Functional improvement and maturation of human cardiomyocytes derived from human pluripotent stem cells by barbaloin preconditioning

2019 ◽  
Vol 51 (10) ◽  
pp. 1041-1048
Author(s):  
Hui Yang ◽  
Weiyi Zhong ◽  
Mohammad Rafi Hamidi ◽  
Gaojun Zhou ◽  
Chen Liu
Keyword(s):  
Disease Modeling ◽  
Calcium Handling ◽  
Functional Improvement ◽  
Electrophysiological Properties ◽  
Human Cardiomyocytes ◽  
Cardiac Gene Expression ◽  
Cardiac Gene ◽  
Novel Strategy ◽  
Regenerative Therapies

Abstract The development of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is a significant advancement in our ability to obtain cardiomyocytes in vitro for regenerative therapies and drug discovery. However, hPSC-CMs obtained via existing protocols usually exhibit a markedly immature phenotype, compared with adult cardiomyocytes, thereby limiting their application. Here we report that barbaloin preconditioning dramatically improves the morphology, structure-related cardiac gene expression, calcium handling, and electrophysiological properties of hPSC-CMs, which means that barbaloin may have the potential to induce the maturation of hPSC-CMs, providing a novel strategy to generate more adult-like cardiomyocytes and promoting the application of hPSC-CMs in regenerative medicine, drug development, and disease modeling.

Multifactorial Regulation of Cardiac Gene Expression: an In Vivo and In Vitro Analysis

1995 ◽  
Vol 752 (1 Cardiac Growt) ◽  
pp. 370-386 ◽  
Author(s):  
J. L. SAMUEL ◽  
I. DUBUS ◽  
F. FARHADIAN ◽  
F. MAROTTE ◽  
P. OLIVIERO ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cardiac Gene Expression ◽  
Cardiac Gene ◽  
Vitro Analysis ◽  
In Vitro Analysis

Abstract 142: Defining the Non-Myocyte Compartment is Key for Enhanced Maturation of Human Engineered Heart Muscle

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Malte Tiburcy ◽  
James E Hudson ◽  
Dirk Ziebolz ◽  
Wolfram H Zimmermann
Keyword(s):  
Heart Muscle ◽  
Muscle Development ◽  
Disease Modeling ◽  
Cardiac Differentiation ◽  
Inotropic Response ◽  
Double Positive ◽  
Human Cardiomyocytes ◽  
Functional Maturation ◽  
And Function

Background: Tissue engineering of heart muscle from human pluripotent stem cells holds great potential for in vitro studies, disease modeling, and cardiac replacement therapy. A number of variables may however affect maturation and function of human cardiomyocytes (CM) in tissue engineered heart muscle (EHM). Here, we hypothesized that defined non-myocyte (NM) populations support structural and functional maturation of EHM. Methods and Results: To investigate the role of non-myocytes (NM) for heart muscle assembly in vitro we generated EHM from purified CM (93±1.5% actinin+) and a mixture of CM and NM (70/30%). Notably, only the NM-supplemented EHM generated measurable forces (0.8±0.1 mN, n=9) with anisotropically aligned cardiomyocytes. Depending on pluripotent stem cell line and differentiation protocol the NM compartment may vary considerably. To further define the influence of the NM compartment we generated EHM from HES2-derived CM with undefined NM, i.e the NM typically derived during cardiac differentiation, and defined NM (fibroblasts). Defined EHM were more mature with higher forces and lower variability between experimental series (defined: 9.8±0.9 nN/CM, undefined: 4.7±1.4 nN/CM, n=10/9), higher EC50 for calcium, and enhanced inotropic response to isoprenaline despite comparable CM:NM composition of 1:1. Increased actinin protein per CM, a reduction of MLC2V/2A double positive CM, and evidence of CM cycle withdrawal indicated enhanced ventricular maturation in defined EHM. Next, we tested whether defining cell composition and NM in iPS-derived EHM will yield a comparable functional phenotype to HES2-EHM. In agreement with the above data, defined iPS-EHM displayed advanced functional maturation with high specific forces, comparable calcium EC50, and inotropic response to isoprenaline. Summary and Conclusions: Here we demonstrate that defining the NM compartment is essential for optimized human heart muscle formation and maturation in vitro. Moreover, our data provide (1) evidence for the applicability of EHM in modelling of heart muscle development and (2) a strong rationale for the need to define CM and NM compartments in tissue engineered myocardium to reduce variability in applications such as disease modelling.

