Our knowledge of pluripotent stem cell (PSC) biology has advanced to the point where we now can generate most cells of the human body in the laboratory. PSC-derived cardiomyocytes can be generated ...routinely with high yield and purity for disease research and drug development, and these cells are now gradually entering the clinical research phase for the testing of heart regeneration therapies. However, a major hurdle for their applications is the immature state of these cardiomyocytes. In this Review, we describe the structural and functional properties of cardiomyocytes and present the current approaches to mature PSC-derived cardiomyocytes. To date, the greatest success in maturation of PSC-derived cardiomyocytes has been with transplantation into the heart in animal models and the engineering of 3D heart tissues with electromechanical conditioning. In conventional 2D cell culture, biophysical stimuli such as mechanical loading, electrical stimulation and nanotopology cues all induce substantial maturation, particularly of the contractile cytoskeleton. Metabolism has emerged as a potent means to control maturation with unexpected effects on electrical and mechanical function. Different interventions induce distinct facets of maturation, suggesting that activating multiple signalling networks might lead to increased maturation. Despite considerable progress, we are still far from being able to generate PSC-derived cardiomyocytes with adult-like phenotypes in vitro. Future progress will come from identifying the developmental drivers of maturation and leveraging them to create more mature cardiomyocytes for research and regenerative medicine.
Developing vascular networks that integrate with the host circulation and support cells engrafted within engineered tissues remains a key challenge in tissue engineering. Most previous work in this ...field has focused on developing new methods to build human vascular networks within engineered tissues prior to their implant in vivo, with substantively less attention paid to the role of the host in tissue vascularization and engraftment. Here, we assessed the role that different host animal models and anatomic implant locations play in vascularization and cardiomyocyte survival within engineered tissues. We found major differences in the formation of graft-derived blood vessels and survival of cardiomyocytes after implantation of identical tissues in immunodeficient athymic nude mice versus rats. Athymic mice supported robust guided vascularization of human microvessels carrying host blood but relatively sparse cardiac grafts within engineered tissues, regardless of implant site. Conversely, athymic rats produced substantive inflammatory changes that degraded grafts (abdomen) or disrupted vascular patterning (heart). Despite disrupted vascular patterning, athymic rats supported > 3-fold larger human cardiomyocyte grafts compared to athymic mice. This work demonstrates the critical importance of the host for vascularization and engraftment of engineered tissues, which has broad translational implications across regenerative medicine.
It has been appreciated for some time that cardiovascular disease involves large-scale transcriptional changes in various cell types. What has become increasingly clear only in the past few years, ...however, is the role of chromatin remodeling in cardiovascular phenotypes in normal physiology, as well as in development and disease. This review summarizes the state of the chromatin field in terms of distinct mechanisms to regulate chromatin structure in vivo, identifying when these modes of regulation have been demonstrated in cardiovascular tissues. We describe areas in which a better understanding of chromatin structure is leading to new insights into the fundamental biology of cardiovascular disease. (Circ J 2013; 77: 1389–1398)
Human induced pluripotent stem cells (hiPSCs) offer opportunities to study human biology where primary cell types are limited. CRISPR technology allows forward genetic screens using engineered ...Cas9-expressing cells. Here, we sought to generate a CRISPR activation (CRISPRa) hiPSC line to activate endogenous genes during pluripotency and differentiation. We first targeted catalytically inactive Cas9 fused to VP64, p65 and Rta activators (dCas9-VPR) regulated by the constitutive CAG promoter to the
AAVS1
safe harbor site. These CRISPRa hiPSC lines effectively activate target genes in pluripotency, however the dCas9-VPR transgene expression is silenced after differentiation into cardiomyocytes and endothelial cells. To understand this silencing, we systematically tested different safe harbor sites and different promoters. Targeting to safe harbor sites
hROSA26
and
CLYBL
loci also yielded hiPSCs that expressed dCas9-VPR in pluripotency but silenced during differentiation. Muscle-specific regulatory cassettes, derived from cardiac troponin T or muscle creatine kinase promoters, were also silent after differentiation when dCas9-VPR was introduced. In contrast, in cell lines where the dCas9-VPR sequence was replaced with cDNAs encoding fluorescent proteins, expression persisted during differentiation in all loci and with all promoters. Promoter DNA was hypermethylated in CRISPRa-engineered lines, and demethylation with 5-azacytidine enhanced dCas9-VPR gene expression. In summary, the dCas9-VPR cDNA is readily expressed from multiple loci during pluripotency but induces silencing in a locus- and promoter-independent manner during differentiation to mesoderm derivatives. Researchers intending to use this CRISPRa strategy during stem cell differentiation should pilot their system to ensure it remains active in their population of interest.
Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined.
To investigate the mechanisms of ...epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload-induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes.
Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements.
These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy.
Methods to differentiate stem cells into cardiomyocytes have been well established. However, a limitation for the successful application of these cells for research and medicine has been their ...fetal‐like phenotype with respect to cell size, contractility, calcium handling, metabolism, and electrophysiology. We sought to increase the maturity of human pluripotent stem cell derived cardiomyocytes (hPSC‐CMs) through metabolic pathway regulation. We hypothesized that switching the main metabolic substrates from glucose to fatty acids, mimicking the switch from placental to breast milk nutrient consumption that occurs during development, will increase hPSC‐CM maturation. RUES2 embryonic stem cells were differentiated into cardiomyocytes and then treated with base media with varying glucose and calcium concentrations and fatty acid supplementation. Using qRT‐PCR, we measured an inverse relationship between glucose levels and markers of cardiomyocyte maturation: increased expression of cardiac troponin I relative to skeletal troponin I isoforms (TNNI3:TNNI1), increased expression of metabolic (CPT1B, PPARGC1A) and electrical maturity markers (KCNJ2, RYR2). These maturation markers were not influenced with fatty acid supplementation but were enhanced upon the addition of thyroid hormone and dexamethasone. To understand the mechanisms by which media nutrients and signaling molecules cause phenotypic changes we investigated the relationship between maturation and global epigenetic state. Western blot revealed increases in global acetylation levels, measured by histone H3 acetylation, linked to maturation signaling. These findings depict a direct relationship between glucose metabolism and the development of a mature phenotype in hPSC‐CMs, mediated by epigenetic mechanisms.
