Cardiac Differentiation Research Articles

Maternal early overfeeding negatively impacts cardiac progenitor cells differentiation and cardiomyocyte maturation in the neonatal offspring.

Maternal obesity has been positively correlated with an increased cardiometabolic risk in the offspring throughout life, implying intergenerational transmission. However, little is known about the early life cardiac cell modifications that imply the onset of heart diseases later in life. This study analyzed cardiac progenitor cells and cardiomyocyte differentiation on day of birth in the offspring born to obese dams. The litter size reduction model was used to induce obesity in female Swiss mice. Both maternal groups, the Small Litter Dams (SLD-F1), which were overfed during lactation, and the Normal Litter Dams (NLD-F1), control group, were mated to healthy male mice. Their first generation offspring (SLD-F2 and NLD-F2, n=6 by group) were euthanized on birth. Mothers from SLD had increased body mass, Lee Index, fat deposits, hyperglycemia, and glucose intolerance, confirming the obese phenotype. The offspring born from SLD-F1 had also increased body mass, Lee Index, and fasting hyperglycemia. The heart of SLD-F2 showed decreased cardiac mass/body mass ratio, increased cardiac collagen deposits, and a greater number of undifferentiated cardiac c-kit+ and Sca-1+ progenitor cells, and increased NKX2.5+ cardiomyoblasts compared to control. In addition, SLD-F2 demonstrated immature cardiomyocytes. Obese dams negatively impact its offspring, leading to altered biometric and metabolic parameters, along with an immature heart already at birth, with extracellular matrix adverse remodeling, delayed cardiac progenitor cells differentiation and restrained cardiomyocyte maturation, which can be related with the development of cardiometabolic disease in the adulthood.

A Thorough Navigation of miRNA's Blueprint in Crafting Cardiovascular Fate.

Cardiovascular diseases contribute significantly to global morbidity and mortality. MicroRNAs are crucial in the development and progression of these diseases by regulating gene expression in various cells and tissues. Their roles in conditions like atherosclerosis, heart failure, myocardial infarction, and arrhythmias have been widely researched. The present study provides an overview of existing evidence regarding miRNAs' role in cardiovascular disease pathogenesis. Furthermore, the study examines current state-of-the-art technologies used in the study of miRNAs in cardiovascular disease. As a final point, we examine how miRNAs may serve as disease biomarkers, therapeutic targets, and prognostic indicators. In cardiology, microRNAs, small noncoding RNA molecules, are crucial to the posttranscriptional regulation of genes. Their role in regulating cardiac cell differentiation and maturation is critical during the development of the heart. They maintain the cardiac function of an adult heart by contributing to its electrical and contractile activity. By binding to messenger RNA molecules, they inhibit protein translation or degrade mRNA. Several cardiovascular diseases are associated with dysregulation of miRNAs, including arrhythmias, hypertension, atherosclerosis, and heart failure. miRNAs can be used as biomarkers to diagnose and predict diseases as well as therapeutic targets. A variety of state-of-the-art technologies have aided researchers in discovering, profiling, and analyzing miRNAs, including microarray analysis, next-generation sequencing, and others. Developing new diagnostics and therapeutic approaches is becoming more feasible as researchers refine their understanding of miRNA function. Ultimately, this will reduce the burden of cardiovascular disease around the world.

Epigenetic modifications in transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