scholarly journals Metabolically-Driven Maturation of hiPSC-Cell Derived Cardiac Chip

2018 ◽  
Author(s):  
Nathaniel Huebsch ◽  
Berenice Charrez ◽  
Brian Siemons ◽  
Steven C. Boggess ◽  
Samuel Wall ◽  
...  
Keyword(s):  
Pluripotent Stem Cell ◽  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Left Ventricular ◽  
Calcium Handling ◽  
Acid Oxidation ◽  
Microphysiological Systems ◽  
Induced Pluripotent ◽  
Drug Responsiveness

AbstractHuman induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) are a promising in vitro tool for drug development and disease modeling, but their immature electrophysiology limits diagnostic utility. Tissue engineering approaches involving aligned 3D cultures enhance hiPSC-CM structural maturation but are insufficient to induce mature electrophysiology. We hypothesized that mimicking post-natal switching of the heart’s primary ATP source from glycolysis to fatty acid oxidation could enhance electrophysiological maturation of hiPSC-CM. We combined hiPSC-CM with microfabricated culture chambers to form 3D cardiac microphysiological systems (MPS) that enhanced immediate microtissue alignment and tissue specific extracellular matrix (ECM) production. Using Robust Experimental design, we identified a maturation media that improved calcium handling in MPS derived from two genetically distinct hiPSC sources. Although calcium handling and metabolic maturation were improved in both genotypes, there was a divergent effect on action potential duration (APD): MPS that started with abnormally prolonged APD exhibited shorter APD in response to maturation media, whereas the same media prolonged the APD in MPS that started with aberrantly short APD. Importantly, the APD of both genotypes was brought near the range of 270-300ms observed in human left ventricular cardiomyocytes. Mathematical modeling explained these divergent phenotypes, and further predicted the response of matured MPS to drugs with known pro-arrhythmic effects. These results suggest that systematic combination of biophysical stimuli and metabolic cues can enhance the electrophysiological maturation of hiPSC-derived cardiomyocytes. However, they also reveal that maturation-inducing cues can have differential effects on electrophysiology depending on the baseline phenotype of hiPSC-CM. In silico models provide a valuable tool for predicting how changes in cellular maturation will manifest in drug responsiveness.

scholarly journals Effects of Circulating HMGB-1 and Histones on Cardiomyocytes–Hemadsorption of These DAMPs as Therapeutic Strategy after Multiple Trauma

2020 ◽  
Vol 9 (5) ◽  
pp. 1421
Author(s):  
Birte Weber ◽  
Ina Lackner ◽  
Meike Baur ◽  
Giorgio Fois ◽  
Florian Gebhard ◽  
...  
Keyword(s):  
Cell Viability ◽  
Ex Vivo ◽  
Therapeutic Option ◽  
Substantial Reduction ◽  
Calcium Handling ◽  
Human Cardiomyocytes ◽  
Multiple Injured ◽  
Extracellular Histones ◽  
Injured Patients

Background and purpose: The aim of the study was to determine the effects of post-traumatically released High Mobility Group Box-1 protein (HMGB1) and extracellular histones on cardiomyocytes (CM). We also evaluated a therapeutic option to capture circulating histones after trauma, using a hemadsorption filter to treat CM dysfunction. Experimental Approach: We evaluated cell viability, calcium handling and mitochondrial respiration of human cardiomyocytes in the presence of HMGB-1 and extracellular histones. In a translational approach, a hemadsorption filter was applied to either directly eliminate extracellular histones or to remove them from blood samples obtained from multiple injured patients. Key results: Incubation of human CM with HMGB-1 or histones is associated with changes in calcium handling, a reduction of cell viability and a substantial reduction of the mitochondrial respiratory capacity. Filtrating plasma from injured patients with a hemadsorption filter reduces histone concentration ex vivo and in vitro, depending on dosage. Conclusion and implications: Danger associated molecular patterns such as HMGB-1 and extracellular histones impair human CM in vitro. A hemadsorption filter could be a therapeutic option to reduce high concentrations of histones.

