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1
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Seo SJ, Jin Y. Enhancing Cardiomyocyte Purity through Lactate-Based Metabolic Selection. Tissue Eng Regen Med 2025; 22:249-260. [PMID: 39820961 PMCID: PMC11794935 DOI: 10.1007/s13770-024-00696-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2024] [Revised: 12/20/2024] [Accepted: 12/22/2024] [Indexed: 01/19/2025] Open
Abstract
BACKGROUND Direct reprogramming of fibroblasts into chemically induced cardiomyocyte-like cells (CiCMs) through small molecules presents a promising cell source for cardiac regeneration and therapeutic development. However, the contaminating non-cardiomyocytes, primarily unconverted fibroblasts, reduce the effectiveness of CiCMs in various applications. This study investigated a metabolic selection approach using lactate to enrich CiCMs by exploiting the unique metabolic capability of cardiomyocytes to utilize lactate as an alternative energy source. METHODS Primary mouse embryonic fibroblasts (pMEFs) were reprogrammed into CiCMs and subjected to a glucose-depleted, lactate-supplemented medium for 4 days. Afterward, cell viability was analyzed, and cardiomyocyte efficiency was assessed through the expression of cardiac-specific markers. Additionally, electrophysiological function was evaluated by examining drug-induced responses. RESULTS The lactate treatment led to a significant decrease in the viability of non-cardiomyocytes (pMEF-LAC), while CiCMs (CiCM-LAC) showed minimal cell death. Specifically, the expression of all cardiac-related markers was increased in CiCM-LAC. Metabolically purified CiCMs exhibited enhanced contractile force and increased contraction frequency compared to non-purified CiCMs, as well as an elevated responsiveness to drugs. CONCLUSION This study demonstrates that lactate-based metabolic selection is an effective and practical approach for enriching CiCMs, offering a cost-effective alternative to other purification methods. The application of this strategy could potentially broaden the accessibility and utility of reprogrammed cardiomyocytes in cardiac regeneration and therapeutic development.
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Affiliation(s)
- Seung Ju Seo
- Department of Physiology, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, Seoul, 03722, Republic of Korea
| | - Yoonhee Jin
- Department of Physiology, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, Seoul, 03722, Republic of Korea.
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2
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Soma Y, Tani H, Morita-Umei Y, Kishino Y, Fukuda K, Tohyama S. Pluripotent stem cell-based cardiac regenerative therapy for heart failure. J Mol Cell Cardiol 2024; 187:90-100. [PMID: 38331557 DOI: 10.1016/j.yjmcc.2023.12.001] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/11/2023] [Revised: 12/03/2023] [Accepted: 12/04/2023] [Indexed: 02/10/2024]
Abstract
Cardiac regenerative therapy using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is expected to become an alternative to heart transplantation for severe heart failure. It is now possible to produce large numbers of human pluripotent stem cells (hPSCs) and eliminate non-cardiomyocytes, including residual undifferentiated hPSCs, which can cause teratoma formation after transplantation. There are two main strategies for transplanting hPSC-CMs: injection of hPSC-CMs into the myocardium from the epicardial side, and implantation of hPSC-CM patches or engineered heart tissues onto the epicardium. Transplantation of hPSC-CMs into the myocardium of large animals in a myocardial infarction model improved cardiac function. The engrafted hPSC-CMs matured, and microvessels derived from the host entered the graft abundantly. Furthermore, as less invasive methods using catheters, injection into the coronary artery and injection into the myocardium from the endocardium side have recently been investigated. Since transplantation of hPSC-CMs alone has a low engraftment rate, various methods such as transplantation with the extracellular matrix or non-cardiomyocytes and aggregation of hPSC-CMs have been developed. Post-transplant arrhythmias, imaging of engrafted hPSC-CMs, and immune rejection are the remaining major issues, and research is being conducted to address them. The clinical application of cardiac regenerative therapy using hPSC-CMs has just begun and is expected to spread widely if its safety and efficacy are proven in the near future.
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Affiliation(s)
- Yusuke Soma
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Hidenori Tani
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan; Joint Research Laboratory for Medical Innovation in Heart Disease, Keio University School of Medicine, Tokyo, Japan
| | - Yuika Morita-Umei
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan; Kanagawa Institute of Industrial Science and Technology (KISTEC), Kanagawa, Japan
| | - Yoshikazu Kishino
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Keiichi Fukuda
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan.
| | - Shugo Tohyama
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan.
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Łoboda A, Kłaptocz M, Kaziród K, Wolnik J, Biniecka M, Stępniewski J, Dulak J. Comparison of Two Differentiation Protocols of Human-Induced Pluripotent Stem Cells into Cardiomyocytes. Methods Mol Biol 2024; 2835:69-82. [PMID: 39105907 DOI: 10.1007/978-1-0716-3995-5_7] [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] [Indexed: 08/07/2024]
Abstract
Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) offer an attractive platform to evaluate the mechanisms of cardiovascular-related incidents and to develop and test new drugs for heart diseases. This work focuses on the comparison of two hiPSC-CM differentiation protocols: the GiWi method based on temporal modulation of the Wnt/β-catenin pathway and the commercially available PSC Cardiomyocyte Differentiation Kit. We underlined the need to optimize several parameters such as cell density or small molecule concentration (CHIR-99021, IWR-1) to obtain functional hiPSC-CMs. Both protocols yield a similar differentiation efficiency; therefore, the choice of a particular procedure may depend on the preferences of the experimenter.
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Affiliation(s)
- Agnieszka Łoboda
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, Kraków, Poland.
| | - Monika Kłaptocz
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, Kraków, Poland
| | - Katarzyna Kaziród
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, Kraków, Poland
| | - Jan Wolnik
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, Kraków, Poland
| | - Monika Biniecka
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, Kraków, Poland
| | - Jacek Stępniewski
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, Kraków, Poland
| | - Józef Dulak
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Krakow, Kraków, Poland
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4
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Kishino Y, Tohyama S, Morita Y, Soma Y, Tani H, Okada M, Kanazawa H, Fukuda K. Cardiac Regenerative Therapy Using Human Pluripotent Stem Cells for Heart Failure: A State-of-the-Art Review. J Card Fail 2023; 29:503-513. [PMID: 37059512 DOI: 10.1016/j.cardfail.2022.10.433] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2022] [Revised: 10/07/2022] [Accepted: 10/09/2022] [Indexed: 04/16/2023]
Abstract
Heart transplantation (HT) is the only definitive treatment available for patients with end-stage heart failure who are refractory to medical and device therapies. However, HT as a therapeutic option, is limited by a significant shortage of donors. To overcome this shortage, regenerative medicine using human pluripotent stem cells (hPSCs), such as human embryonic stem cells and human-induced pluripotent stem cells (hiPSCs), has been considered an alternative to HT. Several issues, including the methods of large-scale culture and production of hPSCs and cardiomyocytes, the prevention of tumorigenesis secondary to contamination of undifferentiated stem cells and non-cardiomyocytes, and the establishment of an effective transplantation strategy in large-animal models, need to be addressed to fulfill this unmet need. Although post-transplantation arrhythmia and immune rejection remain problems, the ongoing rapid technological advances in hPSC research have been directed toward the clinical application of this technology. Cell therapy using hPSC-derived cardiomyocytes is expected to serve as an integral component of realistic medicine in the near future and is being potentially viewed as a treatment that would revolutionize the management of patients with severe heart failure.
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Affiliation(s)
- Yoshikazu Kishino
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Shugo Tohyama
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan.
| | - Yuika Morita
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Yusuke Soma
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Hidenori Tani
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Marina Okada
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Hideaki Kanazawa
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Keiichi Fukuda
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
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5
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Kishino Y, Fukuda K. Unlocking the Pragmatic Potential of Regenerative Therapies in Heart Failure with Next-Generation Treatments. Biomedicines 2023; 11:biomedicines11030915. [PMID: 36979894 PMCID: PMC10046277 DOI: 10.3390/biomedicines11030915] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2023] [Revised: 02/28/2023] [Accepted: 02/28/2023] [Indexed: 03/18/2023] Open
Abstract
Patients with chronic heart failure (HF) have a poor prognosis due to irreversible impairment of left ventricular function, with 5-year survival rates <60%. Despite advances in conventional medicines for HF, prognosis remains poor, and there is a need to improve treatment further. Cell-based therapies to restore the myocardium offer a pragmatic approach that provides hope for the treatment of HF. Although first-generation cell-based therapies using multipotent cells (bone marrow-derived mononuclear cells, mesenchymal stem cells, adipose-derived regenerative cells, and c-kit-positive cardiac cells) demonstrated safety in preclinical models of HF, poor engraftment rates, and a limited ability to form mature cardiomyocytes (CMs) and to couple electrically with existing CMs, meant that improvements in cardiac function in double-blind clinical trials were limited and largely attributable to paracrine effects. The next generation of stem cell therapies uses CMs derived from human embryonic stem cells or, increasingly, from human-induced pluripotent stem cells (hiPSCs). These cell therapies have shown the ability to engraft more successfully and improve electromechanical function of the heart in preclinical studies, including in non-human primates. Advances in cell culture and delivery techniques promise to further improve the engraftment and integration of hiPSC-derived CMs (hiPSC-CMs), while the use of metabolic selection to eliminate undifferentiated cells will help minimize the risk of teratomas. Clinical trials of allogeneic hiPSC-CMs in HF are now ongoing, providing hope for vast numbers of patients with few other options available.
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Affiliation(s)
| | - Keiichi Fukuda
- Correspondence: ; Tel.: +81-3-5363-3874; Fax: +81-3-5363-3875
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Sart S, Liu C, Zeng EZ, Xu C, Li Y. Downstream bioprocessing of human pluripotent stem cell-derived therapeutics. Eng Life Sci 2022; 22:667-680. [PMID: 36348655 PMCID: PMC9635003 DOI: 10.1002/elsc.202100042] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2021] [Revised: 07/08/2021] [Accepted: 08/16/2021] [Indexed: 11/30/2022] Open
Abstract
With the advancement in lineage-specific differentiation from human pluripotent stem cells (hPSCs), downstream cell separation has now become a critical step to produce hPSC-derived products. Since differentiation procedures usually result in a heterogeneous cell population, cell separation needs to be performed either to enrich the desired cell population or remove the undesired cell population. This article summarizes recent advances in separation processes for hPSC-derived cells, including the standard separation technologies, such as magnetic-activated cell sorting, as well as the novel separation strategies, such as those based on adhesion strength and metabolic flux. Specifically, the downstream bioprocessing flow and the identification of surface markers for various cell lineages are discussed. While challenges remain for large-scale downstream bioprocessing of hPSC-derived cells, the rational quality-by-design approach should be implemented to enhance the understanding of the relationship between process and the product and to ensure the safety of the produced cells.
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Affiliation(s)
- Sebastien Sart
- Laboratory of Physical Microfluidics and BioengineeringDepartment of Genome and GeneticsInstitut PasteurParisFrance
| | - Chang Liu
- Department of Chemical and Biomedical EngineeringFAMU‐FSU College of EngineeringFlorida State UniversityTallahasseeFLUSA
| | - Eric Z. Zeng
- Department of Chemical and Biomedical EngineeringFAMU‐FSU College of EngineeringFlorida State UniversityTallahasseeFLUSA
| | - Chunhui Xu
- Department of PediatricsEmory University School of Medicine and Children's Healthcare of AtlantaAtlantaGAUSA
| | - Yan Li
- Department of Chemical and Biomedical EngineeringFAMU‐FSU College of EngineeringFlorida State UniversityTallahasseeFLUSA
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Schwach V, Cofiño-Fabres C, ten Den SA, Passier R. Improved Atrial Differentiation of Human Pluripotent Stem Cells by Activation of Retinoic Acid Receptor Alpha (RARα). J Pers Med 2022; 12:jpm12040628. [PMID: 35455744 PMCID: PMC9032956 DOI: 10.3390/jpm12040628] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Revised: 03/24/2022] [Accepted: 04/10/2022] [Indexed: 11/16/2022] Open
Abstract
Human pluripotent stem cell (hPSC)-derived cardiomyocytes have proven valuable for modeling disease and as a drug screening platform. Here, we depict an optimized protocol for the directed differentiation of hPSCs toward cardiomyocytes with an atrial identity by modulating the retinoic acid signaling cascade in spin embryoid bodies. The crucial steps of the protocol, including hPSC maintenance, embryoid body (EB) differentiation, the induction of cardiac mesoderm, direction toward the atrial phenotype, as well as molecular and functional characterization of the cardiomyocytes, are described. Atrial cardiomyocytes (AMs) can be generated within 14 days. Most importantly, we show that induction of the specific retinoic acid receptor alpha (RARα) increased the efficiency of atrial differentiation to 72% compared with 45% after modulating the retinoic acid (RA) pathway with all-trans RA (atRA). In contrast, the induction of RARβ signaling only had a minor impact on the efficiency of atrial differentiation (from about 45% to 50%). Similarly, the total yield of AM per EB of 5000 hPSCs was increased from 10,350 (2.07 per hPSC) to 16,120 (3.22 per hPSC) while selectively modulating RARα signaling. For further purification of the AMs, we describe a metabolic selection procedure that enhanced the AM percentage to more than 90% without compromising the AM yield (15,542 per EB, equal to 3.11 per hPSC) or functionality of the AMs as evaluated by RNAseq, immunostaining, and optical action potential measurement. Cardiomyocytes with distinct atrial and ventricular properties can be applied for selective pharmacology, such as the development of novel atrial-specific anti-arrhythmic agents, and disease modeling, including atrial fibrillation, which is the most common heart rhythm disorder. Moreover, fully characterized and defined cardiac subtype populations are of the utmost importance for potential cell-based therapeutic approaches.