Abstract Background Adipose tissue-derived stromal vascular fraction (adipose SVF) contains pluripotent mesenchymal stem cells and rarely transdifferentiate into beating cardiomyocytes. We developed a simple culture protocol under which the adult murine inguinal adipose SVF reproductively transdifferentiates into beating cardiomyocyte-like cells (beating CM) without inductions in the primary culture. We also reported that Mef2c is a crucial factor in this conversion. However, the characteristics of beating CM and the factors that regulate the differentiation of adipose SVF toward the cardiac lineage are unknown. Purpose To investigate sequential changes in global gene expression profiles of adipose SVF in primary culture and determine the mechanism of differentiation into beating CM. Methods Adipose SVF cells were isolated from adult mice's inguinal subcutaneous fat pad and cultured using the previously established beating CM induction method (Sci Rep. 2021). At 6 time points (days 0, 3, 7, 14, 21, and 28) in primary culture, total RNA was extracted, and RNA-sequencing was performed (n = 3). The data was analyzed using Subio Platform and MetaCore software. To assess the candidate gene and proteins, adipose SVF cells were transduced with the gene using a lentivirus vector and supplemented with the specific inhibitors. The cardiac differentiation was evaluated by quantitative PCR on day 28. Results The beating CM was confirmed on days 14, 21, and 28 in the primary culture. Among prefiltered 14,574 genes, the expression level of 749 genes were significantly changed (153 genes upregulated vs. 596 genes downregulated) between days 7 and 14 (2-fold, P < 0.05), in which beating CM appeared. The GO molecular functions analysis showed that O-GluNAC transferase and histone deacetylase (HDAC)-associated genes were expressed dominantly before day 7, and KLF4-associated genes were expressed prominently after day 14 in primary culture. The expression of cardiac troponin T (Tnnt2) and myosin light chain ventricular type (Myl2) increased on day 3 and day 14, respectively. The HDAC7, an epigenetic regulator, decreased the expression on day 14 or later. Treatment with the HDAC inhibitor (150 nmol/L) increased the expression of Tnnt2 (5-fold vs. control) on day 28 in adipose SVF. Furthermore, treatment of Mef2c-transduced adipose SVFs with HDAC inhibitors increased expression of cardiac troponin T (29-fold vs. control, P < 0.05). Conclusion The time course analysis demonstrated that a remarkable change occurred in the regulation of transcription on day 14 in adipose SVF primary culture. Alterations in epigenetic modifications increased beating CM in adipose SVFs. Adipose SVF could be applied to new cardiac regeneration therapies by increasing the efficiency of introduction into the beating CM.

The transcription factor foxo3 regulates cardiac remodeling after myocardial infarction

Abstract Background Myofibroblast and myeloid cell differentiation following acute myocardial infarction (MI) are important for myocardial fibrosis but also cause adverse remodeling leading to cardiac dysfunction and heart failure. The transcription factor forkhead box O 3 (Foxo3) inhibits hypertrophic cardiac remodeling. We hypothesized that Foxo3, a key regulator of cell differentiation and immunity might inhibit differentiation of fibroblasts and myoloid cells and therefore cardiac fibrosis after MI. Methods MI was induced in Foxo3-/- and wild-type mice by permanent LAD ligation. Cardiac function was determined by transthoracic echocardiography. Cell differentiation was investigated by single nucleus RNA-sequencing (snRNA-seq). Moreover, transdifferentiation assays ex vivo were performed. Results Foxo3-/- mice showed significantly improved survival post- MI (p<0.01) that was in part due to reduced cardiac ruptures (p<0.05). TTE showed reduced LV function, cardiac output, and global strain 4 days post-MI in both groups of animals. However, LV function was significantly attenuated in Foxo3-/- mice 14 d post-MI while diastolic parameters indicated enhanced wall stiffness. snRNA-seq on day 4 post-MI revealed significantly increased numbers of myofibroblasts (p<0.05) transdifferentiating from Progenitor-like state fibroblasts and epicardial derived fibroblasts in Foxo3-/- mice. Myofibroblasts upregulated genes important for extracellular matrix structure and cell migration. Moreover, an upregulation of pro-fibrotic genes was observed in other fibroblast clusters in Foxo3-/- mice and corroborated by significantly elevated collagen type 1 and 3 as well as periostin protein expression. Mechanistically, fibroblasts from Foxo3a-/- mice exhibited enhanced Smad3 dependent myofibroblast marker expression after TGF-ß stimulation. Cell chat analysis indicated enhanced number of interactions and interaction strength of myeloid cells with fibroblasts in Foxo3-/- animals. In line with these observations, differences in cardiac myeloid cell differentiation characterized by reduced center-log ratios of cardiac resident macrophages (Lyve1hiMHCIIlo and Lyve1loMHCIIhi) and increase in monocyte-derived macrophages (Ccr2hi MΦ and Trem2hi MΦ) on day 4 post-MI that was enhanced in Foxo3-/- mice were determined. Differential gene expression patterns on day 4 in pro-inflammatory macrophages (Ccr2hi MΦ) showed upregulation of genes in the interleukin-12 pathway while reparative macrophages (Trem2hi MΦ) revealed an increased expression of genes for extracellular matrix proteins and ECM-collagen interactions in Foxo3-/- mice. Conclusion Our data identify Foxo3 as a master regulator of cardiac remodeling inhibiting myofibroblast and myeloid cell differentiation after acute MI, and suggesting that deficiency of Foxo3 promotes early scar formation but also contributes to adverse matricellular remodeling resulting in impaired cardiac function and enhanced fibrosis.

