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1
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Franco D, Sánchez-Fernández C, García-Padilla C, Lozano-Velasco E. Exploring the role non-coding RNAs during myocardial cell fate. Biochem Soc Trans 2024; 52:1339-1348. [PMID: 38775188 DOI: 10.1042/bst20231216] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2024] [Revised: 05/02/2024] [Accepted: 05/07/2024] [Indexed: 06/27/2024]
Abstract
Myocardial cell fate specification takes place during the early stages of heart development as the precardiac mesoderm is configured into two symmetrical sets of bilateral precursor cells. Molecular cues of the surrounding tissues specify and subsequently determine the early cardiomyocytes, that finally matured as the heart is completed at early postnatal stages. Over the last decade, we have greatly enhanced our understanding of the transcriptional regulation of cardiac development and thus of myocardial cell fate. The recent discovery of a novel layer of gene regulation by non-coding RNAs has flourished their implication in epigenetic, transcriptional and post-transcriptional regulation of cardiac development. In this review, we revised the current state-of-the-art knowledge on the functional role of non-coding RNAs during myocardial cell fate.
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Affiliation(s)
- Diego Franco
- Cardiovascular Development Group, Department of Experimental Biology, University of Jaen, Jaen 23071, Spain
- Fundación Medina, Granada, Spain
| | - Cristina Sánchez-Fernández
- Cardiovascular Development Group, Department of Experimental Biology, University of Jaen, Jaen 23071, Spain
- Fundación Medina, Granada, Spain
| | - Carlos García-Padilla
- Cardiovascular Development Group, Department of Experimental Biology, University of Jaen, Jaen 23071, Spain
- Fundación Medina, Granada, Spain
| | - Estefania Lozano-Velasco
- Cardiovascular Development Group, Department of Experimental Biology, University of Jaen, Jaen 23071, Spain
- Fundación Medina, Granada, Spain
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2
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Zhu Y, You J, Wei W, Gu J, Xu C, Gu X. Downregulated lncRNA RCPCD promotes differentiation of embryonic stem cells into cardiac pacemaker-like cells by suppressing HCN4 promoter methylation. Cell Death Dis 2021; 12:667. [PMID: 34215719 PMCID: PMC8253811 DOI: 10.1038/s41419-021-03949-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2020] [Revised: 06/16/2021] [Accepted: 06/16/2021] [Indexed: 12/25/2022]
Abstract
Long non-coding RNA (lncRNA) is receiving increasing attention in embryonic stem cells (ESCs) research. However, the roles of lncRNA in the differentiation of ESCs into pacemaker-like cells are still unclear. Therefore, the present study aims to explore the roles and mechanisms of lncRNA in the differentiation of ESCs into pacemaker-like cells. ESCs were cultured and induced differentiation to pacemaker-like cells. RNA sequencing was used to identify the differential expression lncRNAs during the differentiation of ESCs into pacemaker-like cells. Cell morphology observation, flow cytometry, quantitative real-time polymerase chain reaction, western blot, and immunofluorescence were used to detect the differentiation of ESCs into pacemaker-like cells. LncRNA and genes overexpression or knockdown through transfected adenovirus in the differentiation process. The fluorescence in situ hybridization (FISH) detected the lncRNA location in the differentiated ESCs. Luciferase reporter gene assay, methylation-specific PCR, chromatin immunoprecipitation assay, and RNA immunoprecipitation assay were performed to reveal the mechanism of lncRNA-regulating HCN4 expression. Rescue experiments were used to confirm that lncRNA regulates the differentiation of ESCs into pacemaker-like cells through HCN4. We cultured the ESCs and induced the differentiation of ESCs into pacemaker-like cells successfully. The expression of lncRNA RCPCD was significantly decreased in the differentiation of ESCs into pacemaker-like cells. Overexpression of RCPCD inhibited the differentiation of ESCs into pacemaker-like cells. RCPCD inhibited the expression of HCN4 by increasing HCN4 methylation at the promoter region through DNMT1, DNMT2, and DNMT3. RCPCD inhibited the differentiation of ESCs into pacemaker-like cells by inhibiting the expression of HCN4. Our results confirm the roles and mechanism of lncRNA RCPCD in the differentiation of ESCs into pacemaker-like cells, which could pave the path for the development of a cell-based biological pacemaker.
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Affiliation(s)
- Ye Zhu
- Clinical Medical College of Yangzhou University, Yangzhou, China. .,Department of Cardiology, Northern Jiangsu People's Hospital, Yangzhou, China.
| | - Jia You
- Department of Internal Medicine, Yangzhou Maternal and Child Health Care Hospital, Yangzhou, Jiangsu, 225001, China
| | - Wei Wei
- Clinical Medical College of Yangzhou University, Yangzhou, China.,Department of Cardiology, Northern Jiangsu People's Hospital, Yangzhou, China
| | - Jianjun Gu
- Clinical Medical College of Yangzhou University, Yangzhou, China.,Department of Cardiology, Northern Jiangsu People's Hospital, Yangzhou, China
| | - Chao Xu
- Department of Biostatistics and Epidemiology, University of Oklahoma Health Science Center, Oklahoma City, OK, 73104, US
| | - Xiang Gu
- Clinical Medical College of Yangzhou University, Yangzhou, China.,Department of Cardiology, Northern Jiangsu People's Hospital, Yangzhou, China
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3
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Zhang Y, Zhang L, Wang Y, Wang K. A Simulation Study on the Pacing and Driving of the Biological Pacemaker. BIOMED RESEARCH INTERNATIONAL 2020; 2020:4803172. [PMID: 32596315 PMCID: PMC7273435 DOI: 10.1155/2020/4803172] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/04/2019] [Revised: 02/05/2020] [Accepted: 02/20/2020] [Indexed: 11/30/2022]
Abstract
The research on the biological pacemaker has been very active in recent years. And turning nonautomatic ventricular cells into pacemaking cells is believed to hold the key to making a biological pacemaker. In the study, the inward-rectifier K+ current (I K1) is depressed to induce the automaticity of the ventricular myocyte, and then, the effects of the other membrane ion currents on the automaticity are analyzed. It is discovered that the L-type calcium current (I CaL) plays a major part in the rapid depolarization of the action potential (AP). A small enough I CaL would lead to the failure of the automaticity of the ventricular myocyte. Meanwhile, the background sodium current (I bNa), the background calcium current (I bCa), and the Na+/Ca2+ exchanger current (I NaCa) contribute significantly to the slow depolarization, indicating that these currents are the main supplementary power of the pacing induced by depressing I K1, while in the 2D simulation, we find that the weak electrical coupling plays a more important role in the driving of a biological pacemaker.
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Affiliation(s)
- Yue Zhang
- College of Computer Science and Technology, Harbin Engineering University, Harbin 150001, China
- School of Computer Science and Engineering, University of New South Wales, Sydney, NSW 2052, Australia
| | - Lei Zhang
- Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland, Baltimore, MD 21201, USA
| | - Yong Wang
- College of Computer Science and Technology, Harbin Engineering University, Harbin 150001, China
| | - Kuanquan Wang
- School of Computer Science and Technology, Harbin Institute of Technology, Harbin 150001, China
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4
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Lemme M, Ulmer BM, Lemoine MD, Zech ATL, Flenner F, Ravens U, Reichenspurner H, Rol-Garcia M, Smith G, Hansen A, Christ T, Eschenhagen T. Atrial-like Engineered Heart Tissue: An In Vitro Model of the Human Atrium. Stem Cell Reports 2018; 11:1378-1390. [PMID: 30416051 PMCID: PMC6294072 DOI: 10.1016/j.stemcr.2018.10.008] [Citation(s) in RCA: 115] [Impact Index Per Article: 16.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2018] [Revised: 10/09/2018] [Accepted: 10/10/2018] [Indexed: 12/20/2022] Open
Abstract
Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are under investigation for their suitability as human models in preclinical drug development. Antiarrhythmic drug development focuses on atrial biology for the treatment of atrial fibrillation. Here we used recent retinoic acid-based protocols to generate atrial CMs from hiPSCs and establish right atrial engineered heart tissue (RA-EHT) as a 3D model of human atrium. EHT from standard protocol-derived hiPSC-CMs (Ctrl-EHT) and intact human muscle strips served as comparators. RA-EHT exhibited higher mRNA and protein concentrations of atrial-selective markers, faster contraction kinetics, lower force generation, shorter action potential duration, and higher repolarization fraction than Ctrl-EHTs. In addition, RA-EHTs but not Ctrl-EHTs responded to pharmacological manipulation of atrial-selective potassium currents. RA- and Ctrl-EHTs’ behavior reflected differences between human atrial and ventricular muscle preparations. Taken together, RA-EHT is a model of human atrium that may be useful in preclinical drug screening.
