Cardiac differentiation of chimpanzee induced pluripotent stem cell lines with different subspecies backgrounds.

The comparative analysis between humans and non-human primates is an instrumental approach for elucidating the evolutional traits and disease propensity of humans. However, in primates, cross-species analyses of their developmental events have encountered constraints because of the ethical and technical limitations in available sample collection, sequential monitoring, and manipulations. In an endeavor to surmount these challenges, in recent years, induced pluripotent stem cells (iPSCs) have garnered escalating interest as an in vitro tool for cross-species analyses between humans and non-human primates. Meanwhile, compared to humans, there is less information on in vitro differentiation of non-human primate iPSCs, and their genetic diversity including subspecies may cause different eligibility to in vitro differentiation methods. Therefore, antecedent to embarking on a comparative analysis to humans, it is a prerequisite to develop the efficacious methodologies for in vitro differentiation regardless of the intraspecies genetic background in non-human primates. In this study, we executed the in vitro differentiation of cardiomyocytes from four chimpanzee iPSC lines with different subspecies and individual backgrounds. To induce cardiomyocytes from chimpanzee iPSCs, we evaluated our methodology for in vitro cardiac differentiation of human iPSCs. Eventually, with minor alterations, our cardiac differentiation method was applicable to all chimpanzee iPSC lines tested as assessed by the expression of cardiac marker genes and the beating ability. Hence, our in vitro differentiation method will advance iPSC-based research of chimpanzee cardiac development and also hold possible utility to cross-species analyses among primate species.

Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells.

BACKGROUND: Embryonic stem cells (ESCs) serve as a crucial ex vivo model, representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos. ESCs exhibit a unique combination of self-renewal potency, unlimited proliferation, and pluripotency. The latter is evident by the ability of the isolated cells to differentiate spontaneously into multiple cell lineages, representing the three primary embryonic germ layers. Multiple regulatory networks guide ESCs, directing their self-renewal and lineage-specific differentiation. Apoptosis, or programmed cell death, emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development. However, the molecular mechanisms underlying the dynamic interplay between differentiation and apoptosis remain poorly understood. AIM: To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells, using mouse ESC (mESC) models - mESC-B-cell lymphoma 2 (BCL-2), mESC-PIM-2, and mESC-metallothionein-1 (MET-1) - which overexpress the anti-apoptotic genes Bcl-2, Pim-2, and Met-1, respectively. METHODS: mESC-T2 (wild-type), mESC-BCL-2, mESC-PIM-2, and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation. The hanging drop method was adopted to generate embryoid bodies (EBs) and induce terminal differentiation of mESCs. The size of the generated EBs was measured in each condition compared to the wild type. At the functional level, the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control. At the molecular level, quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers: Troponin T, GATA4, and NKX2.5. Additionally, troponin T protein expression was evaluated through immunofluorescence and western blot assays. RESULTS: Our findings showed that the upregulation of Bcl-2, Pim-2, and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs, in comparison with their wild-type counterpart. Additionally, a decrease in the count of beating cardiomyocytes among differentiated cells was observed. Furthermore, the mRNA expression of three cardiac markers - troponin T, GATA4, and NKX2.5 - was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line. Moreover, the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression. CONCLUSION: Our findings revealed that the upregulation of Bcl-2, Pim-2, and Met-1 genes altered cardiac differentiation, providing insight into the intricate interplay between apoptosis and ESC fate determination.

Influence of acidic metabolic environment on differentiation of stem cell-derived cardiomyocytes.

Stem cell-based myocardial regeneration is a frontier topic in the treatment of myocardial infarction. Manipulating the metabolic microenvironment of stem cells can influence their differentiation into cardiomyocytes, which have promising clinical applications. pH is an important indicator of the metabolic environment during cardiomyocyte development. And lactate, as one of the main acidic metabolites, is a major regulator of the acidic metabolic environment during early cardiomyocyte development. Here, we summarize the progress of research into the influence of pH value and lactate on cardiomyocyte survival and differentiation, as well as related mechanisms.

The impact of high nicotine concentrations on the viability and cardiac differentiation of mesenchymal stromal cells: a barrier to regenerative therapy for smokers.