Oxytocin, the Love Hormone, in Stem Cell Differentiation

Oxytocin (OXT) is a neurohypophysial nonapeptide that exerts its effects mainly through the oxytocin receptor (OXTR). Several studies have pointed out the role of OXT in the modulation of stem cell (SC) fate and properties. SCs are undifferentiated cells characterized by a remarkable ability to self-renew and differentiate into various cell types of the body. In this review, we focused on the role of OXT in SC differentiation. Specifically, we summarize and discuss the scientific research examining the effects of OXT on mesodermal SC-derived lineages, including cardiac, myogenic, adipogenic, osteogenic, and chondrogenic differentiation. The available studies related to the effects of OXT on SC differentiation provide little insights about the molecular mechanism mediated by the OXT–OXTR pathway. Further research is needed to fully elucidate these pathways to effectively modulate SC differentiation and develop potential therapeutic applications in regenerative medicine.

Evolution and development of the conduction system in the vertebrate heart: a role for hemodynamics and the epicardium.

Development of the heart is a very intricate and multiplex process as it involves not only the three spatial dimensions but also the fourth or time dimension. Over time, the heart of an embryo needs to adapt its function to serve the increasing complexity of differentiation and growth towards adulthood. It becomes even more perplexing by expanding time into millions of years, allocating related species in the tree of life. As the evolution of soft tissues can hardly be studied, we have to rely on comparative embryology, supported heavily by genetic and molecular approaches. These techniques provide insight into relationships, not only between species, but also between cell populations, signaling mechanisms, molecular interactions and physical factors such as hemodynamics. Heart development depends on differentiation of a mesodermal cell population that - in more derived taxa - continues in segmentation of the first and second heart field. These fields deliver not only the cardiomyocytes, forming the three-dimensionally looping cardiac tube as a basis for the chambered heart, but also the enveloping epicardium. The synchronized beating of the heart is then organized by the conduction system. In this Review, the epicardium is introduced as an important player in cardiac differentiation, including the conduction system.

SMYD1 modulates the proliferation of multipotent cardiac progenitor cells derived from human pluripotent stem cells during myocardial differentiation through GSK3β/β-catenin&ERK signaling.

The histone-lysine N-methyltransferase SMYD1, which is specific to striated muscle, plays a crucial role in regulating early heart development. Its deficiency has been linked to the occurrence of congenital heart disease. Nevertheless, the precise mechanism by which SMYD1 deficiency contributes to congenital heart disease remains unclear. We established a SMYD1 knockout pluripotent stem cell line and a doxycycline-inducible SMYD1 expression pluripotent stem cell line to investigate the functions of SMYD1 utilizing an in vitro-directed myocardial differentiation model. Cardiomyocytes lacking SMYD1 displayed drastically diminished differentiation efficiency, concomitant with heightened proliferation capacityof cardiac progenitor cells during the early cardiac differentiation stage. These cellular phenotypes were confirmed through experiments inducing the re-expression of SMYD1. Transcriptome sequencing and small molecule inhibitor intervention suggested that the GSK3β/β-catenin&ERK signaling pathway was involved in the proliferation of cardiac progenitor cells. Chromatin immunoprecipitation demonstrated that SMYD1 acted as a transcriptional activator of GSK3β through histone H3 lysine 4 trimethylation. Additionally, dual-luciferase analyses indicated that SMYD1 could interact with the promoter region of GSK3β, thereby augmenting its transcriptional activity. Moreover, administering insulin and Insulin-like growth factor 1 can enhance the efficacy of myocardial differentiation in SMYD1 knockout cells. Our research indicated that the participation of SMYD1 in the GSK3β/β-catenin&ERK signaling cascade modulated the proliferation of cardiac progenitor cells during myocardial differentiation. This process was partly reliant on the transcription of GSK3β. Our research provided a novel insight into the genetic modification effect of SMYD1 during early myocardial differentiation. The findings were essential to the molecular mechanism and potential interventions for congenital heart disease.

The G4 resolvase Dhx36 modulates cardiomyocyte differentiation and ventricular conduction system development.

