Targeting HDAC6 to treat heart failure with preserved ejection fraction in mice.

Heart failure with preserved ejection fraction (HFpEF) poses therapeutic challenges due to the limited treatment options. Building upon our previous research that demonstrates the efficacy of histone deacetylase 6 (HDAC6) inhibition in a genetic cardiomyopathy model, we investigate HDAC6's role in HFpEF due to their shared mechanisms of inflammation and metabolism. Here, we show that inhibiting HDAC6 with TYA-018 effectively reverses established heart failure and its associated symptoms in male HFpEF mouse models. Additionally, in male mice lacking Hdac6 gene, HFpEF progression is delayed and they are resistant to TYA-018's effects. The efficacy of TYA-018 is comparable to a sodium-glucose cotransporter 2 (SGLT2) inhibitor, and the combination shows enhanced effects. Mechanistically, TYA-018 restores gene expression related to hypertrophy, fibrosis, and mitochondrial energy production in HFpEF heart tissues. Furthermore, TYA-018 also inhibits activation of human cardiac fibroblasts and enhances mitochondrial respiratory capacity in cardiomyocytes. In this work, our findings show that HDAC6 impacts on heart pathophysiology and is a promising target for HFpEF treatment.

Phenotypic screening with deep learning identifies HDAC6 inhibitors as cardioprotective in a BAG3 mouse model of dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by reduced cardiac output, as well as thinning and enlargement of left ventricular chambers. These characteristics eventually lead to heart failure. Current standards of care do not target the underlying molecular mechanisms associated with genetic forms of heart failure, driving a need to develop novel therapeutics for DCM. To identify candidate therapeutics, we developed an in vitro DCM model using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) deficient in B-cell lymphoma 2 (BCL2)-associated athanogene 3 (BAG3). With these BAG3-deficient iPSC-CMs, we identified cardioprotective drugs using a phenotypic screen and deep learning. From a library of 5500 bioactive compounds and siRNA validation, we found that inhibiting histone deacetylase 6 (HDAC6) was cardioprotective at the sarcomere level. We translated this finding to a BAG3 cardiomyocyte-knockout (BAG3cKO) mouse model of DCM, showing that inhibiting HDAC6 with two isoform-selective inhibitors (tubastatin A and a novel inhibitor TYA-018) protected heart function. In BAG3cKO and BAG3E455K mice, HDAC6 inhibitors improved left ventricular ejection fraction and reduced left ventricular diameter at diastole and systole. In BAG3cKO mice, TYA-018 protected against sarcomere damage and reduced Nppb expression. Based on integrated transcriptomics and proteomics and mitochondrial function analysis, TYA-018 also enhanced energetics in these mice by increasing expression of targets associated with fatty acid metabolism, protein metabolism, and oxidative phosphorylation. Our results demonstrate the power of combining iPSC-CMs with phenotypic screening and deep learning to accelerate drug discovery, and they support developing novel therapies that address underlying mechanisms associated with heart disease.

Quantifying drug-induced structural toxicity in hepatocytes and cardiomyocytes derived from hiPSCs using a deep learning method.

Cardiac and hepatic toxicity result from induced disruption of the functioning of cardiomyocytes and hepatocytes, respectively, which is tightly related to the organization of their subcellular structures. Cellular structure can be analyzed from microscopy imaging data. However, subtle or complex structural changes that are not easily perceived may be missed by conventional image-analysis techniques. Here we report the evaluation of PhenoTox, an image-based deep-learning method of quantifying drug-induced structural changes using human hepatocytes and cardiomyocytes derived from human induced pluripotent stem cells. We assessed the ability of the deep learning method to detect variations in the organization of cellular structures from images of fixed or live cells. We also evaluated the power and sensitivity of the method for detecting toxic effects of drugs by conducting a set of experiments using known toxicants and other methods of screening for cytotoxic effects. Moreover, we used PhenoTox to characterize the effects of tamoxifen and doxorubicin-which cause liver toxicity-on hepatocytes. PhenoTox revealed differences related to loss of cytochrome P450 3A4 activity, for which it showed greater sensitivity than a caspase 3/7 assay. Finally, PhenoTox detected structural toxicity in cardiomyocytes, which was correlated with contractility defects induced by doxorubicin, erlotinib, and sorafenib. Taken together, the results demonstrated that PhenoTox can capture the subtle morphological changes that are early signs of toxicity in both hepatocytes and cardiomyocytes.