Background: Current treatment methods are not successful in restoring the lost cardiomyocytes after injury. Stem cell-based strategies have attracted much attention in this regard. Smoking, as a strong cardiovascular risk factor, not only affects the cardiac cells adversely but also deteriorates the function of stem cells. Since mesenchymal stem cells (MSCs) are one of the popular candidates in cardiovascular disease (CVD) clinical trials, we investigated the impact of nicotine on the regenerative properties (viability and cardiac differentiation) of these cells. Methods: MSCs were isolated from rat bone marrow and characterized based on morphology, differentiation capability, and the expression of specific mesenchymal markers. The MTT assay was used to assess the viability of MSCs after being exposed to different concentrations of nicotine. Based on MTT findings and according to the concentration of nicotine in smokers' blood, the growth curve and population doubling time were investigated for eight consecutive days. Cells were treated with 5-azacytidine (an inducer of cardiac differentiation), and then the expressions of cardiac-specific markers were calculated by qPCR. Results: MSCs were spindle-shaped, capable of differentiating into adipocyte and osteocyte, and expressed CD73 and CD90. The viability of MSCs was reduced upon exposure to nicotine in a concentration- and time-dependent manner. The growth curve showed that nicotine reduced the proliferation of MSCs, and treated cells needed more time to double. In addition, the expressions of GATA4 and troponin were downregulated in nicotine-treated cells on day 3. However, these two cardiac markers were overexpressed on day 7. Conclusion: Nicotine decreased normal growth and reduced the expression of cardiac markers in MSCs. This aspect is of eminent importance to smokers with cardiovascular disease who are candidates for stem cell therapy.

Transgene-Free Cynomolgus Monkey iPSCs Generated under Chemically Defined Conditions.

Non-human primates (NHPs) are pivotal animal models for translating novel cell replacement therapies into clinical applications, including validating the safety and efficacy of induced pluripotent stem cell (iPSC)-derived products. Preclinical development and the testing of cell-based therapies ideally comprise xenogeneic (human stem cells into NHPs) and allogenic (NHP stem cells into NHPs) transplantation studies. For the allogeneic approach, it is necessary to generate NHP-iPSCs with generally equivalent quality to the human counterparts that will be used later on in patients. Here, we report the generation and characterization of transgene- and feeder-free cynomolgus monkey (Macaca fascicularis) iPSCs (Cyno-iPSCs). These novel cell lines have been generated according to a previously developed protocol for the generation of rhesus macaque, baboon, and human iPSC lines. Beyond their generation, we demonstrate the potential of the novel Cyno-iPSCs to differentiate into two clinically relevant cell types, i.e., cardiomyocytes and neurons. Overall, we provide a resource of novel iPSCs from the most frequently used NHP species in the regulatory testing of biologics and classical pharmaceutics to expand our panel of iPSC lines from NHP species with high relevance in preclinical testing and translational research.

Differentiation of Pluripotent Stem Cells for Disease Modeling: Learning from Heart Development.

Heart disease is a pressing public health problem and the leading cause of death worldwide. The heart is the first organ to gain function during embryogenesis in mammals. Heart development involves cell determination, expansion, migration, and crosstalk, which are orchestrated by numerous signaling pathways, such as the Wnt, TGF-ß, IGF, and Retinoic acid signaling pathways. Human-induced pluripotent stem cell-based platforms are emerging as promising approaches for modeling heart disease in vitro. Understanding the signaling pathways that are essential for cardiac development has shed light on the molecular mechanisms of congenital heart defects and postnatal heart diseases, significantly advancing stem cell-based platforms to model heart diseases. This review summarizes signaling pathways that are crucial for heart development and discusses how these findings improve the strategies for modeling human heart disease in vitro.

Identification of miR-20b-5p as an inhibitory regulator in cardiac differentiation via TET2 and DNA hydroxymethylation.

