohort of developmental genes in embryonic stem cells (ESCs). Loss of H3K27me3 leads to a failure of ESCs to properly differentiate, making it difficult to determine the precise roles of PRC2 during lineage commitment. Moreover, while studies suggest that PRC2 prevents DNA methylation, how these two epigenetic regulators coordinate to regulate lineage programs is poorly understood. Using several PRC2 mutant ESC lines that maintain varying levels of H3K27me3, we found that partial maintenance of H3K27me3 allowed for proper temporal activation of lineage genes during directed differentiation of ESCs to spinal motor neurons (SMNs). In contrast, genes that function to specify other lineages failed to be repressed in these cells, suggesting that PRC2 is also necessary for lineage fidelity. We also found that loss of H3K27me3 leads to a modest gain in DNA methylation at PRC2 target regions in both ESCs and in SMNs. Our study demonstrates a critical role for PRC2 in safeguarding lineage decisions and in protecting genes against inappropriate DNA methylation.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD.

Advances in genome research have provided an unprecedented opportunity to investigate the function of non-coding DNA regulatory regions that control transcription. Large-scale studies have recently identified hundreds of thousands of distal enhancer elements; their discovery has revealed new insights into the mechanistic details of how tissue-specific gene expression patterns are established and maintained during development. Emerging evidence indicates that lineage-specific transcription factors and chromatin regulators coordinate the activation of distal enhancers to ensure robust control of gene expression programs in a cell type-specific manner. We discuss recent progress in the field and emphasize examples related to the cardiac lineage, where possible, as a model for understanding the contribution of enhancer biology to development and how disruption of enhancer function leads to disease., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Hox genes controlling motor neuron subtype identity are expressed in rostrocaudal patterns that are spatially and temporally collinear with their chromosomal organization. Here we demonstrate that Hox chromatin is subdivided into discrete domains that are controlled by rostrocaudal patterning signals that trigger rapid, domain-wide clearance of repressive histone H3 Lys27 trimethylation (H3K27me3) polycomb modifications. Treatment of differentiating mouse neural progenitors with retinoic acid leads to activation and binding of retinoic acid receptors (RARs) to the Hox1-Hox5 chromatin domains, which is followed by a rapid domain-wide removal of H3K27me3 and acquisition of cervical spinal identity. Wnt and fibroblast growth factor (FGF) signals induce expression of the Cdx2 transcription factor that binds and clears H3K27me3 from the Hox1-Hox9 chromatin domains, leading to specification of brachial or thoracic spinal identity. We propose that rapid clearance of repressive modifications in response to transient patterning signals encodes global rostrocaudal neural identity and that maintenance of these chromatin domains ensures the transmission of positional identity to postmitotic motor neurons later in development.

Cardiogenesis in mammals requires exquisite control of gene expression and faulty regulation of transcriptional programs underpins congenital heart disease (CHD), the most common defect among live births. Similarly, many adult cardiac diseases involve transcriptional changes and sometimes have a developmental basis. Long non-coding RNAs (lncRNAs) are a novel class of transcripts that regulate cellular processes by controlling gene expression; however, detailed insights into their biological and mechanistic functions are only beginning to emerge. Here, we discuss recent findings suggesting that lncRNAs are important factors in regulation of mammalian cardiogenesis and in the pathogenesis of CHD as well as adult cardiac disease. We also outline potential methodological and conceptual considerations for future studies of lncRNAs in the heart and other contexts.

Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm toward a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of mesoderm posterior 1 (MesP1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of polycomb-repressive complex 2 (PRC2), during cardiomyocyte differentiation, suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The histone H2A variant H2A.Z is essential for embryonic development and for proper control of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions of amino acid sequence of H2A.Z likely determine its functional specialization compared to core histone H2A. For example, H2A.Z contains three divergent residues in the essential C-terminal acidic patch that reside on the surface of the histone octamer as an uninterrupted acidic patch domain; however, we know little about how these residues contribute to chromatin structure and function. Here, we show that the divergent amino acids Gly92, Asp97, and Ser98 in the H2A.Z C-terminal acidic patch (H2A.Z(AP3)) are critical for lineage commitment during ESC differentiation. H2A.Z is enriched at most H3K4me3 promoters in ESCs including poised, bivalent promoters that harbor both activating and repressive marks, H3K4me3 and H3K27me3 respectively. We found that while H2A.Z(AP3) interacted with its deposition complex and displayed a highly similar distribution pattern compared to wild-type H2A.Z, its enrichment levels were reduced at target promoters. Further analysis revealed that H2A.Z(AP3) was less tightly associated with chromatin, suggesting that the mutant is more dynamic. Notably, bivalent genes in H2A.Z(AP3) ESCs displayed significant changes in expression compared to active genes. Moreover, bivalent genes in H2A.Z(AP3) ESCs gained H3.3, a variant associated with higher nucleosome turnover, compared to wild-type H2A.Z. We next performed single cell imaging to measure H2A.Z dynamics. We found that H2A.Z(AP3) displayed higher mobility in chromatin compared to wild-type H2A.Z by fluorescent recovery after photobleaching (FRAP). Moreover, ESCs treated with the transcriptional inhibitor flavopiridol resulted in a decrease in the H2A.Z(AP3) mobile fraction and an increase in its occupancy at target genes indicating that the mutant can be properly incorporated into chromatin. Collectively, our work suggests that the divergent residues in the H2A.Z acidic patch comprise a unique domain that couples control of chromatin dynamics to the regulation of developmental gene expression patterns during lineage commitment., Competing Interests: The authors have declared that no competing interests exist.

SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs); however, we currently lack a detailed understanding of how SOX2 controls these distinct stem cell populations. Here we show by genome-wide analysis that, while SOX2 bound to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 bound the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. We found that, similar to its association with OCT4 (Pou5f1) in ESCs, the related POU family member BRN2 (Pou3f2) co-occupied a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs led to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. Our findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors., Competing Interests: The authors have declared that no competing interests exist.

Heart development is exquisitely sensitive to the precise temporal regulation of thousands of genes that govern developmental decisions during differentiation. However, we currently lack a detailed understanding of how chromatin and gene expression patterns are coordinated during developmental transitions in the cardiac lineage. Here, we interrogated the transcriptome and several histone modifications across the genome during defined stages of cardiac differentiation. We find distinct chromatin patterns that are coordinated with stage-specific expression of functionally related genes, including many human disease-associated genes. Moreover, we discover a novel preactivation chromatin pattern at the promoters of genes associated with heart development and cardiac function. We further identify stage-specific distal enhancer elements and find enriched DNA binding motifs within these regions that predict sets of transcription factors that orchestrate cardiac differentiation. Together, these findings form a basis for understanding developmentally regulated chromatin transitions during lineage commitment and the molecular etiology of congenital heart disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Developmental programs are controlled by transcription factors and chromatin regulators, which maintain specific gene expression programs through epigenetic modification of the genome. These regulatory events at enhancers contribute to the specific gene expression programs that determine cell state and the potential for differentiation into new cell types. Although enhancer elements are known to be associated with certain histone modifications and transcription factors, the relationship of these modifications to gene expression and developmental state has not been clearly defined. Here we interrogate the epigenetic landscape of enhancer elements in embryonic stem cells and several adult tissues in the mouse. We find that histone H3K27ac distinguishes active enhancers from inactive/poised enhancer elements containing H3K4me1 alone. This indicates that the amount of actively used enhancers is lower than previously anticipated. Furthermore, poised enhancer networks provide clues to unrealized developmental programs. Finally, we show that enhancers are reset during nuclear reprogramming.

Precise control of gene expression patterns is critical for the specification of cellular diversity during metazoan development. Polycomb group (PcG) proteins comprise a class of transcriptional modifiers that have dynamic and essential roles in regulating a number of key processes including lineage commitment. How this is accomplished during mammalian development is incompletely understood. Here, we discuss recent studies in embryonic stem cells (ESCs) that provide critical new insights into how PcG proteins may be targeted to genomic sites as well as the mechanisms by which these regulators influence gene expression and multilineage differentiation in mammals., (Copyright 2010 Elsevier Inc. All rights reserved.)

The recent generation of induced pluripotent stem cells (iPSCs) from somatic cells provides an invaluable resource for drug or toxicology screening, medical research, and patient-specific cell therapy. However, there are currently a number of obstacles including virus integration and the genetic alteration of iPSCs that will need to be overcome before these cells may be considered safe for clinical applications. Here, we highlight the potential and challenges of iPSC research and review advances in reprogramming methods that have rapidly moved the field closer to realizing the goal of generating safe iPSCs for transplantation.

