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Laboratory for the use of their path clamp equipment. Circ. Received 2018 Oct 30; Revised 2018 Nov 24; Accepted 2019 Jan 10. Biol. In addition, sarcomeres lengthen15. Kawamura M, Miyagawa S, Fukushima S, Saito A, Miki K, Ito E, Sougawa N, Kawamura T, Daimon T, Shimizu T, Okano T, Toda K, Sawa Y. Proteins (30g) were loaded onto 4 to 12% NuPAGE gels (Thermo Fisher Scientific) and transferred to nitrocellulose membranes. In 2006, Brundel et al[35] showed the use of an in vitro system to model alterations in the contractility of cardiomyocytes by displaying how electrical pacing can induce tachycardia. Google Scholar. Differentiated hiPSC-CMs were seeded onto a wire substrate containing a template polydimethylsiloxane channel and collagen gels. Tani, H., Tohyama, S., Kishino, Y., Kanazawa, H. & Fukuda, K. Production of functional cardiomyocytes and cardiac tissue from human induced pluripotent stem cells for regenerative therapy. Drawnel FM, Boccardo S, Prummer M, Delobel F, Graff A, Weber M, Grard R, Badi L, Kam-Thong T, Bu L, Jiang X, Hoflack JC, Kiialainen A, Jeworutzki E, Aoyama N, Carlson C, Burcin M, Gromo G, Boehringer M, Stahlberg H, Hall BJ, Magnone MC, Kolaja K, Chien KR, Bailly J, Iacone R. Disease modeling and phenotypic drug screening for diabetic cardiomyopathy using human induced pluripotent stem cells. Use the Previous and Next buttons to navigate the slides or the slide controller buttons at the end to navigate through each slide. Compensation was performed by FlowJo. J. Mol. The importance of our finding is specificity. Lemoine MD, Mannhardt I, Breckwoldt K, Prondzynski M, Flenner F, Ulmer B, Hirt MN, Neuber C, Horvth A, Kloth B, Reichenspurner H, Willems S, Hansen A, Eschenhagen T, Christ T. Human iPSC-derived cardiomyocytes cultured in 3D engineered heart tissue show physiological upstroke velocity and sodium current density. Novel method of differentiating human induced pluripotent stem cells to mature cardiomyocytes via Sfrp2. j Cardiovasc Transl Res 14, 556572. Cardiomyocytes from human pluripotent stem cells: From laboratory curiosity to industrial biomedical platform. The most efficient method to generate iPSc-derived cardiomyocytes adopts a two-staged approach. Specialty type: Cell and tissue engineering, P- Reviewer: Kiselev SL, Liu L, Saeki K, Tanabe S S- Editor: Ma YJ L- Editor: A E- Editor: Tan WW. However, these methods are only applied after differentiation is attained, requiring specialized equipment or materials and prolonged culture to obtain adult-like cardiomyocytes. (b) Representative image of a Sfrp2-derived cardiomyocyte after 40days in culture. Goversen B, van der Heyden MAG, van Veen TAB, de Boer TP. Human iPSC-derived cardiomyocytes (iPSC-CMs) are relatively immature and have the potential to increase the population of cells that advance to physiological hypertrophy in the presence of extracellular stimuli. 121, 13231330. J. Mol. As electrical and mechanical stimulation becomes more prevalent as a tool for hiPSC-CM maturation, private biotechnology companies have been developing electrical and mechanical devices commercially. In another study, a tissue-engineered cardiac patch was used to promote the maturation of hiPSC-CMs[48]. Federal government websites often end in .gov or .mil. Heat shock protein upregulation protects against pacing-induced myolysis in HL-1 atrial myocytes and in human atrial fibrillation. Sci Rep 13, 3920 (2023). Previous studies elucidated the effects electrical pacing can have on cultured cardiomyocytes. Traditionally, hiPSC-CM generation has been characterised through flow cytometry staining for Troponin T (TNNT2), a cardiac-specific protein, in addition to visual qualification of spontaneously beating cell clusters. For example, in addition to immature calcium handling, hiPSC-CMs display immature ultrastructural and electrophysiological features, low expression of key maturation markers, and rely on glycolysis for their metabolism as opposed to fatty acid metabolism[2,20,22]. 