Extensive genetic studies have elucidated cardiomyocyte differentiation and associated gene networks using single-cell RNA-seq, yet the intricate transcriptional mechanisms governing cardiac conduction system (CCS) development and working cardiomyocyte differentiation remain largely unexplored. Here we show that mice deleted for Dhx36 (encoding the Dhx36 helicase) in the embryonic or neonatal heart develop overt dilated cardiomyopathy, surface ECG alterations related to cardiac impulse propagation, and (in the embryonic heart) a lack of a ventricular conduction system (VCS). Heart snRNA-seq and snATAC-seq reveal the role of Dhx36 in CCS development and in the differentiation of working cardiomyocytes. Dhx36 deficiency directly influences cardiomyocyte gene networks by disrupting the resolution of promoter G-quadruplexes in key cardiac genes, impacting cardiomyocyte differentiation and CCS morphogenesis, and ultimately leading to dilated cardiomyopathy and atrioventricular block. These findings further identify crucial genes and pathways that regulate the development and function of the VCS/Purkinje fiber (PF) network.

Hydrogel microsphere stem cell encapsulation enhances cardiomyocyte differentiation and functionality in scalable suspension system

A reliable suspension-based platform for scaling engineered cardiac tissue (ECT) production from human induced pluripotent stem cells (hiPSCs) is crucial for regenerative therapies. Here, we compared the production and functionality of ECTs formed using our scaffold-based, engineered tissue microsphere differentiation approach with those formed using the prevalent scaffold-free aggregate platform. We utilized a microfluidic system for the rapid (1 million cells/min), high density (30, 40, 60 million cells/ml) encapsulation of hiPSCs within PEG-fibrinogen hydrogel microspheres. HiPSC-laden microspheres and aggregates underwent suspension-based cardiac differentiation in chemically defined media. In comparison to aggregates, microspheres maintained consistent size and shape initially, over time, and within and between batches. Initial size and shape coefficients of variation for microspheres were eight and three times lower, respectively, compared to aggregates. On day 10, microsphere cardiomyocyte (CM) content was 27 % higher and the number of CMs per initial hiPSC was 250 % higher than in aggregates. Contraction and relaxation velocities of microspheres were four and nine times higher than those of aggregates, respectively. Microsphere contractile functionality also improved with culture time, whereas aggregate functionality remained unchanged. Additionally, microspheres displayed improved β-adrenergic signaling responsiveness and uniform calcium transient propagation. Transcriptomic analysis revealed that while both microspheres and aggregates demonstrated similar gene regulation patterns associated with cardiomyocyte differentiation, heart development, cardiac muscle contraction, and sarcomere organization, the microspheres exhibited more pronounced transcriptional changes over time. Taken together, these results highlight the capability of the microsphere platform for scaling up biomanufacturing of ECTs in a suspension-based culture platform.

A new paradigm for generating high-quality cardiac pacemaker cells from mouse pluripotent stem cells

Cardiac biological pacing (BP) is one of the future directions for bradyarrhythmias intervention. Currently, cardiac pacemaker cells (PCs) used for cardiac BP are mainly derived from pluripotent stem cells (PSCs). However, the production of high-quality cardiac PCs from PSCs remains a challenge. Here, we developed a cardiac PC differentiation strategy by adopting dual PC markers and simulating the developmental route of PCs. First, two PC markers, Shox2 and Hcn4, were selected to establish Shox2:EGFP; Hcn4:mCherry mouse PSC reporter line. Then, by stepwise guiding naïve PSCs to cardiac PCs following naïve to formative pluripotency transition and manipulating signaling pathways during cardiac PCs differentiation, we designed the FSK method that increased the yield of SHOX2+; HCN4+ cells with typical PC characteristics, which was 12 and 42 folds higher than that of the embryoid body (EB) and the monolayer M10 methods respectively. In addition, the in vitro cardiac PCs differentiation trajectory was mapped by single-cell RNA sequencing (scRNA-seq), which resembled in vivo PCs development, and ZFP503 was verified as a key regulator of cardiac PCs differentiation. These PSC-derived cardiac PCs have the potential to drive advances in cardiac BP technology, help with the understanding of PCs (patho)physiology, and benefit drug discovery for PC-related diseases as well.

