Maturation Of 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.

Characterizing Mitochondrial Maturation in the Heart

Cardiovascular disease is the leading cause of death around the world and the burden is projected to become worse. Because of their ability to supplant the heart’s weak regenerative capabilities, stem cells and their derivatives have been investigated as a potential therapy. However, stem cell-derived cardiomyocytes are less mature than the cells they would be replacing in a clinical setting, causing many functional shortcomings such as unorganized and weak contraction. An in-depth study of how maturation normally occurs is necessary to identify targets to enhance it. As one of the best-known aspects of cardiomyocyte maturation, the mitochondria, and their substrate preferences, dictate the amount of energy a cell produces as well as modulate a cell’s contraction, differentiation, and survival. Similar to the cell itself, the mitochondria mature over time. However, to what extent mitochondrial maturation drives cardiomyocyte maturation is not clear. In this study, we used mass spectrometry to identify the developmental changes across normal cardiac maturation in mitochondria from mice. We detail key shifts in the mitochondrial proteome over time and identify possible regulators of mitochondrial and cardiomyocyte maturation. Pathway and transcription factor analyses highlight transitions over time to more organized, productive mitochondria. Mechanistic evaluation of identified pathways might reveal current bottlenecks for complete maturation of stem cell-derived cardiomyocytes. This work was supported by RMM091621DS008, NIH155993, and NIH160665. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Maturation of Stem Cell-Derived Cardiomyocytes: Foe in Translation Medicine.

With the in-depth study of heart development, many human cardiomyocytes (CMs) have been generated in a laboratory environment. CMs derived from pluripotent stem cells (PSCs) have been widely used for a series of applications such as laboratory studies, drug toxicology screening, cardiac disease models, and as an unlimited resource for cell-based cardiac regeneration therapy. However, the low maturity of the induced CMs significantly impedes their applicability. Scientists have been committed to improving the maturation of CMs to achieve the purpose of heart regeneration in the past decades. In this review, we take CMs maturation as the main object of discussion, describe the characteristics of CMs maturation, summarize the key regulatory mechanism of regulating maturation and address the approaches to promote CMs maturation. The maturation of CM is gradually improving due to the incorporation of advanced technologies and is expected to continue.

Approaches to Optimize Stem Cell-Derived Cardiomyocyte Maturation and Function

Approaches to Optimize Stem Cell-Derived Cardiomyocyte Maturation and Function

Transcriptomic entropy benchmarks stem cell-derived cardiomyocyte maturation against endogenous tissue at single cell level.

The immaturity of pluripotent stem cell (PSC)-derived tissues has emerged as a universal problem for their biomedical applications. While efforts have been made to generate adult-like cells from PSCs, direct benchmarking of PSC-derived tissues against in vivo development has not been established. Thus, maturation status is often assessed on an ad-hoc basis. Single cell RNA-sequencing (scRNA-seq) offers a promising solution, though cross-study comparison is limited by dataset-specific batch effects. Here, we developed a novel approach to quantify PSC-derived cardiomyocyte (CM) maturation through transcriptomic entropy. Transcriptomic entropy is robust across datasets regardless of differences in isolation protocols, library preparation, and other potential batch effects. With this new model, we analyzed over 45 scRNA-seq datasets and over 52,000 CMs, and established a cross-study, cross-species CM maturation reference. This reference enabled us to directly compare PSC-CMs with the in vivo developmental trajectory and thereby to quantify PSC-CM maturation status. We further found that our entropy-based approach can be used for other cell types, including pancreatic beta cells and hepatocytes. Our study presents a biologically relevant and interpretable metric for quantifying PSC-derived tissue maturation, and is extensible to numerous tissue engineering contexts.

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.

Role of Parkin-mediated mitophagy in glucocorticoid-induced cardiomyocyte maturation