BACKGROUND: Congenital heart disease (CHD) is a prevalent congenital cardiac malformation, which lacks effective early biological diagnosis and intervention. MicroRNAs, as epigenetic regulators of cardiac development, provide potential biomarkers for the diagnosis and treatment of CHD. However, the mechanisms underlying miRNAs-mediated regulation of cardiac development and CHD malformation remain to be further elucidated. This study aimed to explore the function of microRNA-20b-5p (miR-20b-5p) in cardiac development and CHD pathogenesis. METHODS AND RESULTS: miRNA expression profiling identified that miR-20b-5p was significantly downregulated during a 12-day cardiac differentiation of human embryonic stem cells (hESCs), whereas it was markedly upregulated in plasma samples of atrial septal defect (ASD) patients. Our results further revealed that miR-20b-5p suppressed hESCs-derived cardiac differentiation by targeting tet methylcytosine dioxygenase 2 (TET2) and 5-hydroxymethylcytosine, leading to a reduction in key cardiac transcription factors including GATA4, NKX2.5, TBX5, MYH6 and cTnT. Additionally, knockdown of TET2 significantly inhibited cardiac differentiation, which could be partially restored by miR-20b-5p inhibition. CONCLUSIONS: Collectively, this study provides compelling evidence that miR-20b-5p functions as an inhibitory regulator in hESCs-derived cardiac differentiation by targeting TET2, highlighting its potential as a biomarker for ASD.

Reprogramming of DNA methylation patterns mediates perfluorooctane sulfonate-induced fetal cardiac dysplasia.

Prenatal exposure to perfluorooctane sulfonate (PFOS) is associated with adverse health effects, including congenital heart disease, yet the underlying mechanisms remain elusive. Herein, we aimed to evaluate the embryotoxicity of PFOS using C57BL/6 J mice to characterize fetal heart defects after PFOS exposure, with the induction of human embryonic stem cells (hESC) into cardiomyocytes (CMs) as a model of early-stage heart development. We also performed DNA methylation analysis to clarify potential underlying mechanisms and identify targets of PFOS. Our results revealed that PFOS caused septal defects and excessive ventricular trabeculation cardiomyopathy at 5 mg/kg/day in embryonic mice and inhibited the proliferation and pluripotency of ESCs at concentrations >20 µM. Moreover, it decreased the beating rate and the population of CMs during cardiac differentiation. Decreases were observed in the abundances of NPPA+ trabecular and HEY2+ compact CMs. Additionally, DNA methyl transferases and ten-eleven translocation (TET) dioxygenases were regulated dynamically by PFOS, with TETs inhibitor treatment inducing significant decreases similar as PFOS. 850 K DNA methylation analysis combined with expression analysis revealed several potential targets of PFOS, including SORBS2, FHOD1, SLIT2, SLIT3, ADCY9, and HDAC9. In conclusion, PFOS may reprogram DNA methylation, especially demethylation, to induce cardiac toxicity, causing ventricular defects in vivo and abnormal cardiac differentiation in vitro.

Single-cell RNA sequencing reveals the role of mitochondrial dysfunction in the cardiogenic toxicity of perfluorooctane sulfonate in human embryonic stem cells.

Perfluorooctane sulfonate (PFOS), an endocrine-disrupting chemical pollutant, affects embryonic heart development; however, the mechanisms underlying its toxicity have not been fully elucidated. Here, Single-cell RNA sequencing (scRNA-seq) was used to investigate the overall effects of PFOS on myocardial differentiation from human embryonic stem cells (hESCs). Additionally, apoptosis, mitochondrial membrane potential, and ATP assays were performed. Downregulated cardiogenesis-related genes and inhibited cardiac differentiation were observed after PFOS exposure in vitro. The percentages of cardiomyocyte and cardiac progenitor cell clusters decreased significantly following exposure to PFOS, while the proportion of primitive endoderm cell was increased in PFOS group. Moreover, PFOS inhibited myocardial differentiation and blocked cellular development at the early- and middle-stage. A Gene Ontology analysis and pseudo-time trajectory illustrated that PFOS disturbed multiple processes related to cardiogenesis and oxidative phosphorylation in the mitochondria. Furthermore, PFOS decreased mitochondrial membrane potential and induced apoptosis. These results offer meaningful insights into the cardiogenic toxicity of PFOS exposure during heart formation as well as the adverse effects of PFOS on mitochondria.

JAK2 as a surface marker for enrichment of human pluripotent stem cells-derived ventricular cardiomyocytes.