Refined home-brew media for cost-effective, weekend-free hiPSC culture and genetic engineering

Background Cost-effective, practical, and reproducible culture of human induced pluripotent stem cells (hiPSCs) is required for both basic and translational research. This is especially crucial for large-scale expansion of hiPSCs for cell therapy, which should be made accessible to many patients regardless of their socioeconomic background. Basal 8 (B8) has emerged as a cost-effective solution for weekend-free and chemically-defined hiPSC culture. However, homebrewing of some recombinant growth factors for B8 can be a bottleneck towards both access and reproducibility of this technology. Moreover, we found the published B8 formulation to be suboptimal in normoxic hiPSC culture, which is widely used. Lastly, the suitability of B8 for applications such as genome editing or organoid differentiation remains to be assessed. Methods We formulated B8 with commercially available, animal-free growth factors, refined its composition to support normoxic culture of the widely-used WTC11 hiPSC line, and compared it to commercial Essential 8 (E8) and a home-made, weekend-free E8 formulation (hE8). We measured pluripotency marker expression and cell cycle with flow cytometry, and investigated the transcriptional profiles by bulk RNA sequencing. We also assessed the efficiency of gene editing, single-cell sorting, and cardiac differentiation in both monolayer and organoids. Results hE8 performed similarly to commercial E8 in all the assays. Despite morphological changes, cells in B8+, our optimised variant of B8, expressed the pluripotency marker NANOG at the highest level. At the same time, cells grown in B8+ were primed towards a mesendodermal fate. B8+ outperformed other media with regard to genome editing via homology directed recombination, and was on par with other media in other assays. Conclusions Overall, optimised weekend-free media formulations promise to democratise the generation of engineered cells for a wide range of applications.

Medium acidosis drives cardiac differentiation during mesendoderm cell fate specification from human pluripotent stem cells

Effective lineage-specific differentiation is essential to fulfilling the great potentials of human pluripotent stem cells (hPSCs). In this report, we investigate how modulation of medium pH and associated metabolic changes influence mesendoderm differentiation from hPSCs. We show that daily medium pH fluctuations are critical for the heterogeneity of cell fates in the absence of exogenous inducers. Acidic environment alone leads to cardiomyocyte generation without other signaling modulators. In contrast, medium alkalinization is inhibitory to cardiac fate even in the presence of classic cardiac inducers. We then demonstrate that acidic environment suppresses glycolysis to facilitate cardiac differentiation, while alkaline condition promotes glycolysis and diverts the differentiation toward other cell types. We further show that glycolysis inhibition or AMPK activation can rescue cardiac differentiation under alkalinization, and glycolysis inhibition alone can drive cardiac cell fate. This study highlights that pH changes remodel metabolic patterns and modulate signaling pathways to control cell fate.

Optimizing Cardiomyocyte Differentiation: Comparative Analysis of Bone Marrow and Adipose-Derived Mesenchymal Stem Cells in Rats Using 5-Azacytidine and Low-Dose FGF and IGF Treatment.

Mesenchymal stem cells (MSCs) exhibit multipotency, self-renewal, and immune-modulatory properties, making them promising in regenerative medicine, particularly in cardiovascular treatments. However, optimizing the MSC source and induction method of cardiac differentiation is challenging. This study compares the cardiomyogenic potential of bone marrow (BM)-MSCs and adipose-derived (AD)-MSCs using 5-Azacytidine (5-Aza) alone or combined with low doses of Fibroblast Growth Factor (FGF) and Insulin-like Growth Factor (IGF). BM-MSCs and AD-MSCs were differentiated using two protocols: 10 μmol 5-Aza alone and 10 μmol 5-Aza with 1 ng/mL FGF and 10 ng/mL IGF. Morphological, transcriptional, and translational analyses, along with cell viability assessments, were performed. Both the MSC types exhibited similar morphological changes; however, AD-MSCs achieved 70-80% confluence faster than BM-MSCs. Surface marker profiling confirmed CD29 and CD90 positivity and CD45 negativity. The differentiation protocols led to cell flattening and myotube formation, with earlier differentiation in AD-MSCs. The combined protocol reduced cell mortality in BM-MSCs and enhanced the expression of cardiac markers (MEF2c, Troponin I, GSK-3β), particularly in BM-MSCs. Immunofluorescence confirmed cardiac-specific protein expression in all the treated groups. Both MSC types exhibited the expression of cardiac-specific markers indicative of cardiomyogenic differentiation, with the combined treatment showing superior efficiency for BM-MSCs.