Retinoic acid induced differentiation of hiPSCs into atrial-like myocytes 3D engineered heart tissue format favored atrial specificity compared with 2D culture Atrial-like engineered heart tissue can be used as a model of human atrium
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Affiliation(s)
- Marta Lemme
- Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany; Clyde Biosciences Ltd, BioCity Scotland, Bo'Ness Road, Newhouse, Lanarkshire ML1 5UH, UK
| | - Bärbel M Ulmer
- Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany
| | - Marc D Lemoine
- Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany; Department of Cardiology-Electrophysiology, University Heart Center, 20246 Hamburg, Germany
| | - Antonia T L Zech
- Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany
| | - Frederik Flenner
- Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany
| | - Ursula Ravens
- Institute of Experimental Cardiovascular Medicine, University Heart Center Freiburg-Bad Krozingen, 79106 Freiburg, Germany; Institute of Physiology, Medical Faculty Carl Gustav Carus, TU Dresden, 01307 Dresden, Germany
| | - Hermann Reichenspurner
- DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany; Department of Cardiovascular Surgery, University Heart Center, 20246 Hamburg, Germany
| | - Miriam Rol-Garcia
- Clyde Biosciences Ltd, BioCity Scotland, Bo'Ness Road, Newhouse, Lanarkshire ML1 5UH, UK
| | - Godfrey Smith
- Clyde Biosciences Ltd, BioCity Scotland, Bo'Ness Road, Newhouse, Lanarkshire ML1 5UH, UK
| | - Arne Hansen
- Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany
| | - Torsten Christ
- Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany
| | - Thomas Eschenhagen
- Institute of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany.
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5
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Dorn T, Goedel A, Lam JT, Haas J, Tian Q, Herrmann F, Bundschu K, Dobreva G, Schiemann M, Dirschinger R, Guo Y, Kühl SJ, Sinnecker D, Lipp P, Laugwitz KL, Kühl M, Moretti A. Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity. Stem Cells 2016; 33:1113-29. [PMID: 25524439 PMCID: PMC6750130 DOI: 10.1002/stem.1923] [Citation(s) in RCA: 67] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2014] [Revised: 10/29/2014] [Accepted: 11/08/2014] [Indexed: 12/31/2022]
Abstract
During cardiogenesis, most myocytes arise from cardiac progenitors expressing the transcription factors Isl1 and Nkx2-5. Here, we show that a direct repression of Isl1 by Nkx2-5 is necessary for proper development of the ventricular myocardial lineage. Overexpression of Nkx2-5 in mouse embryonic stem cells (ESCs) delayed specification of cardiac progenitors and inhibited expression of Isl1 and its downstream targets in Isl1(+) precursors. Embryos deficient for Nkx2-5 in the Isl1(+) lineage failed to downregulate Isl1 protein in cardiomyocytes of the heart tube. We demonstrated that Nkx2-5 directly binds to an Isl1 enhancer and represses Isl1 transcriptional activity. Furthermore, we showed that overexpression of Isl1 does not prevent cardiac differentiation of ESCs and in Xenopus laevis embryos. Instead, it leads to enhanced specification of cardiac progenitors, earlier cardiac differentiation, and increased cardiomyocyte number. Functional and molecular characterization of Isl1-overexpressing cardiomyocytes revealed higher beating frequencies in both ESC-derived contracting areas and Xenopus Isl1-gain-of-function hearts, which associated with upregulation of nodal-specific genes and downregulation of transcripts of working myocardium. Immunocytochemistry of cardiomyocyte lineage-specific markers demonstrated a reduction of ventricular cells and an increase of cells expressing the pacemaker channel Hcn4. Finally, optical action potential imaging of single cardiomyocytes combined with pharmacological approaches proved that Isl1 overexpression in ESCs resulted in normally electrophysiologically functional cells, highly enriched in the nodal subtype at the expense of the ventricular lineage. Our findings provide an Isl1/Nkx2-5-mediated mechanism that coordinately regulates the specification of cardiac progenitors toward the different myocardial lineages and ensures proper acquisition of myocyte subtype identity.
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Affiliation(s)
- Tatjana Dorn
- I. Medizinische Klinik und Poliklinik, Klinikum rechts der Isar der Technischen Universität München, Munich, Germany
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6
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Jin Y, Cao J, Xu X, Ye X, Chen Y, Yang J, Feng Q, Zhu L, Qian X, Yang C. Effects of C-Reactive Protein on the Cardiac Differentiation of Mouse Embryonic Stem Cells. Biol Pharm Bull 2015; 38:1361-7. [PMID: 26328491 DOI: 10.1248/bpb.b15-00311] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
A major challenge in stem cell therapy for cardiac repair is how to obtain normally functioning stem cell-derived cardiomyocytes. We aim to address the effects of C-reactive protein (CRP) on the cardiac differentiation of embryonic stem (ES) cells. Immunostaining, Western blotting and electrophysiology were employed. A hundred fifty milligran/liters CRP significantly reduced the percentage of cardiomyocytes differentiated from mouse ES cells, while it may also promote sarcomere development compared to 30 mg/L CRP treatment. Further examination of the action potential (AP) in individual ES cell-derived cardiomyocytes showed that there exist three types of cardiomyocytes: artial-like (A-like), ventricular-like (V-like), and pacemaker-like (P-like). A hundred fifty milligran/liters CRP treatment decreased the P-like cardiomyocytes, whereas it increased the A-like. Such inhibitory effect and alteration were not significant at 30 mg/L CRP treatment. Moreover, 150 mg/L CRP significantly increased the APD90 (90% of duration of AP) and decreased the spontaneous firing rate of AP in P-like cells, while had little effect on other electrophysiological characteristics, including APA (AP amplitude) and MDP (maximum diastolic potential). This study revealed the effect of CRP on the cardiac differentiation of ES cells. It provides an in vitro pathological model and may be of importance to the future work of ES cell-based therapy in clinical applications and in vivo pathological studies.
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Affiliation(s)
- Yan Jin
- Department of Cardiology, Nanjing Medical University Affiliated Wuxi Second Hospital
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7
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Dell’Era P, Benzoni P, Crescini E, Valle M, Xia E, Consiglio A, Memo M. Cardiac disease modeling using induced pluripotent stem cell-derived human cardiomyocytes. World J Stem Cells 2015; 7:329-342. [PMID: 25815118 PMCID: PMC4369490 DOI: 10.4252/wjsc.v7.i2.329] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2014] [Revised: 10/27/2014] [Accepted: 11/19/2014] [Indexed: 02/06/2023] Open
Abstract
Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alteration has been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes (CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells (iPSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since iPSC preserve the entire patients’ genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with iPSC.