4a). Sci. In conclusion, we demonstrate that substitution of broad-spectrum Wnt pharmacological inhibitors with Sfrp2 improves the standard method of iPSc-derived cardiomyocyte generation by improving cardiomyocyte maturity. Despite this, there have been significant challenges in developing 3D model systems. PubMed Soma, Y. et al. In this article, Knollmann discusses the most important limitations of iPSC technology in the field of cardiac arrhythmias in its current state as follows: the immature phenotype of the iPSC-derived cardiomyocytes, the mixture of cardiomyocytes with different action potential characteristics generated by the most commonly used protocols, the . One published study used macaque monkeys as a model for cardiomyocyte transplant outcomes. Authors In immature cardiomyocytes, sarcomeres are patchy. 4b & 4c). Modeling hypertrophic cardiomyopathy with human cardiomyocytes derived As such, modelling these diseases in vitro is of paramount importance to advance our understanding of disease and allow the development of new drug therapies. This result suggested that Sfrp2 is potentially influencing cardiac differentiation via a -Catenin dependent signaling pathway. https://doi.org/10.1002/term.2117 (2017). Zhang D, Shadrin IY, Lam J, Xian HQ, Snodgrass HR, Bursac N. Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes. In cardiomyocytes generated via the broad-spectrum Wnt inhibitor standard protocol, gap junctions were absent (Fig. In contrast, hiPSC-CMs mainly rely on glucose and lactate but do possess some capacity to metabolize fatty acids[2,22,27]. Several methods have been used in attempt to improve the maturation of iPSc-derived cardiomyocytes. After two days incubation with the agents, the media was replaced with differentiation media from day 5 to day 9. El-Hattab AW, Scaglia F. Mitochondrial Cardiomyopathies. Enhanced survival of transplanted human induced pluripotent stem cell-derived cardiomyocytes by the combination of cell sheets with the pedicled omental flap technique in a porcine heart. In the first instance, cardiomyocyte differentiation was analyzed by flow cytometry of cardiac troponin-T (cTnT) immunostained cells. ImageJ was used to quantify band density. Goldfracht, I. et al. Development of appropriate fatty acid formulations to raise the contractility of constructed myocardial tissues. We tested the in vitro efficacy of Mavacamten in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) exhibiting hypercontractility and . This review outlined several strategies for the maturation of hiPSC-CMs. Consequently, we wanted to determine if replacement of broad-spectrum pharmacological Wnt inhibitors by Sfrp2 would give rise to mature iPSc-derived cardiomyocytes. Cells were analyzed on day 14 of differentiation protocol. Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Other 3D cardiomyocyte studies have displayed similar results[49,50]. Treated hiPSC-CMs exhibited a contractile force of 12.3 0.7 nmol/L/cell while control cells were significantly lower at 7.5 0.4 nmol/L/cell[30]. The recent development of tissue-culture plates such as AggreWell (STEMCELL Technologies), and cardiomyocyte recovery/dissociation medium (STEMCELL Technologies) may prove useful in regulating cardiac organoid cell size and optimizing cell recovery; however, further research in this area must be done to definitively address these concerns. Triiodothyronine and dexamethasone alter potassium channel expression and promote electrophysiological maturation of human-induced pluripotent stem cell-derived cardiomyocytes. Chemically defined generation of human cardiomyocytes. https://doi.org/10.15283/ijsc21077 (2021). In contrast, hiPSC-CMs display high levels of MYH6 (myosin heavy chain 6) as opposed to MYH7, predominantly display the N2A isoform of cardiac titin instead of N2B[22] and have lower expression of other genes that are highly expressed in adult cardiomyocytes[26,27]. The use of a multicellular 3D cultures potentially allow for higher accuracy in disease modelling and drug testing as 3D cardiomyocyte aggregates are closer to in vivo morphology[2,20,22]. As such, induced pluripotent stem cells differentiated into cardiomyocytes offer a potential tool for the understanding of disease and the development of life-saving therapeutics. Frontiers | Cardiomyocyte Proliferation and Maturation: Two Sides of Sample traces indicated that the iPSc-derived cardiomyocytes were physiologically normal (Fig. Nat. Representative traces are shown. Structural Immaturity of Human iPSC-Derived Cardiomyocytes: Metabolically driven maturation of human-induced-pluripotent-stem-cell Development of a reentrant arrhythmia model in human pluripotent stem cell-derived cardiac cell sheets. View PDF View article View in Scopus Google Scholar. FOIA https://doi.org/10.1093/cvr/cvab115 (2022). The mechanism by which Ascorbic acid improves the efficacy of cardiac differentiation is unclear. By submitting a comment you agree to abide by our Terms and Community Guidelines. N=15 (WntC59) or 19 (Sfrp2). Drouin, E., Charpentier, F., Gauthier, C., Laurent, K. & Le Marec, H. Electrophysiologic characteristics of cells spanning the left ventricular wall of human heart: Evidence for presence of M cells. This result suggested that Sfrp2 gave rise to more mature cardiomyocytes. 