Enhancement of glycolysis in cardiomyocytes elevates endothelin-1 expression through the transcriptional factor hypoxia-inducible factor-1 α

2002 ◽  
Vol 103 (s2002) ◽  
pp. 210S-214S ◽  
Author(s):  
Yoshihiko KAKINUMA ◽  
Takashi MIYAUCHI ◽  
Takahiko SUZUKI ◽  
Koichi YUKI ◽  
Nobuyuki MURAKOSHI ◽  
...  
Keyword(s):  
Gene Expression ◽  
Electrical Stimulation ◽  
Energy Metabolism ◽  
Hypoxia Inducible Factor ◽  
Endothelin 1 ◽  
Cardiac Gene Expression ◽  
Cardiac Gene ◽  
Cultured Cardiomyocytes

We investigated whether the type of energy metabolism directly affects cardiac gene expression. During development, the heart switches from glycolysis to fatty acid β-oxidation in vivo, as demonstrated by the developmental switching of the major isoform of myosin heavy chain (MHC) from β to α. However, the β-MHC isoform predominates in monocrotaline-induced pulmonary hypertension, a model of right ventricular hypertrophy in vivo. Cultured cardiomyocytes showed a predominance of β-MHC expression over that of α-MHC, the same pattern as in the hypertrophied heart, suggesting that the in vitro condition itself causes the energy metabolism of cardiomyocytes to be switched to glycolysis. Electrical stimulation of cultured cardiomyocytes decreased the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxia-inducible factor-1α (HIF-1α), but not that of peroxisome-proliferator-activated receptor-γ co-activator, suggesting that electrical stimulation suppresses the glycolytic system. Furthermore, a higher oxygen content (50%) decreased drastically the expression of GAPDH, HIF-1α and endothelin-1 (ET-1), and increased [3H]palmitate uptake. These findings indicate that the intrinsic energy metabolic system in cultured cardiomyocytes in vitro is predominantly glycolysis, and that the gene expression of cardiac ET-1 parallels the state of the glycolytic system. An antisense oligonucleotide against HIF-1α greatly decreased the gene expression of ET-1 and GAPDH, suggesting that cardiac ET-1 gene expression is regulated by cardiac energy metabolism through HIF-1α. In conclusion, it is suggested that the pattern of gene expression of ET-1 reflects the level of the glycolytic system in cardiomyocytes, and that enhanced glycolysis regulates the cardiac gene expression of ET-1 via HIF-1α.

scholarly journals m6A-mRNA methylation regulates cardiac gene expression and cellular growth

2019 ◽  
Vol 2 (2) ◽  
pp. e201800233 ◽  
Author(s):  
Vivien Kmietczyk ◽  
Eva Riechert ◽  
Laura Kalinski ◽  
Etienne Boileau ◽  
Ellen Malovrh ◽  
...  
Keyword(s):  
Cardiac Tissue ◽  
Cellular Growth ◽  
Translational Efficiency ◽  
Transcript Stability ◽  
Cardiac Gene Expression ◽  
Powerful Approach ◽  
Mrna Methylation ◽  
Cardiac Gene