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Affiliation(s)
- Verena Schwach
- Department of Applied Stem Cell Technologies, TechMed Centre, University of Twente, 7522 NB Enschede, The Netherlands; (C.C.-F.); (S.A.t.D.)
- Correspondence: (V.S.); (R.P.)
| | - Carla Cofiño-Fabres
- Department of Applied Stem Cell Technologies, TechMed Centre, University of Twente, 7522 NB Enschede, The Netherlands; (C.C.-F.); (S.A.t.D.)
| | - Simone A. ten Den
- Department of Applied Stem Cell Technologies, TechMed Centre, University of Twente, 7522 NB Enschede, The Netherlands; (C.C.-F.); (S.A.t.D.)
| | - Robert Passier
- Department of Applied Stem Cell Technologies, TechMed Centre, University of Twente, 7522 NB Enschede, The Netherlands; (C.C.-F.); (S.A.t.D.)
- Department of Anatomy and Embryology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands
- Correspondence: (V.S.); (R.P.)
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8
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Hou X, Ma S, Fan W, Li F, Xu M, Yang C, Liu F, Yan Y, Wan J, Lan F, Liao B. Chemically defined and small molecules-based generation of sinoatrial node-like cells. Stem Cell Res Ther 2022; 13:158. [PMID: 35410454 PMCID: PMC8996538 DOI: 10.1186/s13287-022-02834-y] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2021] [Accepted: 03/25/2022] [Indexed: 11/10/2022] Open
Abstract
Background Existing methods for in vitro differentiation of human pluripotent stem cells (hPSCs) into sinoatrial node-like cells (SANLCs) require complex and undefined medium constituents. This might hinder the elucidation of the molecular mechanisms involved in cardiac subtype specification and prevent translational application. In our study, we aimed to establish a chemically defined differentiation methods to generate SANLCs effectively and stably. Methods We induced human embryonic stem cells (hESCs)/induced PSCs (hiPSCs) to pan-cardiomyocytes by temporal modulation of the WNT/β-catenin (WNT) signaling pathway with GSK3 inhibitor and WNT inhibitor. During cardiac mesoderm stage of the differentiation process, signaling of WNT, retinoid acid (RA), and fibroblast growth factor (FGF) was manipulated by three specific molecules. Moreover, metabolic selection was designed to improve the enrichment of SANLCs. Finally, RT-PCR, immunofluorescence, flow cytometry, and whole cell patch clamp were used to identify the SANLCs.
Results WNT, RA, and FGF signaling promote the differentiation of hPSCs into SANLCs in a concentration- and time window-sensitive manner, respectively. Synergetic modulation of WNT, FGF, and RA signaling pathways enhance the pacemaker phenotype and improve the differentiation efficiency of SANLCs (up to 45%). Moreover, the purification based on lactate metabolism and glucose starvation further reached approximately 50% of SANLCs. Finally, the electrophysiological data demonstrate that cells differentiated with the proposed protocol produce a considerable number of SANLCs that display typical electrophysiological characteristics of pacemaker cells in vitro. Conclusion We provide an optimized and chemically defined protocol to generate SANLCs by combined modulation of WNT, RA, and FGF signaling pathways and metabolic selection by lactate enrichment and glucose starvation. This chemically defined method for generating SANLCs might provide a platform for disease modeling, drug discovery, predictive toxicology, and biological pacemaker construction. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-022-02834-y.
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Affiliation(s)
- Xiaojie Hou
- Department of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical University, Luzhou, 646000, China.,Metabolic Vascular Diseases Key Laboratory of Sichuan Province, Luzhou, 646000, China.,Key Laboratory of Medical Electrophysiology, Ministry of Education & Medical Electrophysiological Key Laboratory of Sichuan Province, (Collaborative Innovation Center for Prevention of Cardiovascular Diseases) Institute of Cardiovascular Research, Southwest Medical University, Luzhou, 646000, China
| | - Shuhong Ma
- Shenzhen Key Laboratory of Cardiovascular Disease, Fuwai Hospital Chinese Academy of Medical Sciences, State Key Laboratory of Cardiovascular Disease, Key Laboratory of Pluripotent Stem Cells in Cardiac Repair and Regeneration, Chinese Academy of Medical Sciences and Peking Union Medical College, Shenzhen, 518057, China
| | - Wei Fan
- Department of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical University, Luzhou, 646000, China.,Metabolic Vascular Diseases Key Laboratory of Sichuan Province, Luzhou, 646000, China.,Key Laboratory of Medical Electrophysiology, Ministry of Education & Medical Electrophysiological Key Laboratory of Sichuan Province, (Collaborative Innovation Center for Prevention of Cardiovascular Diseases) Institute of Cardiovascular Research, Southwest Medical University, Luzhou, 646000, China
| | - Fang Li
- Key Laboratory of Medical Electrophysiology, Ministry of Education & Medical Electrophysiological Key Laboratory of Sichuan Province, (Collaborative Innovation Center for Prevention of Cardiovascular Diseases) Institute of Cardiovascular Research, Southwest Medical University, Luzhou, 646000, China.,Department of Cardiology, Jianyang City People's Hospital, Jianyang, 641499, China
| | - Miaomiao Xu
- Shenzhen Key Laboratory of Cardiovascular Disease, Fuwai Hospital Chinese Academy of Medical Sciences, State Key Laboratory of Cardiovascular Disease, Key Laboratory of Pluripotent Stem Cells in Cardiac Repair and Regeneration, Chinese Academy of Medical Sciences and Peking Union Medical College, Shenzhen, 518057, China
| | - Chao Yang
- Department of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical University, Luzhou, 646000, China.,Metabolic Vascular Diseases Key Laboratory of Sichuan Province, Luzhou, 646000, China.,Key Laboratory of Medical Electrophysiology, Ministry of Education & Medical Electrophysiological Key Laboratory of Sichuan Province, (Collaborative Innovation Center for Prevention of Cardiovascular Diseases) Institute of Cardiovascular Research, Southwest Medical University, Luzhou, 646000, China
| | - Feng Liu
- Department of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical University, Luzhou, 646000, China.,Metabolic Vascular Diseases Key Laboratory of Sichuan Province, Luzhou, 646000, China.,Key Laboratory of Medical Electrophysiology, Ministry of Education & Medical Electrophysiological Key Laboratory of Sichuan Province, (Collaborative Innovation Center for Prevention of Cardiovascular Diseases) Institute of Cardiovascular Research, Southwest Medical University, Luzhou, 646000, China
| | - Ying Yan
- Department of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical University, Luzhou, 646000, China.,Metabolic Vascular Diseases Key Laboratory of Sichuan Province, Luzhou, 646000, China.,Key Laboratory of Medical Electrophysiology, Ministry of Education & Medical Electrophysiological Key Laboratory of Sichuan Province, (Collaborative Innovation Center for Prevention of Cardiovascular Diseases) Institute of Cardiovascular Research, Southwest Medical University, Luzhou, 646000, China
| | - Juyi Wan
- Department of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical University, Luzhou, 646000, China. .,Metabolic Vascular Diseases Key Laboratory of Sichuan Province, Luzhou, 646000, China. .,Key Laboratory of Medical Electrophysiology, Ministry of Education & Medical Electrophysiological Key Laboratory of Sichuan Province, (Collaborative Innovation Center for Prevention of Cardiovascular Diseases) Institute of Cardiovascular Research, Southwest Medical University, Luzhou, 646000, China.
| | - Feng Lan
- Shenzhen Key Laboratory of Cardiovascular Disease, Fuwai Hospital Chinese Academy of Medical Sciences, State Key Laboratory of Cardiovascular Disease, Key Laboratory of Pluripotent Stem Cells in Cardiac Repair and Regeneration, Chinese Academy of Medical Sciences and Peking Union Medical College, Shenzhen, 518057, China.
| | - Bin Liao
- Department of Cardiovascular Surgery, Affiliated Hospital of Southwest Medical University, Luzhou, 646000, China. .,Metabolic Vascular Diseases Key Laboratory of Sichuan Province, Luzhou, 646000, China. .,Key Laboratory of Medical Electrophysiology, Ministry of Education & Medical Electrophysiological Key Laboratory of Sichuan Province, (Collaborative Innovation Center for Prevention of Cardiovascular Diseases) Institute of Cardiovascular Research, Southwest Medical University, Luzhou, 646000, China.
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Jiang CL, Goyal Y, Jain N, Wang Q, Truitt RE, Coté AJ, Emert B, Mellis IA, Kiani K, Yang W, Jain R, Raj A. Cell type determination for cardiac differentiation occurs soon after seeding of human-induced pluripotent stem cells. Genome Biol 2022; 23:90. [PMID: 35382863 PMCID: PMC8985385 DOI: 10.1186/s13059-022-02654-6] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2021] [Accepted: 03/16/2022] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND Cardiac differentiation of human-induced pluripotent stem (hiPS) cells consistently produces a mixed population of cardiomyocytes and non-cardiac cell types, even when using well-characterized protocols. We sought to determine whether different cell types might result from intrinsic differences in hiPS cells prior to the onset of differentiation. RESULTS By associating individual differentiated cells that share a common hiPS cell precursor, we tested whether expression variability is predetermined from the hiPS cell state. In a single experiment, cells that shared a progenitor were more transcriptionally similar to each other than to other cells in the differentiated population. However, when the same hiPS cells were differentiated in parallel, we did not observe high transcriptional similarity across differentiations. Additionally, we found that substantial cell death occurs during differentiation in a manner that suggested all cells were equally likely to survive or die, suggesting that there is no intrinsic selection bias for cells descended from particular hiPS cell progenitors. We thus wondered how cells grow spatially during differentiation, so we labeled cells by expression of marker genes and found that cells expressing the same marker tended to occur in patches. Our results suggest that cell type determination across multiple cell types, once initiated, is maintained in a cell-autonomous manner for multiple divisions. CONCLUSIONS Altogether, our results show that while substantial heterogeneity exists in the initial hiPS cell population, it is not responsible for the variability observed in differentiated outcomes; instead, factors specifying the various cell types likely act during a window that begins shortly after the seeding of hiPS cells for differentiation.
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Affiliation(s)
- Connie L Jiang
- Genetics and Epigenetics, Cell and Molecular Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Yogesh Goyal
- Department of Cell and Developmental Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
- Center for Synthetic Biology, Northwestern University, Evanston, IL, USA
- Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania, Philadelphia, PA, USA
| | - Naveen Jain
- Genetics and Epigenetics, Cell and Molecular Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Qiaohong Wang
- Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA
| | - Rachel E Truitt
- Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Allison J Coté
- Cell Biology, Physiology, and Metabolism, Cell and Molecular Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Benjamin Emert
- Genomics and Computational Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Ian A Mellis
- Genomics and Computational Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Karun Kiani
- Genetics and Epigenetics, Cell and Molecular Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Wenli Yang
- Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, USA
- Penn Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Rajan Jain
- Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
- Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA.
- Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, USA.
- Penn Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
| | - Arjun Raj
- Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania, Philadelphia, PA, USA.
- Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
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10
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Soma Y, Morita Y, Kishino Y, Kanazawa H, Fukuda K, Tohyama S. The Present State and Future Perspectives of Cardiac Regenerative Therapy Using Human Pluripotent Stem Cells. Front Cardiovasc Med 2021; 8:774389. [PMID: 34957258 PMCID: PMC8692665 DOI: 10.3389/fcvm.2021.774389] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2021] [Accepted: 10/25/2021] [Indexed: 12/13/2022] Open
Abstract
The number of patients with heart failure (HF) is increasing with aging in our society worldwide. Patients with HF who are resistant to medication and device therapy are candidates for heart transplantation (HT). However, the shortage of donor hearts is a serious issue. As an alternative to HT, cardiac regenerative therapy using human pluripotent stem cells (hPSCs), such as human embryonic stem cells and induced pluripotent stem cells, is expected to be realized. Differentiation of hPSCs into cardiomyocytes (CMs) is facilitated by mimicking normal heart development. To prevent tumorigenesis after transplantation, it is important to eliminate non-CMs, including residual hPSCs, and select only CMs. Among many CM selection systems, metabolic selection based on the differences in metabolism between CMs and non-CMs is favorable in terms of cost and efficacy. Large-scale culture systems have been developed because a large number of hPSC-derived CMs (hPSC-CMs) are required for transplantation in clinical settings. In large animal models, hPSC-CMs transplanted into the myocardium improved cardiac function in a myocardial infarction model. Although post-transplantation arrhythmia and immune rejection remain problems, their mechanisms and solutions are under investigation. In this manner, the problems of cardiac regenerative therapy are being solved individually. Thus, cardiac regenerative therapy with hPSC-CMs is expected to become a safe and effective treatment for HF in the near future. In this review, we describe previous studies related to hPSC-CMs and discuss the future perspectives of cardiac regenerative therapy using hPSC-CMs.
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Affiliation(s)
- Yusuke Soma
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Yuika Morita
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Yoshikazu Kishino
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Hideaki Kanazawa
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Keiichi Fukuda
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Shugo Tohyama
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
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11
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Disease Modeling of Mitochondrial Cardiomyopathy Using Patient-Specific Induced Pluripotent Stem Cells. BIOLOGY 2021; 10:biology10100981. [PMID: 34681080 PMCID: PMC8533352 DOI: 10.3390/biology10100981] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Revised: 09/25/2021] [Accepted: 09/26/2021] [Indexed: 12/15/2022]
Abstract
Mitochondrial cardiomyopathy (MCM) is characterized as an oxidative phosphorylation disorder of the heart. More than 100 genetic variants in nuclear or mitochondrial DNA have been associated with MCM. However, the underlying molecular mechanisms linking genetic variants to MCM are not fully understood due to the lack of appropriate cellular and animal models. Patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) provide an attractive experimental platform for modeling cardiovascular diseases and predicting drug efficacy to such diseases. Here we introduce the pathological and therapeutic studies of MCM using iPSC-CMs and discuss the questions and latest strategies for research using iPSC-CMs.