2e). The problem is that these cells remain largely immature, and in order to utilize them, they must reach a functional degree of maturity. Both Sfrp2 and the broad-spectrum Wnt pharmacological inhibitor (WntC59) were found to inhibit the -Catenin pathway as evidence by reduced total -Catenin (Fig. The authors of this study treated hiPSC-CMs with 20 ng/ml of T3 and noticed key morphological differences. One study noted larger cell sizes and increased sarcomere length, in addition to higher contractile force and increased mitochondrial respiration capacity post-T3 treatment. To transition hiPSC-CMs into a mature electrical state, the use of electrical and/or mechanical stimulation is being extensively explored. PubMed Taken together, these results indicated that Sfrp2 protocol improves cardiac maturation. Thyroid and glucocorticoid hormones promote functional T-tubule development in human-induced pluripotent stem cell-derived cardiomyocytes. Get the most important science stories of the day, free in your inbox. This study then implicated VEGF, bFGF, SDF-1, and GM-CSF as secreted factors from hMSCs that are key in hiPSC-CM maturation. (b) Cardiac differentiation was determined by flow cytometry of the cardiomyocyte marker cardiac troponin-T (cTnT). PubMed Scale bar 20 microns. In this review, we discuss how cardiac proliferation and maturation are regulated during embryonic development and postnatal growth . Novel method of differentiating human induced pluripotent stem cells to mature cardiomyocytes via Sfrp2, https://doi.org/10.1038/s41598-023-31144-3. Cardiomyocytes-derived from induced pluripotent stem cells (iPSc) are a potential cell source to replace heart tissue lost after infarction1,2,3. Adult cardiomyocytes display Z-discs, I-, H-, A- and M- bands. Maturing hiPSC-CMs is key to fully realizing the potential of these cells. Qi XY, Yeh YH, Xiao L, Burstein B, Maguy A, Chartier D, Villeneuve LR, Brundel BJ, Dobrev D, Nattel S. Cellular signaling underlying atrial tachycardia remodeling of L-type calcium current. The colored boxes indicate the various stages of cardiomyocyte differentiation, while the clear boxes indicate the regulatory molecules. For example, treatment of hiPSC-CMs with both T3 and dexamethasone has shown success in furthering the maturational state of hiPSC-CMs[31]. https://doi.org/10.1007/s00424-021-02536-z (2021). Yoshida S, Miyagawa S, Fukushima S, Kawamura T, Kashiyama N, Ohashi F, Toyofuku T, Toda K, Sawa Y. Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes by Soluble Factors from Human Mesenchymal Stem Cells. 3a, top panels). Yang, X., Pabon, L. & Murry, C. E. Engineering adolescence: Maturation of human pluripotent stem cell-derived cardiomyocytes. 1 is non-linear data. This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. Hsueh, YC., Pratt, R.E., Dzau, V.J. Extant protocols induce cardiac mesoderm specification through the use of broad-spectrum inhibitors. Immature hiPSC-CMs show remarkable flexibility in adapting to growth conditions. Another promising approach involves the addition of conductive materials to the cell substrate or matrix. Biochim. Following blocking, cells were incubated overnight at 4C with cardiac troponin-T antibody (abcam, ab45932, 1:200), -Actinin antibody (Sigma, A7811, 1:800), and/or Connexin 43 (Cell Signaling Technology, 1:100) in antibody buffer. Co-culturing hiPSC-CMs with other cell types has also been shown to further the maturation state of these cells[34]. Med 8, 7743. https://doi.org/10.3389/fcvm.2021.774389 (2021). Human iPScs were differentiated into cardiomyocytes via a standard protocol6. https://doi.org/10.1136/heartjnl-2011-301317 (2012). When hiPSC-CMs were cultured with both chemicals for 15 days, an extensive