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8
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SHOX2 overexpression favors differentiation of embryonic stem cells into cardiac pacemaker cells, improving biological pacing ability. Stem Cell Reports 2014; 4:129-142. [PMID: 25533636 PMCID: PMC4297875 DOI: 10.1016/j.stemcr.2014.11.004] [Citation(s) in RCA: 88] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2014] [Revised: 11/20/2014] [Accepted: 11/20/2014] [Indexed: 01/01/2023] Open
Abstract
When pluripotency factors are removed, embryonic stem cells (ESCs) undergo spontaneous differentiation, which, among other lineages, also gives rise to cardiac sublineages, including chamber cardiomyocytes and pacemaker cells. Such heterogeneity complicates the use of ESC-derived heart cells in therapeutic and diagnostic applications. We sought to direct ESCs to differentiate specifically into cardiac pacemaker cells by overexpressing a transcription factor critical for embryonic patterning of the native cardiac pacemaker (the sinoatrial node). Overexpression of SHOX2 during ESC differentiation upregulated the pacemaker gene program, resulting in enhanced automaticity in vitro and induced biological pacing upon transplantation in vivo. The accentuated automaticity is accompanied by temporally evolving changes in the effectors and regulators of Wnt signaling. Our findings provide a strategy for enriching the cardiac pacemaker cell population from ESCs.
SHOX2 accentuates the molecular profile of pacemaker cells in differentiating ESCs SHOX2 increases the frequency and rate of spontaneously active cardiac derivatives SHOX2-overexpressing EBs function as biopacemakers when transplanted in vivo Wnt signaling underlies SHOX2-mediated pacemaker cell specification
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Sa S, Wong L, McCloskey KE. Combinatorial fibronectin and laminin signaling promote highly efficient cardiac differentiation of human embryonic stem cells. Biores Open Access 2014; 3:150-61. [PMID: 25126479 PMCID: PMC4120929 DOI: 10.1089/biores.2014.0018] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Cardiomyocytes (CMs) differentiated from human embryonic stem cells (hESCs) are a promising and potentially unlimited cell source for myocardial repair and regeneration. Recently, multiple methodologies-primarily based on the optimization of growth factors-have been described for efficient cardiac differentiation of hESCs. However, the role of extracellular matrix (ECM) signaling in CM differentiation has not yet been explored fully. This study examined the role of ECM signaling in the efficient generation of CMs from both H7 and H9 ESCs. The hESCs were differentiated on ECM substrates composed of a range of fibronectin (FN) and laminin (LN) ratios and gelatin and evaluated by the fluorescence activated cell scanning (FACS) analysis on day 14. Of the ECM substrates examined, the 70:30 FN:LN reproducibly generated the greatest numbers of CMs from both hESC lines. Moreover, the LN receptor integrin β4 (ITGB4) and FN receptor integrin β5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin. Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway. Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4(+) and ITGB5(+) cells.
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Affiliation(s)
- Silin Sa
- Graduate Group in Biological Engineering & Small-Scale Technologies, University of California, Merced, California
| | - Lian Wong
- Graduate Group in Biological Engineering & Small-Scale Technologies, University of California, Merced, California
| | - Kara E. McCloskey
- Graduate Group in Biological Engineering & Small-Scale Technologies, University of California, Merced, California
- School of Engineering, University of California, Merced, California
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10
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Hashem SI, Lam ML, Mihardja SS, White SM, Lee RJ, Claycomb WC. Shox2 regulates the pacemaker gene program in embryoid bodies. Stem Cells Dev 2013; 22:2915-26. [PMID: 23767866 DOI: 10.1089/scd.2013.0123] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
The pacemaker tissues of the heart are a complex set of specialized cells that initiate the rhythmic heartbeat. The sinoatrial node (SAN) serves as the primary pacemaker, whereas the atrioventricular node can serve as a subsidiary pacemaker in cases of SAN failure or block. The elucidation of genetic networks regulating the development of these tissues is crucial for understanding the mechanisms underlying arrhythmias and for the design of targeted therapies. Here we report temporal and spatial self-organized formation of the pacemaker and contracting tissues in three-dimensional aggregate cultures of mouse embryonic stem cells termed embryoid bodies (EBs). Using genetic marker expression and electrophysiological analyses we demonstrate that in EBs the pacemaker potential originates from a localized population of cells and propagates into the adjacent contracting region forming a functional syncytium. When Shox2, a major determinant of the SAN genetic pathway, was ablated we observed substantial slowing of spontaneous contraction rates and an altered gene expression pattern including downregulation of HCN4, Cx45, Tbx2, Tbx3, and bone morphogenetic protein 4 (BMP4); and upregulation of Cx40, Cx43, Nkx2.5, and Tbx5. This phenotype could be rescued by adding BMP4 to Shox2 knockout EBs in culture from days 6 to 16 of differentiation. When wild-type EBs were treated with Noggin, a potent BMP4 inhibitor, we observed a phenotype consistent with the Shox2 knockout EB. Altogether, we have generated a reproducible in vitro model that will be an invaluable tool for studying the molecular pathways regulating the development of cardiac pacemaker tissues.
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Affiliation(s)
- Sherin I Hashem
- 1 Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center , New Orleans, Louisiana
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11
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He JQ, January CT, Thomson JA, Kamp TJ. Human embryonic stem cell-derived cardiomyocytes: drug discovery and safety pharmacology. Expert Opin Drug Discov 2013; 2:739-53. [PMID: 23488962 DOI: 10.1517/17460441.2.5.739] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Human embryonic stem cells (hESCs) can provide potentially unlimited quantities of a wide range of human cell types that can be used in drug discovery and development, basic research and regenerative medicine. In this review, the authors describe the differentiation of hESCs into cardiomyocytes and outline the properties of hESC-derived cardiomyocytes (hESC-CMs), including their cardiac-type action potentials and contractile characteristics. In vitro cellular assays using hESC-CMs, which can be genetically engineered to create target-specific reporters as well as human disease models, will have applications at multiple stages of the drug discovery process. Furthermore, cardiac safety pharmacology assays evaluating the risk of proarrhythmic side effects associated with QT prolongation may be enhanced in their predictive value with the use of hESC-CMs.
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Affiliation(s)
- Jia-Qiang He
- Cellular Dynamics International, Inc., 525 Science Drive, Suite 200, Madison, WI 53711, USA +1 608 263 4856 ; +1 608 263 0405 ;
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12
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Malan D, Fleischmann BK. Functional expression and modulation of the L-type Ca2+ current in embryonic heart cells. Pediatr Cardiol 2012; 33:907-15. [PMID: 22639002 DOI: 10.1007/s00246-012-0360-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/19/2012] [Accepted: 04/30/2012] [Indexed: 11/28/2022]
Abstract
Voltage-dependent L-type Ca2+ channels (VDCCs) are critically involved in excitation contraction coupling and regulation of the force of contraction. An important mechanism contributing to the adaptation of heart function is modulation of the L-type Ca2+ current (I(Ca-L)) by hormones of the autonomous nervous system. The signaling pathways underlying this regulation in the adult heart are well understood. However, VDCC expression and its regulation in the embryonic heart are less understood. This report therefore provides a short overview of the current knowledge on this topic using embryonic stem cells and the mouse as model systems.
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Affiliation(s)
- Daniela Malan
- Institute of Physiology I, Life and Brain Center, University of Bonn, Sigmund-Freud-Strasse 25, 53105, Bonn, Germany.
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13
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Lu TY, Yang L. Uses of cardiomyocytes generated from induced pluripotent stem cells. Stem Cell Res Ther 2011; 2:44. [PMID: 22099214 PMCID: PMC3340553 DOI: 10.1186/scrt85] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Affiliation(s)
- Tung-Ying Lu
- Department of Developmental Biology, University of Pittsburgh, Rangos Research Center, Pittsburgh, PA 15201, USA
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14
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Young DA, DeQuach JA, Christman KL. Human cardiomyogenesis and the need for systems biology analysis. WILEY INTERDISCIPLINARY REVIEWS-SYSTEMS BIOLOGY AND MEDICINE 2010; 3:666-80. [PMID: 21197666 DOI: 10.1002/wsbm.141] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Cardiovascular disease remains the leading cause of death in the Western world and myocardial infarction is one of the primary facets of this disease. The limited natural self-renewal of cardiac muscle following injury and restricted supply of heart transplants has encouraged researchers to investigate other means to stimulate regeneration of damaged myocardium. The plasticity of stem cells toward multiple lineages offers the potential to repair the heart following injury. Embryonic stem cells have been extensively studied for their ability to differentiate into early cardiomyocytes, however, the pathway has only been partially defined and inadequate efficiency limits their clinical applicability. Some studies have shown cardiomyogenesis from adult mesenchymal stem cells, from both bone marrow and adipose tissue, but their differentiation pathway remains poorly detailed and these results remain controversial. Despite promising results using stem cells in animal models of cardiac injury, the driving mechanisms behind their differentiation down a cardiomyogenic pathway have yet to be determined. Currently, there is a paucity of information regarding cardiomyogenesis on the systemic level. Stem cell differentiation results from multiple signaling parameters operating in a tightly regulated spatiotemporal pattern. Investigating this phenomenon from a systems biology perspective could unveil the abstruse mechanisms controlling cardiomyogenesis that would otherwise require extensive in vitro testing.