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12
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Efficient Method to Dissociate Induced Pluripotent Stem Cell-Derived Cardiomyocyte Aggregates into Single Cells. Methods Mol Biol 2021. [PMID: 34302645 DOI: 10.1007/978-1-0716-1484-6_4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register]
Abstract
The human adult heart consists of approximately four billion cardiomyocytes, which do not possess self-renewal abilities. Severe myocardial infarction and dilated cardiomyopathy result in the loss of more than a billion cardiomyocytes. Induced pluripotent stem cells (iPSCs) can differentiate into various types of cells. Due to this ability, these cells could potentially serve as a new resource for cell therapy. Many studies have utilized cardiomyocytes derived from iPSCs for myocardial regeneration therapy. To obtain large number of cardiomyocytes for transplantation, we need to develop effective methods that would allow us to dissociate multiple cardiomyocyte aggregates simultaneously. Here, we describe a method to efficiently dissociate large number of iPSC-derived cardiomyocyte aggregates.
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13
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Cheng W, Li X, Yang S, Wang H, Li Y, Feng Y, Wang Y. Low doses of BPF-induced hypertrophy in cardiomyocytes derived from human embryonic stem cells via disrupting the mitochondrial fission upon the interaction between ERβ and calcineurin A-DRP1 signaling pathway. Cell Biol Toxicol 2021; 38:409-426. [PMID: 34023961 DOI: 10.1007/s10565-021-09615-y] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Accepted: 04/29/2021] [Indexed: 12/16/2022]
Abstract
Bisphenol F (BPF) is a replacement to bisphenol A, which has been extensively used in industrial manufacturing. Its wide detection in various human samples raises increasing concern on its safety. Currently, whether a low dose of BPF compromises cardiac function is still unknown. This study provides the first evidence that low-dose BPF can induce cardiac hypertrophy by using cardiomyocytes derived from human embryonic stem cells (hES). Non-cytotoxic BPF increased cytosolic Ca 2+ influx ([Ca2+ ]c), which was most remarkable at low dose (7 ng/ml) rather than at higher doses. Significant changes in the morphological parameters of mitochondria and significant decreases in ATP production were induced by 7 ng/ml BPF, representing a classic hypertrophic cardiomyocyte. After eliminating the direct effects on mitochondrial fission-related DRP1 by administration of the DRP1 inhibitor Mdivi-1, we examined the changes in [Ca 2+ ]c levels induced by BPF, which enhanced the calcineurin (Cn) activity and induced the abnormal mitochondrial fission via the CnAβ-DRP1 signaling pathway. BPF triggered excessive Ca 2+ influx by disrupting the L-type Ca 2+channel in cardiomyocytes. The interaction between ERβ and CnAβ cooperatively involved in the BPF-induced Ca 2+ influx, which resulted in the abnormal mitochondrial fission and compromised the cardiac function. Our findings provide a feasible molecular mechanism for explaining low-dose BPF-induced cardiac hypertrophy in vitro, preliminarily suggesting that BPF may not be as safe as assumed in humans.
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Affiliation(s)
- Wei Cheng
- School of Public Health, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China, 200025
| | - Xiaolan Li
- School of Public Health, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China, 200025
| | - Shoufei Yang
- School of Public Health, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China, 200025
| | - Hui Wang
- Center for Single-Cell Omics, School of Public Health, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China, 200025
| | - Yan Li
- School of Public Health, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China, 200025
| | - Yan Feng
- School of Public Health, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China, 200025
| | - Yan Wang
- School of Public Health, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China, 200025. .,The Ninth People's Hospital of Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China, 200011. .,Shanghai Collaborative Innovation Center for Translational Medicine, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China, 200025.
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14
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Salicylic diamines selectively eliminate residual undifferentiated cells from pluripotent stem cell-derived cardiomyocyte preparations. Sci Rep 2021; 11:2391. [PMID: 33504837 PMCID: PMC7841182 DOI: 10.1038/s41598-021-81351-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2018] [Accepted: 12/14/2020] [Indexed: 02/06/2023] Open
Abstract
Clinical translation of pluripotent stem cell (PSC) derivatives is hindered by the tumorigenic risk from residual undifferentiated cells. Here, we identified salicylic diamines as potent agents exhibiting toxicity to murine and human PSCs but not to cardiomyocytes (CMs) derived from them. Half maximal inhibitory concentrations (IC50) of small molecules SM2 and SM6 were, respectively, 9- and 18-fold higher for human than murine PSCs, while the IC50 of SM8 was comparable for both PSC groups. Treatment of murine embryoid bodies in suspension differentiation cultures with the most effective small molecule SM6 significantly reduced PSC and non-PSC contamination and enriched CM populations that would otherwise be eliminated in genetic selection approaches. All tested salicylic diamines exerted their toxicity by inhibiting the oxygen consumption rate (OCR) in PSCs. No or only minimal and reversible effects on OCR, sarcomeric integrity, DNA stability, apoptosis rate, ROS levels or beating frequency were observed in PSC-CMs, although effects on human PSC-CMs seemed to be more deleterious at higher SM-concentrations. Teratoma formation from SM6-treated murine PSC-CMs was abolished or delayed compared to untreated cells. We conclude that salicylic diamines represent promising compounds for PSC removal and enrichment of CMs without the need for other selection strategies.
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15
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Branco MA, Cabral JM, Diogo MM. From Human Pluripotent Stem Cells to 3D Cardiac Microtissues: Progress, Applications and Challenges. Bioengineering (Basel) 2020; 7:E92. [PMID: 32785039 PMCID: PMC7552661 DOI: 10.3390/bioengineering7030092] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Revised: 07/30/2020] [Accepted: 08/06/2020] [Indexed: 12/19/2022] Open
Abstract
The knowledge acquired throughout the years concerning the in vivo regulation of cardiac development has promoted the establishment of directed differentiation protocols to obtain cardiomyocytes (CMs) and other cardiac cells from human pluripotent stem cells (hPSCs), which play a crucial role in the function and homeostasis of the heart. Among other developments in the field, the transition from homogeneous cultures of CMs to more complex multicellular cardiac microtissues (MTs) has increased the potential of these models for studying cardiac disorders in vitro and for clinically relevant applications such as drug screening and cardiotoxicity tests. This review addresses the state of the art of the generation of different cardiac cells from hPSCs and the impact of transitioning CM differentiation from 2D culture to a 3D environment. Additionally, current methods that may be employed to generate 3D cardiac MTs are reviewed and, finally, the adoption of these models for in vitro applications and their adaptation to medium- to high-throughput screening settings are also highlighted.
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Affiliation(s)
| | | | - Maria Margarida Diogo
- iBB-Institute for Bioengineering and Biosciences and Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal; (M.A.B.); (J.M.S.C.)
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16
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Choi JS, Smith AST, Williams NP, Matsubara T, Choi M, Kim JW, Kim HJ, Choi S, Kim DH. Nanopatterned Nafion microelectrode arrays for in vitro cardiac electrophysiology. ADVANCED FUNCTIONAL MATERIALS 2020; 30:1910660. [PMID: 33244297 PMCID: PMC7688058 DOI: 10.1002/adfm.201910660] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/23/2019] [Accepted: 03/09/2020] [Indexed: 05/03/2023]
Abstract
In this study, we report nanopatterned Nafion microelectrode arrays for in vitro cardiac electrophysiology. With the aim of defining sophisticated Nafion nanostructures with highly ionic conductivity, fabrication parameters such as Nafion concentration and curing temperature were optimized. By increasing curing temperature and Nafion concentration, we were able to control the replication fidelity of Nafion nanopatterns when copied from a PDMS master mold. We also found that cross-sectional morphology and ion current density of nanopatterned Nafion strongly depends on the fabrication parameters. To investigate this dependency, current-voltage analysis was conducted using organic electrochemical transistors (OECT) overlaid with patterned Nafion substrates. Nanopatterned Nafion was found to allow higher ion current densities than unpatterned surfaces. Furthermore, higher curing temperatures were found to render Nafion layers with higher ion/electrical transfer properties. To optimize nanopattern dimensions, electrical current flows, and film uniformity, a final configuration consisting of 5% nanopatterned Nafion cured at 65°C was chosen. Multielectrode arrays (MEAs) were then covered with optimized Nafion nanopatterns and used for electrophysiological analysis of two types of induced pluripotent stem cell-derived cardiomyocytes (iPSCs-CMs). These data highlight the suitability of nanopatterned Nafion, combined with MEAs, for enhancing the cellular environment of iPSC-CMs for use in electrophysiological analysis in vitro.
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Affiliation(s)
- Jong Seob Choi
- Department of Biomedical Engineering and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, United States
| | - Alec S T Smith
- Department of Bioengineering, University of Washington, 850 Republican Street, Seattle, WA 98109, United States
| | - Nisa P Williams
- Department of Bioengineering, University of Washington, 850 Republican Street, Seattle, WA 98109, United States
| | - Tatsuya Matsubara
- Department of Mechanical Engineering, Tokyo Institute of Technology, 226-85603, Japan
| | - Minji Choi
- Convergence Medical Device Research Center, Gumi Electronics and Information Technology Research Institute (GERI), 350-27, Gumidaero, Gumi, Gyeongbuk 39253, South Korea
| | - Joon-Wan Kim
- Laboratoryfor Future Interdisciplinary Research of Science and Technology (FIRST), Institute of Innovative Research(IIR), Tokyo Institute of Technology,J3-12, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan
| | - Hyung Jin Kim
- Convergence Medical Device Research Center, Gumi Electronics and Information Technology Research Institute (GERI), 350-27, Gumidaero, Gumi, Gyeongbuk 39253, South Korea
| | - Seungkeun Choi
- Division of Engineering and Mathematics, Electrical Engineering, University of Washington, 18115 Campus Way NE, Bothell, WA 98011, United States
| | - Deok-Ho Kim
- Department of Biomedical Engineering and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, United States
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17
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Lee HY, Hong IS. Metabolic Regulation and Related Molecular Mechanisms in Various Stem Cell Functions. Curr Stem Cell Res Ther 2020; 15:531-546. [PMID: 32394844 DOI: 10.2174/1574888x15666200512105347] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2019] [Revised: 02/11/2020] [Accepted: 03/02/2020] [Indexed: 02/07/2023]
Abstract
Recent studies on the mechanisms that link metabolic changes with stem cell fate have deepened our understanding of how specific metabolic pathways can regulate various stem cell functions during the development of an organism. Although it was originally thought to be merely a consequence of the specific cell state, metabolism is currently known to play a critical role in regulating the self-renewal capacity, differentiation potential, and quiescence of stem cells. Many studies in recent years have revealed that metabolic pathways regulate various stem cell behaviors (e.g., selfrenewal, migration, and differentiation) by modulating energy production through glycolysis or oxidative phosphorylation and by regulating the generation of metabolites, which can modulate multiple signaling pathways. Therefore, a more comprehensive understanding of stem cell metabolism could allow us to establish optimal culture conditions and differentiation methods that would increase stem cell expansion and function for cell-based therapies. However, little is known about how metabolic pathways regulate various stem cell functions. In this context, we review the current advances in metabolic research that have revealed functional roles for mitochondrial oxidative phosphorylation, anaerobic glycolysis, and oxidative stress during the self-renewal, differentiation and aging of various adult stem cell types. These approaches could provide novel strategies for the development of metabolic or pharmacological therapies to promote the regenerative potential of stem cells and subsequently promote their therapeutic utility.
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Affiliation(s)
- Hwa-Yong Lee
- Department of Biomedical Science, Jungwon University, 85 Goesan-eup, Munmu-ro, Goesan-gun, Chungcheongbuk-do 367-700, Korea
| | - In-Sun Hong
- Department of Health Sciences and Technology, GAIHST, Gachon University, Incheon 21999, Korea
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18
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Ramachandra CJA, Chua J, Cong S, Kp MMJ, Shim W, Wu JC, Hausenloy DJ. Human-induced pluripotent stem cells for modelling metabolic perturbations and impaired bioenergetics underlying cardiomyopathies. Cardiovasc Res 2020; 117:694-711. [PMID: 32365198 DOI: 10.1093/cvr/cvaa125] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/15/2020] [Revised: 03/23/2020] [Accepted: 04/24/2020] [Indexed: 12/17/2022] Open
Abstract
Normal cardiac contractile and relaxation functions are critically dependent on a continuous energy supply. Accordingly, metabolic perturbations and impaired mitochondrial bioenergetics with subsequent disruption of ATP production underpin a wide variety of cardiac diseases, including diabetic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, anthracycline cardiomyopathy, peripartum cardiomyopathy, and mitochondrial cardiomyopathies. Crucially, there are no specific treatments for preventing the onset or progression of these cardiomyopathies to heart failure, one of the leading causes of death and disability worldwide. Therefore, new treatments are needed to target the metabolic disturbances and impaired mitochondrial bioenergetics underlying these cardiomyopathies in order to improve health outcomes in these patients. However, investigation of the underlying mechanisms and the identification of novel therapeutic targets have been hampered by the lack of appropriate animal disease models. Furthermore, interspecies variation precludes the use of animal models for studying certain disorders, whereas patient-derived primary cell lines have limited lifespan and availability. Fortunately, the discovery of human-induced pluripotent stem cells has provided a promising tool for modelling cardiomyopathies via human heart tissue in a dish. In this review article, we highlight the use of patient-derived iPSCs for studying the pathogenesis underlying cardiomyopathies associated with metabolic perturbations and impaired mitochondrial bioenergetics, as the ability of iPSCs for self-renewal and differentiation makes them an ideal platform for investigating disease pathogenesis in a controlled in vitro environment. Continuing progress will help elucidate novel mechanistic pathways, and discover novel therapies for preventing the onset and progression of heart failure, thereby advancing a new era of personalized therapeutics for improving health outcomes in patients with cardiomyopathy.