T-tubule network was generated, a key indicator of adult-like cardiomyocytes as the extensions are crucial in contractility[31]. Their experiments improved the metabolic, structural, and electrophysiological state of hiPSC-CMs. In addition, while Sfrp2 was found to produce both ventricular and atrial cardiomyocytes there was a clear preference for the former (Fig. Our findings with do not exist in a vacuum and other groups have also been successful in promoting cardiomyocyte maturation. Jackson, A. O., Rahman, G. A., Yin, K. & Long, S. Enhancing matured stem-cardiac cell generation and transplantation: A novel strategy for heart failure therapy. Biophys. Cardiovascular disease is the greatest cause of mortality worldwide[10]. In contrast, gap junctions were present in cardiomyocytes derived via the Sfrp2 protocol. This work was supported by the NIH grant R01HL139718 as well as by the The resulting cardiac progenitors can differentiate into several different cell-types. The https:// ensures that you are connecting to the Thomas, D., Cunningham, N. J., Shenoy, S. & Wu, J. C. Human-induced pluripotent stem cells in cardiovascular research: Current approaches in cardiac differentiation, maturation strategies, and scalable production. For each experiment, 3 wells were analyzed. This was confirmed by staining for the ventricular isoform of the myosin light chain 2 (MLC2v) (Fig. However, this limitation is widely recognized, and numerous groups have made substantial progress in addressing this problem. The .gov means its official. Previous reports from our laboratory have shown that Sfrp2 influences cell behavior via Wnt3a. Importantly, replacement of broad spectrum pharmacological inhibitors with Sfrp2 gave rise to mature cardiomyocytes as evidenced by their sarcomere structure, electrophysiological profiles, and ability to form gap junctions. In contrast, in mature cardiomyocytes sarcomeres are present across the entire cell. We chose to analyze the immunostaining for two markers of maturation: circularity and sarcomere length. N=3. 3b). Kamakura T, Makiyama T, Sasaki K, Yoshida Y, Wuriyanghai Y, Chen J, Hattori T, Ohno S, Kita T, Horie M, Yamanaka S, Kimura T. Ultrastructural maturation of human-induced pluripotent stem cell-derived cardiomyocytes in a long-term culture. 246, 18161828. Proc. Jacot JG, McCulloch AD, Omens JH. Methods evaluated to date include electrical stimulation, mechanical stimulation, modulation of carbon source, growth on various substrates, and the development of 3D culture conditions or organoids. USA 112, E2785-2794. The forkhead box (FOX) family of transcription factors has previously been shown to regulate metabolic phenotype in neonatal cardiomyocytes through a balance between FOXO and FOXM proteins. However, these approaches seem to be important for the maturation of hiPSC-CMs and should be considered when developing a maturation protocol. Efficient dissociation and re-plating of 3D hiPSC-CMs is a known problem as many cells do not survive post-dissociation. Cite this article. *P<0.05. sharing sensitive information, make sure youre on a federal 1a). : Conception and design, Supervision, Manuscript writing, Final Approval of Manuscript; C.P.H. Maroli, G. & Braun, T. The long and winding road of cardiomyocyte maturation. Current methods to generate cardiomyocytes from induced pluripotent stem cells (iPSc) utilize broad-spectrum pharmacological inhibitors. Strategies for Improving the Maturity of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Google Scholar. Wang G, McCain ML, Yang L, He A, Pasqualini FS, Agarwal A, Yuan H, Jiang D, Zhang D, Zangi L, Geva J, Roberts AE, Ma Q, Ding J, Chen J, Wang DZ, Li K, Wang J, Wanders RJ, Kulik W, Vaz FM, Laflamme MA, Murry CE, Chien KR, Kelley RI, Church GM, Parker KK, Pu WT. Chong JJ, Yang X, Don CW, Minami E, Liu YW, Weyers JJ, Mahoney WM, Van Biber B, Cook SM, Palpant NJ, Gantz JA, Fugate JA, Muskheli V, Gough GM, Vogel KW, Astley CA, Hotchkiss CE, Baldessari A, Pabon L, Reinecke H, Gill EA, Nelson V, Kiem HP, Laflamme MA, Murry CE. They then exposed multiple cardiac wires to electrical stimuli and stimulated cells displayed improved calcium handling properties, increased myofibril organization, and higher conduction velocity[47]. The authors created engineered tissue with the use of computational modelling and polydimethylsiloxane reservoirs