BACKGROUND: Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) hold great promise for cardiac disease modelling, drug discovery and regenerative medicine. Despite the advancement in various differentiation protocols, the heterogeneity of the generated population composed of diverse cardiac subtypes poses a significant challenge to their practical applications. Mixed populations of cardiac subtypes can compromise disease modelling and drug discovery, while transplanting them may lead to undesired arrhythmias as they may not integrate and synchronize with the host tissue's contractility. It is therefore crucial to identify cell surface markers that could enable high purity of ventricular CMs for subsequent applications. METHODS: By exploiting the fact that immature CMs expressing myosin light chain 2A (MLC2A) will gradually express myosin light chain 2 V (MLC2V) protein as they mature towards ventricular fate, we isolated signal regulatory protein alpha (SIRPA)-positive CMs expressing intracellular MLC2A or MLC2V using MARIS (method for analysing RNA following intracellular sorting). Subsequently, RNA sequencing analysis was performed to examine the gene expression profile of MLC2A + and MLC2V + sorted CMs. We identified genes that were significantly up-regulated in MLC2V + samples to be potential surface marker candidates for ventricular specification. To validate these surface markers, we performed immunostaining and western blot analysis to measure MLC2A and MLC2V protein expressions in SIRPA + CMs that were either positive or negative for the putative surface markers, JAK2 (Janus kinase 2) or CD200. We then characterized the electrophysiological properties of surface marker-sorted CMs, using fluo-4 AM, a green-fluorescent calcium indicator, to measure the cellular calcium transient at the single cell level. For functional validation, we investigated the response of the surface marker-sorted CMs to vernakalant, an atrial-selective anti-arrhythmic agent. RESULTS: In this study, while JAK2 and CD200 were identified as potential surface markers for the purification of ventricular-like CMs, the SIRPA+/JAK2+ population showed a higher percentage of MLC2V-expressing cells (~ 90%) compared to SIRPA+/CD200+ population (~ 75%). SIRPA+/JAK2+ sorted CMs exhibited ventricular-like electrophysiological properties, including slower beating rate, slower calcium depolarization and longer calcium repolarization duration. Importantly, vernakalant had limited to no significant effect on the calcium repolarization duration of SIRPA+/JAK2+ population, indicating their enrichment for ventricular-like CMs. CONCLUSION: Our study lays the groundwork for the identification of cardiac subtype surface markers that allow purification of cardiomyocyte sub-populations. Our findings suggest that JAK2 can be employed as a cell surface marker for enrichment of hPSC-derived ventricular-like CMs.

The role of c-Jun for beating cardiomycyte formation in prepared embryonic body.

BACKGROUND: Morbidity and mortality associated with cardiovascular diseases, such as myocardial infarction, stem from the inability of terminally differentiated cardiomyocytes to regenerate, and thus repair the damaged myocardial tissue structure. The molecular biological mechanisms behind the lack of regenerative capacity for those cardiomyocytes remains to be fully elucidated. Recent studies have shown that c-Jun serves as a cell cycle regulator for somatic cell fates, playing a key role in multiple molecular pathways, including the inhibition of cellular reprogramming, promoting angiogenesis, and aggravation of cardiac hypertrophy, but its role in cardiac development is largely unknown. This study aims to delineate the role of c-Jun in promoting early-stage cardiac differentiation. METHODS: The c-Jun gene in mouse embryonic stem cells (mESCs) was knocked out with CRISPR-Cas9, and the hanging drop method used to prepare the resulting embryoid bodies. Cardiac differentiation was evaluated up to 9 days after c-Jun knockout (ko) via immunofluorescence, flow cytometric, and qPCR analyses. RESULTS: Compared to the wild-type control group, obvious beating was observed among the c-Jun-ko mESCs after 6 days, which was also associated with significant increases in myocardial marker expression. Additionally, markers associated with mesoderm and endoderm cell layer development, essential for further differentiation of ESCs into cardiomyocytes, were also up-regulated in the c-Jun-ko cell group. CONCLUSIONS: Knocking out c-Jun directs ESCs toward a meso-endodermal cell lineage fate, in turn leading to generation of beating myocardial cells. Thus, c-Jun plays an important role in regulating early cardiac cell development.

Lethal Phenotype-Based Database Screening Identifies Ceramide as a Negative Regulator of Primitive Streak Formation.

In early embryogenesis, the primitive streak (PrS) generates the mesendoderm and is essential for organogenesis. However, because the PrS is a minute and transient tissue, elucidating the mechanism of its formation has been challenging. We performed comprehensive screening of 2 knockout mouse databases based on the fact that failure of PrS formation is lethal. We identified 812 genes involved in various cellular functions and responses that might be linked to PrS formation, with the category of greatest abundance being "Metabolism." In this study, we focused on genes of sphingolipid metabolism and investigated their roles in PrS formation using an in vitro mouse ES cell differentiation system. We show here that elevated intracellular ceramide negatively regulates gene expression essential for PrS formation and instead induces neurogenesis. In addition, sphingosine-1-phosphate (a ceramide derivative) positively regulates neural maturation. Our results indicate that ceramide regulates both PrS formation and the induction of neural differentiation.