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Affiliation(s)
- Chrishan J A Ramachandra
- National Heart Research Institute Singapore, National Heart Centre Singapore, 5 Hospital Drive, Singapore 169609, Singapore.,Cardiovascular and Metabolic Disorders Programme, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore
| | - Jasper Chua
- National Heart Research Institute Singapore, National Heart Centre Singapore, 5 Hospital Drive, Singapore 169609, Singapore.,Faculty of Science, National University of Singapore, 6 Science Drive 2, Singapore 117546, Singapore
| | - Shuo Cong
- National Heart Research Institute Singapore, National Heart Centre Singapore, 5 Hospital Drive, Singapore 169609, Singapore.,Department of Cardiac Surgery, Zhongshan Hospital, Fudan University, 111 Yixueyuan Road, Xuhui District, Shanghai 200032, China
| | - Myu Mai Ja Kp
- National Heart Research Institute Singapore, National Heart Centre Singapore, 5 Hospital Drive, Singapore 169609, Singapore
| | - Winston Shim
- Health and Social Sciences Cluster, Singapore Institute of Technology, 10 Dover Drive, Singapore 138683, Singapore
| | - Joseph C Wu
- Cardiovascular Institute, Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305, USA.,Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.,Department of Medicine, Stanford University, Stanford, CA 94305, USA.,Department of Radiology, Stanford University, Stanford, CA 94305, USA
| | - Derek J Hausenloy
- National Heart Research Institute Singapore, National Heart Centre Singapore, 5 Hospital Drive, Singapore 169609, Singapore.,Cardiovascular and Metabolic Disorders Programme, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore.,Yong Loo Lin Medical School, National University of Singapore, 10 Medical Drive, Singapore 11759, Singapore.,The Hatter Cardiovascular Institute, University College London, 67 Chenies Mews, Bloomsbury, London WC1E 6HX, UK.,Cardiovascular Research Centre, College of Medical and Health Sciences, Asia University, No. 500, Liufeng Road, Wufeng District, Taichung City 41354,Taiwan
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19
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Jiang B, Yan L, Shamul JG, Hakun M, He X. Stem cell therapy of myocardial infarction: a promising opportunity in bioengineering. ADVANCED THERAPEUTICS 2020; 3:1900182. [PMID: 33665356 PMCID: PMC7928435 DOI: 10.1002/adtp.201900182] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Indexed: 02/06/2023]
Abstract
Myocardial infarction (MI) is a life-threatening disease resulting from irreversible death of cardiomyocytes (CMs) and weakening of the heart blood-pumping function. Stem cell-based therapies have been studied for MI treatment over the last two decades with promising outcome. In this review, we critically summarize the past work in this field to elucidate the advantages and disadvantages of treating MI using pluripotent stem cells (PSCs) including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), adult stem cells, and cardiac progenitor cells. The main advantage of the latter is their cytokine production capability to modulate immune responses and control the progression of healing. However, human adult stem cells have very limited (if not 'no') capacity to differentiate into functional CMs in vitro or in vivo. In contrast, PSCs can be differentiated into functional CMs although the protocols for the cardiac differentiation of PSCs are mainly for adherent cells under 2D culture. Derivation of PSC-CMs in 3D, allowing for large-scale production of CMs via modulation of the Wnt/β-catenin signal pathway with defined chemicals and medium, may be desired for clinical translation. Furthermore, the technology of purification and maturation of the PSC-CMs may need further improvements to eliminate teratoma formation after in vivo implantation of the PSC-CMs for treating MI. In addition, in vitro derived PSC-CMs may have mechanical and electrical mismatch with the patient's cardiac tissue, which causes arrhythmia. This supports the use of PSC-derived cells committed to cardiac lineage without beating for implantation to treat MI. In this case, the PSC derived cells may utilize the mechanical, electrical, and chemical cues in the heart to further differentiate into mature/functional CMs in situ. Another major challenge facing stem cell therapy of MI is the low retention/survival of stem cells or their derivatives (e.g., PSC-CMs) in the heart for MI treatment after injection in vivo. This may be resolved by using biomaterials to engineer stem cells for reduced immunogenicity, immobilization of the cells in the heart, and increased integration with the host cardiac tissue. Biomaterials have also been applied in the derivation of CMs in vitro to increase the efficiency and maturation of differentiation. Collectively, a lot has been learned from the past failure of simply injecting intact stem cells or their derivatives in vivo for treating MI, and bioengineering stem cells with biomaterials is expected to be a valuable strategy for advancing stem cell therapy towards its widespread application for treating MI in the clinic.
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Affiliation(s)
- Bin Jiang
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
| | - Li Yan
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
| | - James G Shamul
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
| | - Maxwell Hakun
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
| | - Xiaoming He
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States
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20
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Williams NP, Rhodehamel M, Yan C, Smith AST, Jiao A, Murry CE, Scatena M, Kim DH. Engineering anisotropic 3D tubular tissues with flexible thermoresponsive nanofabricated substrates. Biomaterials 2020; 240:119856. [PMID: 32105818 DOI: 10.1016/j.biomaterials.2020.119856] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2019] [Revised: 02/08/2020] [Accepted: 02/08/2020] [Indexed: 12/19/2022]
Abstract
Tissue engineering aims to capture the structural and functional aspects of diverse tissue types in vitro. However, most approaches are limited in their ability to produce complex 3D geometries that are essential for tissue function. Tissues, such as the vasculature or chambers of the heart, often possess curved surfaces and hollow lumens that are difficult to recapitulate given their anisotropic architecture. Cell-sheet engineering techniques using thermoresponsive substrates provide a means to stack individual layers of cells with spatial control to create dense, scaffold-free tissues. In this study, we developed a novel method to fabricate complex 3D structures by layering multiple sheets of aligned cells onto flexible scaffolds and casting them into hollow tubular geometries using custom molds and gelatin hydrogels. To enable the fabrication of 3D tissues, we adapted our previously developed thermoresponsive nanopatterned cell-sheet technology by applying it to flexible substrates that could be folded as a form of tissue origami. We demonstrated the versatile nature of this platform by casting aligned sheets of smooth and cardiac muscle cells circumferentially around the surfaces of gelatin hydrogel tubes with hollow lumens. Additionally, we patterned skeletal muscle in the same fashion to recapitulate the 3D curvature that is observed in the muscles of the trunk. The circumferential cell patterning in each case was maintained after one week in culture and even encouraged organized skeletal myotube formation. Additionally, with the application of electrical field stimulation, skeletal myotubes began to assemble functional sarcomeres that could contract. Cardiac tubes could spontaneously contract and be paced for up to one month. Our flexible cell-sheet engineering approach provides an adaptable method to recapitulate more complex 3D geometries with tissue specific customization through the addition of different cell types, mold shapes, and hydrogels. By enabling the fabrication of scaled biomimetic models of human tissues, this approach could potentially be used to investigate tissue structure-function relationships, development, and maturation in the dish.
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Affiliation(s)
- Nisa P Williams
- Department of Bioengineering, University of Washington, Seattle, WA, 98109, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, 98109, USA
| | - Marcus Rhodehamel
- Department of Bioengineering, University of Washington, Seattle, WA, 98109, USA
| | - Calysta Yan
- Department of Bioengineering, University of Washington, Seattle, WA, 98109, USA
| | - Alec S T Smith
- Department of Bioengineering, University of Washington, Seattle, WA, 98109, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, 98109, USA
| | - Alex Jiao
- Department of Bioengineering, University of Washington, Seattle, WA, 98109, USA
| | - Charles E Murry
- Department of Bioengineering, University of Washington, Seattle, WA, 98109, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, 98109, USA; Department of Pathology, University of Washington, Seattle, WA, 98109, USA; Department of Medicine/Cardiology, University of Washington, Seattle, WA, 98109, USA; Center for Cardiovascular Biology, University of Washington, Seattle, WA, USA
| | - Marta Scatena
- Department of Bioengineering, University of Washington, Seattle, WA, 98109, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, 98109, USA; Center for Cardiovascular Biology, University of Washington, Seattle, WA, USA
| | - Deok-Ho Kim
- Department of Bioengineering, University of Washington, Seattle, WA, 98109, USA; Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, 98109, USA; Center for Cardiovascular Biology, University of Washington, Seattle, WA, USA; Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, 20205, USA; Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 20205, USA.
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21
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Guo NN, Liu LP, Zheng YW, Li YM. Inducing human induced pluripotent stem cell differentiation through embryoid bodies: A practical and stable approach. World J Stem Cells 2020; 12:25-34. [PMID: 32110273 PMCID: PMC7031760 DOI: 10.4252/wjsc.v12.i1.25] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/01/2019] [Revised: 09/30/2019] [Accepted: 12/13/2019] [Indexed: 02/06/2023] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) are invaluable resources for producing high-quality differentiated cells in unlimited quantities for both basic research and clinical use. They are particularly useful for studying human disease mechanisms in vitro by making it possible to circumvent the ethical issues of human embryonic stem cell research. However, significant limitations exist when using conventional flat culturing methods especially concerning cell expansion, differentiation efficiency, stability maintenance and multicellular 3D structure establishment, differentiation prediction. Embryoid bodies (EBs), the multicellular aggregates spontaneously generated from iPSCs in the suspension system, might help to address these issues. Due to the unique microenvironment and cell communication in EB structure that a 2D culture system cannot achieve, EBs have been widely applied in hiPSC-derived differentiation and show significant advantages especially in scaling up culturing, differentiation efficiency enhancement, ex vivo simulation, and organoid establishment. EBs can potentially also be used in early prediction of iPSC differentiation capability. To improve the stability and feasibility of EB-mediated differentiation and generate high quality EBs, critical factors including iPSC pluripotency maintenance, generation of uniform morphology using micro-pattern 3D culture systems, proper cellular density inoculation, and EB size control are discussed on the basis of both published data and our own laboratory experiences. Collectively, the production of a large quantity of homogeneous EBs with high quality is important for the stability and feasibility of many PSCs related studies.
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Affiliation(s)
- Ning-Ning Guo
- Institute of Regenerative Medicine, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
| | - Li-Ping Liu
- Institute of Regenerative Medicine, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
| | - Yun-Wen Zheng
- Institute of Regenerative Medicine, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
- Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, University of Tsukuba Faculty of Medicine, Tsukuba, Ibaraki 305-8575, Japan
- Yokohama City University School of Medicine, Yokohama, Kanagawa 234-0006, Japan
- Division of Regenerative Medicine, Center for Stem Cell Biology and Regenerative Medicine, The Institute of Medical Science, the University of Tokyo, Tokyo 108-8639, Japan.
| | - Yu-Mei Li
- Institute of Regenerative Medicine, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang 212001, Jiangsu Province, China
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Kishino Y, Fujita J, Tohyama S, Okada M, Tanosaki S, Someya S, Fukuda K. Toward the realization of cardiac regenerative medicine using pluripotent stem cells. Inflamm Regen 2020; 40:1. [PMID: 31938077 PMCID: PMC6956487 DOI: 10.1186/s41232-019-0110-4] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2019] [Accepted: 12/19/2019] [Indexed: 01/08/2023] Open
Abstract
Heart transplantation (HT) is the only radical treatment available for patients with end-stage heart failure that is refractory to optimal medical treatment and device therapies. However, HT as a therapeutic option is limited by marked donor shortage. To overcome this difficulty, regenerative medicine using human-induced pluripotent stem cells (hiPSCs) has drawn increasing attention as an alternative to HT. Several issues including the preparation of clinical-grade hiPSCs, methods for large-scale culture and production of hiPSCs and cardiomyocytes, prevention of tumorigenesis secondary to contamination of undifferentiated stem cells and non-cardiomyocytes, and establishment of an effective transplantation strategy need to be addressed to fulfill this unmet medical need. The ongoing rapid technological advances in hiPSC research have been directed toward the clinical application of this technology, and currently, most issues have been satisfactorily addressed. Cell therapy using hiPSC-derived cardiomyocytes is expected to serve as an integral component of realistic medicine in the near future and is being potentially viewed as a treatment that would revolutionize the management of patients with severe heart failure.