Advancing physiological maturation in human induced pluripotent stem cell-derived cardiac muscle by gene editing an inducible adult troponin isoform switch.

Advancing maturation of stem cell-derived cardiac muscle represents a major barrier to progress in cardiac regenerative medicine. Cardiac muscle maturation involves a myriad of gene, protein, and cell-based transitions, spanning across all aspects of cardiac muscle form and function. We focused here on a key developmentally controlled transition in the cardiac sarcomere, the functional unit of the heart. Using a gene-editing platform, human induced pluripotent stem cell (hiPSCs) were engineered with a drug-inducible expression cassette driving the adult cardiac troponin I (cTnI) regulatory isoform, a transition shown to be a rate-limiting step in advancing sarcomeric maturation of hiPSC cardiac muscle (hiPSC-CM) toward the adult state. Findings show that induction of the adult cTnI isoform resulted in the physiological acquisition of adult-like cardiac contractile function in hiPSC-CMs in vitro. Specifically, cTnI induction accelerated relaxation kinetics at baseline conditions, a result independent of alterations in the kinetics of the intracellular Ca2+ transient. In comparison, isogenic unedited hiPSC-CMs had no cTnI induction and no change in relaxation function. Temporal control of adult cTnI isoform induction did not alter other developmentally regulated sarcomere transitions, including myosin heavy chain isoform expression, nor did it affect expression of SERCA2a or phospholamban. Taken together, precision genetic targeting of sarcomere maturation via inducible TnI isoform switching enables physiologically relevant adult myocardium-like contractile adaptations that are essential for beat-to-beat modulation of adult human heart performance. These findings have relevance to hiPSC-CM structure-function and drug-discovery studies in vitro, as well as for potential future clinical applications of physiologically optimized hiPSC-CM in cardiac regeneration/repair.

Spatiotemporal mosaic self-patterning of pluripotent stem cells using CRISPR interference.

Morphogenesis involves interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events lack control over cell-type co-emergence and offer little capability to selectively perturb specific cell subpopulations. Our in vitro system interrogates cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined effects of induced mosaic knockdown of molecular regulators of cortical tension (ROCK1) and cell-cell adhesion (CDH1) with CRISPR interference. Mosaic knockdown of ROCK1 or CDH1 resulted in differential patterning within hiPSC colonies due to cellular self-organization, while retaining an epithelial pluripotent phenotype. Knockdown induction stimulates a transient wave of differential gene expression within the mixed populations that stabilized in coordination with observed self-organization. Mosaic patterning enables genetic interrogation of emergent multicellular properties, which can facilitate better understanding of the molecular pathways that regulate symmetry-breaking during morphogenesis.

Esophageal Organoids from Human Pluripotent Stem Cells Delineate Sox2 Functions during Esophageal Specification.

Tracheal and esophageal disorders are prevalent in humans and difficult to accurately model in mice. We therefore established a three-dimensional organoid model of esophageal development through directed differentiation of human pluripotent stem cells. Sequential manipulation of bone morphogenic protein (BMP), Wnt, and RA signaling pathways was required to pattern definitive endoderm into foregut, anterior foregut (AFG), and dorsal AFG spheroids. Dorsal AFG spheroids grown in a 3D matrix formed human esophageal organoids (HEOs), and HEO cells could be transitioned into two-dimensional cultures and grown as esophageal organotypic rafts. In both configurations, esophageal tissues had proliferative basal progenitors and a differentiated stratified squamous epithelium. Using HEO cultures to model human esophageal birth defects, we identified that Sox2 promotes esophageal specification in part through repressing Wnt signaling in dorsal AFG and promoting survival. Consistently, Sox2 ablation in mice causes esophageal agenesis. Thus, HEOs present a powerful platform for modeling human pathologies and tissue engineering.