Support or Funding Information
Washington Research Foundation Undergraduate Fellowship
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) offer a promising cell-based therapy for myocardial infarction. However, the presence of transitory ventricular arrhythmias, termed ...engraftment arrhythmias (EAs), hampers clinical applications. We hypothesized that EA results from pacemaker-like activity of hPSC-CMs associated with their developmental immaturity. We characterized ion channel expression patterns during maturation of transplanted hPSC-CMs and used pharmacology and genome editing to identify those responsible for automaticity in vitro. Multiple engineered cell lines were then transplanted in vivo into uninjured porcine hearts. Abolishing depolarization-associated genes HCN4, CACNA1H, and SLC8A1, along with overexpressing hyperpolarization-associated KCNJ2, creates hPSC-CMs that lack automaticity but contract when externally stimulated. When transplanted in vivo, these cells engrafted and coupled electromechanically with host cardiomyocytes without causing sustained EAs. This study supports the hypothesis that the immature electrophysiological prolife of hPSC-CMs mechanistically underlies EA. Thus, targeting automaticity should improve the safety profile of hPSC-CMs for cardiac remuscularization.
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•Ion channel gene expression changes extensively as hPSC-CM grafts mature in vivo•Targeting individual ion channels was insufficient to prevent engraftment arrhythmia•Automaticity of hPSC-CMs relies on calcium and voltage clocks•Graft automaticity is a major contributor to arrhythmias post-hPSC-CM engraftment
Engraftment arrhythmia (EA) compromises the safety of hPSC-CM cell therapy. We hypothesized that spontaneous graft depolarizations are the source of EAs. We used a CRISPR screen to demonstrate that targeting excitatory channels HCN4, CACNA1H, and SLC8A1 and expressing the inhibitory KCNJ2 channel generates quiescent-yet-excitable cardiomyocytes that engraft without resulting in EAs.
Standard transgenic cell line generation requires screening 100–1000s of colonies to isolate correctly edited cells. We describe CRISPRa On-Target Editing Retrieval (CRaTER) which enriches for cells ...with on-target knock-in of a cDNA-fluorescent reporter transgene by transient activation of the targeted locus followed by flow sorting to recover edited cells. We show CRaTER recovers rare cells with heterozygous, biallelic-editing of the transcriptionally-inactive MYH7 locus in human induced pluripotent stem cells (hiPSCs), enriching on average 25-fold compared to standard antibiotic selection. We leveraged CRaTER to enrich for heterozygous knock-in of a library of variants in MYH7, a gene in which missense mutations cause cardiomyopathies, and recovered hiPSCs with 113 different variants. We differentiated these hiPSCs to cardiomyocytes and show MHC-β fusion proteins can localize as expected. Additionally, single-cell contractility analyses revealed cardiomyocytes with a pathogenic, hypertrophic cardiomyopathy-associated MYH7 variant exhibit salient HCM physiology relative to isogenic controls. Thus, CRaTER substantially reduces screening required for isolation of gene-edited cells, enabling generation of functional transgenic cell lines at unprecedented scale.
On-target gene-edited hiPSCs (left) are enriched using CRaTER (middle), enabling rapid, pooled generation of variant libraries for characterization in relevant cell types of interest after directed differentiation (right). Display omitted
•CRISPRa On-Target Editing Retrieval enriches for cells with desired gene editing.•CRaTER enables pooled generation of >100 variants in hiPSCs without colony picking.•After CRaTER enrichment, hiPSCs can differentiate to disease-relevant cell types.
Heart failure is associated with hypertrophying of cardiomyocytes and changes in transcriptional activity. Studies from rapidly dividing cells in culture have suggested that transcription may be ...compartmentalized into factories within the nucleus, but this phenomenon has not been tested in vivo and the role of nuclear architecture in cardiac gene regulation is unknown. While alterations to transcription have been linked to disease, little is known about the regulation of the spatial organization of transcription and its properties in the pathological setting. In the present study, we investigate the structural features of endogenous transcription factories in the heart and determine the principles connecting chromatin structure to transcriptional regulation in vivo. Super-resolution imaging of endogenous RNA polymerase II clusters in neonatal and adult cardiomyocytes revealed distinct properties of transcription factories in response to pathological stress: neonatal nuclei demonstrated changes in number of clusters, with parallel increases in nuclear area, while the adult nuclei underwent changes in size and intensity of RNA polymerase II foci. Fluorescence in situ hybridization-based labeling of genes revealed locus-specific relationships between expression change and anatomical localization—with respect to nuclear periphery and heterochromatin regions, both sites associated with gene silencing—in the nuclei of cardiomyocytes in hearts (but not liver hepatocytes) of mice subjected to pathologic stimuli that induce heart failure. These findings demonstrate a role for chromatin organization and rearrangement of nuclear architecture for cell type-specific transcription in vivo during disease. RNA polymerase II ChIP and chromatin conformation capture studies in the same model system demonstrate formation and reorganization of distinct nuclear compartments regulating gene expression. These findings reveal locus-specific compartmentalization of stress-activated, housekeeping and silenced genes in the anatomical context of the endogenous nucleus, revealing basic principles of global chromatin structure and nuclear architecture in the regulation of gene expression in healthy and diseased conditions.
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