Molecular changes in endometrium origin stromal cells during initiation of cardiomyogenic differentiation induced with Decitabine, Angiotensin II and TGF- ß1.

Stem cells' differentiation toward cardiac lineage is a complex process dependent on various alterations in molecular basis and regulation pathways. The aim of the study is to show that endometrium-derived stromal cells - menstrual, endometrial and endometriotic, could be an attractive source for examination of the mechanisms underlying cardiomyogenesis. After treatment with Decitabine, Angiotensin II and TGF-ß1, cells demonstrated morphological dedifferentiation into early cardiomyocyte-like cells and expressed CD36, CD106, CD172a typically used to sort for human pluripotent stem cell-derived cardiomyocytes. RT-qPCR revealed changed cells' genetic profiles, as majority of cardiac lineage differentiation related genes and cardiac ion channels (calcium, sodium, potassium) coding genes were upregulated after 6 and 13 days of exposure. Additionally, analysis of expression of various signaling proteins (FOXO1, PDGFB, TGFBR1, mTOR, VEGFA, WNT4, Notch1) coding genes showed differences between cell cultures as they seem to employ distinct signaling pathways through differentiation initiation. Early stages of differentiation had biggest impact on cardiomyogenesis related proteins (Nkx-2.5, EZH2, FOXO3a, H3K9Ac) levels, as we noticed after conducting Western blot and as expected, early cardiac transcription factor Nkx-2.5 was highly expressed and localized in nucleus of differentiating cells. These findings led us to assess endometrium origin stromal cells' potential to differentiate towards cardiomyogenic lineage and better understand the regulation of complex differentiation processes in ex vivo model systems.

Transfer of cardiomyocyte-derived extracellular vesicles to neighboring cardiac cells requires tunneling nanotubes during heart development.

Rationale: Extracellular vesicles (EVs) are thought to mediate intercellular communication during development and disease. Yet, biological insight to intercellular EV transfer remains elusive, also in the heart, and is technically challenging to demonstrate. Here, we aimed to investigate biological transfer of cardiomyocyte-derived EVs in the neonatal heart. Methods: We exploited CD9 as a marker of EVs, and generated two lines of cardiomyocyte specific EV reporter mice: Tnnt2-Cre; double-floxed inverted CD9/EGFP and αMHC-MerCreMer; double-floxed inverted CD9/EGFP. The two mouse lines were utilized to determine whether developing cardiomyocytes transfer EVs to other cardiac cells (non-myocytes and cardiomyocytes) in vitro and in vivo and investigate the intercellular transport pathway of cardiomyocyte-derived EVs. Results: Genetic tagging of cardiomyocytes was confirmed in both reporter mouse lines and proof of concept in the postnatal heart showed that, a fraction of EGFP+/MYH1- non-myocytes exist firmly demonstrating in vivo cardiomyocyte-derived EV transfer. However, two sets of direct and indirect EGFP +/- cardiac cell co-cultures showed that cardiomyocyte-derived EGFP+ EV transfer requires cell-cell contact and that uptake of EGFP+ EVs from the medium is limited. The same was observed when co-cultiring with mouse macrophages. Further mechanistic insight showed that cardiomyocyte EV transfer occurs through type I tunneling nanotubes. Conclusion: While the current notion assumes that EVs are transferred through secretion to the surroundings, our data show that cardiomyocyte-derived EV transfer in the developing heart occurs through nanotubes between neighboring cells. Whether these data are fundamental and relate to adult hearts and other organs remains to be determined, but they imply that the normal developmental process of EV transfer goes through cell-cell contact rather than through the extracellular compartment.

Generation and Characterization of hiPSC-Derived Vascularized-, Perfusable Cardiac Microtissues-on-Chip.

