Introductions to the Community: Early-Career Researchers in the Time of COVID-19
CELL STEM CELL
2021; 28 (1): 17?19
COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.
View details for Web of Science ID 000606536600007
View details for PubMedID 33417867
Network-based screen in iPSC-derived cells reveals therapeutic candidate for heart valve disease.
Science (New York, N.Y.)
Mapping the gene regulatory networks dysregulated in human disease would allow the design of network-correcting therapies that treat the core disease mechanism. However, small molecules are traditionally screened for their effects on one to several outputs at most, biasing discovery and limiting the likelihood of true disease-modifying drug candidates. Here, we developed a machine learning approach to identify small molecules that broadly correct gene networks dysregulated in a human induced pluripotent stem cell (iPSC) disease model of a common form of heart disease involving the aortic valve. Gene network correction by the most efficacious therapeutic candidate, XCT790, generalized to patient-derived primary aortic valve cells and was sufficient to prevent and treat aortic valve disease in vivo in a mouse model. This strategy, made feasible by human iPSC technology, network analysis, and machine learning, may represent an effective path for drug discovery.
View details for DOI 10.1126/science.abd0724
View details for PubMedID 33303684
Context-Specific Transcription Factor Functions Regulate Epigenomic and Transcriptional Dynamics during Cardiac Reprogramming.
Cell stem cell
2019; 25 (1): 87?102.e9
Ectopic expression of combinations of transcription factors (TFs) can drive direct lineage conversion, thereby reprogramming a somatic cell's identity. To determine the molecular mechanisms by which Gata4, Mef2c, and Tbx5 (GMT) induce conversion from a cardiac fibroblast toward an induced cardiomyocyte, we performed comprehensive transcriptomic, DNA-occupancy, and epigenomic interrogation throughout the reprogramming process. Integration of these datasets identified new TFs involved in cardiac reprogramming and revealed context-specific roles for GMT, including the ability of Mef2c and Tbx5 to independently promote chromatin remodeling at previously inaccessible sites. We also find evidence for cooperative facilitation and refinement of each TF's binding profile in a combinatorial setting. A reporter assay employing newly defined regulatory elements confirmed that binding of a single TF can be sufficient for gene activation, suggesting that co-binding events do not necessarily reflect synergy. These results shed light on fundamental mechanisms by which combinations of TFs direct lineage conversion.
View details for DOI 10.1016/j.stem.2019.06.012
View details for PubMedID 31271750
View details for PubMedCentralID PMC6632093
Oligogenic inheritance of a human heart disease involving a genetic modifier.
Science (New York, N.Y.)
2019; 364 (6443): 865?70
Complex genetic mechanisms are thought to underlie many human diseases, yet experimental proof of this model has been elusive. Here, we show that a human cardiac anomaly can be caused by a combination of rare, inherited heterozygous mutations. Whole-exome sequencing of a nuclear family revealed that three offspring with childhood-onset cardiomyopathy had inherited three missense single-nucleotide variants in the MKL2, MYH7, and NKX2-5 genes. The MYH7 and MKL2 variants were inherited from the affected, asymptomatic father and the rare NKX2-5 variant (minor allele frequency, 0.0012) from the unaffected mother. We used CRISPR-Cas9 to generate mice encoding the orthologous variants and found that compound heterozygosity for all three variants recapitulated the human disease phenotype. Analysis of murine hearts and human induced pluripotent stem cell-derived cardiomyocytes provided histologic and molecular evidence for the NKX2-5 variant's contribution as a genetic modifier.
View details for DOI 10.1126/science.aat5056
View details for PubMedID 31147515
View details for PubMedCentralID PMC6557373
Single-cell analysis of cardiogenesis reveals basis for organ-level developmental defects.
2019; 572 (7767): 120?24
Organogenesis involves integration of diverse cell types; dysregulation of cell-type-specific gene networks results in birth defects, which affect 5% of live births. Congenital heart defects are the most common malformations, and result from disruption of discrete subsets of cardiac progenitor cells1, but the transcriptional changes in individual progenitors that lead to organ-level defects remain unknown. Here we used single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular subpopulations has catastrophic consequences. A network-based computational method for single-cell RNA-sequencing analysis that predicts lineage-specifying transcription factors2,3 identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite the failure of right ventricular formation in Hand2-null mice4. Temporal single-cell-transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium was specified but failed to properly differentiate and migrate. Loss of Hand2 also led to dysregulation of retinoic acid signalling and disruption of anterior-posterior patterning of cardiac progenitors. This work reveals transcriptional determinants that specify fate and differentiation in individual cardiac progenitor cells, and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework for investigating congenital heart defects.