Recent advances in regulating the proliferation or maturation of human-induced pluripotent stem cell-derived cardiomyocytes.

In the last decade, human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM)-based cell therapy has drawn broad attention as a potential therapy for treating injured hearts. However, mass production of hiPSC-CMs remains challenging, limiting their translational potential in regenerative medicine. Therefore, multiple strategies including cell cycle regulators, small molecules, co-culture systems, and epigenetic modifiers have been used to improve the proliferation of hiPSC-CMs. On the other hand, the immaturity of these proliferative hiPSC-CMs could lead to lethal arrhythmias due to their limited ability to functionally couple with resident cardiomyocytes. To achieve functional maturity, numerous methods such as prolonged culture, biochemical or biophysical stimulation, in vivo transplantation, and 3D culture approaches have been employed. In this review, we summarize recent approaches used to promote hiPSC-CM proliferation, and thoroughly review recent advances in promoting hiPSC-CM maturation, which will serve as the foundation for large-scale production of mature hiPSC-CMs for future clinical applications.

The ad hoc chemical design of random PBS-based copolymers influences the activation of cardiac differentiation while altering the HYPPO pathway target genes in hiPSCs.

Cardiac tissue engineering is a cutting-edge technology aiming to replace irreversibly damaged cardiac tissue and restore contractile functionality. However, cardiac tissue engineering porous and perfusable scaffolds to enable oxygen supply in vitro and eventually promote angiogenesis in vivo are still desirable. Two fully-aliphatic random copolymers of poly(butylene succinate) (PBS), poly(butylene succinate/Pripol), P(BSBPripol), and poly(butylene/neopentyl glycol succinate), P(BSNS), containing two different subunits, neopentyl glycol and Pripol 1009, were successfully synthesized and then electrospun in tridimentional fibrous mats. The copolymers show different thermal and mechanical behaviours as result of their chemical structure. In particular, copolymerization led to a reduction in crystallinity and consequently PBS stiffness, reaching values of elastic modulus very close to those of soft tissues. Then, to check the biological suitability, human induced Pluripotent Stem Cells (hiPSCs) were directly seeded on both PBS-based copolymeric scaffolds. The results confirmed the ability of both the scaffolds to sustain cell viability and to maintain their stemness during cell expansion. Furthermore, gene expression and immunofluorescence analysis showed that P(BSBPripol) scaffold promoted an upregulation of the early cardiac progenitor and later-stage markers with a simultaneously upregulation of HYPPO pathway gene expression, crucial for mechanosensing of cardiac progenitor cells. These results suggest that the correct ad-hoc chemical design and, in turn, the mechanical properties of the matrix, such as substrate stiffness, together with surface porosity, play a critical role in regulating the behaviour of cardiac progenitors, which ultimately offers valuable insights into the development of novel bio-inspired scaffolds for cardiac tissue regeneration.

Cripto Is Targeted by miR-1a-3p in a Mouse Model of Heart Development.

During cardiac differentiation, numerous factors contribute to the development of the heart. Understanding the molecular mechanisms underlying cardiac development will help combat cardiovascular disorders, among the leading causes of morbidity and mortality worldwide. Among the main mechanisms, we indeed find Cripto. Cripto is found in both the syncytiotrophoblast of ampullary pregnancies and the inner cell mass along the primitive streak as the second epithelial-mesenchymal transformation event occurs to form the mesoderm and the developing myocardium. At the same time, it is now known that cardiac signaling pathways are intimately intertwined with the expression of myomiRNAs, including miR-1. This miR-1 is one of the muscle-specific miRs; aberrant expression of miR-1 plays an essential role in cardiac diseases. Given this scenario, our study aimed to evaluate the inverse correlation between Cripto and miR-1 during heart development. We used in vitro models of the heart, represented by embryoid bodies (EBs) and embryonic carcinoma cell lines derived from an embryo-derived teratocarcinoma in mice (P19 cells), respectively. First, through a luciferase assay, we demonstrated that Cripto is a target of miR-1. Following this result, we observed that as the days of differentiation increased, the Cripto gene expression decreased, while the level of miR-1 increased; furthermore, after silencing miR-1 in P19 cells, there was an increase in Cripto expression. Moreover, inducing damage with a cobra cardiotoxin (CTX) in post-differentiation cells, we noted a decreased miR-1 expression and increased Cripto. Finally, in mouse cardiac biopsies, we observed by monitoring gene expression the distribution of Cripto and miR-1 in the right and left ventricles. These results allowed us to detect an inverse correlation between miR-1 and Cripto that could represent a new pharmacological target for identifying new therapies.