In the heart in vivo, vasculature forms a semi-permeable endothelial barrier for selective nutrient and (immune) cell delivery to the myocardium and removal of waste products. Crosstalk between the vasculature and the heart cells regulates homeostasis in health and disease. To model heart development and disease in vitro it is important that essential features of this crosstalk are captured. Cardiac organoid and microtissue models often integrate endothelial cells (ECs) to form microvascular networks inside the 3D structure. However, in static culture without perfusion, these networks may fail to show essential functionality. Here, we describe a protocol to generate an in vitro model of human induced pluripotent stem cell (hiPSC)-derived vascularized cardiac microtissues on a microfluidic organ-on-chip platform (VMToC) in which the blood vessels are perfusable. First, prevascularized cardiac microtissues (MT) are formed by combining hiPSC-derived cardiomyocytes, ECs, and cardiac fibroblasts in a pre-defined ratio. Next, these prevascularized MTs are integrated in the chips in a fibrin hydrogel containing additional vascular cells, which self-organize into tubular structures. The MTs become vascularized through anastomosis between the pre-existing microvasculature in the MT and the external vascular network. The VMToCs are then ready for downstream structural and functional assays and basic characterization. Using this protocol, cardiac MTs can be efficiently and robustly vascularized and perfused within 7 days. In vitro vascularized organoid and MT models have the potential to transition current 3D cardiac models to more physiologically relevant organ models that allow the role of the endothelial barrier in drug and inflammatory response to be investigated. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Generation of VMToC Support Protocol 1: Functional Characterization of VMToC Support Protocol 2: Structural Characterization of VMToC.

Improving iPSC Differentiation Using a Nanodot Platform.

Differentiation of induced pluripotent stem cells (iPSCs) is an extremely complex process that has proven difficult to study. In this research, we utilized nanotopography to elucidate details regarding iPSC differentiation by developing a nanodot platform consisting of nanodot arrays of increasing diameter. Subjecting iPSCs cultured on the nanodot platform to a cardiomyocyte (CM) differentiation protocol revealed several significant gene expression profiles that were associated with poor differentiation. The observed expression trends were used to select existing small-molecule drugs capable of modulating differentiation efficiency. BRD K98 was repurposed to inhibit CM differentiation, while iPSCs treated with NSC-663284, carmofur, and KPT-330 all exhibited significant increases in not only CM marker expression but also spontaneous beating, suggesting improved CM differentiation. In addition, quantitative polymerase chain reaction was performed to determine the gene regulation responsible for modulating differentiation efficiency. Multiple genes involved in extracellular matrix remodeling were correlated with a CM differentiation efficiency, while genes involved in the cell cycle exhibited contrasting expression trends that warrant further studies. The results suggest that expression profiles determined via short time-series expression miner analysis of nanodot-cultured iPSC differentiation can not only reveal drugs capable of enhancing differentiation efficiency but also highlight crucial sets of genes related to processes such as extracellular matrix remodeling and the cell cycle that can be targeted for further investigation. Our findings confirm that the nanodot platform can be used to reveal complex mechanisms behind iPSC differentiation and could be an indispensable tool for optimizing iPSC technology for clinical applications.

Pharmacological activation of mesenchymal stem cells increases gene expression pattern of cell adhesion molecules and fusion with neonatal cardiomyocytes.

Cellular therapy is considered a better option for the treatment of degenerative disorders. Different cell types are being used for tissue regeneration. Despite extensive research in this field, several issues remain to be addressed concerning cell transplantation. One of these issues is the survival and homing of administered cells in the injured tissue, which depends on the ability of these cells to adhere. To enhance cell adherence and survival, Rap1 GTPase was activated in mesenchymal stem cells (MSCs) as well as in cardiomyocytes (CMs) by using 8-pCPT-2'-O-Me-cAMP, and the effect on gene expression dynamics was determined through quantitative reverse transcriptase-polymerase chain reaction analysis. Pharmacological activation of MSCs and CMs resulted in the upregulation of connexin-43 and cell adhesion genes, which increased the cell adhesion ability of MSCs and CMs, and increased the fusion of MSCs with neonatal CMs. Treating stem cells with a pharmacological agent that activates Rap1a before transplantation can enhance their fusion with CMs and increase cellular regeneration.

