Sharon Paige, MD, PhD

Abstract

Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.

View details for DOI 10.1038/s41598-021-81860-x

View details for PubMedID 33542270

Abstract

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have become critically important for the detailed study of cardiac development, disease modeling, and drug screening. However, directed differentiation of hiPSCs into cardiomyocytes often results in mixed populations of cardiomyocytes and other cell types, which may confound experiments that require pure populations of cardiomyocytes. Here, we detail the use of a CRISPR/Cas9 genome editing strategy to develop cardiomyocyte-specific reporters that allow for the isolation of hiPSC-derived cardiomyocytes and chamber-specific myocytes. Moreover, we describe a cardiac differentiation protocol to derive cardiomyocytes from hiPSCs, as well as a strategy to use fluorescence-activated cell sorting to isolate pure populations of fluorescently labeled cardiomyocytes for downstream applications.

View details for DOI 10.1007/978-1-0716-0668-1_17

View details for PubMedID 32857377

Abstract

Background: In patients with complex congenital heart disease, such as those with tetralogy of Fallot, the right ventricle (RV) is subject to pressure overload stress, leading to RV hypertrophy and eventually RV failure. The role of lipid peroxidation, a potent form of oxidative stress, in mediating RV hypertrophy and failure in congenital heart disease is unknown. Methods: Lipid peroxidation and mitochondrial function and structure were assessed in RV myocardium collected from patients with RV hypertrophy with normal RV systolic function (RV FAC 47.33.8%) and in patients with RV failure showing decreased RV systolic function (RV FAC 26.63.1%). The mechanism of the effect of lipid peroxidation, mediated by 4-hydroxynonenal (4HNE; a byproduct of lipid peroxidation) on mitochondrial function and structure was assessed in HL1 murine cardiomyocytes and human induced pluripotent stem cellderived cardiomyocytes. Results: RV failure was characterized by an increase in 4HNE adduction of metabolic and mitochondrial proteins (16/27 identified proteins), in particular electron transport chain proteins. Sarcomeric (myosin) and cytoskeletal proteins (desmin, tubulin) also underwent 4HNEadduction. RV failure showed lower oxidative phosphorylation [moderate RV hypertrophy 287.619.75 vs. RV failure 137.811.57 pmol/(sec*ml), p=0.0004], and mitochondrial structural damage. Using a cell model, we show that 4HNE decreases cell number and oxidative phosphorylation (control 388.123.54 vs. 4HNE 143.711.64 pmol/(sec*ml), p<0.0001). Carvedilol, a known antioxidant did not decrease 4HNE adduction of metabolic and mitochondrial proteins and did not improve oxidative phosphorylation. Conclusions: Metabolic, mitochondrial, sarcomeric and cytoskeletal proteins are susceptible to 4HNE-adduction in patients with RV failure. 4HNE decreases mitochondrial oxygen consumption by inhibiting electron transport chain complexes. Carvedilol did not improve the 4HNE-mediated decrease in oxygen consumption. Strategies to decrease lipid peroxidation could improve mitochondrial energy generation and cardiomyocyte survival and improve RV failure in patients with congenital heart disease.

View details for DOI 10.1161/CIRCULATIONAHA.120.045470

View details for PubMedID 32806952

Abstract

Hypoplastic left heart syndrome (HLHS) is a complex congenital heart disease characterized by abnormalities in the left ventricle, associated valves, and ascending aorta. Studies have shown intrinsic myocardial defects but do not sufficiently explain developmental defects in the endocardial-derived cardiac valve, septum, and vasculature. Here, we identify a developmentally impaired endocardial population in HLHS through single-cell RNA profiling of hiPSC-derived endocardium and human fetal heart tissue with an underdeveloped left ventricle. Intrinsic endocardial defects contribute to abnormal endothelial-to-mesenchymal transition, NOTCH signaling, and extracellular matrix organization, key factors in valve formation. Endocardial abnormalities cause reduced cardiomyocyte proliferation and maturation by disrupting fibronectin-integrin signaling, consistent with recently described de novo HLHS mutations associated with abnormal endocardial gene and fibronectin regulation. Together, these results reveal a critical role for endocardium in HLHS etiology and provide a rationale for considering endocardial function in regenerative strategies.