Chemically defined production of engineered cardiac tissue microspheres from hydrogel-encapsulated pluripotent stem cells.

Chemically defined, suspension culture conditions are a key requirement in realizing clinical translation of engineered cardiac tissues (ECTs). Building on our previous work producing functional ECT microspheres through differentiation of biomaterial encapsulated human induced pluripotent stem cells (hiPSCs), here we establish the ability to use chemically defined culture conditions, including stem cell media (E8) and cardiac differentiation media (chemically defined differentiation media with three components, CDM3). A custom microfluidic cell encapsulation system was used to encapsulate hiPSCs at a range of initial cell concentrations and diameters in the hybrid biomaterial, poly(ethylene glycol)-fibrinogen (PF), for the formation of highly spherical and uniform ECT microspheres for subsequent cardiac differentiation. Initial microsphere diameter could be tightly controlled, and microspheres could be produced with an initial diameter between 400 and 800 µm. Three days after encapsulation, cardiac differentiation was initiated through small molecule modulation of Wnt signaling in CDM3. Cardiac differentiation occurred resulting in in situ ECTformation; results showed that this differentiation protocol could be used to achieve cardiomyocyte (CM) contents greater than 90%, although there was relatively high variability in CM content and yield between differentiation batches. Spontaneous contraction of ECT microspheres initiated between Days 7 and 10 of differentiation and ECT microspheres responded to electrical pacing up to 1.5 Hz. Resulting CMs had well-defined sarcomeres and the gap junction protein, connexin 43, and had appropriate temporal changes in gene expression. In summary, this study demonstrated the proof-of-concept to produce functional ECT microspheres with chemically defined media in suspension culture in combination with biomaterial support of microsphere encapsulated hiPSCs.

Abstract We091: Maternal hyperglycemia disrupts human cardiac cell lineage differentiation

Maternal diabetes is associated with a 4-fold increased risk of offspring developing congenital heart defects (CHDs). It remains unknown how maternal diabetes interferes with cardiac cell lineage determination and leads to malformations in the heart. In this study, we aim to elucidate the cellular mechanisms by which maternal hyperglycemia causes high risk of CHDs in newborns. Here we leverage an in vitro hyperglycemic model using human induced pluripotent stem cells (iPSCs), which could recapture cardiac differentiation and cell lineage commitment during embryonic heart development. We collected differentiating cells at D5 (cardiac mesoderm), D10 (cardiac progenitors), and D14 (early cardiomyocytes) during cardiac differentiation under normal and hyperglycemic conditions, and performed single-cell transcriptomic analysis. We found that hyperglycemia significantly impedes cardiac differentiation of human iPSCs as robust cardiac differentiation is rarely observed in multiple iPSC lines (n=10) under hyperglycemia. At the cellular level, hyperglycemia interferes with cardiac differentiation of human iPSCs in response to WNT signaling activation, which is manifested by reduced number of cardiac mesoderm (MESP1+ PDGFRA+) cells at D5 of differentiation. In contrast, neural differentiation is enhanced under hyperglycemia, with a high proportion of neural cell lineage (SOX2+ PAX6+). At D10, differentiated neural cell lineage dominates the cell population under hyperglycemia at the expense of cardiac progenitors and early cardiomyocytes. At D14, the prevalence of neural lineage persist whereas early cardiomyocytes only accounts for a small portion of cell population under hyperglycemia. Moreover, we treated D30 iPSC-derived cardiomyocytes (iPSC-CMs) with high glucose concentration (25 mM) for 7 days and found that iPSC-CMs show reduced mitochondria respiration and ATP production, but elevated apoptosis and reactive oxygen species (ROS) generation. Together, our data suggest that maternal hyperglycemia could interrupt human embryonic heart morphogenesis through overriding WNT-medicated cardiac differentiation and promoting neural cell lineage determination by default.

