Abstract:Single-cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq) is rapidly becoming a powerful technology for assessing the epigenetic landscape of thousands of cells. However, the sparsity of the resulting data poses significant challenges to their interpretability and informativeness. Different computational methods are available, proposing ways to generate significant features from accessibility data and process them to obtain meaningful results. Foremost among them is the peak calling, which interprets the raw scATAC-seq data generating the peaks as features. However, scATAC-seq data are not trivially comparable with single-cell RNA sequencing (scRNA-seq) data, an increasingly pressing challenge since the necessity of multimodal experiments integration. For this reason, this study wants to improve the concept of the Gene Activity Matrix (GAM), which links the accessibility data to the genes, by proposing an improved version of the Genomic-Annotated Gene Activity Matrix (GAGAM) concept. Specifically, this paper presents GAGAM v1.2, a new and better version of GAGAM v1.0. GAGAM aims to label the peaks and link them to the genes through functional annotation of the whole genome. Using genes as features in scATAC-seq datasets makes different datasets comparable and allows linking gene accessibility and expression. This link is crucial for gene regulation understanding and fundamental for the increasing impact of multi-omics data. Results confirm that our method performs better than the previous GAMs and shows a preliminary comparison with scRNA-seq data.Keywords: epigenomic single-cell data; gene activity matrix; bioinformatics
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Our understanding of the spatiotemporal regulation of cardiogenesis is hindered by the difficulties in modeling this complex organ currently by in vitro models. Here we develop a method to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies. Consecutive morphological changes proceed in a self-organizing manner in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4), and the resulting in vitro heart organoid possesses atrium- and ventricle-like parts containing cardiac muscle, conducting tissues, smooth muscle and endothelial cells that exhibited myocardial contraction and action potentials. The heart organoids exhibit ultrastructural, histochemical and gene expression characteristics of considerable similarity to those of developmental hearts in vivo. Our results demonstrate that this method not only provides a biomimetic model of the developing heart-like structure with simplified differentiation protocol, but also represents a promising research tool with a broad range of applications, including drug testing.
For 3D culture methods using ESCs or adult stem cells, ECMs including Matrigel and laminin were used for in vitro organogenesis, including the development of the brain16, stomach17, kidney18, and intestinal tissues19. FGF and FGFR1 are required for cardiac gene expression, particularly during SHF proliferation and chamber formation in cooperation with the Sonic hedgehog gene during heart development20,21. Therefore, it may be beneficial to consider the role of the extracellular matrix (ECM) environment and FGF signal, which may promote the self-organizing process reconstructing in vivo cardiogenesis. The mouse model system is effective because the embryonic period of mice is shorter than that of humans and advantageous because embryonic heart samples are easily obtained for comparison, and many ESC lines with mutations in genes related to heart development are available for comprehensive analysis.
Here, we report the generation of a heart organoid with atrium- and ventricle-like structures showing the ultrastructural, histochemical and gene expression profile similarities to embryonic hearts in the presence of the laminin-entactin (LN/ET) complex22 in the ECM and exogenous fibroblast growth factor 4 (FGF4)20 via consecutive morphological changes like self-organization and, therefore, without the requirement of complex differentiation protocols.
Next, we investigated whether in vitro heart organoids faithfully exhibit cardiac properties. The ultrastructural and structural compartments of heart organoids, as well as their development, were evaluated through comparison with embryonic mouse hearts. Transmission electron microscopy revealed that the heart organoids exhibited the CM-like ultrastructures that appear during embryogenesis, including sarcomere structures with Z-bands, mitochondria, and desmosomes (Fig. 2a). Additionally, further distinctive characteristics, such as the honeycomb structure of the sarcoplasmic reticulum (SR) (Fig. 2b, left) and intercalated disc (ID, and especially wavy ventricular myocyte IDs; Fig. 2b, right)4, were observed in the heart organoids. The ID is a cardiac muscle-specific structure required for coordinated muscle contraction, indicating that these heart organoids might possess contractile cardiac muscle cell properties.