Monkey see, monkey do: Tracking iPS-cardiomyocyte survival and maturation in autografts.

Induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) therapy has emerged as a highly promising field of heart repair. Lin et al.1 presented compelling evidence on the long-term engraftment and maturation of autologous iPSC-CMs in two rhesus macaques, demonstrating unprecedented cardiac autografting data in large animal models without the need of immunosuppressants.

Protein-free media for cardiac differentiation of hPSCs in 2000 mL suspension culture.

BACKGROUND: Commonly used media for the differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) contain high concentrations of proteins, in particular albumin, which is prone to quality variations and presents a substantial cost factor, hampering the clinical translation of in vitro-generated cardiomyocytes for heart repair. To overcome these limitations, we have developed chemically defined, entirely protein-free media based on RPMI, supplemented with L-ascorbic acid 2-phosphate (AA-2P) and either the non-ionic surfactant Pluronic F-68 or a specific polyvinyl alcohol (PVA). METHODS AND RESULTS: Both media compositions enable the efficient, directed differentiation of embryonic and induced hPSCs, matching the cell yields and cardiomyocyte purity ranging from 85 to 99% achieved with the widely used protein-based CDM3 medium. The protein-free differentiation approach was readily up-scaled to a 2000 mL process scale in a fully controlled stirred tank bioreactor in suspension culture, producing > 1.3 × 109 cardiomyocytes in a single process run. Transcriptome analysis, flow cytometry, electrophysiology, and contractile force measurements revealed that the mass-produced cardiomyocytes differentiated in protein-free medium exhibit the expected ventricular-like properties equivalent to the well-established characteristics of CDM3-control cells. CONCLUSIONS: This study promotes the robustness and upscaling of the cardiomyogenic differentiation process, substantially reduces media costs, and provides an important step toward the clinical translation of hPSC-CMs for heart regeneration.

Efficient and reproducible generation of human iPSC-derived cardiomyocytes and cardiac organoids in stirred suspension systems.

Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and regeneration. Challenges with quality, inter-batch consistency, cryopreservation and scale remain, reducing experimental reproducibility and clinical translation. Here, we report a robust stirred suspension cardiac differentiation protocol, and we perform extensive morphological and functional characterization of the resulting bioreactor-differentiated iPSC-CMs (bCMs). Across multiple different iPSC lines, the protocol produces 1.2E6/mL bCMs with ~94% purity. bCMs have high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs, bCMs are more reproducible across batches and have more mature functional properties. The protocol also works with magnetically stirred spinner flasks, which are more economical and scalable than bioreactors. Minor protocol modifications generate cardiac organoids fully in suspension culture. These reproducible, scalable, and resource-efficient approaches to generate iPSC-CMs and organoids will expand their applications, and our benchmark data will enable comparison to cells produced by other cardiac differentiation protocols.

Generation of cardiomyocytes from stem cells cultured on nanofibrous scaffold: Experimental approach for attenuation of myocardial infarction.

The current study was constructed to fabricate polyamide based nanofibrous scaffolds (NS) and to define the most promising one for the generation of cardiomyocytes from adipose tissue derived mesenchymal stem cells (ADMSCs). This purpose was extended to assess the potentiality of the generated cardiomyocytes in relieving myocardial infarction (MI) in rats. Production and characterization of NSs were carried out. ADMSCs were cultured on NS and induced to differentiate into cardiomyocytes by specific growth factors. Molecular analysis for myocyte-specific enhancer factor 2 C (MEF2C) and alpha sarcomeric actin (α-SCA) expression was done to confirm the differentiation of ADMSCs into cardiomyocytes for further transplantation into MI induced rats. Implantation of cells in MI afflicted rats boosted heart rate, ST height and PR interval and lessened P duration, RR, QTc and QRS intervals. Also, this type of medication minified serum lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) enzymes activity as well as serum and cardiac troponin T (Tn-T) levels and upraised serum and cardiac α-SCA and cardiac connexin 43 (CX 43) levels. Microscopic feature of cardiac tissue sections of rats in the treated groups revealed great renovation in the cardiac microarchitecture. Conclusively, this attempt gains insight into a realistic strategy for recovery of MI through systemic employment of in vitro generated cardiomyocytes.