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Affiliation(s)
- Yoshikazu Kishino
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, 160-8582 Japan
| | - Jun Fujita
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, 160-8582 Japan
| | - Shugo Tohyama
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, 160-8582 Japan
| | - Marina Okada
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, 160-8582 Japan
| | - Sho Tanosaki
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, 160-8582 Japan
| | - Shota Someya
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, 160-8582 Japan
| | - Keiichi Fukuda
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo, 160-8582 Japan
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Cheng W, Yang S, Li X, Liang F, Zhou R, Wang H, Feng Y, Wang Y. Low doses of BPA induced abnormal mitochondrial fission and hypertrophy in human embryonic stem cell-derived cardiomyocytes via the calcineurin-DRP1 signaling pathway: A comparison between XX and XY cardiomyocytes. Toxicol Appl Pharmacol 2019; 388:114850. [PMID: 31830493 DOI: 10.1016/j.taap.2019.114850] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2019] [Revised: 11/28/2019] [Accepted: 12/03/2019] [Indexed: 02/08/2023]
Abstract
Humans are inevitably exposed to bisphenol A (BPA) via multiple exposure ways. Thus, attention should be raised to the possible adverse effects related to low doses of BPA. Epidemiological studies have outlined BPA exposure and the increased risk of cardiovascular diseases (such as cardiac hypertrophy), which has been confirmed to be sex-specific in rodent animals and present in few in vitro studies, although the molecular mechanism is still unclear. However, whether BPA at low doses equivalent to human internal exposure level could induce cardiac hypertrophy via the calcineurin-DRP1 signaling pathway by disrupting calcium homeostasis is unknown. To address this, human embryonic stem cell (H1, XY karyotype and H9, XX karyotype)-derived cardiomyocytes (CM) were purified and applied to study the low-dose effects of BPA on cardiomyocyte hypertrophy. In our study, when H1- and H9-CM were exposed to noncytotoxic BPA (8 ng/ml), markedly elevated hypertrophic-related mRNA expression levels (such as NPPA and NPPB), enhanced cellular area and reduced ATP supplementation, demonstrated the hypertrophic cardiomyocyte phenotype in vitro. The excessive fission produced by BPA was promoted by CnAβ-mediated dephosphorylation of DRP1. At the molecular level, the increase in cytosolic Ca2+ levels by low doses of BPA could discriminate between H1- and H9-CM, which may suggest a potential sex-specific hypertrophic risk in cardiomyocytes in terms of abnormal mitochondrial fission and ATP production by impairing CnAβ-DRP1 signaling. In CnAβ-knockdown cardiomyocytes, these changes were highly presented in XX-karyotyped cells, rather than in XY-karyotyped cells.
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Affiliation(s)
- Wei Cheng
- School of Public Health, Shanghai Jiaotong University, School of Medicine, Shanghai 200025, PR China
| | - Shoufei Yang
- School of Public Health, Shanghai Jiaotong University, School of Medicine, Shanghai 200025, PR China
| | - Xiaolan Li
- School of Public Health, Shanghai Jiaotong University, School of Medicine, Shanghai 200025, PR China
| | - Fan Liang
- School of Public Health, Shanghai Jiaotong University, School of Medicine, Shanghai 200025, PR China
| | - Ren Zhou
- The Ninth People's Hospital of Shanghai Jiao Tong University, School of Medicine, Shanghai 200011, PR China
| | - Hui Wang
- Center for Single-Cell Omics, School of Public Health, Shanghai Jiao Tong University, School of Medicine, Shanghai 200025, PR China
| | - Yan Feng
- School of Public Health, Shanghai Jiaotong University, School of Medicine, Shanghai 200025, PR China
| | - Yan Wang
- School of Public Health, Shanghai Jiaotong University, School of Medicine, Shanghai 200025, PR China; The Ninth People's Hospital of Shanghai Jiao Tong University, School of Medicine, Shanghai 200011, PR China; Shanghai Collaborative Innovation Center for Translational Medicine, Shanghai Jiaotong University, School of Medicine, Shanghai 200025, PR China.
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24
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An effective detachment system for human induced pluripotent stem cells cultured on multilayered cultivation substrates using resonance vibrations. Sci Rep 2019; 9:15655. [PMID: 31666563 PMCID: PMC6821886 DOI: 10.1038/s41598-019-51944-w] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2018] [Accepted: 10/08/2019] [Indexed: 01/14/2023] Open
Abstract
Clinical application of human induced pluripotent stem cells (hiPSCs) has been hampered by the lack of a practical, scalable culture system. Stacked culture plates (SCPs) have recently attracted attention. However, final cell yields depend on the efficiency of cell detachment, and inefficient cell recovery from SCPs presents a major challenge to their use. We have developed an effective detachment method using resonance vibrations (RVs) of substrates with sweeping driving frequency. By exciting RVs that have 1–3 antinodes with ultra-low-density enzyme spread on each substrate of SCPs, 87.8% of hiPSCs were successfully detached from a 5-layer SCP compared to 30.8% detached by the conventional enzymatic method. hiPSC viability was similar after either method. Moreover, hiPSCs detached by the RV method maintained their undifferentiated state. Additionally, hiPSCs after long-term culture (10 passages) kept excellent detachment efficiency, had the normal karyotypes, and maintained the undifferentiated state and pluripotency. These results indicated that the RV method has definite advantages over the conventional enzymatic method in the scalable culture of hiPSCs using SCPs.
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Brodehl A, Ebbinghaus H, Deutsch MA, Gummert J, Gärtner A, Ratnavadivel S, Milting H. Human Induced Pluripotent Stem-Cell-Derived Cardiomyocytes as Models for Genetic Cardiomyopathies. Int J Mol Sci 2019; 20:ijms20184381. [PMID: 31489928 PMCID: PMC6770343 DOI: 10.3390/ijms20184381] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2019] [Revised: 08/29/2019] [Accepted: 09/03/2019] [Indexed: 12/17/2022] Open
Abstract
In the last few decades, many pathogenic or likely pathogenic genetic mutations in over hundred different genes have been described for non-ischemic, genetic cardiomyopathies. However, the functional knowledge about most of these mutations is still limited because the generation of adequate animal models is time-consuming and challenging. Therefore, human induced pluripotent stem cells (iPSCs) carrying specific cardiomyopathy-associated mutations are a promising alternative. Since the original discovery that pluripotency can be artificially induced by the expression of different transcription factors, various patient-specific-induced pluripotent stem cell lines have been generated to model non-ischemic, genetic cardiomyopathies in vitro. In this review, we describe the genetic landscape of non-ischemic, genetic cardiomyopathies and give an overview about different human iPSC lines, which have been developed for the disease modeling of inherited cardiomyopathies. We summarize different methods and protocols for the general differentiation of human iPSCs into cardiomyocytes. In addition, we describe methods and technologies to investigate functionally human iPSC-derived cardiomyocytes. Furthermore, we summarize novel genome editing approaches for the genetic manipulation of human iPSCs. This review provides an overview about the genetic landscape of inherited cardiomyopathies with a focus on iPSC technology, which might be of interest for clinicians and basic scientists interested in genetic cardiomyopathies.
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Affiliation(s)
- Andreas Brodehl
- Erich and Hanna Klessmann Institute, Heart and Diabetes Center NRW, University Hospital of the Ruhr-University Bochum, Georgstrasse 11, D-32545 Bad Oeynhausen, Germany.
| | - Hans Ebbinghaus
- Erich and Hanna Klessmann Institute, Heart and Diabetes Center NRW, University Hospital of the Ruhr-University Bochum, Georgstrasse 11, D-32545 Bad Oeynhausen, Germany.
| | - Marcus-André Deutsch
- Department of Thoracic and Cardiovascular Surgery, Heart and Diabetes Center NRW, University Hospital Ruhr-University Bochum, Georgstrasse 11, D-32545 Bad Oeynhausen, Germany.
| | - Jan Gummert
- Erich and Hanna Klessmann Institute, Heart and Diabetes Center NRW, University Hospital of the Ruhr-University Bochum, Georgstrasse 11, D-32545 Bad Oeynhausen, Germany.
- Department of Thoracic and Cardiovascular Surgery, Heart and Diabetes Center NRW, University Hospital Ruhr-University Bochum, Georgstrasse 11, D-32545 Bad Oeynhausen, Germany.
| | - Anna Gärtner
- Erich and Hanna Klessmann Institute, Heart and Diabetes Center NRW, University Hospital of the Ruhr-University Bochum, Georgstrasse 11, D-32545 Bad Oeynhausen, Germany.
| | - Sandra Ratnavadivel
- Erich and Hanna Klessmann Institute, Heart and Diabetes Center NRW, University Hospital of the Ruhr-University Bochum, Georgstrasse 11, D-32545 Bad Oeynhausen, Germany.
| | - Hendrik Milting
- Erich and Hanna Klessmann Institute, Heart and Diabetes Center NRW, University Hospital of the Ruhr-University Bochum, Georgstrasse 11, D-32545 Bad Oeynhausen, Germany.
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Fujita J, Tohyama S, Kishino Y, Okada M, Morita Y. Concise Review: Genetic and Epigenetic Regulation of Cardiac Differentiation from Human Pluripotent Stem Cells. Stem Cells 2019; 37:992-1002. [PMID: 31021504 DOI: 10.1002/stem.3027] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2018] [Accepted: 04/15/2019] [Indexed: 12/28/2022]
Abstract
Human pluripotent stem cells (hPSCs), including both embryonic stem cells and induced pluripotent stem cells, are the ideal cell sources for disease modeling, drug discovery, and regenerative medicine. In particular, regenerative therapy with hPSC-derived cardiomyocytes (CMs) is an unmet medical need for the treatment of severe heart failure. Cardiac differentiation protocols from hPSCs are made on the basis of cardiac development in vivo. However, current protocols have yet to yield 100% pure CMs, and their maturity is low. Cardiac development is regulated by the cardiac gene network, including transcription factors (TFs). According to our current understanding of cardiac development, cardiac TFs are sequentially expressed during cardiac commitment in hPSCs. Expression levels of each gene are strictly regulated by epigenetic modifications. DNA methylation, histone modification, and noncoding RNAs significantly influence cardiac differentiation. These complex circuits of genetic and epigenetic factors dynamically affect protein expression and metabolic changes in cardiac differentiation and maturation. Here, we review cardiac differentiation protocols and their molecular machinery, closing with a discussion of the future challenges for producing hPSC-derived CMs. Stem Cells 2019;37:992-1002.
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Affiliation(s)
- Jun Fujita
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Shugo Tohyama
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Yoshikazu Kishino
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Marina Okada
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
| | - Yuika Morita
- Department of Cardiology, Keio University School of Medicine, Tokyo, Japan
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27
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Sadahiro T, Isomi M, Muraoka N, Kojima H, Haginiwa S, Kurotsu S, Tamura F, Tani H, Tohyama S, Fujita J, Miyoshi H, Kawamura Y, Goshima N, Iwasaki YW, Murano K, Saito K, Oda M, Andersen P, Kwon C, Uosaki H, Nishizono H, Fukuda K, Ieda M. Tbx6 Induces Nascent Mesoderm from Pluripotent Stem Cells and Temporally Controls Cardiac versus Somite Lineage Diversification. Cell Stem Cell 2018; 23:382-395.e5. [PMID: 30100166 DOI: 10.1016/j.stem.2018.07.001] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2017] [Revised: 05/08/2018] [Accepted: 07/02/2018] [Indexed: 10/28/2022]
Abstract
The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification.
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Affiliation(s)
- Taketaro Sadahiro
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Mari Isomi
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Naoto Muraoka
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Hidenori Kojima
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Sho Haginiwa
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Shota Kurotsu
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Fumiya Tamura
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Hidenori Tani
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Shugo Tohyama
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Jun Fujita
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Hiroyuki Miyoshi
- Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Yoshifumi Kawamura
- Japan Biological Informatics Consortium (JBiC), Koto-ku, Tokyo 135-8073, Japan
| | - Naoki Goshima
- Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Koto-ku, Tokyo 135-0064, Japan
| | - Yuka W Iwasaki
- Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Kensaku Murano
- Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Kuniaki Saito
- Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; Invertebrate Genetics Laboratory, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Mishima, Shizuoka 411-8540, Japan; Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Hayama, Kanagawa 240-0193, Japan
| | - Mayumi Oda
- Department of Systems Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Peter Andersen
- Division of Cardiology, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Chulan Kwon
- Division of Cardiology, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Hideki Uosaki
- Division of Cardiology, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan
| | - Hirofumi Nishizono
- Life Science Research Center, University of Toyama, Sugitani, Toyama 930-0194, Japan
| | - Keiichi Fukuda
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Masaki Ieda
- Department of Cardiology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba City, Ibaraki 305-8575, Japan.
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Ting S, Lam A, Tong G, Chen A, Wei H, Wu J, Lam YN, Reuveny S, Oh S. Meticulous optimization of cardiomyocyte yields in a 3-stage continuous integrated agitation bioprocess. Stem Cell Res 2018; 31:161-173. [DOI: 10.1016/j.scr.2018.07.020] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/26/2018] [Revised: 07/12/2018] [Accepted: 07/23/2018] [Indexed: 01/15/2023] Open
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Abou-Saleh H, Zouein FA, El-Yazbi A, Sanoudou D, Raynaud C, Rao C, Pintus G, Dehaini H, Eid AH. The march of pluripotent stem cells in cardiovascular regenerative medicine. Stem Cell Res Ther 2018; 9:201. [PMID: 30053890 PMCID: PMC6062943 DOI: 10.1186/s13287-018-0947-5] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Cardiovascular disease (CVD) continues to be the leading cause of global morbidity and mortality. Heart failure remains a major contributor to this mortality. Despite major therapeutic advances over the past decades, a better understanding of molecular and cellular mechanisms of CVD as well as improved therapeutic strategies for the management or treatment of heart failure are increasingly needed. Loss of myocardium is a major driver of heart failure. An attractive approach that appears to provide promising results in reducing cardiac degeneration is stem cell therapy (SCT). In this review, we describe different types of stem cells, including embryonic and adult stem cells, and we provide a detailed discussion of the properties of induced pluripotent stem cells (iPSCs). We also present and critically discuss the key methods used for converting somatic cells to pluripotent cells and iPSCs to cardiomyocytes (CMs), along with their advantages and limitations. Integrating and non-integrating reprogramming methods as well as characterization of iPSCs and iPSC-derived CMs are discussed. Furthermore, we critically present various methods of differentiating iPSCs to CMs. The value of iPSC-CMs in regenerative medicine as well as myocardial disease modeling and cardiac regeneration are emphasized.