Contractile deficits in engineered cardiac microtissues as a result of MYBPC3 deficiency and mechanical overload.

The integration of in vitro cardiac tissue models, human induced pluripotent stem cells (hiPSCs) and genome-editing tools allows for the enhanced interrogation of physiological phenotypes and recapitulation of disease pathologies. Here, using a cardiac tissue model consisting of filamentous three-dimensional matrices populated with cardiomyocytes derived from healthy wild-type (WT) hiPSCs (WT hiPSC-CMs) or isogenic hiPSCs deficient in the sarcomere protein cardiac myosin-binding protein C (MYBPC3-/- hiPSC-CMs), we show that the WT microtissues adapted to the mechanical environment with increased contraction force commensurate to matrix stiffness, whereas the MYBPC3-/- microtissues exhibited impaired force development kinetics regardless of matrix stiffness and deficient contraction force only when grown on matrices with high fibre stiffness. Under mechanical overload, the MYBPC3-/- microtissues had a higher degree of calcium transient abnormalities, and exhibited an accelerated decay of calcium dynamics as well as calcium desensitization, which accelerated when contracting against stiffer fibres. Our findings suggest that MYBPC3 deficiency and the presence of environmental stresses synergistically lead to contractile deficits in cardiac tissues.

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress.

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.

Multi-Imaging Method to Assay the Contractile Mechanical Output of Micropatterned Human iPSC-Derived Cardiac Myocytes.

RATIONALE: During each beat, cardiac myocytes (CMs) generate the mechanical output necessary for heart function through contractile mechanisms that involve shortening of sarcomeres along myofibrils. Human-induced pluripotent stem cells (hiPSCs) can be differentiated into CMs (hiPSC-CMs) that model cardiac contractile mechanical output more robustly when micropatterned into physiological shapes. Quantifying the mechanical output of these cells enables us to assay cardiac activity in a dish. OBJECTIVE: We sought to develop a computational platform that integrates analytic approaches to quantify the mechanical output of single micropatterned hiPSC-CMs from microscopy videos. METHODS AND RESULTS: We micropatterned single hiPSC-CMs on deformable polyacrylamide substrates containing fluorescent microbeads. We acquired videos of single beating cells, of microbead displacement during contractions, and of fluorescently labeled myofibrils. These videos were independently analyzed to obtain parameters that capture the mechanical output of the imaged single cells. We also developed novel methods to quantify sarcomere length from videos of moving myofibrils and to analyze loss of synchronicity of beating in cells with contractile defects. We tested this computational platform by detecting variations in mechanical output induced by drugs and in cells expressing low levels of myosin-binding protein C. CONCLUSIONS: Our method can measure the cardiac function of single micropatterned hiPSC-CMs and determine contractile parameters that can be used to elucidate mechanisms that underlie variations in CM function. This platform will be amenable to future studies of the effects of mutations and drugs on cardiac function.

CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells.

The human genome produces thousands of long noncoding RNAs (lncRNAs)-transcripts >200 nucleotides long that do not encode proteins. Although critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. We developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in seven diverse cell lines, including six transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth-modifying function exclusively in one cell type. We further found that lncRNA knockdown can perturb complex transcriptional networks in a cell type-specific manner. These data underscore the functional importance and cell type specificity of many lncRNAs.

Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.

Miniaturized iPS-Cell-Derived Cardiac Muscles for Physiologically Relevant Drug Response Analyses.