Advancing Cardiovascular Drug Screening Using Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as a promising tool for studying cardiac physiology and drug responses. However, their use is largely limited by an immature phenotype and lack of high-throughput analytical methodology. In this study, we developed a high-throughput testing platform utilizing hPSC-CMs to assess the cardiotoxicity and effectiveness of drugs. Following an optimized differentiation and maturation protocol, hPSC-CMs exhibited mature CM morphology, phenotype, and functionality, making them suitable for drug testing applications. We monitored intracellular calcium dynamics using calcium imaging techniques to measure spontaneous calcium oscillations in hPSC-CMs in the presence or absence of test compounds. For the cardiotoxicity test, hPSC-CMs were treated with various compounds, and calcium flux was measured to evaluate their effects on calcium dynamics. We found that cardiotoxic drugs withdrawn due to adverse drug reactions, including encainide, mibefradil, and cetirizine, exhibited toxicity in hPSC-CMs but not in HEK293-hERG cells. Additionally, in the effectiveness test, hPSC-CMs were exposed to ATX-II, a sodium current inducer for mimicking long QT syndrome type 3, followed by exposure to test compounds. The observed changes in calcium dynamics following drug exposure demonstrated the utility of hPSC-CMs as a versatile model system for assessing both cardiotoxicity and drug efficacy. Overall, our findings highlight the potential of hPSC-CMs in advancing drug discovery and development, which offer a physiologically relevant platform for the preclinical screening of novel therapeutics.

[Sanshentongmai Mixture Improves Oxidative Damage in Rat Cardiomyocytes H9C2 via Upregulation of microRNA-146a]. / 三参通脉合剂通过上调microRNA-146aæ”¹å–„å¤§é¼ å¿ƒè‚Œç»†èƒžH9C2的氧化损伤.

Objective: To investigate the effect of Sanshentongmai (SSTM) mixture on the regulation of oxidative damage to rat cardiomyocytes (H9C2) through microRNA-146a and its mechanism. Methods: H9C2 were cultured in vitro, H2O2 was used as an oxidant to create an oxidative damage model in H9C2 cells. SSTM intervention was administered to the H9C2 cells. Then, the changes in H2O2-induced oxidative damage in H9C2 cells and the expression of microRNA-146a were observed to explore the protective effect of SSTM on H9C2 and its mechanism. H9C2 cells cultured i n vitro were divided into 3 groups, including a control group, a model group of H2O2-induced oxidative damage (referred to hereafter as the model group), and a group given H2O2 modeling plus SSTM intervention at 500 µg/L for 72 h (referred to hereafter as the treatment group). The cell viability was measured by CCK8 assay. In addition, the levels of N-terminal pro-brain natriuretic peptide (Nt-proBNP), nitric oxide (NO), high-sensitivity C-reactive protein (Hs-CRP), and angiotensin were determined by enzyme-linked immunosorbent assay (ELISA). The expression level of microRNA-146a was determined by real-time PCR (RT-PCR). Result: H9C2 cells were pretreated with SSTM at mass concentrations ranging from 200 to 1500 µg/L. Then, CCK8 assay was performed to measure cell viability and the findings showed that the improvement in cell proliferation reached its peak when the mass concentration of SSTM was 500 µg/L, which was subsequently used as the intervention concentration. ELISA was performed to measure the indicators related to heart failure, including Nt-proBNP, NO, Hs-CRP, and angiotensin Ⅱ. Compared with those of the control group, the expressions of Nt-proBNP and angiotensin Ⅱ in the treatment group were up-regulated (P<0.05), while the expression of NO was down-regulated (P<0.05). There was no significant difference in the expression of Hs-CRP between the treatment group and the control group. These findings indicate that SSTM could effectively ameliorate oxidative damage in H9C2 rat cardiomyocytes. Finally, according to the RT-PCR findings for the expression of microRNA-146a in each group, H2O2 treatment at 15 µmol/L could significantly reduce the expression of microRNA-146a, and the expression of microRNA-146a in the treatment group was nearly doubled compared with that in the model group. There was no significant difference between the treatment group and the control group. Conclusion: SSTM can significantly resist the H2O2-induced oxidative damage of H9C2 cells and may play a myocardial protective role by upregulating microRNA-146a.