Glucocorticoids can promote cardiomyocyte maturation. However, the mechanism underlying glucocorticoid-mediated cardiomyocyte maturation is still unclear. Mitophagy plays a key role in cardiomyocyte maturation. Based on current knowledge, our study evaluated the effects of the glucocorticoid dexamethasone (100 nM) on the maturation of mouse embryonic stem cell-derived cardiomyocytes and the role of mitophagy in this maturation. The results showed that dexamethasone can promote embryonic stem cell-derived cardiomyocyte maturation, inhibit cardiomyocyte proliferation, and promote myocardial fiber arrangement. However, dexamethasone did not affect mitochondrial morphology in cardiomyocytes. Glucocorticoid receptor inhibitors (RU486, 1 nM) can inhibit dexamethasone-mediated cardiomyocyte maturation. Additionally, dexamethasone can promote mitophagy in embryonic stem cell-derived cardiomyocytes and induce LC3 and lysosomal aggregation in mitochondria. The inhibition of mitophagy can inhibit the cardiomyocyte maturation effect of dexamethasone. Furthermore, our research found that dexamethasone may mediate the occurrence of mitophagy in cardiomyocytes through Parkin. The siRNA-mediated inhibition of Parkin expression can inhibit mitochondrial autophagy caused by dexamethasone, thus inhibiting cardiomyocyte maturation. Overall, our study found that dexamethasone can promote embryonic stem cell-derived cardiomyocyte maturation through Parkin-mediated mitophagy.

Learn from Your Elders: Developmental Biology Lessons to Guide Maturation of Stem Cell-Derived Cardiomyocytes.

Human pluripotent stem cells (hPSCs) offer a multifaceted platform to study cardiac developmental biology, understand disease mechanisms, and develop novel therapies. Remarkable progress over the last two decades has led to methods to obtain highly pure hPSC-derived cardiomyocytes (hPSC-CMs) with reasonable ease and scalability. Nevertheless, a major bottleneck for the translational application of hPSC-CMs is their immature phenotype, resembling that of early fetal cardiomyocytes. Overall, bona fide maturation of hPSC-CMs represents one of the most significant goals facing the field today. Developmental biology studies have been pivotal in understanding the mechanisms to differentiate hPSC-CMs. Similarly, evaluation of developmental cues such as electrical and mechanical activities or neurohormonal and metabolic stimulations revealed the importance of these pathways in cardiomyocyte physiological maturation. Those signals cooperate and dictate the size and the performance of the developing heart. Likewise, this orchestra of stimuli is important in promoting hPSC-CM maturation, as demonstrated by current in vitro maturation approaches. Different shades of adult-like phenotype are achieved by prolonging the time in culture, electromechanical stimulation, patterned substrates, microRNA manipulation, neurohormonal or metabolic stimulation, and generation of human-engineered heart tissue (hEHT). However, mirroring this extremely dynamic environment is challenging, and reproducibility and scalability of these approaches represent the major obstacles for an efficient production of mature hPSC-CMs. For this reason, understanding the pattern behind the mechanisms elicited during the late gestational and early postnatal stages not only will provide new insights into postnatal development but also potentially offer new scalable and efficient approaches to mature hPSC-CMs.

Human Stem Cell Derived Cardiomyocyte Maturation is Regulated by Glucose Levels and Metabolic Hormone Supplementation

Methods to differentiate stem cells into cardiomyocytes have been well established. However, a limitation for the successful application of these cells for research and medicine has been their fetal‐like phenotype with respect to cell size, contractility, calcium handling, metabolism, and electrophysiology. We sought to increase the maturity of human pluripotent stem cell derived cardiomyocytes (hPSC‐CMs) through metabolic pathway regulation. We hypothesized that switching the main metabolic substrates from glucose to fatty acids, mimicking the switch from placental to breast milk nutrient consumption that occurs during development, will increase hPSC‐CM maturation. RUES2 embryonic stem cells were differentiated into cardiomyocytes and then treated with base media with varying glucose and calcium concentrations and fatty acid supplementation. Using qRT‐PCR, we measured an inverse relationship between glucose levels and markers of cardiomyocyte maturation: increased expression of cardiac troponin I relative to skeletal troponin I isoforms (TNNI3:TNNI1), increased expression of metabolic (CPT1B, PPARGC1A) and electrical maturity markers (KCNJ2, RYR2). These maturation markers were not influenced with fatty acid supplementation but were enhanced upon the addition of thyroid hormone and dexamethasone. To understand the mechanisms by which media nutrients and signaling molecules cause phenotypic changes we investigated the relationship between maturation and global epigenetic state. Western blot revealed increases in global acetylation levels, measured by histone H3 acetylation, linked to maturation signaling. These findings depict a direct relationship between glucose metabolism and the development of a mature phenotype in hPSC‐CMs, mediated by epigenetic mechanisms.Support or Funding InformationWashington Research Foundation Undergraduate FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Changing Metabolism in Differentiating Cardiac Progenitor Cells-Can Stem Cells Become Metabolically Flexible Cardiomyocytes?

