The discovery of human pluripotent stem cells (hPSCs), including both human embryonic stem cells and human-induced pluripotent stem cells, has opened up novel paths for a wide range of scientific ...studies. The capability to direct the differentiation of hPSCs into functional cardiomyocytes has provided a platform for regenerative medicine, development, tissue engineering, disease modeling, and drug toxicity testing. Despite exciting progress, achieving the optimal benefits has been hampered by the immature nature of these cardiomyocytes. Cardiac maturation has long been studied in vivo using animal models; however, finding ways to mature hPSC cardiomyocytes is only in its initial stages. In this review, we discuss progress in promoting the maturation of the hPSC cardiomyocytes, in the context of our current knowledge of developmental cardiac maturation and in relation to in vitro model systems such as rodent ventricular myocytes. Promising approaches that have begun to be examined in hPSC cardiomyocytes include long-term culturing, 3-dimensional tissue engineering, mechanical loading, electric stimulation, modulation of substrate stiffness, and treatment with neurohormonal factors. Future studies will benefit from the combinatorial use of different approaches that more closely mimic nature’s diverse cues, which may result in broader changes in structure, function, and therapeutic applicability.
The investment of nearly 2 decades of clinical investigation into cardiac cell therapy has yet to change cardiovascular practice. Recent insights into the mechanism of cardiac regeneration help ...explain these results and provide important context in which we can develop next-generation therapies. Non-contractile cells such as bone marrow or adult heart derivatives neither engraft long-term nor induce new muscle formation. Correspondingly, these cells offer little functional benefit to infarct patients. In contrast, preclinical data indicate that transplantation of bona fide cardiomyocytes derived from pluripotent stem cells induces direct remuscularization. This new myocardium beats synchronously with the host heart and induces substantial contractile benefits in macaque monkeys, suggesting that regeneration of contractile myocardium is required to fully recover function. Through a review of the preclinical and clinical trials of cardiac cell therapy, distinguishing the primary mechanism of benefit as either contractile or non-contractile helps appreciate the barriers to cardiac repair and establishes a rational path to optimizing therapeutic benefit.
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.
The potential to generate virtually any differentiated cell type from embryonic stem cells (ESCs) offers the possibility to establish new models of mammalian development and to create new sources of ...cells for regenerative medicine. To realize this potential, it is essential to be able to control ESC differentiation and to direct the development of these cells along specific pathways. Embryology has offered important insights into key pathways regulating ESC differentiation, resulting in advances in modeling gastrulation in culture and in the efficient induction of endoderm, mesoderm, and ectoderm and many of their downstream derivatives. This has led to the identification of new multipotential progenitors for the hematopoietic, neural, and cardiovascular lineages and to the development of protocols for the efficient generation of a broad spectrum of cell types including hematopoietic cells, cardiomyocytes, oligodendrocytes, dopamine neurons, and immature pancreatic β cells. The next challenge will be to demonstrate the functional utility of these cells, both in vitro and in preclinical models of human disease.
Directed differentiation of human embryonic stem cells (ESCs) into cardiovascular cells provides a model for studying molecular mechanisms of human cardiovascular development. Although it is known ...that chromatin modification patterns in ESCs differ markedly from those in lineage-committed progenitors and differentiated cells, the temporal dynamics of chromatin alterations during differentiation along a defined lineage have not been studied. We show that differentiation of human ESCs into cardiovascular cells is accompanied by programmed temporal alterations in chromatin structure that distinguish key regulators of cardiovascular development from other genes. We used this temporal chromatin signature to identify regulators of cardiac development, including the homeobox gene MEIS2. Using the zebrafish model, we demonstrate that MEIS2 is critical for proper heart tube formation and subsequent cardiac looping. Temporal chromatin signatures should be broadly applicable to other models of stem cell differentiation to identify regulators and provide key insights into major developmental decisions.
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► Genome-wide chromatin modification dynamics during lineage-specific differentiation ► Chromatin modification dynamics distinguish structural from regulatory genes ► A temporal chromatin signature predicts regulators of cardiac development ► Temporal chromatin signatures can identify regulators of other cell fates
During ESC differentiation into cardiovascular cells, a specific dynamic pattern of histone modification distinguishes loci encoding regulators of cardiac development from those encoding structural proteins, allowing identification of additional cardiac development regulators.
Heart regeneration Laflamme, Michael A; Murry, Charles E
Nature (London),
05/2011, Letnik:
473, Številka:
7347
Journal Article
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Heart failure plagues industrialized nations, killing more people than any other disease. It usually results from a deficiency of specialized cardiac muscle cells known as cardiomyocytes, and a ...robust therapy to regenerate lost myocardium could help millions of patients every year. Heart regeneration is well documented in amphibia and fish and in developing mammals. After birth, however, human heart regeneration becomes limited to very slow cardiomyocyte replacement. Several experimental strategies to remuscularize the injured heart using adult stem cells and pluripotent stem cells, cellular reprogramming and tissue engineering are in progress. Although many challenges remain, these interventions may eventually lead to better approaches to treat or prevent heart failure.