Effect and mechanism of T lymphocytes on human induced pluripotent stem cell-derived cardiomyocytes via Proteomics.

BACKGROUND: Abnormalities in T cell activation play an important role in the pathogenesis of myocarditis, and persistent T cell responses can lead to autoimmunity and chronic cardiac inflammation, as well as even dilated cardiomyopathy. Although previous work has examined the role of T cells in myocarditis in animal models, the specific mechanism for human cardiomyocytes has not been investigated. METHODS: In this study, we constructed the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and established the T cell-mediated cardiac injury model by co-culturing with activated CD4 + T or CD8 + T cells that were isolated from peripheral mononuclear blood to elucidate the pathogenesis of myocardial cell injury caused by inflammation. RESULTS: By combination of quantitative proteomics with tissue and cell immunofluorescence examination, we established a proteome profile of inflammatory myocardia from hiPSC-CMs with obvious cardiomyocyte injury and increased levels of lactate dehydrogenase content, creatine kinase isoenzyme MB and cardiac troponin. A series of molecular dysfunctions of hiPSC-CMs was observed and indicated that CD4 + cells could produce direct cardiomyocyte injury by activating the NOD-like receptor signals pathway. CONCLUSIONS: The data presented in our study established a proteome map of inflammatory myocardial based on hiPSC-CMs injury model. These results can provide guidance in the discovery of improved clinical treatments for myocarditis.

Engineered Heart Tissues for Standard 96-Well Tissue Culture Plates.

Engineered heart tissues (EHTs) have been shown to be a valuable platform for disease investigation and therapeutic testing by increasing human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturity and better recreating the native cardiac environment. The protocol detailed in this chapter describes the generation of miniaturized EHTs (mEHTs) incorporating hiPSC-CMs and human stromal cells in a fibrin hydrogel. This platform utilizes an array of silicone posts designed to fit in a standard 96-well tissue culture plate. Stromal cells and hiPSC-CMs are cast in a fibrin matrix suspended between two silicone posts, forming an mEHT that produces synchronous muscle contractions. The platform presented here has the potential to be used for high throughput characterization and screening of disease phenotypes and novel therapeutics through measurements of the myocardial function, including contractile force and calcium handling, and its compatibility with immunostaining.

Establishment of human embryonic stem cell lines carrying LQT1 mutations by CRISPR base editing.

The KCNQ1 gene encodes a voltage-gated potassium channel required for cardiac action potentials. Mutations in this gene have been associated with hereditary long QT syndrome 1, Jervell and Lange-Nielsen syndromes, and familial atrial fibrillation. The NM_000218.3(KCNQ1): c.604 + 2T > C mutation has been categorized as the causative variant leading to LQT1. In this study, we generated a KCNQ1 (c.644 + 2T > C) mutation human embryonic stem cell line WAe009-A-1L based on CRISPR base editing system. WAe009-A-1L cell has the potential to differentiate cardiomyocytes and would be used as an in vitro disease model for mechanism exploration and drug screening.

Sex-Based Mechanisms of Cardiac Development and Function: Applications for Induced-Pluripotent Stem Cell Derived-Cardiomyocytes.