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Affiliation(s)
- Haissam Abou-Saleh
- Department of Biological and Environmental Sciences, Qatar University, Doha, Qatar
| | - Fouad A. Zouein
- Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
| | - Ahmed El-Yazbi
- Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
- Department of Pharmacology and Toxicology, Alexandria University, Alexandria, Egypt
| | - Despina Sanoudou
- Clinical Genomics and Pharmacogenomics Unit, 4th Department of Internal Medicine, “Attikon” Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
| | | | - Christopher Rao
- Department of Surgery, Queen Elizabeth Hospital, Woolwich, London, UK
| | - Gianfranco Pintus
- Department of Biomedical Sciences, College of Health Sciences, Qatar University, Doha, Qatar
| | - Hassan Dehaini
- Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
| | - Ali H. Eid
- Department of Biological and Environmental Sciences, Qatar University, Doha, Qatar
- Department of Pharmacology and Toxicology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
- Department of Biomedical Sciences, College of Health Sciences, Qatar University, Doha, Qatar
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Musunuru K, Sheikh F, Gupta RM, Houser SR, Maher KO, Milan DJ, Terzic A, Wu JC. Induced Pluripotent Stem Cells for Cardiovascular Disease Modeling and Precision Medicine: A Scientific Statement From the American Heart Association. CIRCULATION. GENOMIC AND PRECISION MEDICINE 2018; 11:e000043. [PMID: 29874173 PMCID: PMC6708586 DOI: 10.1161/hcg.0000000000000043] [Citation(s) in RCA: 135] [Impact Index Per Article: 19.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Induced pluripotent stem cells (iPSCs) offer an unprece-dented opportunity to study human physiology and disease at the cellular level. They also have the potential to be leveraged in the practice of precision medicine, for example, personalized drug testing. This statement comprehensively describes the provenance of iPSC lines, their use for cardiovascular disease modeling, their use for precision medicine, and strategies through which to promote their wider use for biomedical applications. Human iPSCs exhibit properties that render them uniquely qualified as model systems for studying human diseases: they are of human origin, which means they carry human genomes; they are pluripotent, which means that in principle, they can be differentiated into any of the human body's somatic cell types; and they are stem cells, which means they can be expanded from a single cell into millions or even billions of cell progeny. iPSCs offer the opportunity to study cells that are genetically matched to individual patients, and genome-editing tools allow introduction or correction of genetic variants. Initial progress has been made in using iPSCs to better understand cardiomyopathies, rhythm disorders, valvular and vascular disorders, and metabolic risk factors for ischemic heart disease. This promising work is still in its infancy. Similarly, iPSCs are only just starting to be used to identify the optimal medications to be used in patients from whom the cells were derived. This statement is intended to (1) summarize the state of the science with respect to the use of iPSCs for modeling of cardiovascular traits and disorders and for therapeutic screening; (2) identify opportunities and challenges in the use of iPSCs for disease modeling and precision medicine; and (3) outline strategies that will facilitate the use of iPSCs for biomedical applications. This statement is not intended to address the use of stem cells as regenerative therapy, such as transplantation into the body to treat ischemic heart disease or heart failure.
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Kim HS, Yoon JW, Li H, Jeong GO, Park JJ, Shin SE, Jang IH, Kim JH, Park WS. Functional expression and pharmaceutical efficacy of cardiac-specific ion channels in human embryonic stem cell-derived cardiomyocytes. Sci Rep 2017; 7:13821. [PMID: 29062050 PMCID: PMC5653792 DOI: 10.1038/s41598-017-14198-y] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2017] [Accepted: 07/21/2017] [Indexed: 01/08/2023] Open
Abstract
Cardiomyocytes differentiated from human pluripotent stem cells provide promising tools for screening of cardiotoxic drugs. For evaluation of human pluripotent stem cell-derived cardiomyocytes for cardiotoxicity test, in the present study, human embryonic stem cells (hESCs) were differentiated to cardiomyocytes, followed by metabolic selection to enrich the differentiated cardiomyocytes. The highly purified hESC-derived cardiomyocytes (hESC-CMs) expressed several cardiomyocyte-specific markers including cTnT, MLC2a, and α-SA, but not pluripotency markers, such as OCT4 and NANOG. Patch clamp technique and RT-PCR revealed the expression of cardiomyocyte-specific Na+, Ca2+, and K+ channels and cardiac action potential in hESC-CMs. To explore the potential use of hESC-CMs as functional cardiomyocytes for drug discovery and cardiotoxicity screening, we examined the effects of bisindolylmaleimide (BIM) (I), which inhibits native cardiac Ca2+ channels, on the Ca2+ channel activity of hESC-CMs. We observed a similar response for the BIM (I)-induced modulation of Ca2+ channels between hESC-CMs and native cardiomyocytes through L-type Ca2+ channel current. These results suggest that hESC-CMs can be useful for evaluation of pharmaceutical efficacy and safety of novel drug candidate in cardiac research.
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Affiliation(s)
- Han Sol Kim
- Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 24341, Republic of Korea
| | - Jung Won Yoon
- Department of Physiology, School of Medicine, Pusan National University, Yangsan, 50612, Republic of Korea
| | - Hongliang Li
- Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 24341, Republic of Korea
| | - Geun Ok Jeong
- Department of Physiology, School of Medicine, Pusan National University, Yangsan, 50612, Republic of Korea
| | - Jin Ju Park
- Department of Physiology, School of Medicine, Pusan National University, Yangsan, 50612, Republic of Korea
| | - Sung Eun Shin
- Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 24341, Republic of Korea
| | - Il Ho Jang
- Department of Oral Biochemistry and Molecular Biology, Pusan National University School of Dentistry, Yangsan, 50612, Republic of Korea
| | - Jae Ho Kim
- Department of Physiology, School of Medicine, Pusan National University, Yangsan, 50612, Republic of Korea. .,Research Institute of Convergence Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan, 50612, Republic of Korea.
| | - Won Sun Park
- Department of Physiology, Kangwon National University School of Medicine, Chuncheon, 24341, Republic of Korea.
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Tohyama S, Fujita J, Fujita C, Yamaguchi M, Kanaami S, Ohno R, Sakamoto K, Kodama M, Kurokawa J, Kanazawa H, Seki T, Kishino Y, Okada M, Nakajima K, Tanosaki S, Someya S, Hirano A, Kawaguchi S, Kobayashi E, Fukuda K. Efficient Large-Scale 2D Culture System for Human Induced Pluripotent Stem Cells and Differentiated Cardiomyocytes. Stem Cell Reports 2017; 9:1406-1414. [PMID: 28988990 PMCID: PMC5829307 DOI: 10.1016/j.stemcr.2017.08.025] [Citation(s) in RCA: 87] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2016] [Revised: 08/30/2017] [Accepted: 08/31/2017] [Indexed: 01/08/2023] Open
Abstract
Cardiac regenerative therapies utilizing human induced pluripotent stem cells (hiPSCs) are hampered by ineffective large-scale culture. hiPSCs were cultured in multilayer culture plates (CPs) with active gas ventilation (AGV), resulting in stable proliferation and pluripotency. Seeding of 1 × 106 hiPSCs per layer yielded 7.2 × 108 hiPSCs in 4-layer CPs and 1.7 × 109 hiPSCs in 10-layer CPs with pluripotency. hiPSCs were sequentially differentiated into cardiomyocytes (CMs) in a two-dimensional (2D) differentiation protocol. The efficiency of cardiac differentiation using 10-layer CPs with AGV was 66%–87%. Approximately 6.2–7.0 × 108 cells (4-layer) and 1.5–2.8 × 109 cells (10-layer) were obtained with AGV. After metabolic purification with glucose- and glutamine-depleted and lactate-supplemented media, a massive amount of purified CMs was prepared. Here, we present a scalable 2D culture system using multilayer CPs with AGV for hiPSC-derived CMs, which will facilitate clinical applications for severe heart failure in the near future.
Efficient mass production of hiPSCs by multilayer culture plates with AGV Efficient mass production of hiPSC-CMs using a massive 2D culture system with AGV Mass production of pure hiPSC-CMs via metabolic selection
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Affiliation(s)
- Shugo Tohyama
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; Department of Organ Fabrication, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Jun Fujita
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
| | - Chihana Fujita
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Miho Yamaguchi
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Sayaka Kanaami
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Rei Ohno
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Kazuho Sakamoto
- Department of Pharmacology, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan; Department of Bio-Informational Pharmacology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
| | - Masami Kodama
- Department of Bio-informational Pharmacology, Medical Research Institute, National University Corporation Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Junko Kurokawa
- Department of Bio-Informational Pharmacology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
| | - Hideaki Kanazawa
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Tomohisa Seki
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Yoshikazu Kishino
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Marina Okada
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Kazuaki Nakajima
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Sho Tanosaki
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Shota Someya
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Akinori Hirano
- Department of Cardiovascular Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Shinji Kawaguchi
- Department of Cardiovascular Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Eiji Kobayashi
- Department of Organ Fabrication, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
| | - Keiichi Fukuda
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
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(Re-)programming of subtype specific cardiomyocytes. Adv Drug Deliv Rev 2017; 120:142-167. [PMID: 28916499 DOI: 10.1016/j.addr.2017.09.005] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2017] [Revised: 08/29/2017] [Accepted: 09/07/2017] [Indexed: 01/10/2023]
Abstract
Adult cardiomyocytes (CMs) possess a highly restricted intrinsic regenerative potential - a major barrier to the effective treatment of a range of chronic degenerative cardiac disorders characterized by cellular loss and/or irreversible dysfunction and which underlies the majority of deaths in developed countries. Both stem cell programming and direct cell reprogramming hold promise as novel, potentially curative approaches to address this therapeutic challenge. The advent of induced pluripotent stem cells (iPSCs) has introduced a second pluripotent stem cell source besides embryonic stem cells (ESCs), enabling even autologous cardiomyocyte production. In addition, the recent achievement of directly reprogramming somatic cells into cardiomyocytes is likely to become of great importance. In either case, different clinical scenarios will require the generation of highly pure, specific cardiac cellular-subtypes. In this review, we discuss these themes as related to the cardiovascular stem cell and programming field, including a focus on the emergent topic of pacemaker cell generation for the development of biological pacemakers and in vitro drug testing.
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Potassium as a pluripotency-associated element identified through inorganic element profiling in human pluripotent stem cells. Sci Rep 2017; 7:5005. [PMID: 28694442 PMCID: PMC5504050 DOI: 10.1038/s41598-017-05117-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2016] [Accepted: 05/24/2017] [Indexed: 12/20/2022] Open
Abstract
Despite their well-known function in maintaining normal cell physiology, how inorganic elements are relevant to cellular pluripotency and differentiation in human pluripotent stem cells (hPSCs) has yet to be systematically explored. Using total reflection X-ray fluorescence (TXRF) spectrometry and inductively coupled plasma mass spectrometry (ICP-MS), we analyzed the inorganic components of human cells with isogenic backgrounds in distinct states of cellular pluripotency. The elemental profiles revealed that the potassium content of human cells significantly differs when their cellular pluripotency changes. Pharmacological treatment that alters cell membrane permeability to potassium affected the maintenance and establishment of cellular pluripotency via multiple mechanisms in bona fide hPSCs and reprogrammed cells. Collectively, we report that potassium is a pluripotency-associated inorganic element in human cells and provide novel insights into the manipulation of cellular pluripotency in hPSCs by regulating intracellular potassium.
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Jacobson EF, Tzanakakis ES. Human pluripotent stem cell differentiation to functional pancreatic cells for diabetes therapies: Innovations, challenges and future directions. J Biol Eng 2017; 11:21. [PMID: 28680477 PMCID: PMC5494890 DOI: 10.1186/s13036-017-0066-3] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2017] [Accepted: 06/02/2017] [Indexed: 12/18/2022] Open
Abstract
Recent advances in the expansion and directed pancreatogenic differentiation of human pluripotent stem cells (hPSCs) have intensified efforts to generate functional pancreatic islet cells, especially insulin-secreting β-cells, for cell therapies against diabetes. However, the consistent generation of glucose-responsive insulin-releasing cells remains challenging. In this article, we first present basic concepts of pancreatic organogenesis, which frequently serves as a basis for engineering differentiation regimens. Next, past and current efforts are critically discussed for the conversion of hPSCs along pancreatic cell lineages, including endocrine β-cells and α-cells, as well as exocrine cells with emphasis placed on the later stages of commitment. Finally, major challenges and future directions are examined, such as the identification of factors for in vivo maturation, large-scale culture and post processing systems, cell loss during differentiation, culture economics, efficiency, and efficacy and exosomes and miRNAs in pancreatic differentiation.