View details for DOI 10.1038/s41586-019-1414-x
View details for PubMedID 31341279
View details for PubMedCentralID PMC6719697
Genetic determinants and epigenetic effects of pioneer-factor occupancy
2018; 50 (2): 250-+
Transcription factors (TFs) direct developmental transitions by binding to target DNA sequences, influencing gene expression and establishing complex gene-regultory networks. To systematically determine the molecular components that enable or constrain TF activity, we investigated the genomic occupancy of FOXA2, GATA4 and OCT4 in several cell types. Despite their classification as pioneer factors, all three TFs exhibit cell-type-specific binding, even when supraphysiologically and ectopically expressed. However, FOXA2 and GATA4 can be distinguished by low enrichment at loci that are highly occupied by these factors in alternative cell types. We find that expression of additional cofactors increases enrichment at a subset of these sites. Finally, FOXA2 occupancy and changes to DNA accessibility can occur in G1-arrested cells, but subsequent loss of DNA methylation requires DNA replication.
View details for DOI 10.1038/s41588-017-0034-3
View details for Web of Science ID 000424519200016
View details for PubMedID 29358654
View details for PubMedCentralID PMC6517675
Differentiation of V2a interneurons from human pluripotent stem cells.
Proceedings of the National Academy of Sciences of the United States of America
2017; 114 (19): 4969?74
The spinal cord consists of multiple neuronal cell types that are critical to motor control and arise from distinct progenitor domains in the developing neural tube. Excitatory V2a interneurons in particular are an integral component of central pattern generators that control respiration and locomotion; however, the lack of a robust source of human V2a interneurons limits the ability to molecularly profile these cells and examine their therapeutic potential to treat spinal cord injury (SCI). Here, we report the directed differentiation of CHX10+ V2a interneurons from human pluripotent stem cells (hPSCs). Signaling pathways (retinoic acid, sonic hedgehog, and Notch) that pattern the neural tube were sequentially perturbed to identify an optimized combination of small molecules that yielded ?25% CHX10+ cells in four hPSC lines. Differentiated cultures expressed much higher levels of V2a phenotypic markers (CHX10 and SOX14) than other neural lineage markers. Over time, CHX10+ cells expressed neuronal markers [neurofilament, NeuN, and vesicular glutamate transporter 2 (VGlut2)], and cultures exhibited increased action potential frequency. Single-cell RNAseq analysis confirmed CHX10+ cells within the differentiated population, which consisted primarily of neurons with some glial and neural progenitor cells. At 2 wk after transplantation into the spinal cord of mice, hPSC-derived V2a cultures survived at the site of injection, coexpressed NeuN and VGlut2, extended neurites >5 mm, and formed putative synapses with host neurons. These results provide a description of V2a interneurons differentiated from hPSCs that may be used to model central nervous system development and serve as a potential cell therapy for SCI.
View details for DOI 10.1073/pnas.1608254114
View details for PubMedID 28438991
View details for PubMedCentralID PMC5441696
Transcriptional and Chromatin Dynamics of Muscle Regeneration after Severe Trauma
STEM CELL REPORTS
2016; 7 (5): 983?97
Following injury, adult skeletal muscle undergoes a well-coordinated sequence of molecular and physiological events to promote repair and regeneration. However, a thorough understanding of the in vivo epigenomic and transcriptional mechanisms that control these reparative events is lacking. To address this, we monitored the in vivo dynamics of three histone modifications and coding and noncoding RNA expression throughout the regenerative process in a mouse model of traumatic muscle injury. We first illustrate how both coding and noncoding RNAs in tissues and sorted satellite cells are modified and regulated during various stages after trauma. Next, we use chromatin immunoprecipitation followed by sequencing to evaluate the chromatin state of cis-regulatory elements (promoters and enhancers) and view how these elements evolve and influence various muscle repair and regeneration transcriptional programs. These results provide a comprehensive view of the central factors that regulate muscle regeneration and underscore the multiple levels through which both transcriptional and epigenetic patterns are regulated to enact appropriate repair and regeneration.
View details for DOI 10.1016/j.stemcr.2016.09.009
View details for Web of Science ID 000389509000013
View details for PubMedID 27773702
View details for PubMedCentralID PMC5106515
Heart disease modelling adds a Notch to its belt.