To confirm whether the heart organoids (Fig. 2c, left) expressed cardiac transcription factors (TFs) and/or structural genes expressed by the in vivo embryonic heart, we assessed the expression of T-box transcriptional factor 5 (Tbx5, an important marker for proper development of both atria and the LV27,28,29,30,31) and SM-myosin heavy chain (SM-MHC, an SM marker). Notably, the spatial pattern of Tbx5 expression in the heart organoids resembled that of in vivo embryonic hearts (E12.5H and E13.5H) after chamber formation (i.e., positive in the left atrium-, right atrium- and left ventricle-like parts) and also detected SM-MHC expression (Fig. 2c, right).
Taken together, these results suggested that FGF4 and the gelated LN/ET complex are required for effective in vitro generation of heart organoids with similar cardiac properties to those of embryonic hearts.
In the cardiogenesis, several types of cardiac cells and neuronal elements are differentiated from undifferentiated cells. To assess whether undifferentiated stem cells remained in the heart organoids cultured for 11 days, we performed immunostaining assays to detect the pluripotent stem cell marker Oct3/4. Concerning undifferentiated stem cell markers the heart organoids did not exhibit any Oct3/4 expression, in contrast to the EBs prior to culture, indicating that no pluripotent stem cells remained in the 11-day cultured heart organoid (Supplementary Fig. 5a), whereas Nestin which is expressed in the fetal heart as well as in brain tissue was detected in both of the heart organoids and E12.5 hearts.
Based on these results, the heart organoids appeared to exhibit the organized structural properties of the embryonic hearts (CMs, SMs and ECs) as well as the cytological and histological maturity, such as the presence of Purkinje fibers, the association of SR/T tubule junction and the tight neuronal contact with the myocytes.
Next, we investigated the expressions of coronary circulation-related genes in the heart organoids. The development of coronary circulation is heralded by the appearance of budding and canalized venous sprouts from the sinus venosus which contains vein cells undergo an early cell fate switch to create pre-artery population to build coronary arteries4. Especially, Nr2f2 (Coup-tf2), a venous transcription factor, regulates the expression of artery genes. Notably, the gene expression level of the venous marker Nr2f2 in the heart organoids was similar to that in in vivo embryonic hearts (Supplementary Fig. 5c, top). Similarly, in almost all of the heart organoids (regardless of the culture period), the gene expression patterns of the artery markers Cxcr4, Jag2 and Msx1 were similar to those of in vivo embryonic hearts from E9.5 to E12.5, although other artery markers, including Notch4, Dll4 and Gja4, were only expressed at low levels in heart organoids cultured for 6 days (Supplementary Fig. 5c, bottom).
These results indicated that, overall, the heart organoid mimics the specific features of in vivo embryonic hearts in gene expressions, reflecting the diverse property of mammalian cardiac development including coronary circulation.
Also, the expression of Nestin (fetal heart and brain marker) was observed in both heart organoids and in E12.5 hearts, suggesting that Nestin-positive cells derived from NCCs may contribute to cardiac innervation and conduction through the cardiac autonomic nervous system25,53.
Despite major improvements in the treatment of virtually all cardiac disorders, heart failure (HF) is an exception, in that its prevalence is rising, and only small prolongations in survival are occurring. An increasing fraction, especially older women with diabetes, obesity, and atrial fibrillation exhibit HF with preserved systolic function. Several pathogenetic mechanisms appear to be operative in HF. These include increased hemodynamic overload, ischemia-related dysfunction, ventricular remodeling, excessive neurohumoral stimulation, abnormal myocyte calcium cycling, excessive or inadequate proliferation of the extracellular matrix, accelerated apoptosis, and genetic mutations. Biomarkers released as a consequence of myocardial stretch, imbalance between formation and breakdown of extracellular matrix, inflammation, and renal failure are useful in the identification of the pathogenetic mechanism and, when used in combination, may become helpful in estimating prognosis and selecting appropriate therapy. Promising new therapies that are now undergoing intensive investigation include an angiotensin receptor neprilysin inhibitor, a naturally-occurring vasodilator peptide, a myofilament sensitizer and several drugs that enhance Ca++ uptake by the sarcoplasmic reticulum. Cell therapy, using autologous bone marrow and cardiac progenitor cells, appears to be promising, as does gene therapy. Chronic left ventricular assistance with continuous flow pumps is being applied more frequently and successfully as destination therapy, as a bridge to transplantation, and even as a bridge to recovery and explantation. While many of these therapies will improve the care of patients with HF, significant reductions in prevalence will require vigorous, multifaceted, preventive approaches. 781b155fdc