Maturation Of Pluripotent Stem Cell-derived Cardiomyocytes Research Articles

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.

Mapping the landscape of PSC-CM research through bibliometric analysis.

The discovery of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) has not only deepened our understanding of the pathogenesis and progression of heart disease, but also advanced the development of engineered cardiac tissues, cardiac regenerative therapy, drug discovery and the cardiotoxicity assessment of drugs. This study aims to visualize the developmental trajectory of PSC-CM research over the past 18 years to identify the emerging research frontiers and challenges. The literature on PSC-CMs from 2007 to 2024 was retrieved from the Web of Science and PubMed databases. Bibliometrix, VOSviewer and CiteSpace software were used for statistical analysis and visualization of scientific literature. Previous clinical trials were summarized using data from the ClinicalTrials.gov database. A total of 29,660 authors from 81 countries and regions published 6,406 papers on PSC-CMs over the past 18 years. The annual output of PSC-CM research experienced a general upward trend from 2007 to 2021, reaching its peak in 2021, followed by a notable decline in 2022 and 2023. The United States has emerged as the most influential nation in this field, with Stanford University being the most prolific institution and Joseph C. Wu standing out as the most productive and highly cited scholar. Circulation Research, Circulation, and Nature have been identified as the most co-cited journals. Organ-on-a-chip, 3D bio-printing, cardiac microtissue, extracellular vesicle, inflammation, energy metabolism, atrial fibrillation, personalized medicine etc., with a longer burst period, and maturation of PSC-CMs, with the highest burst strength of 27.19, are the major research focuses for rigorous investigation in recent years. Cardiac organoid is emerging as a promising key research frontier. While the clinical trials of stem-cell-mediated treatment for heart diseases shows promise, significant challenges remain. Further research is imperative to optimize protocols, enhance cell delivery methods, and establish standardized practices to improve clinical outcomes. In conclusion, several major research hotspots, including engineered cardiac tissue and maturation, exosome-based regenerative therapy, inflammation response, energy metabolism, atrial fibrillation, and personalized medicine etc. will continue to attract substantial interest from investigators worldwide. Cardiac organoids to in vitro recapitulate the intricate human heart is emerging as a promising key research frontier. Significant challenges persist in the clinical trials of stem-cell-mediated therapies for heart diseases.

Topological butterfly wings for human induced pluripotent stem cell-derived cardiomyocyte maturation and myocardial infarction treatment

Myocardial infarction (MI) causes irreversible myocardial cell loss; however, current treatments are limited to slowing disease progression rather than restoring lost myocardium. Cardiac tissue engineering, based on inexhaustible human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), becomes an ideal treatment method for supplying exogenous cardiomyocytes (CMs) to help contractile function recovery. However, hiPSC-CMs currently used are usually immature, which might trigger ventricular arrhythmias in MI treatments and limit their application in myocardial tissue engineering. The topological structure, as one of the most effective cues in controlling cellular functions, may benefit hiPSC-CM maturation. Here, the natural topological Morpho Menelaus butterfly wings (BFW) were chosen to regulate hiPSC-CM maturation and form the grafts for MI treatment. Immunofluorescence and calcium imaging tests confirmed that the parallel nanoridges on the surface of BFW oriented cellular growth and enhanced gap junction formation and improved the calcium handling ability of hiPSC-CMs, which demonstrated the maturation of hiPSC-CMs. BFW-matured hiPSC-CMs were transplanted as a patch for MI treatment evaluation, and twenty-eight days after engraftment, the histological destruction and contractile function of MI mice were improved obviously. This work provides an effective method for promoting hiPSC-CM maturation and potentially expands the application of natural materials in myocardial tissue engineering.

Metabolic Regulation of Human Pluripotent Stem Cell-Derived Cardiomyocyte Maturation.