Conceptually similar to modifications of DNA, mRNAs undergo chemical modifications, which can affect their activity, localization, and stability. The most prevalent internal modification in mRNA is the methylation of adenosine at the N6-position (m6A). This returns mRNA to a role as a central hub of information within the cell, serving as an information carrier, modifier, and attenuator for many biological processes. Still, the precise role of internal mRNA modifications such as m6A in human and murine-dilated cardiac tissue remains unknown. Transcriptome-wide mapping of m6A in mRNA allowed us to catalog m6A targets in human and murine hearts. Increased m6A methylation was found in human cardiomyopathy. Knockdown and overexpression of the m6A writer enzyme Mettl3 affected cell size and cellular remodeling both in vitro and in vivo. Our data suggest that mRNA methylation is highly dynamic in cardiomyocytes undergoing stress and that changes in the mRNA methylome regulate translational efficiency by affecting transcript stability. Once elucidated, manipulations of methylation of specific m6A sites could be a powerful approach to prevent worsening of cardiac function.

Abstract 156: Hydrogel Mattress, an in vitro Platform to Enhance Maturation and Evaluate Contractile Function of Individual hiPSC-CMs

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Tromondae K Feaster ◽  
Charles H Williams ◽  
Adrian G Cadar ◽  
Young W Chun ◽  
Lili Wang ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Disease Modeling ◽  
Contractile Function ◽  
Traction Force ◽  
Induced Pluripotent Stem Cell ◽  
Contractile Properties ◽  
Calcium Handling ◽  
Cell Shortening ◽  
Induced Pluripotent

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have great potential as tools for human heart disease modeling and drug discovery. However, their contractile properties have not been routinely evaluated; as current methods are not accessible for most laboratories. We sought to develop a more efficient method to evaluate hiPSC-CM mechanical properties, at the single cell level. Individual hiPSC-CMs were cultured on a hydrogel based platform, termed the “hydrogel mattress,” and their cellular contractile properties evaluated using video-based edge detection. We found that hiPSC-CMs maintained on the mattress reproducibly exhibited robust cell shortening, in dramatic contrast to hiPSC-CMs maintained in a standard manner. We further found that contraction and peak cell shortening amplitude of hiPSC-CMs on mattress was comparable to that of freshly isolated adult ventricular mouse CM. Importantly, hiPSC-CMs maintained on the mattress exhibited several characteristics of a native CM, in terms of myocyte elongation, calcium handling and pharmacological response. Finally, using this platform, we could calculate the traction force generated by individual CMs. In summary, the Hydrogel mattress platform is a simple and reliable in vitro platform that not only enables the quantification of contractile performance of isolated hiPSC-CMs, but also enhances CM maturation. This flexible platform can be extended to in vitro disease modeling, drug discovery and cardiotoxicity testing.

scholarly journals Human perinatal stem cell derived extracellular matrix enables rapid maturation of hiPSC-CM structural and functional phenotypes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Travis Block ◽  
Jeffery Creech ◽  
Andre Monteiro da Rocha ◽  
Milos Marinkovic ◽  
Daniela Ponce-Balbuena ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Stem Cell ◽  
Disease Modeling ◽  
Toxicity Testing ◽  
Induced Pluripotent Stem Cell ◽  
Torsades De Pointes ◽  
Mouse Cell ◽  
Human Cardiomyocytes ◽  
Cell Assays

Abstract The immature phenotype of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) is a major limitation to the use of these valuable cells for pre-clinical toxicity testing and for disease modeling. Here we tested the hypothesis that human perinatal stem cell derived extracellular matrix (ECM) promotes hiPSC-CM maturation to a greater extent than mouse cell derived ECM. We refer to the human ECM as Matrix Plus (Matrix Plus) and compare effects to commercially available mouse ECM (Matrigel). hiPSC-CMs cultured on Matrix Plus mature functionally and structurally seven days after thaw from cryopreservation. Mature hiPSC-CMs showed rod-shaped morphology, highly organized sarcomeres, elevated cTnI expression and mitochondrial distribution and function like adult cardiomyocytes. Matrix Plus also promoted mature hiPSC-CM electrophysiological function and monolayers’ response to hERG ion channel specific blocker was Torsades de Pointes (TdP) reentrant arrhythmia activations in 100% of tested monolayers. Importantly, Matrix Plus enabled high throughput cardiotoxicity screening using mature human cardiomyocytes with validation utilizing reference compounds recommended for the evolving Comprehensive In Vitro Proarrhythmia Assay (CiPA) coordinated by the Health and Environmental Sciences Institute (HESI). Matrix Plus offers a solution to the commonly encountered problem of hiPSC-CM immaturity that has hindered implementation of these human based cell assays for pre-clinical drug discovery.