Cardiac tissue engineering is a promising approach to provide large-scale tissues for transplantation to regenerate the heart after ischemic injury, however, integration with the host myocardium will ...be required to achieve electromechanical benefits. To test the ability of engineered heart tissues to electrically integrate with the host, 10 million human embryonic stem cell (hESC)-derived cardiomyocytes were used to form either scaffold-free tissue patches implanted on the epicardium or micro-tissue particles (~1000 cells/particle) delivered by intramyocardial injection into the left ventricular wall of the ischemia/reperfusion injured athymic rat heart. Results were compared to intramyocardial injection of 10 million dispersed hESC-cardiomyocytes. Graft size was not significantly different between treatment groups and correlated inversely with infarct size. After implantation on the epicardial surface, hESC-cardiac tissue patches were electromechanically active, but they beat slowly and were not electrically coupled to the host at 4 weeks based on ex vivo fluorescent imaging of their graft-autonomous GCaMP3 calcium reporter. Histologically, scar tissue physically separated the patch graft and host myocardium. In contrast, following intramyocardial injection of micro-tissue particles and suspended cardiomyocytes, 100% of the grafts detected by fluorescent GCaMP3 imaging were electrically coupled to the host heart at spontaneous rate and could follow host pacing up to a maximum of 300-390 beats per minute (5-6.5 Hz). Gap junctions between intramyocardial graft and host tissue were identified histologically. The extensive coupling and rapid response rate of the human myocardial grafts after intramyocardial delivery suggest electrophysiological adaptation of hESC-derived cardiomyocytes to the rat heart's pacemaking activity. These data support the use of the rat model for studying electromechanical integration of human cardiomyocytes, and they identify lack of electrical integration as a challenge to overcome in tissue engineered patches.
BACKGROUND:Tissue engineering enables the generation of functional human cardiac tissue with cells derived in vitro in combination with biocompatible materials. Human-induced pluripotent stem ...cell-derived cardiomyocytes provide a cell source for cardiac tissue engineering; however, their immaturity limits their potential applications. Here we sought to study the effect of mechanical conditioning and electric pacing on the maturation of human-induced pluripotent stem cell-derived cardiac tissues.
METHODS:Cardiomyocytes derived from human-induced pluripotent stem cells were used to generate collagen-based bioengineered human cardiac tissue. Engineered tissue constructs were subjected to different mechanical stress and electric pacing conditions.
RESULTS:The engineered human myocardium exhibits Frank-Starling–type force-length relationships. After 2 weeks of static stress conditioning, the engineered myocardium demonstrated increases in contractility (0.63±0.10 mN/mm vs 0.055±0.009 mN/mm for no stress), tensile stiffness, construct alignment, and cell size. Stress conditioning also increased SERCA2 (Sarco/Endoplasmic Reticulum Calcium ATPase 2) expression, which correlated with a less negative force-frequency relationship. When electric pacing was combined with static stress conditioning, the tissues showed an additional increase in force production (1.34±0.19 mN/mm), with no change in construct alignment or cell size, suggesting maturation of excitation-contraction coupling. Supporting this notion, we found expression of RYR2 (Ryanodine Receptor 2) and SERCA2 further increased by combined static stress and electric stimulation.
CONCLUSIONS:These studies demonstrate that electric pacing and mechanical stimulation promote maturation of the structural, mechanical, and force generation properties of human-induced pluripotent stem cell-derived cardiac tissues.
The pervasive expression of circular RNA is a recently discovered feature of gene expression in highly diverged eukaryotes, but the functions of most circular RNAs are still unknown. Computational ...methods to discover and quantify circular RNA are essential. Moreover, discovering biological contexts where circular RNAs are regulated will shed light on potential functional roles they may play.
We present a new algorithm that increases the sensitivity and specificity of circular RNA detection by discovering and quantifying circular and linear RNA splicing events at both annotated and un-annotated exon boundaries, including intergenic regions of the genome, with high statistical confidence. Unlike approaches that rely on read count and exon homology to determine confidence in prediction of circular RNA expression, our algorithm uses a statistical approach. Using our algorithm, we unveiled striking induction of general and tissue-specific circular RNAs, including in the heart and lung, during human fetal development. We discover regions of the human fetal brain, such as the frontal cortex, with marked enrichment for genes where circular RNA isoforms are dominant.
The vast majority of circular RNA production occurs at major spliceosome splice sites; however, we find the first examples of developmentally induced circular RNAs processed by the minor spliceosome, and an enriched propensity of minor spliceosome donors to splice into circular RNA at un-annotated, rather than annotated, exons. Together, these results suggest a potentially significant role for circular RNA in human development.
Cellular-state information between generations of developing cells may be propagated via regulatory regions. We report consistent patterns of gain and loss of DNase I-hypersensitive sites (DHSs) as ...cells progress from embryonic stem cells (ESCs) to terminal fates. DHS patterns alone convey rich information about cell fate and lineage relationships distinct from information conveyed by gene expression. Developing cells share a proportion of their DHS landscapes with ESCs; that proportion decreases continuously in each cell type as differentiation progresses, providing a quantitative benchmark of developmental maturity. Developmentally stable DHSs densely encode binding sites for transcription factors involved in autoregulatory feedback circuits. In contrast to normal cells, cancer cells extensively reactivate silenced ESC DHSs and those from developmental programs external to the cell lineage from which the malignancy derives. Our results point to changes in regulatory DNA landscapes as quantitative indicators of cell-fate transitions, lineage relationships, and dysfunction.
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•Cell fate and lineage relationships can be derived from DHS patterns•The proportion of a cell’s DHSs shared with ESCs is a benchmark of maturity•Developmentally stable DHSs encode binding sites for self-regulating TFs•Cancer cells reactivate both ESC DHSs and those from noncognate lineages
Gains and losses of DNase I-hypersensitive sites (DHSs) during cellular differentiation provide a measure of developmental maturity and lineage relationships between cell types. Whereas developing cells exhibit a steady pruning of DHSs shared with ESCs, cancer cells show a disordered acquisition of multiple unrelated DHSs.
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