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Affiliation(s)
- Elena F Jacobson
- Department of Chemical and Biological Engineering, Tufts University, 4 Colby St., Room 276A, Medford, MA 02155 USA
| | - Emmanuel S Tzanakakis
- Department of Chemical and Biological Engineering, Tufts University, 4 Colby St., Room 276A, Medford, MA 02155 USA.,Tufts Clinical and Translational Science Institute, Tufts Medical Center, Boston, MA 02111 USA
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Tohyama S, Tanosaki S, Someya S, Fujita J, Fukuda K. Manipulation of Pluripotent Stem Cell Metabolism for Clinical Application. CURRENT STEM CELL REPORTS 2017; 3:28-34. [PMID: 28261548 PMCID: PMC5315714 DOI: 10.1007/s40778-017-0073-9] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
PURPOSE OF REVIEW Pluripotent stem cells (PSCs) have the capacity to differentiate into various types of cells, and are promising cell sources for regenerative therapy and drug screening. However, to realize the clinical application of PSCs, a large number of highly qualified target cells must be stably prepared with low cost. To achieve this, great improvements in the reprogramming, differentiation, and elimination of residual PSCs will be necessary. In this review, we summarize the updated knowledge about metabolism in PSCs and its application. RECENT FINDINGS Recent studies have shown that PSCs have distinct metabolic profiles compared to differentiated cells. The metabolic profiles of PSCs are indispensable for the maintenance of pluripotency, self-renewal, differentiation capacity, and cell survival. SUMMARY Metabolic approaches show improved simplicity, scalability, and lower cost than conventional methods for differentiation and elimination of residual PSCs. Thus, manipulation of PSC metabolism will lead to new technologies to improve their efficiencies.
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Affiliation(s)
- Shugo Tohyama
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, 160-8582 Japan
- Department of Organ Fabrication, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, 160-8582 Japan
| | - Sho Tanosaki
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, 160-8582 Japan
| | - Shota Someya
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, 160-8582 Japan
| | - Jun Fujita
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, 160-8582 Japan
| | - Keiichi Fukuda
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, 160-8582 Japan
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37
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Mazzotta S, Neves C, Bonner RJ, Bernardo AS, Docherty K, Hoppler S. Distinctive Roles of Canonical and Noncanonical Wnt Signaling in Human Embryonic Cardiomyocyte Development. Stem Cell Reports 2016; 7:764-776. [PMID: 27641648 PMCID: PMC5063467 DOI: 10.1016/j.stemcr.2016.08.008] [Citation(s) in RCA: 86] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2015] [Revised: 08/11/2016] [Accepted: 08/11/2016] [Indexed: 11/27/2022] Open
Abstract
Wnt signaling is a key regulator of vertebrate heart development; however, specific roles for human cardiomyocyte development remain uncertain. Here we use human embryonic stem cells (hESCs) to analyze systematically in human cardiomyocyte development the expression of endogenous Wnt signaling components, monitor pathway activity, and dissect stage-specific requirements for canonical and noncanonical Wnt signaling mechanisms using small-molecule inhibitors. Our analysis suggests that WNT3 and WNT8A, via FZD7 and canonical signaling, regulate BRACHYURY expression and mesoderm induction; that WNT5A/5B, via ROR2 and noncanonical signaling, regulate MESP1 expression and cardiovascular development; and that later in development WNT2, WNT5A/5B, and WNT11, via FZD4 and FZD6, regulate functional cardiomyocyte differentiation via noncanonical Wnt signaling. Our findings confirm in human development previously proposed roles for canonical Wnt signaling in sequential stages of vertebrate cardiomyogenesis, and identify more precise roles for noncanonical signaling and for individual Wnt signal and Wnt receptor genes in human cardiomyocyte development.
hESCs were used to study Wnt signaling during human cardiomyocyte development Previously proposed roles for canonical Wnt signaling were confirmed in human Specific roles for noncanonical Wnt signaling were identified in cardiomyogenesis Individual Wnt signal and receptor genes were identified in human cardiomyogenesis
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Affiliation(s)
- Silvia Mazzotta
- Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK
| | - Carlos Neves
- Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK
| | - Rory J Bonner
- Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK
| | - Andreia S Bernardo
- The Francis Crick Institute, Mill Hill Laboratory, London NW7 1AA, UK; Anne McLaren Laboratory for Regenerative Medicine, University of Cambridge, West Forvie Building, Robinson Way, Cambridge CB2 0SZ, UK
| | - Kevin Docherty
- Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK
| | - Stefan Hoppler
- Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK.
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38
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Fonoudi H, Ansari H, Abbasalizadeh S, Blue GM, Aghdami N, Winlaw DS, Harvey RP, Bosman A, Baharvand H. Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol. J Vis Exp 2016. [PMID: 27500408 DOI: 10.3791/54276] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-β, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing > 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.
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Affiliation(s)
- Hananeh Fonoudi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR; Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute; St. Vincent´s Clinical School, Faculty of Medicine, University of New South Wales; Department of Developmental Biology, University of Science and Culture, Tehran, Iran
| | - Hassan Ansari
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR; Department of Developmental Biology, University of Science and Culture, Tehran, Iran
| | - Saeed Abbasalizadeh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR
| | - Gillian M Blue
- Heart Centre for Children, The Children´s Hospital at Westmead; Sydney Medical School, University of Sydney
| | - Nasser Aghdami
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR
| | - David S Winlaw
- Heart Centre for Children, The Children´s Hospital at Westmead; Sydney Medical School, University of Sydney
| | - Richard P Harvey
- Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute; St. Vincent´s Clinical School, Faculty of Medicine, University of New South Wales; School of Biotechnology and Biomolecular Sciences, University of New South Wales
| | - Alexis Bosman
- Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute; St. Vincent´s Clinical School, Faculty of Medicine, University of New South Wales;
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR; Department of Developmental Biology, University of Science and Culture
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39
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Fatima A, Xu G, Nguemo F, Kuzmenkin A, Burkert K, Hescheler J, Šarić T. Murine transgenic iPS cell line for monitoring and selection of cardiomyocytes. Stem Cell Res 2016; 17:266-272. [PMID: 27879210 DOI: 10.1016/j.scr.2016.07.007] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/01/2016] [Revised: 06/20/2016] [Accepted: 07/19/2016] [Indexed: 10/21/2022] Open
Abstract
We report here a transgenic murine induced pluripotent stem cell (iPSC) line expressing puromycin N-acetyltransferase (PAC) and enhanced green fluorescent protein (EGFP) under the control of α-myosin heavy chain promoter. This transgenic cell line reproducibly differentiates into EGFP-expressing cardiomyocytes (CMs) which can be generated at high purity with puromycin treatment and exhibit molecular and functional properties of immature heart muscle cells. This genetically modified iPSC line can be used for assessment of the utility of CMs for myocardial repair, pharmacological and toxicological applications and development of improved cardiac differentiation protocols.
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Affiliation(s)
- Azra Fatima
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany
| | - Guoxing Xu
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany
| | - Filomain Nguemo
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany
| | - Alexey Kuzmenkin
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany
| | - Karsten Burkert
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany
| | - Jürgen Hescheler
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany
| | - Tomo Šarić
- Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany.
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40
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Taegtmeyer H, Young ME, Lopaschuk GD, Abel ED, Brunengraber H, Darley-Usmar V, Des Rosiers C, Gerszten R, Glatz JF, Griffin JL, Gropler RJ, Holzhuetter HG, Kizer JR, Lewandowski ED, Malloy CR, Neubauer S, Peterson LR, Portman MA, Recchia FA, Van Eyk JE, Wang TJ. Assessing Cardiac Metabolism: A Scientific Statement From the American Heart Association. Circ Res 2016; 118:1659-701. [PMID: 27012580 DOI: 10.1161/res.0000000000000097] [Citation(s) in RCA: 199] [Impact Index Per Article: 22.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
In a complex system of interrelated reactions, the heart converts chemical energy to mechanical energy. Energy transfer is achieved through coordinated activation of enzymes, ion channels, and contractile elements, as well as structural and membrane proteins. The heart's needs for energy are difficult to overestimate. At a time when the cardiovascular research community is discovering a plethora of new molecular methods to assess cardiac metabolism, the methods remain scattered in the literature. The present statement on "Assessing Cardiac Metabolism" seeks to provide a collective and curated resource on methods and models used to investigate established and emerging aspects of cardiac metabolism. Some of those methods are refinements of classic biochemical tools, whereas most others are recent additions from the powerful tools of molecular biology. The aim of this statement is to be useful to many and to do justice to a dynamic field of great complexity.
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Park KM, Hussein KH, Hong SH, Ahn C, Yang SR, Park SM, Kweon OK, Kim BM, Woo HM. Decellularized Liver Extracellular Matrix as Promising Tools for Transplantable Bioengineered Liver Promotes Hepatic Lineage Commitments of Induced Pluripotent Stem Cells. Tissue Eng Part A 2016; 22:449-60. [PMID: 26801816 DOI: 10.1089/ten.tea.2015.0313] [Citation(s) in RCA: 80] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Liver transplantation is the last resort for liver failure patients. However, due to the shortage of donor organs, bioengineered liver generated from decellularized whole liver scaffolds and induced pluripotent stem cell (iPSC)-derived hepatocytes (iPSC-Heps) is being studied as an alternative approach to treat liver disease. Nevertheless, there has been no report on both the interaction of iPSC-Heps with a liver extracellular matrix (ECM) and the analysis of recellularized iPSC-Heps into the whole liver scaffolds. In this study, we produced porcine iPSC-Heps, which strongly expressed the hepatic markers α-fetoprotein and albumin and exhibited hepatic functionalities, including glycogen storage, lipid accumulation, low-density lipoprotein uptake, and indocyanine green metabolism. Supplementation of ECM from porcine decellularized liver containing liver-derived growth factors stimulated the albumin expression of porcine iPSC-Heps during differentiation procedures. The iPSC-Heps were reseeded into decellularized liver scaffolds, and the recellularized liver was cultured using a continuous perfusion system. The recellularized liver scaffolds were transplanted into rats for a short term, and the grafts expressed hepatocyte markers and did not rupture. These results provide a foundation for development of bioengineered liver using stem cell and decellularized scaffolds.
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Affiliation(s)
- Kyung-Mee Park
- 1 Stem Cell Institute-KNU, Kangwon National University , Chuncheon, Korea.,2 School of Veterinary Medicine, Kangwon National University , Chuncheon, Korea
| | - Kamal Hany Hussein
- 1 Stem Cell Institute-KNU, Kangwon National University , Chuncheon, Korea.,2 School of Veterinary Medicine, Kangwon National University , Chuncheon, Korea
| | - Seok-Ho Hong
- 1 Stem Cell Institute-KNU, Kangwon National University , Chuncheon, Korea.,3 School of Medicine, Kangwon National University , Chuncheon, Korea
| | - Cheol Ahn
- 1 Stem Cell Institute-KNU, Kangwon National University , Chuncheon, Korea.,4 School of Biomedical Technology, Kangwon National University , Chuncheon, Korea
| | - Se-Ran Yang
- 1 Stem Cell Institute-KNU, Kangwon National University , Chuncheon, Korea.,3 School of Medicine, Kangwon National University , Chuncheon, Korea
| | - Sung-Min Park
- 1 Stem Cell Institute-KNU, Kangwon National University , Chuncheon, Korea.,3 School of Medicine, Kangwon National University , Chuncheon, Korea
| | - Oh-Kyeong Kweon
- 5 School of Veterinary Medicine, Seoul National University , Seoul, Korea
| | - Byeong-Moo Kim
- 6 Departments of Medicine/GI Unit, Massachusetts General Hospital , Harvard Medical School, Boston, Massachusetts
| | - Heung-Myong Woo
- 1 Stem Cell Institute-KNU, Kangwon National University , Chuncheon, Korea.,2 School of Veterinary Medicine, Kangwon National University , Chuncheon, Korea
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42
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Human cardiomyocyte generation from pluripotent stem cells: A state-of-art. Life Sci 2015; 145:98-113. [PMID: 26682938 DOI: 10.1016/j.lfs.2015.12.023] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2015] [Revised: 11/08/2015] [Accepted: 12/09/2015] [Indexed: 12/11/2022]
Abstract
The human heart is considered a non-regenerative organ. Worldwide, cardiovascular diseases continue to be the leading cause of death. Despite advances in cardiac treatment, myocardial repair remains severely limited by the lack of an appropriate source of viable cardiomyocytes (CMs) to replace damaged tissue. Human pluripotent stem cells (hPSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can efficiently be differentiated into functional CMs necessary for cell replacement therapy and other potential applications. The number of protocols that derive CMs from hPSCs has increased exponentially over the past decade following observation of the first human beating CMs. A number of highly efficient, chemical based protocols have been developed to generate human CMs (hCMs) in small-scale and large-scale suspension systems. To reduce the heterogeneity of hPSC-derived CMs, the differentiation protocols were modulated to exclusively generate atrial-, ventricular-, and nodal-like CM subtypes. Recently, remarkable advances have been achieved in hCM generation including chemical-based cardiac differentiation, cardiac subtype specification, large-scale suspension culture differentiation, and development of chemically defined culture conditions. These hCMs could be useful particularly in the context of in vitro disease modeling, pharmaceutical screening and in cellular replacement therapies once the safety issues are overcome. Herein we review recent progress in the in vitro generation of CMs and cardiac subtypes from hPSCs and discuss their potential applications and current limitations.
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43
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Fonoudi H, Ansari H, Abbasalizadeh S, Larijani MR, Kiani S, Hashemizadeh S, Zarchi AS, Bosman A, Blue GM, Pahlavan S, Perry M, Orr Y, Mayorchak Y, Vandenberg J, Talkhabi M, Winlaw DS, Harvey RP, Aghdami N, Baharvand H. A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells. Stem Cells Transl Med 2015; 4:1482-94. [PMID: 26511653 DOI: 10.5966/sctm.2014-0275] [Citation(s) in RCA: 98] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2014] [Accepted: 07/08/2015] [Indexed: 12/15/2022] Open
Abstract
UNLABELLED Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large-scale production of hPSC-CM aggregates in a stirred suspension bioreactor as a single-unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor-β and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size-controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC-CMs successfully expressed canonical lineage-specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC-CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. SIGNIFICANCE Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC-CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end-stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC-CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC-CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high-throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease.