scholarly journals Calcium handling precedes cardiac differentiation to initiate the first heartbeat

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Richard CV Tyser ◽  
Antonio MA Miranda ◽  
Chiann-mun Chen ◽  
Sean M Davidson ◽  
Shankar Srinivas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Contractile Function ◽  
Rapid Development ◽  
Mouse Embryos ◽  
Cardiac Differentiation ◽  
Calcium Handling ◽  
Heart Tube ◽  
Cardiac Gene Expression ◽  
Stage Of Development ◽  
Cardiac Gene

The mammalian heartbeat is thought to begin just prior to the linear heart tube stage of development. How the initial contractions are established and the downstream consequences of the earliest contractile function on cardiac differentiation and morphogenesis have not been described. Using high-resolution live imaging of mouse embryos, we observed randomly distributed spontaneous asynchronous Ca2+-oscillations (SACOs) in the forming cardiac crescent (stage E7.75) prior to overt beating. Nascent contraction initiated at around E8.0 and was associated with sarcomeric assembly and rapid Ca2+ transients, underpinned by sequential expression of the Na+-Ca2+ exchanger (NCX1) and L-type Ca2+ channel (LTCC). Pharmacological inhibition of NCX1 and LTCC revealed rapid development of Ca2+ handling in the early heart and an essential early role for NCX1 in establishing SACOs through to the initiation of beating. NCX1 blockade impacted on CaMKII signalling to down-regulate cardiac gene expression, leading to impaired differentiation and failed crescent maturation.

scholarly journals The impact of in vitro cell culture duration on the maturation of human cardiomyocytes derived from induced pluripotent stem cells of myogenic origin

2018 ◽  
Vol 27 (7) ◽  
pp. 1047-1067 ◽  
Author(s):  
Jarosław Lewandowski ◽  
Natalia Rozwadowska ◽  
Tomasz J. Kolanowski ◽  
Agnieszka Malcher ◽  
Agnieszka Zimna ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
In Vitro Culture ◽  
Troponin I ◽  
Calcium Handling ◽  
Marker Genes ◽  
Human Cardiomyocytes ◽  
Ventricular Cells ◽  
Culture Duration ◽  
The Impact

Ischemic heart disease, also known as coronary artery disease (CAD), poses a challenge for regenerative medicine. iPSC technology might lead to a breakthrough due to the possibility of directed cell differentiation delivering a new powerful source of human autologous cardiomyocytes. One of the factors supporting proper cell maturation is in vitro culture duration. In this study, primary human skeletal muscle myoblasts were selected as a myogenic cell type reservoir for genetic iPSC reprogramming. Skeletal muscle myoblasts have similar ontogeny embryogenetic pathways (myoblasts vs. cardiomyocytes), and thus, a greater chance of myocardial development might be expected, with maintenance of acquired myogenic cardiac cell characteristics, from the differentiation process when iPSCs of myoblastoid origin are obtained. Analyses of cell morphological and structural changes, gene expression (cardiac markers), and functional tests (intracellular calcium transients) performed at two in vitro culture time points spanning the early stages of cardiac development (day 20 versus 40 of cell in vitro culture) confirmed the ability of the obtained myogenic cells to acquire adult features of differentiated cardiomyocytes. Prolonged 40-day iPSC-derived cardiomyocytes (iPSC-CMs) revealed progressive cellular hypertrophy; a better-developed contractile apparatus; expression of marker genes similar to human myocardial ventricular cells, including a statistically significant CX43 increase, an MHC isoform switch, and a troponin I isoform transition; more efficient intercellular calcium handling; and a stronger response to β-adrenergic stimulation.

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