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.
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.
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.
Wnt/beta-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate ...cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.
We tested the hypothesis that Wnt/beta-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/beta-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for beta-myosin heavy chain (beta-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/beta-catenin signaling is required for Smad1 activation by BMP4.
Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/beta-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.
Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes ...(hiPSC-CMs) in vitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3β 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-3β 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.
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•GSK-3β inhibition-mediated hiPSC-cardiomyocyte proliferation is cell density dependent•GSK-3β inhibition with reduced cell-cell contact massively expands hiPSC-cardiomyocytes•LEF/TCF activity inhibits hiPSC-cardiomyocyte maturation without promoting cell cycling•Long-term expansion does not alter cardiomyocyte contractile function
Deriving a large number of hiPSC-cardiomyocytes would be beneficial for large-scale tissue engineering and drug screening applications. Buikema et al. show that GSK-3β inhibition combined with removal of cell-cell contact enables massive expansion of hiPSC-cardiomyocytes with comparable function to non-expanded cells.
Although considerable evidence exists on the association between negative health outcomes and daily experiences of discrimination, less is known about such experiences in the health care system at ...the national level. It is critically necessary to measure and address discrimination in the health care system to mitigate harm to patients and as part of the larger ongoing project of responding to health inequities.
To (1) identify the national prevalence of patient-reported experiences of discrimination in the health care system, the frequency with which they occur, and the main types of discrimination experienced and (2) examine differences in the prevalence of discrimination across demographic groups.
This cross-sectional national survey fielded online in May 2019 used a general population sample from the National Opinion Research Center's AmeriSpeak Panel. Surveys were sent to 3253 US adults aged 21 years or older, including oversamples of African American respondents, Hispanic respondents, and respondents with annual household incomes below 200% of the federal poverty level.
Analyses drew on 3 survey items measuring patient-reported experiences of discrimination, the primary types of discrimination experienced, the frequency with which they occurred, and the demographic and health-related characteristics of the respondents. Weighted bivariable and multivariable logistic regressions were conducted to assess associations between experiences of discrimination and several demographic and health-related characteristics.
Of 2137 US adult respondents who completed the survey (66.3% response rate; unweighted 51.0% female; mean SD age, 49.6 16.3 years), 458 (21.4%) reported that they had experienced discrimination in the health care system. After applying weights to generate population-level estimates, most of those who had experienced discrimination (330 72.0%) reported experiencing it more than once. Of 458 reporting experiences of discrimination, racial/ethnic discrimination was the most common type (79 17.3%), followed by discrimination based on educational or income level (59 12.9%), weight (53 11.6%), sex (52 11.4%), and age (44 9.6%). In multivariable analysis, the odds of experiencing discrimination were higher for respondents who identified as female (odds ratio OR, 1.88; 95% CI, 1.50-2.36) and lower for older respondents (OR, 0.98; 95% CI, 0.98-0.99), respondents earning at least $50 000 in annual household income (OR, 0.76; 95% CI, 0.60-0.95), and those reporting good (OR, 0.59; 95% CI, 0.46-0.75) or excellent (OR, 0.41; 95% CI, 0.31-0.56) health compared with poor or fair health.
The results of this study suggest that experiences of discrimination in the health care system appear more common than previously recognized and deserve considerable attention. These findings contribute to understanding of the scale at which interpersonal discrimination occurs in the US health care system and provide crucial evidence for next steps in assessing the risks and consequences of such discrimination. The findings also point to a need for further analysis of how interpersonal discrimination interacts with structural inequities and social determinants of health to build effective responses.
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 α-actin content, suggesting diminished cell maturation. Cyclic stress-conditioning increased expression of several cardiac markers, including β-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.
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 (hBM-MNCs). 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.
•Transplantation of hBM-MNCs can halt the negative remodeling of the infarcted heart•Both hESC-derived cardiovascular progenitors and definitive cardiomyocytes improve contractility•hBM-MNCs lead to greater vessel number than hESC-derived cells
In the present study, Murry and colleagues compared the impact of three promising cellular sources for cardiac repair on host cardiac remodeling and contractile function of the infarcted rat heart. After transplantation, human bone marrow mononuclear cells halt the deterioration of left ventricular contractile function. In contrast, human embryonic stem cell (hESC)-derived cardiovascular progenitors and definitive cardiomyocytes both improved systolic function and negative remodeling of the infarcted hearts.
With the emergence of new health information technologies, health information can be shared across networks, with or without patients' awareness and/or their consent. It is often argued that there ...can be an ethical obligation to participate in biomedical research, motivated by altruism, particularly when risks are low. In this study, we explore whether altruism contributes to the belief that there is an ethical obligation to share information about one's health as well as how other health care experiences, perceptions, and concerns might be related to belief in such an obligation.
We conducted an online survey using the National Opinion Research Center's (NORC) probability-based, nationally representative sample of U.S. adults. Our final analytic sample included complete responses from 2069 participants. We used multivariable logistic regression to examine how altruism, together with other knowledge, attitudes, and experiences contribute to the belief in an ethical obligation to allow health information to be used for research.
We find in multivariable regression that general altruism is associated with a higher likelihood of belief in an ethical obligation to allow one's health information to be used for research (OR = 1.22, SE = 0.14, p = 0.078). Trust in the health system and in care providers are both associated with a significantly higher likelihood of believing there is an ethical obligation to allow health information to be used (OR = 1.48, SE = 0.76, p<0.001; OR = 1.58, SE = 0.26, p<0.01, respectively).
Belief that there is an ethical obligation to allow one's health information to be used for research is shaped by altruism and by one's experience with, and perceptions of, health care and by general concerns about the use of personal information. Altruism cannot be assumed and researchers must recognize the ways encounters with the health care system influence (un)willingness to share one's health information.
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.
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