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Affiliation(s)
- Hananeh Fonoudi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia
| | - Hassan Ansari
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Saeed Abbasalizadeh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Mehran Rezaei Larijani
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Sahar Kiani
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Shiva Hashemizadeh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Ali Sharifi Zarchi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Alexis Bosman
- Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia
| | - Gillian M Blue
- Kids Heart Research, The Children's Hospital at Westmead, Sydney, New South Wales, Australia The Heart Centre for Children, The Children's Hospital at Westmead, Sydney, New South Wales, Australia Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
| | - Sara Pahlavan
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Matthew Perry
- St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia
| | - Yishay Orr
- Kids Heart Research, The Children's Hospital at Westmead, Sydney, New South Wales, Australia The Heart Centre for Children, The Children's Hospital at Westmead, Sydney, New South Wales, Australia
| | - Yaroslav Mayorchak
- The Heart Centre for Children, The Children's Hospital at Westmead, Sydney, New South Wales, Australia
| | - Jamie Vandenberg
- St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia
| | - Mahmood Talkhabi
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - David S Winlaw
- Kids Heart Research, The Children's Hospital at Westmead, Sydney, New South Wales, Australia The Heart Centre for Children, The Children's Hospital at Westmead, Sydney, New South Wales, Australia Sydney Medical School, University of Sydney, Sydney, New South Wales, Australia
| | - Richard P Harvey
- Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, New South Wales, Australia St. Vincent's Clinical School, Faculty of Medicine, University of New South Wales, Kensington, New South Wales, Australia School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, New South Wales, Australia
| | - Nasser Aghdami
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, Academic Center for Education, Culture and Research, Tehran, Iran Department of Developmental Biology, University of Science and Culture, Academic Center for Education, Culture and Research, Tehran, Iran
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Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S, Ishige N. Hepatocyte selection medium eliminating induced pluripotent stem cells among primary human hepatocytes. World J Methodol 2015; 5:108-114. [PMID: 26413482 PMCID: PMC4572022 DOI: 10.5662/wjm.v5.i3.108] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/10/2015] [Revised: 05/22/2015] [Accepted: 08/30/2015] [Indexed: 02/06/2023] Open
Abstract
Hepatic insufficiency is a fatal liver disease with a significant decrease in functioning hepatocytes. If hepatocytes could be generated from human induced pluripotent stem (hiPS) cells and transplanted into patients with hepatic insufficiency, the disease may become curable. However, a major limitation to this therapeutic strategy is due to the tumorigenicity of hiPS cells and their ability to form cancer. Current methods for eliminating unwanted hiPS cells use genetic manipulation or reagents that are potentially hazardous for hepatocytes; therefore, revised methods are necessary and anticipated. Glucose and arginine are essential cell culture medium ingredients for the survival of most cells, including hiPS cells. However, hepatocytes can produce its own glucose and arginine through galactokinase and ornithine transcarbamylase, respectively. Therefore, it was hypothesized that unwanted hiPS cells could be eliminated in a medium without glucose and arginine, and supplemented with galactose and ornithine instead. This modified medium has been established as hepatocyte selection medium (HSM). So far, attempts to generate a pure colony of mature hepatocytes from hiPS cells have not been successful. After establishment of co-culture in HSM, primary human hepatocytes survive while hiPS cells die within three days. Our latest results regarding a modification of HSM will be introduced in this manuscript.
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Kempf H, Kropp C, Olmer R, Martin U, Zweigerdt R. Cardiac differentiation of human pluripotent stem cells in scalable suspension culture. Nat Protoc 2015; 10:1345-61. [PMID: 26270394 DOI: 10.1038/nprot.2015.089] [Citation(s) in RCA: 115] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are a potential cell source for regenerative therapies, drug discovery and disease modeling. All these applications require a routine supply of relatively large quantities of in vitro-generated CMs. This protocol describes a suspension culture-based strategy for the generation of hPSC-CMs as cell-only aggregates, which facilitates process development and scale-up. Aggregates are formed for 4 d in hPSC culture medium followed by 10 d of directed differentiation by applying chemical Wnt pathway modulators. The protocol is applicable to static multiwell formats supporting fast adaptation to specific hPSC line requirements. We also demonstrate how to apply the protocol using stirred tank bioreactors at a 100-ml scale, providing a well-controlled upscaling platform for CM production. In bioreactors, the generation of 40-50 million CMs per differentiation batch at >80% purity without further lineage enrichment can been achieved within 24 d.
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Affiliation(s)
- Henning Kempf
- 1] Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany. [2] REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany
| | - Christina Kropp
- 1] Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany. [2] REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany
| | - Ruth Olmer
- 1] Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany. [2] REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany. [3] Member of Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany
| | - Ulrich Martin
- 1] Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany. [2] REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany. [3] Member of Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany
| | - Robert Zweigerdt
- 1] Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany. [2] REBIRTH-Cluster of Excellence, Hannover Medical School, Hannover, Germany
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Cardiovascular Disease Modeling Using Patient-Specific Induced Pluripotent Stem Cells. Int J Mol Sci 2015; 16:18894-922. [PMID: 26274955 PMCID: PMC4581278 DOI: 10.3390/ijms160818894] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2015] [Revised: 08/01/2015] [Accepted: 08/03/2015] [Indexed: 12/20/2022] Open
Abstract
The generation of induced pluripotent stem cells (iPSCs) has opened up a new scientific frontier in medicine. This technology has made it possible to obtain pluripotent stem cells from individuals with genetic disorders. Because iPSCs carry the identical genetic anomalies related to those disorders, iPSCs are an ideal platform for medical research. The pathophysiological cellular phenotypes of genetically heritable heart diseases such as arrhythmias and cardiomyopathies, have been modeled on cell culture dishes using disease-specific iPSC-derived cardiomyocytes. These model systems can potentially provide new insights into disease mechanisms and drug discoveries. This review focuses on recent progress in cardiovascular disease modeling using iPSCs, and discusses problems and future perspectives concerning their use.
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Nakajima K, Fujita J, Matsui M, Tohyama S, Tamura N, Kanazawa H, Seki T, Kishino Y, Hirano A, Okada M, Tabei R, Sano M, Goto S, Tabata Y, Fukuda K. Gelatin Hydrogel Enhances the Engraftment of Transplanted Cardiomyocytes and Angiogenesis to Ameliorate Cardiac Function after Myocardial Infarction. PLoS One 2015; 10:e0133308. [PMID: 26186362 PMCID: PMC4505846 DOI: 10.1371/journal.pone.0133308] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2015] [Accepted: 06/25/2015] [Indexed: 11/18/2022] Open
Abstract
Cell transplantation therapy will mean a breakthrough in resolving the donor shortage in cardiac transplantation. Cardiomyocyte (CM) transplantation, however, has been relatively inefficient in restoring cardiac function after myocardial infarction (MI) due to low engraftment of transplanted CM. In order to ameliorate engraftment of CM, the novel transplantation strategy must be invented. Gelatin hydrogel (GH) is a biodegradable water-soluble polymer gel. Gelatin is made of collagen. Although we observed that collagen strongly induced the aggregation of platelets to potentially cause coronary microembolization, GH did not enhance thrombogenicity. Therefore, GH is a suitable biomaterial in the cell therapy after heart failure. To assess the effect of GH on the improvement of cardiac function, fetal rat CM (5×10(6) or 1x10(6) cells) were transplanted with GH (10 mg/ml) to infarcted hearts. We compared this group with sham operated rats, CM in phosphate buffered saline (PBS), only PBS, and only GH-transplanted groups. Three weeks after transplantation, cardiac function was evaluated by echocardiography. The echocardiography confirmed that transplantation of 5×10(6) CM with GH significantly improved cardiac systolic function, compared with the CM+PBS group (fractional area change: 75.1±3.4% vs. 60.7±5.9%, p<0.05), only PBS, and only GH groups (60.1±6.5%, 65.0±2.8%, p<0.05). Pathological analyses demonstrated that in the CM+GH group, CM were efficiently engrafted in infarcted myocardium (p<0.01) and angiogenesis was significantly enhanced (p<0.05) in both central and peripheral areas of the scar. Moreover, quantitative RT-PCR revealed that angiogenic cytokines, such as basic fibroblast growth factor, vascular endothelial growth factor, and hepatocyte growth factor, were significantly enriched in the CM+GH group (p<0.05). Here, we report that GH confined the CM effectively in infarcted myocardium after transplantation, and that CM transplanted with GH improved cardiac function with a direct contraction effect and enhanced angiogenesis.
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Affiliation(s)
- Kazuaki Nakajima
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, Japan
| | - Jun Fujita
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, Japan
- * E-mail:
| | - Makoto Matsui
- Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho Shogoin, Sakyo-ku, Kyoto, Japan
| | - Shugo Tohyama
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, Japan
- Japan Society for the Promotion of Science, Tokyo, Japan
| | - Noriko Tamura
- Department of Medicine (Cardiology), Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, Japan
| | - Hideaki Kanazawa
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, Japan
| | - Tomohisa Seki
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, Japan
| | - Yoshikazu Kishino
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, Japan
| | - Akinori Hirano
- Department of Cardiovascular Surgery, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, Japan
| | - Marina Okada
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, Japan
| | - Ryota Tabei
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, Japan
| | - Motoaki Sano
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, Japan
| | - Shinya Goto
- Department of Medicine (Cardiology), Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, Japan
| | - Yasuhiko Tabata
- Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho Shogoin, Sakyo-ku, Kyoto, Japan
| | - Keiichi Fukuda
- Department of Cardiology, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, Japan
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Generation of highly purified human cardiomyocytes from peripheral blood mononuclear cell-derived induced pluripotent stem cells. PLoS One 2015; 10:e0126596. [PMID: 25970162 PMCID: PMC4430251 DOI: 10.1371/journal.pone.0126596] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2015] [Accepted: 04/06/2015] [Indexed: 11/22/2022] Open
Abstract
Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.
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Parikh A, Wu J, Blanton RM, Tzanakakis ES. Signaling Pathways and Gene Regulatory Networks in Cardiomyocyte Differentiation. TISSUE ENGINEERING PART B-REVIEWS 2015; 21:377-92. [PMID: 25813860 DOI: 10.1089/ten.teb.2014.0662] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Strategies for harnessing stem cells as a source to treat cell loss in heart disease are the subject of intense research. Human pluripotent stem cells (hPSCs) can be expanded extensively in vitro and therefore can potentially provide sufficient quantities of patient-specific differentiated cardiomyocytes. Although multiple stimuli direct heart development, the differentiation process is driven in large part by signaling activity. The engineering of hPSCs to heart cell progeny has extensively relied on establishing proper combinations of soluble signals, which target genetic programs thereby inducing cardiomyocyte specification. Pertinent differentiation strategies have relied as a template on the development of embryonic heart in multiple model organisms. Here, information on the regulation of cardiomyocyte development from in vivo genetic and embryological studies is critically reviewed. A fresh interpretation is provided of in vivo and in vitro data on signaling pathways and gene regulatory networks (GRNs) underlying cardiopoiesis. The state-of-the-art understanding of signaling pathways and GRNs presented here can inform the design and optimization of methods for the engineering of tissues for heart therapies.
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Affiliation(s)
- Abhirath Parikh
- 1 Lonza Walkersville, Inc. , Lonza Group, Walkersville, Maryland
| | - Jincheng Wu
- 2 Department of Chemical and Biological Engineering, Tufts University , Medford, Massachusetts
| | - Robert M Blanton
- 3 Division of Cardiology, Molecular Cardiology Research Institute , Tufts Medical Center, Tufts School of Medicine, Boston, Massachusetts
| | - Emmanuel S Tzanakakis
- 2 Department of Chemical and Biological Engineering, Tufts University , Medford, Massachusetts.,4 Tufts Clinical and Translational Science Institute (CTSI) , Boston, Massachusetts
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Rodrigues GMC, Rodrigues CAV, Fernandes TG, Diogo MM, Cabral JMS. Clinical-scale purification of pluripotent stem cell derivatives for cell-based therapies. Biotechnol J 2015; 10:1103-14. [PMID: 25851544 DOI: 10.1002/biot.201400535] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2014] [Revised: 02/20/2015] [Accepted: 03/04/2015] [Indexed: 01/12/2023]
Abstract
Human pluripotent stem cells (hPSCs) have the potential to revolutionize cell-replacement therapies because of their ability to self renew and differentiate into nearly every cell type in the body. However, safety concerns have delayed the clinical translation of this technology. One cause for this is the capacity that hPSCs have to generate tumors after transplantation. Because of the challenges associated with achieving complete differentiation into clinically relevant cell types, the development of safe and efficient strategies for purifying committed cells is essential for advancing hPSC-based therapies. Several purification strategies have now succeeded in generating non-tumorigenic and homogeneous cell-populations. These techniques typically enrich for cells by either depleting early committed populations from teratoma-initiating hPSCs or by positively selecting cells after differentiation. Here we review the working principles behind separation methods that have facilitated the safe and controlled application of hPSC-derived cells in laboratory settings and pre-clinical research. We underscore the need for improving and integrating purification strategies within differentiation protocols in order to unlock the therapeutic potential of hPSCs.
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Affiliation(s)
- Gonçalo M C Rodrigues
- Department of Bioengineering and IBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
| | - Carlos A V Rodrigues
- Department of Bioengineering and IBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
| | - Tiago G Fernandes
- Department of Bioengineering and IBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
| | - Maria Margarida Diogo
- Department of Bioengineering and IBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
| | - Joaquim M S Cabral
- Department of Bioengineering and IBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal
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