Evaluation of the effect of boron derivatives on cardiac differentiation of mouse pluripotent stem cells.

BACKGROUND: The heart is one of the first organs to form during embryonic development and has a very important place. So much that the formation of a functional heart is completed on the 55th day of human development and the 15th day of mouse development. Myocardial, endocardial and epicardial cells, which are derived from the mesoderm layer, are the cells that form the basis of the heart. Cardiac development, like other embryonic developments, is tightly controlled and regulated by various signaling pathways. The WNT signaling pathway is the most studied of these signaling pathways and the one with the clearest relationship with heart development. It is known that boron compounds and the Wnt/ß-catenin pathway are highly correlated. Therefore, this study aimed to investigate the role of boron compounds in heart development as well as its effect on pluripotency of mouse embryonic stem cells for the first time in the literature. METHODS: Toxicity of boron compounds was evaluated by using MTS analysis and obtained results were supported by morphological pictures, Trypan Blue staining and Annexin V staining. Additionally, the possible boron-related change in pluripotency of embryonic stem cells were analyzed with alkaline phosphatase activity and immunocytochemical staining of Oct4 protein as well as gene expression levels of pluripotency related OCT4, SOX2 and KLF4 genes. The alterations in the embryonic body formation capacity of mouse embryonic stem cells due to the application boron derivatives were also evaluated. Three linage differentiation was conducted to clarify the real impact of boron compounds on embryonic development. Lastly, cardiac differentiation of mESCs was investigated by using morphological pictures, cytosolic calcium measurement, gene expression and immunocytochemical analysis of cardiac differentiation related genes and in the presence of boron compounds. RESULTS: Obtained results show that boron treatment maintains the pluripotency of embryonic stem cells at non-toxic concentrations. Additionally, endodermal, and mesodermal fate was found to be triggered after boron treatment. Also, initiation of cardiomyocyte differentiation by boron derivative treatments caused an increased gene expression levels of cardiac differentiation related TNNT2, Nkx2.5 and ISL-1 gene expression levels. CONCLUSION: This study indicates that boron application, which is responsible for maintaining pluripotency of mESCs, can be used for increased cardiomyocyte differentiation of mESCs.

Stage-specific requirement for m6A RNA methylation during cardiac differentiation of pluripotent stem cells.

Precise spatiotemporal control of gene expression patterns is critical for normal development. Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with the ability of unlimited self-renewal and differentiation into any cell type, provide a unique tool for understanding the underlying mechanism of development and disease in a dish. N6-methyl-adenosine (m6A) modification is the most extensive internal mRNA modification, which regulates almost all aspects of mRNA metabolism and thus extensively participates in gene expression regulation. However, the role of m6A during cardiogenesis still needs to be fully elucidated. Here, we found that core components of m6A methyltransferase decreased during cardiomyocyte differentiation. Impeding m6A deposition, by either deleting the m6A methyltransferase Mettl3 or overexpressing m6A demethylase alkB homolog 5 (Alkbh5), at early stages of cardiac differentiation of mouse pluripotent stem cells, led to inhibition of cardiac gene activation and retardation of the outgrowth of embryoid bodies, whereas interfering m6A modification at later stages of differentiation had minimal effects. Consistently, stage-specific inhibition of METTL3 with METTL3 inhibitor STM2457 during human ESCs (hESCs) cardiac differentiation demonstrated a similarly pivotal role of METTL3 for the induction of mesodermal cells while dispensable function for later stages. In summary, our study reveals a stage-specific requirement of m6A on the cardiac differentiation of pluripotent stem cells and demonstrates that precise tuning of m6A level is critical for cardiac differentiation.

Recapitulating porcine cardiac development in vitro: from expanded potential stem cell to embryo culture models.