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Affiliation(s)
- D Adam Young
- Department of Bioengineering, University of California, San Diego, CA, USA
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15
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zur Nieden NI, Davis LA, Rancourt DE. Comparing three novel endpoints for developmental osteotoxicity in the embryonic stem cell test. Toxicol Appl Pharmacol 2010; 247:91-7. [DOI: 10.1016/j.taap.2010.05.010] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2010] [Revised: 04/21/2010] [Accepted: 05/15/2010] [Indexed: 01/05/2023]
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16
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Abstract
Bradyarrhythmias are common and may be caused by sinus node dysfunction or conduction block. Many of these conditions can be treated by the implantation of electronic cardiac pacemakers that reduce mortality and morbidity in carefully selected patient groups. Implantable electronic pacemakers are small, sophisticated and reliable but not without complication and limitation. Efforts have been made to create a de novo sinus node using gene therapy, the so-called biopacemaker. This approach has potential as permanent cure for bradyarrythmias with greater physiological responsiveness than that provided by rate-responsive electronic pacemakers. This article reviews the current approaches to the problem and gives a perspective on the challenges remaining to bring the therapy to clinical practice.
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Affiliation(s)
- Gwilym M. Morris
- Cardiovascular Medicine, School of Medicine, University of Manchester, Manchester, UK,
| | - Mark R. Boyett
- Mark R. Boyett Cardiovascular Medicine, School of Medicine, University of Manchester, Manchester, UK
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17
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Christoforou N, Miller RA, Hill CM, Jie CC, McCallion AS, Gearhart JD. Mouse ES cell-derived cardiac precursor cells are multipotent and facilitate identification of novel cardiac genes. J Clin Invest 2008; 118:894-903. [PMID: 18246200 DOI: 10.1172/jci33942] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2007] [Accepted: 11/28/2007] [Indexed: 01/13/2023] Open
Abstract
Although the differentiation of ES cells to cardiomyocytes has been firmly established, the extent to which corresponding cardiac precursor cells can contribute to other cardiac populations remains unclear. To determine the molecular and cellular characteristics of cardiac-fated populations derived from mouse ES (mES) cells, we isolated cardiac progenitor cells (CPCs) from differentiating mES cell cultures by using a reporter cell line that expresses GFP under the control of a cardiac-specific enhancer element of Nkx2-5, a transcription factor expressed early in cardiac development. This ES cell-derived CPC population initially expressed genetic markers of both stem cells and mesoderm, while differentiated CPCs displayed markers of 3 distinct cell lineages (cardiomyocytes, vascular smooth muscle cells, and endothelial cells)--Flk1 (also known as Kdr), c-Kit, and Nkx2-5, but not Brachyury--and subsequently expressed Isl1. Clonally derived CPCs also demonstrated this multipotent phenotype. By transcription profiling of CPCs, we found that mES cell-derived CPCs displayed a transcriptional signature that paralleled in vivo cardiac development. Additionally, these studies suggested the involvement of genes that we believe were previously unknown to play a role in cardiac development. Taken together, our data demonstrate that ES cell-derived CPCs comprise a multipotent precursor population capable of populating multiple cardiac lineages and suggest that ES cell differentiation is a valid model for studying development of multiple cardiac-fated tissues.
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Affiliation(s)
- Nicolas Christoforou
- Institute for Cell Engineering, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland 21205, USA
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18
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Sauer H, Ruhe C, Müller JP, Schmelter M, D'Souza R, Wartenberg M. Reactive oxygen species and upregulation of NADPH oxidases in mechanotransduction of embryonic stem cells. Methods Mol Biol 2008; 477:397-418. [PMID: 19082963 DOI: 10.1007/978-1-60327-517-0_30] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
Deciphering the differentiation pathway of embryonic stem (ES) cells is a challenging task not only for basic research, but also for clinicians who intend to use ES cells for cell transplantation approaches. We have shown that reactive oxygen species (ROS) play a primordial role in the differentiation of mouse ES cells toward the cardiovascular cell lineage. During differentiation, ES cells robustly generate ROS, which interfere with signaling pathways that direct cardiac and vascular commitment. Differentiating ES cells expression of Nox-1, Nox-2, and Nox-4 has been demonstrated. We have shown that mechanical strain application to embyoid bodies grown from ES cells initiates the cardiovascular differentiation program. Under these conditions, a burst of ROS generation occurs which is followed by induction of Nox-1 and Nox-4 and a feed-forward upregulation of ROS production.
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Affiliation(s)
- Heinrich Sauer
- Department of Physiology, Justus Liebig University, Giessen, Germany
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19
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Guan K, Wagner S, Unsöld B, Maier LS, Kaiser D, Hemmerlein B, Nayernia K, Engel W, Hasenfuss G. Generation of Functional Cardiomyocytes From Adult Mouse Spermatogonial Stem Cells. Circ Res 2007; 100:1615-25. [PMID: 17478732 DOI: 10.1161/01.res.0000269182.22798.d9] [Citation(s) in RCA: 110] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Stem cell–based therapy is a promising approach for the treatment of heart failure. Adult stem cells with the pluripotency of embryonic stem cells (ESCs) would be an ideal cell source. Recently, we reported the successful establishment of multipotent adult germline stem cells (maGSCs) from mouse testis. These cultured maGSCs show phenotypic characteristics similar to ESCs and can spontaneously differentiate into cells from all 3 germ layers. In the present study, we used the hanging drop method to differentiate maGSCs into cardiomyocytes and analyzed their functional properties. Differentiation efficiency of beating cardiomyocytes from maGSCs was similar to that from ESCs. The maGSC-derived cardiomyocytes expressed cardiac-specific L-type Ca
2+
channels and responded to Ca
2+
channel–modulating drugs. Cx43 was expressed at cell-to-cell contacts in cardiac clusters, and fluorescence recovery after photobleaching assay showed the presence of functional gap junctions among cardiomyocytes. Action potential analyses demonstrated the presence of pacemaker-, ventricle-, atrial-, and Purkinje-like cardiomyocytes. Stimulation with isoproterenol resulted in a significant increase in beating frequency, whereas the addition of cadmium chloride abolished spontaneous electrical activity. Confocal microscopy analysis of intracellular Ca
2+
in maGSC-derived cardiomyocytes showed that calcium increased periodically throughout the cell in a homogenous fashion, pointing to a fine regulated Ca
2+
release from intracellular Ca
2+
stores. By using line-scan mode, we found rhythmic Ca
2+
transients. Furthermore, we transplanted maGSCs into normal hearts of mice and found that maGSCs were able to proliferate and differentiate. No tumor formation was found up to 1 month after cell transplantation. Taken together, we believe that maGSCs provide a new source of distinct types of cardiomyocytes for basic research and potential therapeutic application.