Molecular regulation of embryonic stem cell (ESC) fate involves a coordinated interaction between epigenetic, transcriptional and translational mechanisms. It is unclear how these different molecular regulatory mechanisms interact to regulate changes in stem cell fate. Here we present a dynamic systems-level study of cell fate change in murine ESCs following a well-defined perturbation. Global changes in histone acetylation, chromatin-bound RNA polymerase II, messenger RNA (mRNA), and nuclear protein levels were measured over 5 days after downregulation of Nanog, a key pluripotency regulator. Our data demonstrate how a single genetic perturbation leads to progressive widespread changes in several molecular regulatory layers, and provide a dynamic view of information flow in the epigenome, transcriptome and proteome. We observe that a large proportion of changes in nuclear protein levels are not accompanied by concordant changes in the expression of corresponding mRNAs, indicating important roles for translational and post-translational regulation of ESC fate. Gene-ontology analysis across different molecular layers indicates that although chromatin reconfiguration is important for altering cell fate, it is preceded by transcription-factor-mediated regulatory events. The temporal order of gene expression alterations shows the order of the regulatory network reconfiguration and offers further insight into the gene regulatory network. Our studies extend the conventional systems biology approach to include many molecular species, regulatory layers and temporal series, and underscore the complexity of the multilayer regulatory mechanisms responsible for changes in protein expression that determine stem cell fate.

Two recent studies, including one in this issue of Cell Stem Cell, have identified novel regulators of embryonic stem cell (ESC) self-renewal using genome-wide RNAi screens in mouse ESCs (Ding et al., 2009; Hu et al., 2009) and have further expanded the repertoire of factors that regulate ESC identity.

Elucidating how chromatin influences gene expression patterns and ultimately cell fate is fundamental to understanding development and disease. The histone variant H2AZ has emerged as a key regulator of chromatin function and plays an essential but unknown role during mammalian development. Here, genome-wide analysis reveals that H2AZ occupies the promoters of developmentally important genes in a manner that is remarkably similar to that of the Polycomb group (PcG) protein Suz12. By using RNAi, we demonstrate a role for H2AZ in regulating target gene expression, find that H2AZ and PcG protein occupancy is interdependent at promoters, and further show that H2AZ is necessary for ES cell differentiation. Notably, H2AZ occupies a different subset of genes in lineage-committed cells, suggesting that its dynamic redistribution is necessary for cell fate transitions. Thus, H2AZ, together with PcG proteins, may establish specialized chromatin states in ES cells necessary for the proper execution of developmental gene expression programs.

In metazoans, lineage-specific transcription factors and epigenetic modifiers function to establish and maintain proper gene expression programs during development. Recent landmark studies in both mouse and human have defined a set of transcription factors whose ectopic expression by retroviral transduction is capable of reprogramming a somatic nucleus to the pluripotent state. The identification of factors that are sufficient for the induction of pluripotency suggests that rewiring transcriptional regulatory networks at the molecular level can be used to manipulate cell fate in vitro. These findings have broad implications for understanding development and disease and for the potential use of stem cells in therapeutic applications.

Background: Genome-wide approaches have begun to reveal the transcriptional networks responsible for pluripotency in embryonic stem (ES) cells. Chromatin Immunoprecipitation (ChIP) followed either by hybridization to a microarray platform (ChIP-chip) or by DNA sequencing (ChIP-PET), has identified binding targets of the ES cell transcription factors OCT4 and NANOG in humans and mice, respectively. These studies have provided an outline of the transcriptional framework involved in maintaining pluripotency. Recent evidence with comparing multiple technologies suggests that expanding these datasets using different platforms would be a useful resource for examining the mechanisms underlying pluripotency regulation., Results: We have now identified OCT4 and NANOG genomic targets in mouse ES cells by ChIP-chip and provided the means to compare these data with previously reported ChIP-PET results in mouse ES cells. We have mapped the sequences of OCT4 and NANOG binding events from each dataset to genomic coordinates, providing a valuable resource to facilitate a better understanding of the ES cell regulatory circuitry. Interestingly, although considerable differences are observed in OCT4 and NANOG occupancy as identified by each method, a substantial number of targets in both datasets are enriched for genes that have known roles in cell-fate specification and that are differentially expressed upon Oct4 or Nanog knockdown., Conclusion: This study suggests that each dataset is a partial representation of the overall ES cell regulatory circuitry, and through integrating binding data obtained by ChIP-chip and ChIP-PET, the methods presented here provide a useful means for integrating datasets obtained by different techniques in the future.

We describe the results of a genome-wide analysis of human cells that suggests that most protein-coding genes, including most genes thought to be transcriptionally inactive, experience transcription initiation. We found that nucleosomes with H3K4me3 and H3K9,14Ac modifications, together with RNA polymerase II, occupy the promoters of most protein-coding genes in human embryonic stem cells. Only a subset of these genes produce detectable full-length transcripts and are occupied by nucleosomes with H3K36me3 modifications, a hallmark of elongation. The other genes experience transcription initiation but show no evidence of elongation, suggesting that they are predominantly regulated at postinitiation steps. Genes encoding most developmental regulators fall into this group. Our results also identify a class of genes that are excluded from experiencing transcription initiation, at which mechanisms that prevent initiation must predominate. These observations extend to differentiated cells, suggesting that transcription initiation at most genes is a general phenomenon in human cells.