Males and females exhibit intrinsic differences in the structure and function of the heart, while the prevalence and severity of cardiovascular disease vary in the two sexes. However, the mechanisms of this sex-based dimorphism are yet to be elucidated. Sex chromosomes and sex hormones are the main contributors to sex-based differences in cardiac physiology and pathophysiology. In recent years, the advances in induced pluripotent stem cell-derived cardiac models and multi-omic approaches have enabled a more comprehensive understanding of the sex-specific differences in the human heart. Here, we provide an overview of the roles of these two factors throughout cardiac development and explore the sex hormone signaling pathways involved. We will also discuss how the employment of stem cell-based cardiac models and single-cell RNA sequencing help us further investigate sex differences in healthy and diseased hearts.

Maturation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) on polycaprolactone and polyurethane nanofibrous mats.

Investigating the potential of human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) in in vitro heart models is essential to develop cardiac regenerative medicine. iPSC-CMs are immature with a fetal-like phenotype relative to cardiomyocytes in vivo. Literature indicates methods for enhancing the structural maturity of iPSC-CMs. Among these strategies, nanofibrous scaffolds offer more accurate mimicry of the functioning of cardiac tissue structures in the human body. However, further research is needed on the use of nanofibrous mats to understand their effects on iPSC-CMs. Our research aimed to evaluate the suitability of poly(ε-caprolactone) (PCL) and polyurethane (PU) nanofibrous mats with different elasticities as materials for the maturation of iPSC-CMs. Analysis of cell morphology and orientation and the expression levels of selected genes and proteins were performed to determine the effect of the type of nanofibrous mats on the maturation of iPSC-CMs after long-term (10-day) culture. Understanding the impact of 3D structural properties in in vitro cardiac models on induced pluripotent stem cell-derived cardiomyocyte maturation is crucial for advancing cardiac tissue engineering and regenerative medicine because it can help optimize conditions for obtaining more mature and functional human cardiomyocytes.

A Sample Preparation Procedure for Isobaric Labeling-Based Single-Cell Proteomics.

Mass spectrometry-based proteomics has traditionally been limited by the amount of input material for analysis. Single-cell proteomics has emerged as a challenging discipline due to the ultra-high sensitivity required. Isobaric labeling-based multiplex strategies with a carrier proteome offer an approach to overcome the sensitivity limitations. Following this as the basic strategy, we show here the general workflow for preparing cells for single-cell mass spectrometry-based proteomics. This protocol can also be applied to manually isolated cells when large cells, such as cardiomyocytes, are difficult to isolate properly with conventional fluorescence-activated cell sorting (FACS) sorter methods.

Current Status of Cardiac Regenerative Therapy Using Induced Pluripotent Stem Cells.

Heart failure (HF) is a life-threatening disorder and is treated by drug therapies and surgical interventions such as heart transplantation and left ventricular assist device (LVAD). However, these treatments can lack effectiveness in the long term and are associated with issues such as donor shortage in heart transplantation, and infection, stroke, or gastrointestinal bleeding in LVADs. Therefore, alternative therapeutic strategies are still needed. In this respect, stem cell therapy has been introduced for the treatment of HF and numerous preclinical and clinical studies are employing a range of stem cell varieties. These stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have been shown to improve cardiac function and attenuate left ventricular remodeling. IPSCs, which have a capacity for unlimited proliferation and differentiation into cardiomyocytes, are a promising cell source for myocardial regeneration therapy. In this review, we discuss the following topics: (1) what are iPSCs; (2) the limitations and solutions for the translation of iPSC-CMs practically; and (3) the current therapeutic clinical trials.

Cardiac Progenitor Cells of the First and Second Heart Fields.

The heart forms from the first and second heart fields, which contribute to distinct regions of the myocardium. This is supported by clonal analyses, which identify corresponding first and second cardiac cell lineages in the heart. Progenitor cells of the second heart field and its sub-domains are controlled by a gene regulatory network and signaling pathways, which determine their behavior. Multipotent cells in this field can also contribute cardiac endothelial and smooth muscle cells. Furthermore, the skeletal muscles of the head and neck are clonally related to myocardial cells that form the arterial and venous poles of the heart. These lineage relationships, together with the genes that regulate the heart fields, have major implications for congenital heart disease.