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Affiliation(s)
- Kaomei Guan
- Department of Cardiology and Pneumology, Georg-August-University of Göttingen, Göttingen, Germany
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20
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Buggisch M, Ateghang B, Ruhe C, Strobel C, Lange S, Wartenberg M, Sauer H. Stimulation of ES-cell-derived cardiomyogenesis and neonatal cardiac cell proliferation by reactive oxygen species and NADPH oxidase. J Cell Sci 2007; 120:885-94. [PMID: 17298980 DOI: 10.1242/jcs.03386] [Citation(s) in RCA: 146] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
After birth the proliferation of cardiac cells declines, and further growth of the heart occurs by hypertrophic cell growth. In the present study the cell proliferation capacity of mouse embryonic stem (ES) cells versus neonatal cardiomyocytes and the effects of reactive oxygen species (ROS) on cardiomyogenesis and cardiac cell proliferation of ES cells was investigated. Low levels of hydrogen peroxide stimulated cardiomyogenesis of ES cells and induced proliferation of cardiomyocytes derived from ES cells and neonatal mice, as investigated by nuclear translocation of cyclin D1, downregulation of p27(Kip1), phosphorylation of retinoblastoma (Rb), increase of Ki-67 expression and incorporation of BrdU. The observed effects were blunted by the free radical scavengers vitamin E and 2-mercaptoglycin (NMPG). In ES cells ROS induced expression of the cardiac-specific genes encoding alpha-actin, beta-MHC, MLC2a, MLC2v and ANP as well as the transcription factors GATA-4, Nkx-2.5, MEF2C, DTEF-1 and the growth factor BMP-10. During differentiation ES cells expressed the NADPH oxidase isoforms Nox-1, Nox-2 and Nox-4. Treatment of cardiac cells with ROS increased Nox-1, Nox-4, p22-phox, p47-phox and p67-phox proteins as well as Nox-1 and Nox-4 mRNA, indicating feed-forward regulation of ROS generation. Inhibition of NADPH oxidase with diphenylen iodonium chloride (DPI) and apocynin abolished ROS-induced cardiomyogenesis of ES cells. Our data suggest that proliferation of neonatal and ES-cell-derived cardiac cells involves ROS-mediated signalling cascades and point towards an involvement of NADPH oxidase in cardiovascular differentiation of ES cells.
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Affiliation(s)
- Martina Buggisch
- Department of Physiology, Justus-Liebig-University Giessen, Aulweg 129, 35392 Giessen, Germany
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21
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Marbán E, Cho HC. Creation of a biological pacemaker by gene- or cell-based approaches. Med Biol Eng Comput 2007; 45:133-44. [PMID: 17262203 DOI: 10.1007/s11517-007-0165-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2006] [Accepted: 01/06/2007] [Indexed: 02/06/2023]
Abstract
Cardiac rhythm-associated disorders are caused by mal-functions of impulse generation and conduction. Present therapies for the impulse generation span a wide array of approaches but remain largely palliative. The progress in the understanding of the biology of the diseases with related biological tools beckons for new approaches to provide better alternatives to the present routine. Here, we review the current state of the art in gene- and cell-based approaches to correct cardiac rhythm disturbances. These include genetic suppression of an ionic current, stem cell therapies, adult somatic cell-fusion approach, novel synthetic pacemaker channel, and creating a self-contained pacemaker activity in non-excitable cells. We then conclude by discussing advantages and disadvantages of the new possibilities.
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Affiliation(s)
- Eduardo Marbán
- Institute of Molecular Cardiobiology, Division of Cardiology, Johns Hopkins University School of Medicine, 858 Ross Bldg, Baltimore, MD 21205, USA.
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22
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Sugawara T, Matsubara K, Akagi R, Mori M, Hirata T. Antiangiogenic activity of brown algae fucoxanthin and its deacetylated product, fucoxanthinol. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2006; 54:9805-10. [PMID: 17177505 DOI: 10.1021/jf062204q] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/13/2023]
Abstract
The antiangiogenic effects of fucoxanthin and a deacetylated product, fucoxanthinol, were examined. Fucoxanthin significantly suppressed HUVEC proliferation and tube formation at more than 10 microM, but it had no significant effect on HUVEC chemotaxis. The formation of blood vessel-like structures from CD31-positive cells was evaluated using embryonic stem cell-derived embryoid bodies. Fucoxanthin effectively suppressed the development of these structures at 10-20 microM, suggesting that it could suppress differentiation of endothelial progenitor cells into endothelial cells involving new blood vessel formation. Fucoxanthin and fucoxanthinol suppressed microvessel outgrowth in an ex vivo angiogenesis assay using a rat aortic ring, in a dose-dependent manner. These results imply that fucoxanthin having antiangiogenic activity might be useful in preventing angiogenesis-related diseases.
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Affiliation(s)
- Tatsuya Sugawara
- Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
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23
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Abstract
Coronary artery disease (CAD) remains the leading cause of death in the Western world. The high impact of its main sequelae, acute myocardial infarction and congestive heart failure (CHF), on the quality of life of patients and the cost of health care drives the search for new therapies. The recent finding that stem cells contribute to neovascularization and possibly improve cardiac function after myocardial infarction makes stem cell therapy the most highly active research area in cardiology. Although the concept of stem cell therapy may revolutionize heart failure treatment, several obstacles need to be addressed. To name a few: 1) Which patient population should be considered for stem cell therapy? 2) What type of stem cell should be used? 3) What is the best route for cell delivery? 4) What is the optimum number of cells that should be used to achieve functional effects? 5) Is stem cell therapy safer and more effective than conventional therapies? The published studies vary significantly in design, making it difficult to draw conclusions on the efficacy of this treatment. For example, different models of ischemia, species of donors and recipients, techniques of cell delivery, cell types, cell numbers and timing of the experiments have been used. However, these studies highlight the landmark concept that stem cell therapy may play a major role in treating cardiovascular diseases in the near future. It should be noted that stem cell therapy is not limited to the treatment of ischemic cardiac disease. Non-ischemic cardiomyopathy, peripheral vascular disease, and aging may be treated by stem cells. Stem cells could be used as vehicle for gene therapy and eliminate the use of viral vectors. Finally, stem cell therapy may be combined with pharmacological, surgical, and interventional therapy to improve outcome. Here we attempt a systematic overview of the science of stem cells and their effects when transplanted into ischemic myocardium.
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Affiliation(s)
- Julia N E Sunkomat
- University of Arizona Sarver Heart Center, Section of Cardiology, Tucson, AZ 85624, USA
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24
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White SM, Claycomb WC. Embryonic stem cells form an organized, functional cardiac conduction system in vitro. Am J Physiol Heart Circ Physiol 2005; 288:H670-9. [PMID: 15471973 DOI: 10.1152/ajpheart.00841.2004] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
A functional pacemaking-conduction system is essential for maintaining normal cardiac function. However, no reproducible model system exists for studying the specialized cardiac pacemaking-conduction system in vitro. Although several molecular markers have been shown to delineate components of the cardiac conduction system in vivo, the functional characteristics of the cells expressing these markers remain unknown. The ability to accurately identify cells that function as cardiac pacemaking cells is crucial for being able to study their molecular phenotype. In differentiating murine embryonic stem cells, we demonstrate the development of an organized cardiac pacemaking-conduction system in vitro using the coexpression of the minK-lacZ transgene and the chicken GATA6 (cGATA6) enhancer. These markers identify clusters of pacemaking “nodes” that are functionally coupled with adjacent contracting regions. cGATA6-positive cell clusters spontaneously depolarize, emitting calcium signals to surrounding contracting regions. Physically separating cGATA6-positive cells from nearby contracting regions reduces the rate of spontaneous contraction or abolishes them altogether. cGATA6/ minK copositive cells isolated from embryoid cells display characteristics of specialized pacemaking-conducting cardiac myocytes with regard to morphology, action potential waveform, and expression of a hyperpolarization-activated depolarizing current. Using the cGATA6 enhancer, we have isolated cells that exhibit electrophysiological and genetic properties of cardiac pacemaking myocytes. Using molecular markers, we have generated a novel model system that can be used to study the functional properties of an organized pacemaking-conducting contracting system in vitro. Moreover, we have used a molecular marker to isolate a renewable population of cells that exhibit characteristics of cardiac pacemaking myocytes.