View details for DOI 10.1016/j.stem.2020.07.015

View details for PubMedID 32810435

Abstract

Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation invivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) invitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs invitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3 (GSK-3) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3 inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3 inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.

View details for DOI 10.1016/j.stem.2020.06.001

View details for PubMedID 32619518

View details for DOI 10.1515/jpem-2018-0340

View details for Web of Science ID 000452393300012

Abstract

Palliative surgery for congenital heart disease has allowed patients with previously lethal heart malformations to survive and, in most cases, to thrive. However, these procedures often place pressure and volume loads on the heart, and over time, these chronic loads can cause heart failure. Current therapeutic options for initial surgery and chronic heart failure that results from failed palliation are limited, in part, by the mammalian heart's low inherent capacity to form new cardiomyocytes. Surmounting the heart regeneration barrier would transform the treatment of congenital, as well as acquired, heart disease and likewise would enable development of personalized, in vitro cardiac disease models. Although these remain distant goals, studies of heart development are illuminating the path forward and suggest unique opportunities for heart regeneration, particularly in fetal and neonatal periods. Here, we review major lessons from heart development that inform current and future studies directed at enhancing cardiac regeneration.

View details for DOI 10.1161/CIRCRESAHA.116.309040

View details for Web of Science ID 000397330700007

View details for PubMedID 28302741

Abstract

During normal lifespan, the mammalian heart undergoes limited renewal of cardiomyocytes. While the exact mechanism for this renewal remains unclear, two possibilities have been proposed: differentiated myocyte replication and progenitor/immature cell differentiation. This study aimed to characterize a population of cardiomyocyte precursors in the neonatal heart and to determine their requirement for cardiac development. By tracking the expression of an embryonic Nkx2.5 cardiac enhancer, we identified cardiomyoblasts capable of differentiation into striated cardiomyocytes in vitro. Genome-wide expression profile of neonatal Nkx2.5+cardiomyoblasts showed the absence of sarcomeric gene and the presence of cardiac transcription factors. To determine the lineage contribution of the Nkx2.5+cardiomyoblasts, we generated a doxycycline suppressible Cre transgenic mouse under the regulation of the Nkx2.5 enhancer and showed that neonatal Nkx2.5+cardiomyoblasts mature into cardiomyocytes in vivo. Ablation of neonatal cardiomyoblasts resulted in ventricular hypertrophy and dilation, supporting a functional requirement of the Nkx2.5+cardiomyoblasts. This study provides direct lineage tracing evidence that a cardiomyoblast population contributes to cardiogenesis in the neonatal heart. The cell population identified here may serve as a promising therapeutic for pediatric cardiac regeneration.

View details for PubMedID 28974782

Abstract

Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) can improve the contractility of injured hearts.We hypothesized that mesodermal cardiovascular progenitors (hESC-CVPs), capable of generating vascular cells in addition to cardiomyocytes, would provide superior repair by contributing to multiple components of myocardium. We performed a head-to-head comparison of hESC-CMs and hESC-CVPs and compared these with the most commonly used clinical cell type, human bone marrow mononuclear cells (hBMMNCs). In a nude rat model of myocardial infarction, hESC-CMs and hESC-CVPs generated comparable grafts. Both similarly improved systolic function and ventricular dilation. Furthermore, only rare human vessels formed from hESC-CVPs. hBM-MNCs attenuated ventricular dilation and enhanced host vascularization without engrafting long-term or improving contractility. Thus, hESC-CMs and CVPs show similar efficacy for cardiac repair, and both are more efficient than hBM-MNCs. However, hESC-CVPs do not form larger grafts or more significant numbers of human vessels in the infarcted heart.