Abstract We036: Recapitulating Cardiac Microenvironment: Elucidating the Role of ECM Components and Architecture on Cardiac Differentiation

The discovery of iPSCs was revolutionary for regenerative medicine, especially as a source to replace a cell population with limited regeneration capability, like cardiomyocytes (CMs). However, the exact mechanism(s) governing how the microenvironment influences cardiac differentiation at a cellular scale is still widely unclear. To bridge this gap, we created an in vitro fibrous model from a decellularized porcine cardiac extracellular matrix. The aim is to assess the impact of the microenvironment, specifically the effects of protein composition and micro-architecture, on the cardiac differentiation of iPSCs. To study the role of protein composition, atrial and ventricular regions of the porcine heart are manually identified and separated as their protein composition is observed to be different. To test this, the material was subjected to proteomics analysis with LS-Mass Spectrometry where about 1000 proteins were identified in the ventricular region and only 300 proteins in atria. The decellularized tissue was tested for complete removal of nuclei by staining and further validated with DNA quantification. The dECM was blended with synthetic polymer (polycaprolactone) for electrospinning. The average collected fiber diameter was 14.8 ± 3.9 μm. The fibrous scaffolds were seeded with iPSCs and differentiated towards cardiac lineage. dECM samples show comparable signs of biocompatibility as laminin-521 coated TCPS, forming clusters throughout the differentiation process indicating that the scaffolds encourage cardiac differentiation. Further analysis is being conducted to compare the cellular expressions of differentiation markers including mesoderm formation, cardiac specification, and cardiomyocyte structural and functional markers. The effects of the ECM composition will be checked with myosin light and heavy chain expressions, as their expressions vary in the atrial and ventricular regions of the heart.

Abstract We044: MATURITY OF HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES PROMOTED BY BRACHYURY PRIMING

Introduction: Cardiac differentiation protocols enable derivation of cardiomyocytes (CM). from human induced pluripotent stem cell (iPS). However, limited tools are available to ensure enhanced iPS-CM maturation without the need for prolonged and complex culture. Research Question: This study assessed whether the mesodermal and cardiopoiesis inductor, Brachyury (T), complements standard cardiac differentiation and augments structural and functional maturation of iPS-CM. Aims: The impact of T mRNA introduction prior to initiation of cardiac differentiation was characterized on: i) cardiac transcription factor expression; ii) structural and functional hallmarks of maturity; and iii) cardiotoxicity. Methods: RT-qPCR was employed to monitor the expression of cardiac-specific transcription factors. Transmission electron microscopy enabled the assessment of cellular ultrastructure. Seahorse measurements were used to determine basal respiration. Fluo4 staining was used to measure calcium transients with external field stimulation. Cardiotoxicity was measured as the impact of doxorubicin on calcium handling and cell survival. Results: Transfection with T prompted early and sustained upregulation of the cardiac transcription factors Nkx2.5 (10-fold; FDR<0.001) and MEF2C (7.2-fold; FDR<0.001). Mitochondrial area was increased with T transfection (P<0.001) and T-transfected iPS-CM exhibited greater basal respiration (FDR<0.05), greater calcium handling resilience in response to field stimulation, and a more mature transient morphology. Following doxorubicin exposure T transfected iPS-CM exhibited a greater compromise in calcium handling (FDR<0.001), and exaggerated cell death. Conclusion: Induction of human iPS with T prior to initiation of cardiac differentiation enhanced signatures of structural and functional maturity, and provided more robust readouts of drug toxicity.

Abstract Mo095: Variant Effect Mapping by Combining Base-editing Mutagenesis and Multiplexed Functional Assays Using Human Cardiomyocytes

Introduction: MYBPC3 is the most prevalent gene in hypertrophic cardiomyopathy (HCM). However, the pathogenicity of most MYBPC3 variants remains unknown, and it is still challenging to interpret variants identified in patients. Although multiplexed functional assays using cancer cell lines are powerful approaches to test large number of variants in a single experiment and facilitate the interpretation of variants, there is a limitation of the assessment of genes expressing in specific cell types, such as sarcomeric genes including MYBPC3. Hypothesis: The approach integrating base-editing mutagenesis and multiplexed functional assays using iPSC-derived cardiomyocytes (iPSC-CMs) will enable us to prospectively evaluate MYBPC3 variants in a high-throughput manner and illuminate the pathophysiological mechanism of HCM. Methods: We focused on the intron-exon boundary including intron 11 and exon 12 in MYBPC3 and introduced variants into iPSCs by CRISPR-X, which can target multiple genomic locations and introduce diverse editing, and adenine base editor. After cardiac differentiation, we performed multiplexed functional assays. iPSC-CMs were immunostained for cMyBP-C and BNP expressions and sorted based on those expressions using fluorescence-activated cell sorting. The ubiquitin-proteasome activity associated with cellular proteostasis was analyzed by ubiquitin fusion degradation reporter system. The variant scores were calculated by comparing the frequency of each variant in each population sorted based on phenotype using next-generation sequencing. Results: About 200 variants located at the mutagenesis target region were evaluated via in situ mutagenesis and each functional assay. The pathogenic/likely pathogenic variants from ClinVar exhibited higher variant scores compared to benign/likely benign variants in each assay. Even in variants that have not yet been reported clinically, nonsense variants and several missense and intronic variants including splice site variants showed higher variant scores compared to synonymous variants. Conclusion: This study demonstrates that our novel approach can prospectively evaluate MYBPC3 variant effect at scale and provide clues to elucidate pathophysiological mechanism of HCM.