This review summarizes the important role that metabolism plays in driving maturation of human pluripotent stem cell-derived cardiomyocytes. Human pluripotent stem cell-derived cardiomyocytes provide a model system for human cardiac biology. However, these models have been unable to fully recapitulate the maturity observed in the adult heart. By simulating the glucose to fatty acid transition observed in neonatal mammals, human pluripotent stem cell-derived cardiomyocytes undergo structural and functional maturation also accompanied by transcriptional changes and cell cycle arrest. The role of metabolism in energy production, signaling, and epigenetic modifications illustrates that metabolism and cellular phenotype are intimately linked. Further understanding of key metabolic factors driving cardiac maturation will facilitate the generation of more mature human pluripotent stem cell-derived cardiomyocyte models. This will increase our understanding of cardiac biology and potentially lead to novel therapeutics to enhance heart function.

Naturally Engineered Maturation of Cardiomyocytes.

Ischemic heart disease remains one of the most prominent causes of mortalities worldwide with heart transplantation being the gold-standard treatment option. However, due to the major limitations associated with heart transplants, such as an inadequate supply and heart rejection, there remains a significant clinical need for a viable cardiac regenerative therapy to restore native myocardial function. Over the course of the previous several decades, researchers have made prominent advances in the field of cardiac regeneration with the creation of in vitro human pluripotent stem cell-derived cardiomyocyte tissue engineered constructs. However, these engineered constructs exhibit a functionally immature, disorganized, fetal-like phenotype that is not equivalent physiologically to native adult cardiac tissue. Due to this major limitation, many recent studies have investigated approaches to improve pluripotent stem cell-derived cardiomyocyte maturation to close this large functionality gap between engineered and native cardiac tissue. This review integrates the natural developmental mechanisms of cardiomyocyte structural and functional maturation. The variety of ways researchers have attempted to improve cardiomyocyte maturation in vitro by mimicking natural development, known as natural engineering, is readily discussed. The main focus of this review involves the synergistic role of electrical and mechanical stimulation, extracellular matrix interactions, and non-cardiomyocyte interactions in facilitating cardiomyocyte maturation. Overall, even with these current natural engineering approaches, pluripotent stem cell-derived cardiomyocytes within three-dimensional engineered heart tissue still remain mostly within the early to late fetal stages of cardiomyocyte maturity. Therefore, although the end goal is to achieve adult phenotypic maturity, more emphasis must be placed on elucidating how the in vivo fetal microenvironment drives cardiomyocyte maturation. This information can then be utilized to develop natural engineering approaches that can emulate this fetal microenvironment and thus make prominent progress in pluripotent stem cell-derived maturity toward a more clinically relevant model for cardiac regeneration.

Enhanced stem cell-derived cardiomyocyte differentiation in suspension culture by delivery of nitric oxide using S-nitrosocysteine.