View details for DOI 10.1016/j.stemcr.2015.09.011

View details for Web of Science ID 000364991000008

View details for PubMedID 26607951

View details for PubMedCentralID PMC4649260

Abstract

Recent advances in pluripotent stem cell biology and directed differentiation have identified a population of human cardiovascular progenitors that give rise to cardiomyocytes, smooth muscle, and endothelial cells. Because the heart develops from progenitors in 3D under constant mechanical load, we sought to test the effects of a 3D microenvironment and mechanical stress on differentiation and maturation of human cardiovascular progenitors into myocardial tissue. Progenitors were derived from embryonic stem cells, cast into collagen hydrogels, and left unstressed or subjected to static or cyclic mechanical stress. Compared to 2D culture, the unstressed 3D environment increased cardiomyocyte numbers and decreased smooth muscle numbers. Additionally, 3D culture suppressed smooth muscle -actin content, suggesting diminished cell maturation. Cyclic stress-conditioning increased expression of several cardiac markers, including -myosin heavy chain and cardiac troponin T, and the tissue showed enhanced calcium dynamics and force production. There was no effect of mechanical loading on cardiomyocyte or smooth muscle specification. Thus, 3D growth conditions favor cardiac differentiation from cardiovascular progenitors, whereas 2D conditions promote smooth muscle differentiation. Mechanical loading promotes cardiomyocyte structural and functional maturation. Culture in 3-D facilitates understanding how cues such as mechanical stress affect the differentiation and morphogenesis of distinct cardiovascular cell populations into organized, functional human cardiovascular tissue. Stem Cells 2015;33:2148-2157.

View details for DOI 10.1002/stem.2036

View details for Web of Science ID 000356668200007

View details for PubMedID 25865043

View details for PubMedCentralID PMC4478130

Abstract

The heart is the first organ to form during embryonic development. Given the complex nature of cardiac differentiation and morphogenesis, it is not surprising that some form of congenital heart disease is present in 1 percent of newborns. The molecular determinants of heart development have received much attention over the past several decades. This has been driven in large part by an interest in understanding the causes of congenital heart disease coupled with the potential of using knowledge from developmental biology to generate functional cells and tissues that could be used for regenerative medicine purposes. In this review, we highlight the critical signaling pathways and transcription factor networks that regulate cardiomyocyte lineage specification in both in vivo and in vitro models. Special focus will be given to epigenetic regulators that drive the commitment of cardiomyogenic cells from nascent mesoderm and their differentiation into chamber-specific myocytes, as well as regulation of myocardial trabeculation.

View details for DOI 10.1161/CIRCRESAHA.116.302752

View details for Web of Science ID 000347939000019

View details for PubMedCentralID PMC4299877

Abstract

For cell-based treatments of myocardial infarction, a better understanding of key developmental signaling pathways and more robust techniques for producing cardiomyocytes are required. Manipulation of Notch signaling has promise as it plays an important role during cardiovascular development, but previous studies presented conflicting results that Notch activation both positively and negatively regulates cardiogenesis. We developed surface- and microparticle-based Notch-signaling biomaterials that function in a time-specific activation-tunable manner, enabling precise investigation of Notch activation at specific developmental stages. Using our technologies, a biphasic effect of Notch activation on cardiac differentiation was found: early activation in undifferentiated human embryonic stem cells (hESCs) promotes ectodermal differentiation, activation in specified cardiovascular progenitor cells increases cardiac differentiation. Signaling also induces cardiomyocyte proliferation, and repeated doses of Notch-signaling microparticles further enhance cardiomyocyte population size. These results highlight the diverse effects of Notch activation during cardiac development and provide approaches for generating large quantities of cardiomyocytes.