to create passive stretch. Frontiers | Single-cell transcriptomic profiling reveals specific Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. First, many disease models require the use of single-cells to characterize disease phenotypes. Article It is therefore reasonable to expect that an additional maturation protocol (Figure (Figure2)2) will be necessary to generate cells that truly reflect the in vivo tissue. Coll. Transplanting human embryonic pluripotent stem cell derived-cardiomyocytes (hEPSC-CMs) through an intra-myocardial injection allowed the cells to graft with the host. Further, routine cell sorting may be required to ensure the cellular homogeneity of cultured organoids. Karakikes I, Ameen M, Termglinchan V, Wu JC. Burridge, P. W. et al. In contrast, hiPSC-CMs have lower conduction and upstroke velocities but, due to an increase in the pacemaker current If, are still able to beat spontaneously[2,22]. Culturing these cells in organoid formations could improve cell-cell contact and increase expression of various genes expressed in mature cardiomyocytes although the exact mechanism of maturation is yet to be elucidated. The importance of CASQ2 (calsequestrin 2), COX6A2 (cytochrome oxidase), S100A1 (calcium binding protein 1), SCN5A (sodium voltage-gated channel alpha subunit 5) and MYOM2/3 (myomesin-2/3) as markers of maturation has also been demonstrated[25]. One approach for the maturation of hiPSC-CMs involves the manipulation of growth conditions through the addition of small molecules or changes in culture medium. 2a). In addition, adult cardiomyocytes have sarcomeres that are long (2.2 m) and highly organized[22]. After a week post-seeding, they noticed spontaneous contractions. Indeed, we found that Sfrp2 induced robust cardiac differentiation. Sfrp2 induced cardiac differentiation. Res. These hiPSC-CMs are remarkably powerful as they replicate the genome of the patient donor and allow characterization of various diseases and drugs in a non-invasive manner[2]. https://doi.org/10.1016/j.yjmcc.2014.04.005 (2014). After a decade of research, iPSCs can now be successfully differentiated into hepatocytes[3], cardiomyocytes[4,5], neural cells[6,7], adipocytes[8] and many other cell types[9]. U.S.A. 118, 10. https://doi.org/10.1073/pnas.2103676118 (2021). 8600 Rockville Pike As mentioned, electrical and mechanical stimulation is not without its drawbacks. When compared to the standard protocol, as evidenced by more coverage across the cell, sarcomere structure was more mature under the Sfrp2 protocol (Fig. Since we have recently demonstrated that cardiomyogenesis in vitro and in vivo requires Sfrp2, we asked if Sfrp2 would drive differentiation of human iPSc into cardiomyocytes. This device offers electrical stimulation and mechanical stretch through the use of a control interface and special plates outfitted with electrodes. Sfrp2 induces cardiac differentiation and maturation potentially via -Catenin and Wnt3a. Will iPSC-cardiomyocytes revolutionize the discovery of drugs for heart Lian X, Zhang J, Azarin SM, Zhu K, Hazeltine LB, Bao X, Hsiao C, Kamp TJ, Palecek SP. Moreover, the addition of Wnt3a protein was sufficient to inhibit cardiomyocyte differentiation (Fig. iPSc-derived cardiomyocytes were removed from the tissue culture plate with TrypsinEDTA (Thermo Fisher Scientific, 0.25%) and fixed in a solution containing 1% formaldehyde (Thermo Fisher Scientific) and 90% methanol (Sigma). Importantly, the gap junctions were polarized between cardiomyocytes (Fig. After washing the membranes in TBS-0.1%Tween20, bands were visualized via a Amersham ECL Western Blotting Detection kit and signal detected via a Syngene G:BOX. They found that the tissue displaying a stretch of 7 mm resulted in the hiPSC-CMs showing increased expression of maturation genes involved in the troponin complexes along with potassium ion channels and T-tubule proteins[41]. Departments of Pediatrics and Cardiac Sciences, Alberta Childrens Hospital Research Institute, Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Calgary T2N 4N1, Canada, Departments of Pediatrics, Cardiac Sciences, Biochemistry & Molecular Biology, Alberta Childrens Hospital Research Institute, Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, University of Calgary, Calgary T2N 4N1, Canada.

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