The heart is a metabolic omnivore and the adult heart selects the substrate best suited for each circumstance, with fatty acid oxidation preferred in order to fulfill the high energy demand of the contracting myocardium. The fetal heart exists in an hypoxic environment and obtains the bulk of its energy via glycolysis. After birth, the “fetal switch” to oxidative metabolism of glucose and fatty acids has been linked to the loss of the regenerative phenotype. Various stem cell types have been used in differentiation studies, but most are cultured in high glucose media. This does not change in the majority of cardiac differentiation protocols. Despite the fact that metabolic state affects marker expression and cellular function and activity, the substrate composition is currently being overlooked. In this review we discuss changes in cardiac metabolism during development, the various protocols used to differentiate progenitor cells to cardiomyocytes, what is known about stem cell metabolism and how consideration of metabolism can contribute toward maturation of stem cell-derived cardiomyocytes.

Top-down proteomics for assessing the maturation of stem cell-derived cardiomyocytes

Background: Recent advancements in human pluripotent stem cell (hPSC) technology and cardiomyocyte (CM) differentiation have empowered research in cardiac development and diseases. However, the relative immaturity of stem cell-derived CMs is well documented. Since the expression of sarcomeric protein isoforms, as well as their post-translational modifications (PTMs), change as CMs mature, we sought to develop a method utilizing powerful top-down high-resolution mass spectrometry (MS) to assess the relative maturity of CMs focusing on the expression of sarcomeric protein isoforms and their PTMs. Methods: hPSCs were differentiated to CMs using a small molecule directed protocol and cultured in either 2D monolayer or 3D engineered cardiac tissue (ECT). Sarcomeric proteins were extracted from the CMs and analyzed using a top-down proteomics platform coupling reverse phase chromatography to high-resolution MS. Results: We identified and quantified the major sarcomeric protein isoforms and their PTMs in CMs from 2D monolayer or 3D ECT culture. Slow skeletal troponin I (ssTnI) was highly expressed while cardiac troponin I (cTnI) was under detection limit in 2D-CMs at both day 30 and 60, indicating the relative immaturity of 2D-CMs by day 60. However, the relative expression of the ventricular regulatory light chain versus the atrial isoform in CMs increased by about 7-fold at day 60 compared to the 2D-CMs at day 30, confirming that CMs exhibit maturation with longer 2D culture as anticipated. Tropomyosin phosphorylation decreased in CMs with longer 2D culture, which may serve as a novel marker for assessing CM maturation. CMs cultured in 3D ECT for 42 days after 30 days in 2D culture exhibited a 2-fold increase in cTnI expression relative to ssTnI, compared to day-72 CMs maintained in 2D culture, suggesting that 3D culture can promote CM maturation. These results demonstrate the power of top-down proteomics to assess the maturation of hPSC-derived CMs.

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 Human-Induced Pluripotent Stem Cell Derived Cardiomyocyte Maturation Using a Dual Microgradient Substrate.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) raise many possibilities for cardiac research but they exhibit an immature phenotype, which influences experimental outcomes. The aim of our research is to investigate the effects of a topographical gradient substrate on the morphology and function of commercially available hiPSC-CM. The lateral dimensions the microgrooves on the substrate varied from 8 to 100 μm space between the 8 μm grooves on one axis and from ∼5 nm to ∼1 μm in depth on the other axis. Cells were seeded homogeneously across the substrate and according to the manufacturers protocols. At days 4 and 10, measures of eccentricity, elongation, orientation, sarcomere length (SL), and contractility of the hiPSC-CM were taken. Only the deepest and widest region (8–30 μm wide and 0.85–1 μm deep) showed a significantly higher percentage of hiPSC-CM with an increased eccentricity (31.3 ± 6.4%), elongation (10.4 ± 4.3%), and orientation (<10°) (32.1 ± 2.7%) when compared with the control (flat substrate) (15.8 ± 5.0%, 3.4 ± 2.7%, and 10.6 ± 1.1%, respectively). Additionally, during stimulus-induced contraction, the relaxation phase of the twitch was prolonged (400 ms) compared to nonelongated cells (200 ms). These findings support the potential use of dual microgradient substrates to investigate substrate topographies that stimulate migration and/or maturation of hiPSC-CM.

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.

Enhanced maturation of stem cell-derived cardiomyocytes

Enhanced maturation of stem cell-derived cardiomyocytes

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.