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Affiliation(s)
- Steven M White
- Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA
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25
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Winkler J, Hescheler J, Sachinidis A. Embryonic stem cells for basic research and potential clinical applications in cardiology. Biochim Biophys Acta Mol Basis Dis 2004; 1740:240-8. [PMID: 15949691 DOI: 10.1016/j.bbadis.2004.11.018] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2004] [Revised: 11/09/2004] [Accepted: 11/22/2004] [Indexed: 11/29/2022]
Abstract
Embryonic stem (ES) cells are pluripotent, possessing the unique property to differentiate into any somatic cell type while retaining the ability to proliferate indefinitely. Due to their ability to recapitulate embryonic differentiation, ES cells are an ideal tool to study the process of early embryogenesis in vitro. Signalling cascades and genes involved in differentiation can be easily studied, and functional genomics approaches aim to identify the regulatory networks underlying lineage commitment. Their unique ability to differentiate into any cell type make ES cells a prime candidate for cell replacement therapy (CRT) of various degenerative disorders. Results from various disease models are promising and have demonstrated their principal suitability as a therapeutic agent in diseases such as myocardial infarctions, diabetes mellitus and Parkinson's disease. Prior to clinical trials in humans, two issues remain to be solved: due to their high proliferative potential, ES cells can form teratocarcinomas in the recipient, and depending on the source of the cells, ES cell grafts may be rejected by the host organism. This review discusses the current state of basic ES cell research with a focus on cardiac differentiation and gives an overview of their use in CRT approaches.
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Affiliation(s)
- Johannes Winkler
- Centre of Physiology and Pathophysiology, Institute of Neurophysiology, Robert-Koch-Str. 39, 50931 Cologne, Germany
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26
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Krupnick AS, Balsara KR, Kreisel D, Riha M, Gelman AE, Estives MS, Amin KM, Rosengard BR, Flake AW. Fetal Liver as a Source of Autologous Progenitor Cells for Perinatal Tissue Engineering. ACTA ACUST UNITED AC 2004; 10:723-35. [PMID: 15265289 DOI: 10.1089/1076327041348419] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Mesenchymal progenitor cells, isolated from adult bone marrow, have been shown to have utility for autologous tissue engineering. The possibility of isolating from the fetal hematopoietic system a cell population with similar potential, which could be used for autologous reconstruction of prenatally diagnosed congenital anomalies, has not been explored to date. Liver stromal cells isolated from a portion of the right lateral hepatic lobe of midgestation fetal lambs were expanded in vitro. Passage 1 cells displayed a uniform fibroblast-like morphology but could be induced to differentiate into skeletal muscle, adipocytes, chondrocytes, and endothelial cells by selective medium supplementation. By manipulating the extracellular matrix in vitro, spontaneously contracting cardiac myocyte-like cells could be generated as well. Multilineage differentiation was confirmed by morphology, protein expression, and upregulation of lineage-specific mRNA. The potential for engineering myocardial tissue was then investigated by transplanting early-passage progenitor cells, organized on a three-dimensional matrix, into the ventricle of an immunocompromised rat utilizing a previously described model of left ventricular tissue engineering. Survival, incorporation into the host myocardium, and cardiomyocytic differentiation of the transplanted cells were confirmed. We have demonstrated that mesenchymal progenitor cells with multilineage potential can be isolated from the fetal liver and have potential utility for autologous tissue engineering.
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Affiliation(s)
- Alexander S Krupnick
- Harrison's Department of Surgical Research, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, USA
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27
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Abstract
The study of embryo stem cells began in 1963, initially using disaggregates of cleaving rabbit and mouse embryos. Their differentiation in vitro was modest, and usually curtailed at best to the formation of trophectoderm cells, which attached to plastic. Rabbit morulae and blastocysts adhered more readily, trophectoderm forming a sheet of cells which was overgrown by stem cells from inner cell mass. Whole-blastocyst cultures on collagen-coated surfaces produced a pile of cells, and its outgrowths included neural, blood, neuronal, phagocytic and many other types of cell. When inner cell mass was freed and cultured intact or as cell disaggregates, lines of embryo stem cells (ES) were established which possessed good rates of cleavage, and immense stability in their secretion of enzymes, morphology and chromosomal complement. Developmental capacities of single mouse embryo stem cells were measured by injecting one or more into a recipient blastocyst, and extent of colonization in resulting chimaeras measured their pluripotency. In mouse, cell clumps were termed embryoid bodies, which produced similar outgrowths as in rabbit. Component cells again differentiated widely, depending to a limited extent on their exposure to various cytokines or substrates. Markers for differentiation or pluripotency were established, which revealed how neural, cardiac, haematological and other ES lines could be established in vitro. These have proved useful to study early differentiation and their use in grafting to sick recipients. Displaying similar properties, human ES cells emerged in the late 1990s. Models for the clinical use of ES cells showed how they colonized rapidly, travelled to target tissues via fetal pathways, differentiated and colonized target organs. No signs of inflammation or tissue damage were noted; injured tissues could be repaired including remyelination, and no cancers were formed. ES cells offer wide therapeutic potentials for humans, although extensive clinical trials are still awaited.
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Affiliation(s)
- R G Edwards
- Reproductive BioMedicine Online, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK.
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28
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Zhang YM, Shang L, Hartzell C, Narlow M, Cribbs L, Dudley SC. Characterization and regulation of T-type Ca2+ channels in embryonic stem cell-derived cardiomyocytes. Am J Physiol Heart Circ Physiol 2003; 285:H2770-9. [PMID: 12919937 DOI: 10.1152/ajpheart.01114.2002] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
T-type Ca2+ channels may play a role in cardiac development. We studied the developmental regulation of the T-type currents (ICa,T) in cardiomyocytes (CMs) derived from mouse embryonic stem cells (ESCs). ICa,T was studied in isolated CMs by whole cell patch clamp. Subsequently, CMs were identified by the myosin light chain 2v-driven green fluorescent protein expression, and laser capture microdissection was used to isolate total RNA from groups of cells at various developmental time points. ICa,T showed characteristics of Cav3.1, such as resistance to Ni2+ block, and a transient increase during development, correlating with measures of spontaneous electrical activity. Real-time RT-PCR showed that Cav3.1 mRNA abundance correlated (r2 = 0.81) with ICa,T. The mRNA copy number was low at 7+4 days (2 copies/cell), increased significantly by 7+10 days (27/cell; P < 0.01), peaked at 7+16 days (174/cell), and declined significantly at 7+27 days (25/cell). These data suggest that ICa,T is developmentally regulated at the level of mRNA abundance and that this regulation parallels measures of pacemaker activity, suggesting that ICa,T might play a role in the spontaneous contractions during CM development.
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Affiliation(s)
- Ying Ming Zhang
- Division of Cardiology, Atlanta Veterans Affairs Medical Center, Decatur, GA 30033, USA
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29
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Phillips BW, Vernochet C, Dani C. Differentiation of embryonic stem cells for pharmacological studies on adipose cells. Pharmacol Res 2003; 47:263-8. [PMID: 12644382 DOI: 10.1016/s1043-6618(03)00035-5] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
The ongoing global explosion in the incidence of obesity has focused attention on the development of adipose cells. Severe obesity is the result of an increase in fat cell size in combination with increased fat cell number. New fat cells arise from a pre-existing pool of adipose stem cells that are present irrespective of age. The development of established preadipocyte cell lines has facilitated the study of different steps leading to terminal differentiation. However, these systems are limited for studying early events of differentiation as they represent cells which are already determined for the adipogenic lineage. In vitro differentiation of mouse embryonic stem (ES) cells towards the adipogenic lineage provides an alternative source of adipocytes for study in tissue culture and offers the possibility to investigate regulation of the first steps of adipose cell development. In this review, we describe the sequential requirement of retinoic acid and PPARgamma during adipogenesis in ES cells. Stimulation of ES cells with synthetic retinoids which are selective ligands of the retinoic acid receptor isotypes allowed the investigation of the contribution of the different retinoic receptors on the RA-dependent differentiation. The effects of thiazolidinediones, a new class of pharmacological agents used for the treatment of type 2 diabetes, and of statins, drugs used in therapy for lowering cholesterol, on the differentiation of ES cells into adipocytes or osteoblasts are described. Finally, we propose a model in which PPARgamma plays a key role in the decision of stem cells to undergo differentiation into adipocytes or osteoblasts, two closely related lineages.