View details for DOI 10.1016/j.stemcr.2014.01.011

View details for Web of Science ID 000336647700004

View details for PubMedID 24672751

View details for PubMedCentralID PMC3964284

Abstract

Genetic regulation of the cell fate transition from lateral plate mesoderm to the specification of cardiomyocytes requires suppression of Wnt/-catenin signaling, but the mechanism for this is not well understood. By analyzing gene expression and chromatin dynamics during directed differentiation of human embryonic stem cells (hESCs), we identified a suppressor of Wnt/-catenin signaling, transmembrane protein 88 (TMEM88), as a potential regulator of cardiovascular progenitor cell (CVP) specification. During the transition from mesoderm to the CVP, TMEM88 has a chromatin signature of genes that mediate cell fate decisions, and its expression is highly upregulated in advance of key cardiac transcription factors in vitro and in vivo. In early zebrafish embryos, tmem88a is expressed broadly in the lateral plate mesoderm, including the bilateral heart fields. Short hairpin RNA targeting of TMEM88 during hESC cardiac differentiation increases Wnt/-catenin signaling, confirming its role as a suppressor of this pathway. TMEM88 knockdown has no effect on NKX2.5 or GATA4 expression, but 80% of genes most highly induced during CVP development have reduced expression, suggesting adoption of a new cell fate. In support of this, analysis of later stage cell differentiation showed that TMEM88 knockdown inhibits cardiomyocyte differentiation and promotes endothelial differentiation. Taken together, TMEM88 is crucial for heart development and acts downstream of GATA factors in the pre-cardiac mesoderm to specify lineage commitment of cardiomyocyte development through inhibition of Wnt/-catenin signaling.

View details for DOI 10.1242/dev.094789

View details for Web of Science ID 000323698100009

View details for PubMedID 23924634

View details for PubMedCentralID PMC3754478

Abstract

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.

View details for DOI 10.1016/j.cell.2013.07.020

View details for Web of Science ID 000323202500018

View details for PubMedID 23953118

View details for PubMedCentralID PMC3962256

Abstract

Over the past decade, the ability to culture and differentiate human embryonic stem cells (ESCs) has offered researchers a novel therapeutic that may, for the first time, repair regions of the damaged heart. Studies of cardiac development in lower organisms have led to identification of the transforming growth factor- superfamily (eg, activin A and bone morphogenic protein 4) and the Wnt/-catenin pathway as key inducers of mesoderm and cardiovascular differentiation. These factors act in a context-specific manner (eg, Wnt/-catenin is required initially to form mesoderm but must be antagonized thereafter to make cardiac muscle). Different lines of ESCs produce different levels of agonists and antagonists for these pathways, but with careful optimization, highly enriched populations of immature cardiomyocytes can be generated. These cardiomyocytes survive transplantation to infarcted hearts of experimental animals, where they create new human myocardial tissue and improve heart function. The grafts generated by cell transplantation have been small, however, leading to an exploration of tissue engineering as an alternate strategy. Engineered tissue generated from preparations of human cardiomyocytes survives poorly after transplantation, most likely because of ischemia. Creation of pre-organized vascular networks in the tissue markedly enhances survival, with human capillaries anastomosed to the host coronary circulation. Thus, pathways controlling formation of the human cardiovascular system are emerging, yielding the building blocks for tissue regeneration that may address the root causes of heart failure.

View details for DOI 10.1253/circj.CJ-10-0958

View details for Web of Science ID 000284861400002

View details for PubMedID 21084757

View details for PubMedCentralID PMC3938118

Abstract

Embryonic stem cell (ESC) differentiation is an excellent model to study chromatin changes at developmentally regulated loci. Differentiating mouse and human ESCs increase genome-wide acetylation (euchromatic) and tri-methylation (heterochromatic) of lysine 9 on histone H3. The Oct4 locus is euchromatic when expressed in undifferentiated ESCs and heterochromatic after differentiation. Brachyury T, a mesoderm-specific transcription factor, is not yet expressed in undifferentiated cells, where its locus has "bivalent" tri-methyl lysine 4 and lysine 27 modifications. During directed differentiation to pre-cardiac mesoderm, the activated brachyury locus has high levels of tri-methyl lysine 4 (euchromatin), switching to heterochromatin after gene silencing. Thus, ESC differentiation is accompanied by genome-wide commitment to euchromatin or heterochromatin. Undifferentiated hESCs bivalently modify the brachyury locus, activate it to euchromatin during mesoderm induction, and subsequently repress it to heterochromatin, demonstrating, to our knowledge, the first analysis of chromatin dynamics at a locus essential for mesoderm and endoderm differentiation.

View details for DOI 10.1002/dvdy.21545

View details for Web of Science ID 000255842900015

View details for PubMedID 18425849

View details for PubMedCentralID PMC3075915