Tissue engineering approaches have the potential to increase the physiologic relevance of human iPS-derived cells, such as cardiomyocytes (iPS-CM). However, forming Engineered Heart Muscle (EHM) typically requires >1 million cells per tissue. Existing miniaturization strategies involve complex approaches not amenable to mass production, limiting the ability to use EHM for iPS-based disease modeling and drug screening. Micro-scale cardiospheres are easily produced, but do not facilitate assembly of elongated muscle or direct force measurements. Here we describe an approach that combines features of EHM and cardiospheres: Micro-Heart Muscle (µHM) arrays, in which elongated muscle fibers are formed in an easily fabricated template, with as few as 2,000 iPS-CM per individual tissue. Within µHM, iPS-CM exhibit uniaxial contractility and alignment, robust sarcomere assembly, and reduced variability and hypersensitivity in drug responsiveness, compared to monolayers with the same cellular composition. µHM mounted onto standard force measurement apparatus exhibited a robust Frank-Starling response to external stretch, and a dose-dependent inotropic response to the ß-adrenergic agonist isoproterenol. Based on the ease of fabrication, the potential for mass production and the small number of cells required to form µHM, this system provides a potentially powerful tool to study cardiomyocyte maturation, disease and cardiotoxicology in vitro.

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs.

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.

Efficient CRISPR/Cas9-Based Genome Engineering in Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPS cells) are rapidly emerging as a powerful tool for biomedical discovery. The advent of human induced pluripotent stem cells (hiPS cells) with human embryonic stem (hES)-cell-like properties has led to hPS cells with disease-specific genetic backgrounds for in vitro disease modeling and drug discovery as well as mechanistic and developmental studies. To fully realize this potential, it will be necessary to modify the genome of hPS cells with precision and flexibility. Pioneering experiments utilizing site-specific double-strand break (DSB)-mediated genome engineering tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have paved the way to genome engineering in previously recalcitrant systems such as hPS cells. However, these methods are technically cumbersome and require significant expertise, which has limited adoption. A major recent advance involving the clustered regularly interspaced short palindromic repeats (CRISPR) endonuclease has dramatically simplified the effort required for genome engineering and will likely be adopted widely as the most rapid and flexible system for genome editing in hPS cells. In this unit, we describe commonly practiced methods for CRISPR endonuclease genomic editing of hPS cells into cell lines containing genomes altered by insertion/deletion (indel) mutagenesis or insertion of recombinant genomic DNA.

A non-invasive platform for functional characterization of stem-cell-derived cardiomyocytes with applications in cardiotoxicity testing.

We present a non-invasive method to characterize the function of pluripotent stem-cell-derived cardiomyocytes based on video microscopy and image analysis. The platform, called Pulse, generates automated measurements of beating frequency, beat duration, amplitude, and beat-to-beat variation based on motion analysis of phase-contrast images captured at a fast frame rate. Using Pulse, we demonstrate recapitulation of drug effects in stem-cell-derived cardiomyocytes without the use of exogenous labels and show that our platform can be used for high-throughput cardiotoxicity drug screening and studying physiologically relevant phenotypes.

Automated Video-Based Analysis of Contractility and Calcium Flux in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Cultured over Different Spatial Scales.

Contractile motion is the simplest metric of cardiomyocyte health in vitro, but unbiased quantification is challenging. We describe a rapid automated method, requiring only standard video microscopy, to analyze the contractility of human-induced pluripotent stem cell-derived cardiomyocytes (iPS-CM). New algorithms for generating and filtering motion vectors combined with a newly developed isogenic iPSC line harboring genetically encoded calcium indicator, GCaMP6f, allow simultaneous user-independent measurement and analysis of the coupling between calcium flux and contractility. The relative performance of these algorithms, in terms of improving signal to noise, was tested. Applying these algorithms allowed analysis of contractility in iPS-CM cultured over multiple spatial scales from single cells to three-dimensional constructs. This open source software was validated with analysis of isoproterenol response in these cells, and can be applied in future studies comparing the drug responsiveness of iPS-CM cultured in different microenvironments in the context of tissue engineering.

Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells.

We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications.

An improved technique for chromosomal analysis of human ES and iPS cells.

Prolonged in vitro culture of human embryonic stem (hES) cells can result in chromosomal abnormalities believed to confer a selective advantage. This potential occurrence has crucial implications for the appropriate use of hES cells for research and therapeutic purposes. In view of this, time-point karyotypic evaluation to assess genetic stability is recommended as a necessary control test to be carried out during extensive 'passaging'. Standard techniques currently used for the cytogenetic assessment of ES cells include G-banding and/or Fluorescence in situ Hybridization (FISH)-based protocols for karyotype analysis, including M-FISH and SKY. Critical for both banding and FISH techniques are the number and quality of metaphase spreads available for analysis at the microscope. Protocols for chromosome preparation from hES and human induced pluripotent stem (hiPS) cells published so far appear to differ considerably from one laboratory to another. Here we present an optimized technique, in which both the number and the quality of chromosome metaphase spreads were substantially improved when compared to current standard techniques for chromosome preparations. We believe our protocol represents a significant advancement in this line of work, and has the required attributes of simplicity and consistency to be widely accepted as a reference method for high quality, fast chromosomal analysis of human ES and iPS cells.

The interval between Ins2 and Ascl2 is dispensable for imprinting centre function in the murine Beckwith-Wiedemann region.

Imprinted genes are commonly clustered in domains across the mammalian genome, suggesting a degree of coregulation via long-range coordination of their monoallelic transcription. The distal end of mouse chromosome 7 (Chr 7) contains two clusters of imprinted genes within a approximately 1 Mb domain. This region is conserved on human 11p15.5 where it is implicated in the Beckwith-Wiedemann syndrome. In both species, imprinted regulation requires two critical cis-acting imprinting centres, carrying different germline epigenetic marks and mediating imprinted expression in the proximal and distal sub-domains. The clusters are separated by a region containing the gene for tyrosine hydroxylase (Th) as well as a high density of short repeats and retrotransposons in the mouse. We have used the Cre-loxP recombination system in vivo to engineer an interstitial deletion of this approximately 280-kb intervening region previously proposed to participate in the imprinting mechanism or to act as a boundary between the two sub-domains. The deletion allele, Del(7AI), is silent with respect to epigenetic marking at the two flanking imprinting centres. Reciprocal inheritance of Del(7AI) demonstrates that the deleted region, which represents more than a quarter of the previously defined imprinted domain, is associated with intrauterine growth restriction in maternal heterozygotes. In homozygotes, the deficiency behaves as a Th null allele and can be rescued pharmacologically by bypassing the metabolic requirement for TH in utero. Our results show that the deleted interval is not required for normal imprinting on distal Chr 7 and uncover a new imprinted growth phenotype.

Mitotic recombination counteracts the benefits of genetic segregation.

The ubiquity of sexual reproduction despite its cost has lead to an extensive body of research on the evolution and maintenance of sexual reproduction. Previous work has suggested that sexual reproduction can substantially speed up the rate of adaptation in diploid populations, because sexual populations are able to produce the fittest homozygous genotype by segregation and mating of heterozygous individuals. In contrast, asexual populations must wait for two rare mutational events, one producing a heterozygous carrier and the second converting a heterozygous to a homozygous carrier, before a beneficial mutation can become fixed. By avoiding this additional waiting time, it was shown that the benefits of segregation could overcome a twofold cost of sex. This previous result ignores mitotic recombination (MR), however. Here, we show that MR significantly hastens the spread of beneficial mutations in asexual populations. Indeed, given empirical data on MR, we find that adaptation in asexual populations proceeds as fast as that in sexual populations, especially when beneficial alleles are partially recessive. We conclude that asexual populations can gain most of the benefit of segregation through MR while avoiding the costs associated with sexual reproduction.