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Affiliation(s)
- Blaine W Phillips
- Institute of Signalling, Developmental Biology and Cancer Research, CNRS UMR 6543, Centre de Biochimie, 06108 Nice Cedex 2, France
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30
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He Q, Li J, Bettiol E, Jaconi ME. Embryonic stem cells: new possible therapy for degenerative diseases that affect elderly people. J Gerontol A Biol Sci Med Sci 2003; 58:279-87. [PMID: 12634295 DOI: 10.1093/gerona/58.3.m279] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
The capacity of embryonic stem (ES) cells for virtually unlimited self renewal and differentiation has opened up the prospect of widespread applications in biomedical research and regenerative medicine. The use of these cells would overcome the problems of donor tissue shortage and implant rejection, if the cells are made immunocompatible with the recipient. Since the derivation in 1998 of human ES cell lines from preimplantation embryos, considerable research is centered on their biology, on how differentiation can be encouraged toward particular cell lineages, and also on the means to enrich and purify derivative cell types. In addition, ES cells may be used as an in vitro system not only to study cell differentiation but also to evaluate the effects of new drugs and the identification of genes as potential therapeutic targets. This review will summarize what is known about animal and human ES cells with particular emphasis on their application in four animal models of human diseases. Present studies of mouse ES cell transplantation reveal encouraging results but also technical barriers that have to be overcome before clinical trials can be considered.
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Affiliation(s)
- Qing He
- Biology of Aging Laboratory, Department of Geriatrics, Geneva University Hospitals, Switzerland
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31
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Abstract
Pluripotential embryonic stem (ES) cells have been derived very efficiently from spare human embryos produced by IVF and grown in culture to the nascent blastocyst stage. The inner cell mass (ICM) is isolated by immunosurgery and grown on selected embryonic fibroblast monolayer cultures. ICM cells lose their memory for axis during formation of ES cell colonies and are then unable to integrate tissue formation with a body plan. ES cells form teratomas in vivo with cells and tissues representative of the three major embryonic lineages (ectoderm, mesoderm, endoderm). The ES cells are continuously renewable and can be directed to differentiate into early progenitors of neural stem cells (Noggin cells) and from there into mature neurons and glia (astrocytes and oligodendrocytes). The neural stem cells formed from human ES cells repopulate the brains of newborn mice when injected into the lateral cerebral ventricles, forming astrocytes dominantly in the parenchyma. The human neural cells can be observed migrating from the subventricular areas along the rostral migratory stream. Human neurons can be found in the olfactory bulb. Human ES cells can also be directed into cardiomyocytes when co-cultured with visceral endoderm-like cells (END-2). These observations provide further scope to explore stem cell therapies, gene therapies and drug discovery. For compatible transplantation, ES may need to be derived with a range of HLA types or by nuclear transplantation or stem cell fusion.
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Affiliation(s)
- Alan Trounson
- Institute of Reproduction and Development, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia.
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32
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Abstract
Cell replacement therapy is a promising approach for the treatment of cardiac diseases, but is challenged by a limited supply of appropriate cells. We have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem (hES) cells. Cardiomyocyte differentiation was evaluated using 3 parent (H1, H7, and H9) hES cell lines and 2 clonal (H9.1 and H9.2) hES cell lines. All cell lines examined differentiated into cardiomyocytes, even after long-term culture (50 passages or approximately 260 population doublings). Upon differentiation, beating cells were observed after one week in differentiation conditions, increased in numbers with time, and could retain contractility for over 70 days. The beating cells expressed markers characteristic of cardiomyocytes, such as cardiac alpha-myosin heavy chain, cardiac troponin I and T, atrial natriuretic factor, and cardiac transcription factors GATA-4, Nkx2.5, and MEF-2. In addition, cardiomyocyte differentiation could be enhanced by treatment of cells with 5-aza-2'-deoxycytidine but not DMSO or retinoic acid. Furthermore, the differentiated cultures could be dissociated and enriched by Percoll density centrifugation to give a population containing 70% cardiomyocytes. The enriched population was proliferative and showed appropriate expression of cardiomyocyte markers. The extended replicative capacity of hES cells and the ability to differentiate and enrich for functional human cardiomyocytes warrant further development of these cells for clinical application in heart diseases.
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Affiliation(s)
- Chunhui Xu
- Geron Corporation, Menlo Park, Calif 94025, USA.
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33
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Abstract
The pacemaker cells of the heart initiate the heartbeat, sustain the circulation, and dictate the rate and rhythm of cardiac contraction. Circulatory collapse ensues when these specialized cells are damaged by disease, a situation that currently necessitates the implantation of an electronic pacemaker. Here we report the use of viral gene transfer to convert quiescent heart-muscle cells into pacemaker cells, and the successful generation of spontaneous, rhythmic electrical activity in the ventricle in vivo. Our results indicate that genetically engineered pacemakers could be developed as a possible alternative to implantable electronic devices.
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Affiliation(s)
- Junichiro Miake
- Institute of Molecular Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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Yang HT, Tweedie D, Wang S, Guia A, Vinogradova T, Bogdanov K, Allen PD, Stern MD, Lakatta EG, Boheler KR. The ryanodine receptor modulates the spontaneous beating rate of cardiomyocytes during development. Proc Natl Acad Sci U S A 2002; 99:9225-30. [PMID: 12089338 PMCID: PMC123122 DOI: 10.1073/pnas.142651999] [Citation(s) in RCA: 94] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
In adult myocardium, the heartbeat originates from the sequential activation of ionic currents in pacemaker cells of the sinoatrial node. Ca(2+) release via the ryanodine receptor (RyR) modulates the rate at which these cells beat. In contrast, the mechanisms that regulate heart rate during early cardiac development are poorly understood. Embryonic stem (ES) cells can differentiate into spontaneously contracting myocytes whose beating rate increases with differentiation time. These cells thus offer an opportunity to determine the mechanisms that regulate heart rate during development. Here we show that the increase in heart rate with differentiation is markedly depressed in ES cell-derived cardiomyocytes with a functional knockout (KO) of the cardiac ryanodine receptor (RyR2). KO myocytes show a slowing of the rate of spontaneous diastolic depolarization and an absence of calcium sparks. The depressed rate of pacemaker potential can be mimicked in wild-type myocytes by ryanodine, and rescued in KO myocytes with herpes simplex virus (HSV)-1 amplicons containing full-length RyR2. We conclude that a functional RyR2 is crucial to the progressive increase in heart rate during differentiation of ES cell-derived cardiomyocytes, consistent with a mechanism that couples Ca(2+) release via RyR before an action potential with activation of an inward current that accelerates membrane depolarization.
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Affiliation(s)
- Huang-Tian Yang
- National Institute on Aging, Laboratory of Cardiovascular Science, National Institutes of Health, Baltimore, MD 21224, USA
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Auda-Boucher G, Bernard B, Fontaine-Pérus J, Rouaud T, Mericksay M, Gardahaut MF. Staging of the commitment of murine cardiac cell progenitors. Dev Biol 2000; 225:214-25. [PMID: 10964476 DOI: 10.1006/dbio.2000.9817] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The staging of murine cardiomyocyte specification and determination was investigated in cultures of tissue explants from pre- and postgastrulated embryos and after transplantation of cardiac or cardiogenic tissues from mouse embryos into 2-day-old chick embryos in different locations. The development of transplanted and cultured cells in cardiomyocytes was evaluated by testing the expression of several cardiac transcription factor genes (Nkx 2.5, eHAND, dHAND, GATA-4), alpha-cardiac actin mRNA, and beta-myosin heavy chain protein. In vitro analyses showed that cells with the potential to form cardiac muscle were present prior to gastrulation in 6.5-day postconception (dpc) epiblasts, as indicated by the expression of Nkx 2.5, eHAND, dHAND, and GATA-4 cardiac transcription factors; desmin transgene; alpha-cardiac actin; and beta-myosin heavy chain. Conversely, epiblasts transplanted into the chicken somitic environment did not exhibit full cardiogenic cell differentiation. It was determined that chick host axial structures did not influence cardiogenesis in transplants. Mesoderm from late streak explants was capable of differentiating into the cardiac phenotype in the avian heterotopic environment, indicating that the specification of cardiac precursors (under way by 6.5 dpc) became irreversible at around the late streak stage in mouse embryo. Although in vitro analyses showed that interaction with endoderm is not required for the specification of murine cardiac cells, the presence of endoderm in explant cultures between mid- and late streak stages stimulated emerging mesodermal cells to adopt a myocardial pathway, whereas ectoderm had no influence on cardiomyogenesis.