The development of cell-based treatments for heart disease relies on the creation of functionally mature stem cell-derived cardiomyocytes employing in vitro culture suspension systems, a process which remains a formidable and expensive endeavor. The use of nitric oxide as a signaling molecule during differentiation has demonstrated the potential for creating increased numbers of spontaneously contracting embryoid bodies in culture; however, the effects of nitric oxide signaling on the function and maturation of stem cell-derived cardiomyocytes is not well understood. In this study, the effects of nitric oxide on mouse embryonic stem cell-derived cardiomyocyte contractile activity, protein, and gene expression, and calcium handling were quantified. Embryoid bodies (EBs) formed using the hanging drop method, were treated with the soluble nitric oxide donor S-nitrosocysteine (CysNO) over a period of 18 days in suspension culture and spontaneous contractile activity was assessed. On day 8, selected EBs were dissociated to form monolayers for electrophysiological characterization using calcium transient mapping. Nitric oxide treatment led to increased numbers of stem cell-derived cardiomyocytes (SC-CMs) relative to non-treated EBs after 8 days in suspension culture. Increased incidence of spontaneous contraction and frequency of contraction were observed from days 8-14 in EBs receiving nitric oxide treatment in comparison to control. Expression of cardiac markers and functional proteins was visualized using immunocytochemistry and gene expression was assessed using qPCR. Cardiac-specific proteins were present in both CysNO-treated and control SC-CMs; however, CysNO treatment during differentiation significantly increased βMHC gene expression in SC-CMs relative to control SC-CMs. Furthermore, increased calcium transient velocity and decreased calcium transient duration was observed for CysNO-treated SC-CMs in comparison to control SC-CMs. Soluble nitric oxide donors, including S-nitrosocysteine, have advantages over other bioactive molecules for use in scalable culture systems in driving cardiac differentiation, since they are inexpensive and the diffusivity of nitric oxide is relatively high. By enabling maintenance of spontaneous contraction in suspension culture and progressing electrophysiological function of resulting SC-CMs toward a more mature phenotype, long-term application of S-nitrosocysteine was shown to be beneficial during cardiac differentiation. Taken together, these results demonstrate the efficiency of nitric oxide as a signaling compound, with implications in the improvement of pluripotent stem cell-derived cardiomyocyte maturation in large-scale culture systems.

P343Effect of T3 on human induced pluripotent stem cell-derived cardiomyocyte maturation

Purpose: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold great potential for regenerative medicine and in vitro screening. However, the phenotype is immature and more closely resembles foetal/neonatal cardiomyocytes. The aim of this study is to drive hiPSC-CM to a more mature state using thyroid hormone, triiodothyronine (T3), which has a role in cardiomyocyte maturation during development. Methods: hiPSC-CM (Cellular Dynamics) were treated with 30nM T3 for up to 4 weeks. Expression of β1 adrenergic receptor (β1AR) was assessed by qPCR (Taqman). Beating rates were determined using a Nikon Eclipse TE2000E microscope with Digital sight camera and NIS Elements3.2 software video tracking. Ca2+ dynamics were studied using Fluo-4AM loaded cells imaged with a Nikon Eclipse T6200 and Redshirt NeuroCMOS camera at 0.5KHz with 1Hz, 15V, 5ms field stimulation. Comparative proteomic analysis (4 week T3) was performed by mass spectrometry using isobaric tags. Data expressed as mean ±SEM; statistical significance determined by student's t-test (n=3). Results: 4 week T3 treatment resulted in a 1.5-fold increase in basal beating rate. T3 increased β1AR gene expression and remained stable for up to 2 weeks (p<0.05). CGP 20712A (10μM), a β1AR inverse agonist, decreased the beating rate of T3-treated cells (39.3±9.7bpm) to basal levels in untreated cells (25.7±9bpm). Ca2+ transient analysis revealed alterations in Ca2+ handling. 4 week T3 treatment resulted in decreased time to transient peak from 244±4.0 ms to 90.7±15 (p<0.0006). Time to 50% decay also decreased from 309.2±6.6ms to 287.3±2.3ms (p<0.05) and transient duration (start to 75% decay) decreased from 695.4±6.4ms to 537.9±21.3ms (p<0.002). Proteomic analysis revealed increases in proteins involved in regulating sarcoplasmic reticulum (SR) function including; phospholamban (17.2%), SERCA (27.6%) and calsequestrin2 (303.8%). In addition increases in mitochondrial enzymes, including ATP5B (32.7%), and α/βMHC ratio were detected along with decreases in slow skeletal troponin I (-43.7%) and C (-36.2%) isoforms. Conclusions: T3 treatment induced alterations in expression of proteins involved in SR function, mitochondrial metabolism and cytoskeletal composition. In addition, T3 treatment improved Ca2+ handling and increased β1AR function, with increases in beating rate possibly driven by constitutive β1AR activity. Collectively these data provide evidence for a shift towards a more mature phenotype and indicate that this may be an effective strategy to drive maturation of hiPSC-CM.