Nature cell biology
2016; 18 (1): 3?5
The heart is a complex organ, consisting of multiple cell types that coordinately regulate blood flow. Reciprocal Notch pathway signalling in endocardial and myocardial cells is now shown to promote maturation of the ventricular chambers. These insights reveal mechanisms that, when disrupted, can lead to cardiomyopathies.
View details for DOI 10.1038/ncb3294
View details for PubMedID 26693916
A qPCRCR ScoreCard quantifies the differentiation potential of human pluripotent stem cells
2015; 33 (11): 1182-U117
Research on human pluripotent stem cells has been hampered by the lack of a standardized, quantitative, scalable assay of pluripotency. We previously described an assay called ScoreCard that used gene expression signatures to quantify differentiation efficiency. Here we report an improved version of the assay based on qPCR that enables faster, more quantitative assessment of functional pluripotency. We provide an in-depth characterization of the revised signature panel (commercially available as the TaqMan hPSC Scorecard Assay) through embryoid body and directed differentiation experiments as well as a detailed comparison to the teratoma assay. We further show that the improved ScoreCard enables a wider range of applications, such as screening of small molecules, genetic perturbations and assessment of culture conditions. Our approach can be extended beyond stem cell applications to characterize and assess the utility of other cell types and lineages.
View details for DOI 10.1038/nbt.3387
View details for Web of Science ID 000364916000026
View details for PubMedID 26501952
View details for PubMedCentralID PMC4636964
In vivo Monitoring of Transcriptional Dynamics After Lower-Limb Muscle Injury Enables Quantitative Classification of Healing
2015; 5: 13885
Traumatic lower-limb musculoskeletal injuries are pervasive amongst athletes and the military and typically an individual returns to activity prior to fully healing, increasing a predisposition for additional injuries and chronic pain. Monitoring healing progression after a musculoskeletal injury typically involves different types of imaging but these approaches suffer from several disadvantages. Isolating and profiling transcripts from the injured site would abrogate these shortcomings and provide enumerative insights into the regenerative potential of an individual's muscle after injury. In this study, a traumatic injury was administered to a mouse model and healing progression was examined from 3 hours to 1 month using high-throughput RNA-Sequencing (RNA-Seq). Comprehensive dissection of the genome-wide datasets revealed the injured site to be a dynamic, heterogeneous environment composed of multiple cell types and thousands of genes undergoing significant expression changes in highly regulated networks. Four independent approaches were used to determine the set of genes, isoforms, and genetic pathways most characteristic of different time points post-injury and two novel approaches were developed to classify injured tissues at different time points. These results highlight the possibility to quantitatively track healing progression in situ via transcript profiling using high- throughput sequencing.
View details for DOI 10.1038/srep13885
View details for Web of Science ID 000361376900001
View details for PubMedID 26381351
View details for PubMedCentralID PMC4585378
Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency
2015; 162 (2): 412?24
Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here, we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PAPERCLIP.
View details for DOI 10.1016/j.cell.2015.06.016
View details for Web of Science ID 000358087700020
View details for PubMedID 26186193
View details for PubMedCentralID PMC4511597
Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells
2015; 47 (5): 469?U64
DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases (DNMTs) in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, pluripotent cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to findings in mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome this immediate lethality, we generated a doxycycline-responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1-mutant lines. However, doxycycline-mediated repression of exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT-mutant ESCs, including single-base genome-wide maps of the targets of these enzymes.
View details for DOI 10.1038/ng.3258
View details for Web of Science ID 000353635800009
View details for PubMedID 25822089
View details for PubMedCentralID PMC4414868
Dissecting neural differentiation regulatory networks through epigenetic footprinting
2015; 518 (7539): 355?59
Models derived from human pluripotent stem cells that accurately recapitulate neural development in vitro and allow for the generation of specific neuronal subtypes are of major interest to the stem cell and biomedical community. Notch signalling, particularly through the Notch effector HES5, is a major pathway critical for the onset and maintenance of neural progenitor cells in the embryonic and adult nervous system. Here we report the transcriptional and epigenomic analysis of six consecutive neural progenitor cell stages derived from a HES5::eGFP reporter human embryonic stem cell line. Using this system, we aimed to model cell-fate decisions including specification, expansion and patterning during the ontogeny of cortical neural stem and progenitor cells. In order to dissect regulatory mechanisms that orchestrate the stage-specific differentiation process, we developed a computational framework to infer key regulators of each cell-state transition based on the progressive remodelling of the epigenetic landscape and then validated these through a pooled short hairpin RNA screen. We were also able to refine our previous observations on epigenetic priming at transcription factor binding sites and suggest here that they are mediated by combinations of core and stage-specific factors. Taken together, we demonstrate the utility of our system and outline a general framework, not limited to the context of the neural lineage, to dissect regulatory circuits of differentiation.