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Affiliation(s)
- G Auda-Boucher
- Faculté des Sciences et des Techniques, EP CNRS 1593, Nantes Cedex 03, 44322, France
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Schreiber KL, Calderone A, Rindt H. Distant upstream regulatory domains direct high levels of beta -myosin heavy chain gene expression in differentiated embryonic stem cells. J Mol Cell Cardiol 2000; 32:585-98. [PMID: 10756116 DOI: 10.1006/jmcc.2000.1100] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Eukaryotic gene transcription takes place in the context of chromatin. In order to study the expression of the beta -myosin heavy chain (MyHC) gene in its appropriate cardiac environment in vitro, embryonic stem cell lines were generated and induced to differentiate into the cardiac lineage. We show that the upstream region of the beta -MyHC gene (-5518 to -2490 relative to the transcriptional start site) directed high levels of transcriptional activity only when stably integrated, but not when expressed extrachromosomally in transient assays. These results are consistent with earlier findings using an in vivo transgenic approach. The expression of beta -MyHC reporter gene constructs was strictly correlated to differentiation status and coincided with the expression of endogenous cardiac marker genes and with morphological differentiation of embryoid bodies in vitro. Using populations of stably transfected cell clones, two domains important for high level expression were identified. The analysis of individual cell clones suggested that the positive regulatory domains act according to the graded model of enhancement. These results show that chromosomal integration is necessary for the appropriate function of the beta -MyHC gene's upstream regulatory region.
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Affiliation(s)
- K L Schreiber
- Department of Medicine, University of Montreal and Montreal Heart Institute, 5000 Belanger Street, Montreal, Quebec, H1T 1C8, Canada
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Guan K, Rohwedel J, Wobus AM. Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro. Cytotechnology 1999; 30:211-26. [PMID: 19003371 PMCID: PMC3449946 DOI: 10.1023/a:1008041420166] [Citation(s) in RCA: 75] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called 'embryoid bodies', are able to differentiate in vitro into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm. We established differentiation protocols for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiomyocytes, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells. During differentiation, tissue-specific genes, proteins, ion channels, receptors and action potentials were expressed in a developmentally controlled pattern. This pattern closely recapitulates the developmental pattern during embryogenesis in the living organism. In vitro, the controlled developmental pattern was found to be influenced by differentiation and growth factor molecules or by xenobiotics. Furthermore, the differentiation system has been used for genetic analyses by 'gain of function' and 'loss of function' approaches in vitro.
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Affiliation(s)
- K Guan
- "In Vitro Differentiation" Group, IPK Gatersleben, D-06466, Gatersleben, Germany
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Maltsev VA, Ji GJ, Wobus AM, Fleischmann BK, Hescheler J. Establishment of beta-adrenergic modulation of L-type Ca2+ current in the early stages of cardiomyocyte development. Circ Res 1999; 84:136-45. [PMID: 9933244 DOI: 10.1161/01.res.84.2.136] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
beta-Adrenergic modulation of the L-type Ca2+ current (ICaL) was characterized for different developmental stages in murine embryonic stem cell-derived cardiomyocytes using the whole-cell patch-clamp technique at 37 degreesC. Cardiomyocytes first appeared in embryonic stem cell-derived embryoid bodies grown for 7 days (7d). ICaL was insensitive to isoproterenol, forskolin, and 8-bromo-cAMP in very early developmental stage (VEDS) cardiomyocytes (from 7+1d to 7+2d) but highly stimulated by these substances in late developmental stage (LDS) cardiomyocytes (from 7+9d to 7+12d), indicating that all signaling cascade components became functionally coupled during development. In early developmental stage (EDS) cells (from 7+3d to 7+5d), the stimulatory response to forskolin and 8-bromo-cAMP was relatively weak. The forskolin effect was strongly augmented by ATP-gamma-S. At this stage, basal ICaL was stimulated by the nonselective phosphodiesterase (PDE) inhibitor isobutylmethylxanthine, by PDE inhibitors selective for the PDE II, III, and IV isoforms, as well as by the phosphatase inhibitor okadaic acid. Stimulation of ICaL by the catalytic subunit of the cAMP-dependent protein kinase A (PKA) was found to be similar (about 3 times) throughout development and in adult mouse ventricular cardiomyocytes, indicating that no structural changes of the Ca2+ channel related to phosphorylation occurred during development. ICaL was stimulated by isoproterenol in the presence of a PKA inhibitor and GTP-gamma-S in LDS but not VEDS cardiomyocytes, suggesting the development of a membrane-delimited stimulatory pathway mediated through the stimulatory GTP binding protein, Gs. We conclude that uncoupling and/or low expression of Gs protein accounted for the ICaL insensitivity to beta-adrenergic stimulation in VEDS cardiomyocytes. Furthermore, in EDS cells at the 7+4d stage, the reduced beta-adrenergic response is due, at least in part, to high intrinsic PDE and phosphatase activities.
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Affiliation(s)
- V A Maltsev
- Division of Cardiovascular Medicine, Henry Ford Heart and Vascular Institute, Detroit, MI, USA
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Mueller-Klieser W. Three-dimensional cell cultures: from molecular mechanisms to clinical applications. THE AMERICAN JOURNAL OF PHYSIOLOGY 1997; 273:C1109-23. [PMID: 9357753 DOI: 10.1152/ajpcell.1997.273.4.c1109] [Citation(s) in RCA: 375] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
This article reviews actual advances in the development and application of three-dimensional (3-D) cell culture systems. Recent therapeutically oriented studies include characterization of multicellular-mediated drug resistance, novel ways of quantifying hypoxia, and new approaches to more efficient immunotherapy. Recent progress toward understanding the development of necrosis in tumor spheroids has been made using novel spheroid models. 3-D cultures have been used for studies on molecular mechanisms involved in invasion and metastasis, with a major focus on the role of E-cadherin. Similarly, tumor angiogenesis and the significance of vascular endothelial growth factor have been investigated in a variety of 3-D culture systems. There are many ongoing developments in tissue modeling or remodeling that promise significant progress toward the development of bioartificial liver support and artificial blood. Perhaps one of the most interesting areas of basic research with 3-D cultures is the characterization of embryoid bodies obtained from stable embryonic stem cells. These models have greatly increased the understanding of embryonic development, in particular through the notable exceptional advances in cardiogenesis.
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Affiliation(s)
- W Mueller-Klieser
- Institute of Physiology and Pathophysiology, Johannes Gutenberg-University Mainz, Germany
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Abstract
This review summarizes recent studies of the cellular and molecular events involved in the determination and differentiation of cardiac myocytes in vertebrate embryos. Fate-mapping studies in mouse, chick, amphibian and zebrafish embryos suggest that cardiac muscle precursors are specified shortly before or at the time of gastrulation. Nuclear factors, such as dHAND, aryl hydrocarbon receptor, GATA-6, Nkx-2.3, growth arrest homeobox (Gax) and cardiac adriamycin responsive protein (CARP), which have recently been described as playing a role in the commitment and/or differentiation of cardiac myocytes are discussed.
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Affiliation(s)
- G E Lyons
- Department of Anatomy, University of Wisconsin Medical School, 1300 University Avenue, Madison, 53706, USA.
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