Domestic pigs (Sus scrofa) share many genetic, anatomical, and physiological traits with humans and therefore constitute an excellent preclinical animal model. Fundamental understanding of the cellular and molecular processes governing early porcine cardiogenesis is critical for developing advanced porcine models used for the study of heart diseases and new regenerative therapies. Here, we provide a detailed characterization of porcine cardiogenesis based on fetal porcine hearts at various developmental stages and cardiac cells derived from porcine expanded pluripotent stem cells (pEPSCs), i.e., stem cells having the potential to give rise to both embryonic and extraembryonic tissue. We notably demonstrate for the first time that pEPSCs can differentiate into cardiovascular progenitor cells (CPCs), functional cardiomyocytes (CMs), epicardial cells and epicardial-derived cells (EPDCs) in vitro. Furthermore, we present an enhanced system for whole-embryo culture which allows continuous ex utero development of porcine post-implantation embryos from the cardiac crescent stage (ED14) up to the cardiac looping (ED17) stage. These new techniques provide a versatile platform for studying porcine cardiac development and disease modeling.

Human pluripotent stem cell-based models of heart development and disease.

The heart is a complex organ composed of distinct cell types, such as cardiomyocytes, cardiac fibroblasts, endothelial cells, smooth muscle cells, neuronal cells and immune cells. All these cell types contribute to the structural, electrical and mechanical properties of the heart. Genetic manipulation and lineage tracing studies in mice have been instrumental in gaining critical insights into the networks regulating cardiac cell lineage specification, cell fate and plasticity. Such knowledge has been of fundamental importance for the development of efficient protocols for the directed differentiation of pluripotent stem cells (PSCs) in highly specialized cardiac cell types. In this review, we summarize the evolution and current advances in protocols for cardiac subtype specification, maturation, and assembly in cardiac microtissues and organoids.

Comparative analysis of the cardiomyocyte differentiation potential of induced pluripotent stem cells reprogrammed from human atrial or ventricular fibroblasts.

Background: We had shown that cardiomyocytes (CMs) were more efficiently differentiated from human induced pluripotent stem cells (hiPSCs) when the hiPSCs were reprogrammed from cardiac fibroblasts rather than dermal fibroblasts or blood mononuclear cells. Here, we continued to investigate the relationship between somatic-cell lineage and hiPSC-CM production by comparing the yield and functional properties of CMs differentiated from iPSCs reprogrammed from human atrial or ventricular cardiac fibroblasts (AiPSC or ViPSC, respectively). Methods: Atrial and ventricular heart tissues were obtained from the same patient, reprogrammed into AiPSCs or ViPSCs, and then differentiated into CMs (AiPSC-CMs or ViPSC-CMs, respectively) via established protocols. Results: The time-course of expression for pluripotency genes (OCT4, NANOG, and SOX2), the early mesodermal marker Brachyury, the cardiac mesodermal markers MESP1 and Gata4, and the cardiovascular progenitor-cell transcription factor NKX2.5 were broadly similar in AiPSC-CMs and ViPSC-CMs during the differentiation protocol. Flow-cytometry analyses of cardiac troponin T expression also indicated that purity of the two differentiated hiPSC-CM populations (AiPSC-CMs: 88.23% ± 4.69%, ViPSC-CMs: 90.25% ± 4.99%) was equivalent. While the field-potential durations were significantly longer in ViPSC-CMs than in AiPSC-CMs, measurements of action potential duration, beat period, spike amplitude, conduction velocity, and peak calcium-transient amplitude did not differ significantly between the two hiPSC-CM populations. Yet, our cardiac-origin iPSC-CM showed higher ADP and conduction velocity than previously reported iPSC-CM derived from non-cardiac tissues. Transcriptomic data comparing iPSC and iPSC-CMs showed similar gene expression profiles between AiPSC-CMs and ViPSC-CMs with significant differences when compared to iPSC-CM derived from other tissues. This analysis also pointed to several genes involved in electrophysiology processes responsible for the physiological differences observed between cardiac and non-cardiac-derived cardiomyocytes. Conclusion: AiPSC and ViPSC were differentiated into CMs with equal efficiency. Detected differences in electrophysiological properties, calcium handling activity, and transcription profiles between cardiac and non-cardiac derived cardiomyocytes demonstrated that 1) tissue of origin matters to generate a better-featured iPSC-CMs, 2) the sublocation within the cardiac tissue has marginal effects on the differentiation process.