View details for DOI 10.1038/nature13990
View details for Web of Science ID 000349547400033
View details for PubMedID 25533951
View details for PubMedCentralID PMC4336237
Transcriptional and Epigenetic Dynamics during Specification of Human Embryonic Stem Cells
2013; 153 (5): 1149?63
Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end, we performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole-genome bisulfite sequencing, chromatin immunoprecipitation sequencing, and RNA sequencing reveals unique events associated with specification toward each lineage. Lineage-specific dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements that are frequently bound by pluripotency factors in the undifferentiated hESCs. In addition, we identified germ-layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches, leading to more faithful differentiation strategies as well as providing insights into the rewiring of human regulatory programs during cellular transitions.
View details for DOI 10.1016/j.cell.2013.04.037
View details for Web of Science ID 000319456800020
View details for PubMedID 23664763
View details for PubMedCentralID PMC3709577
Epigenetic obstacles encountered by transcription factors: reprogramming against all odds
CURRENT OPINION IN GENETICS & DEVELOPMENT
2012; 22 (5): 409?15
Reprogramming of a somatic nucleus to an induced pluripotent state can be achieved in vitro through ectopic expression of Oct4 (Pou5f1), Sox2, Klf4 and c-Myc. While the ability of these factors to regulate transcription in a pluripotent context has been studied extensively, their ability to interact with and remodel a somatic genome remains underexplored. Several recent studies have begun to provide mechanistic insights that will eventually lead to a more rational design and improved understanding of nuclear reprogramming.
View details for DOI 10.1016/j.gde.2012.08.002
View details for Web of Science ID 000311470900003
View details for PubMedID 22922161
View details for PubMedCentralID PMC3490009
- Epigenomics and chromatin dynamics. 2012: 313
Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa
NATURE CELL BIOLOGY
2007; 9 (7): 797?U121
Secreted proteins are crucial to the arsenal of bacterial pathogens. Although optimal activity of these proteins is likely to require precise regulation of release, the signalling events that trigger secretion are poorly understood. Here, we identify a threonine phosphorylation event that post-translationally regulates the Hcp secretion island-I-encoded type VI secretion system of Pseudomonas aeruginosa (H-T6SS). We show that a serine-threonine kinase, PpkA, is required for assembly of the H-T6SS and for secretion of Hcp1. PpkA activity is antagonized by PppA, a Ser-Thr phosphatase. These proteins exhibit reciprocal effects on the H-T6SS by acting on an FHA domain-containing protein, termed Fha1. Colocalization experiments with the T6S AAA+ family protein, ClpV1, indicate that Fha1 is a core scaffolding protein of the H-T6SS. Mutations affecting this H-T6S regulatory pathway provide a molecular explanation for the variation in Hcp1 secretion among clinical P. aeruginosa isolates. This mechanism of triggering secretion may be general, as many T6SSs contain orthologues of these proteins. Post-translational regulation of protein secretion by Thr phosphorylation is unprecedented in bacteria, and is likely to reflect the requirement for T6S to respond rapidly and reversibly to its environment.
View details for DOI 10.1038/ncb1605
View details for Web of Science ID 000247760700015
View details for PubMedID 17558395
A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus
2006; 312 (5779): 1526?30
Bacterial pathogens frequently use protein secretion to mediate interactions with their hosts. Here we found that a virulence locus (HSI-I) of Pseudomonas aeruginosa encodes a protein secretion apparatus. The apparatus assembled in discrete subcellular locations and exported Hcp1, a hexameric protein that forms rings with a 40 angstrom internal diameter. Regulatory patterns of HSI-I suggested that the apparatus functions during chronic infections. We detected Hcp1 in pulmonary secretions of cystic fibrosis (CF) patients and Hcp1-specific antibodies in their sera. Thus, HSI-I likely contributes to the pathogenesis of P. aeruginosa in CF patients. HSI-I-related loci are widely distributed among bacterial pathogens and may play a general role in mediating host interactions.
View details for DOI 10.1126/science.1128393
View details for Web of Science ID 000238124100049
View details for PubMedID 16763151
View details for PubMedCentralID PMC2800167