Abstract We080: Mapping the effect of Hedgehog signaling on cardiac differentiation and migration in space and time

Background: Organ morphogenesis requires precise control of organ-specific progenitor cell migration and differentiation. Hedgehog signaling is a major signaling pathway during animal development and is essential for proper cardiac development. Previous work demonstrated that Hedgehog signaling can regulate migration and the timing of differentiation of cardiac progenitors. However, how Hedgehog signaling integrates control of differentiation timing and migration in space to enable proper organ formation during development remains unclear. Hypothesis: We hypothesize that Hedgehog signaling explicitly controls cardiac morphogenesis by coordinating cellular migration and differentiation of cardiac progenitors to determining where and when they turn into cardiomyocytes. Methods: We combine mouse genetics, in-vivo lineage tracing, whole mouse embryo light-sheet imaging, and metanalysis of publicly available spatial transcriptomics datasets to address the hypothesis. Results: We identified the sites of active Hedgehog signaling by studying a reporter for Hedgehog signaling (GBS-GFP). Next, we determined the necessity of Hedgehog signaling for differentiation of cardiomyocytes at the right place and time by tracing the cardiac progenitors over developmental time in mutants in controls compared to Hedgehog signaling mutants. Conclusion and future directions: This work will establish the dual role of Hedgehog signaling in differentiation timing and migration control as a novel paradigm that may be generalizable to other organ systems dependent on Hedgehog signaling (e.g., neural cord and limb patterning).

Abstract We047: Enhanced Vascularization in hPSC-derived Cardiac Organoids by Dynamic Culture

Introduction: Human pluripotent stem cells (hPSC)-derived cardiac organoids (COs) have gained increasing interest in biotechnology due to their characteristics like diverse cardiovascular cell types, 3D complex structure, and function that recapitulates the human heart in early development. However, current methods for vascularizing COs are not systematic and robust. Hypothesis: We hypothesize that culturing hPSC-derived COs dynamically will further enhance vascularization in COs. Materials and Methods: Two conditions were created to compare vascularization in COs: 1) dynamic culturing (Dyn) and 2) static culturing (St). Firstly, hPSCs were cultured for two days in ultra-low attachment plates to develop spheroids. The spheroids were then transferred to pillar plates followed by direct cardiac differentiation the next day. Live-cell imaging was used to monitor the process of vascularization for up to 16 days and the corresponding fluorescence intensity of each cell type from its reporter expression was analyzed. The cardiac function was measured by video-based contractility analysis. Data were presented as mean ± standard error of the mean (SEM). The student’s t-test was used for the statistical analysis. Results: Cardiomyocytes (CMs) and endothelial cells (ECs) were imaged on pillars under the two conditions as shown in Fig. 1A . Successful cardiac differentiation was present, displaying hPSC-CM contraction on day 12 and onwards. Higher intensity values of hPSC-CMs under Dyn were observed in all four days compared to St as shown in Figs. 1B&C . The hPSC-ECs intensity displayed greater intensity values compared to St, demonstrating enhanced vascularization in VCOs due to the active Dyn as shown in Figs. 1D&E . The functionality was improved in Dyn as indicated with higher contraction velocity as shown in Fig. 1F. Conclusion: This study demonstrates the effects of incorporating dynamic culture for enhancing cardiogenesis and vasculogenesis in hPSC-derived COs. The enhanced hPSC-VCOs hold promise for studying early cardiovascular development, congenital heart disease modeling, and drug screening and discovery.