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Almeida M, Inácio JM, Vital CM, Rodrigues MR, Araújo BC, Belo JA. Cell Reprogramming, Transdifferentiation, and Dedifferentiation Approaches for Heart Repair. Int J Mol Sci 2025; 26:3063. [PMID: 40243729 PMCID: PMC11988544 DOI: 10.3390/ijms26073063] [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: 03/01/2025] [Revised: 03/22/2025] [Accepted: 03/24/2025] [Indexed: 04/18/2025] Open
Abstract
Cardiovascular disease (CVD) remains the leading cause of death globally, with myocardial infarction (MI) being a major contributor. The current therapeutic approaches are limited in effectively regenerating damaged cardiac tissue. Up-to-date strategies for heart regeneration/reconstitution aim at cardiac remodeling through repairing the damaged tissue with an external cell source or by stimulating the existing cells to proliferate and repopulate the compromised area. Cell reprogramming is addressed to this challenge as a promising solution, converting fibroblasts and other cell types into functional cardiomyocytes, either by reverting cells to a pluripotent state or by directly switching cell lineage. Several strategies such as gene editing and the application of miRNA and small molecules have been explored for their potential to enhance cardiac regeneration. Those strategies take advantage of cell plasticity by introducing reprogramming factors that regress cell maturity in vitro, allowing for their later differentiation and thus endorsing cell transplantation, or promote in situ cell proliferation, leveraged by scaffolds embedded with pro-regenerative factors promoting efficient heart restoration. Despite notable advancements, important challenges persist, including low reprogramming efficiency, cell maturation limitations, and safety concerns in clinical applications. Nonetheless, integrating these innovative approaches offers a promising alternative for restoring cardiac function and reducing the dependency on full heart transplants.
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Affiliation(s)
| | - José M. Inácio
- Stem Cells and Development Laboratory, iNOVA4Health, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, 1169-056 Lisbon, Portugal; (M.A.); (C.M.V.); (M.R.R.); (B.C.A.)
| | | | | | | | - José A. Belo
- Stem Cells and Development Laboratory, iNOVA4Health, NOVA Medical School|Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, 1169-056 Lisbon, Portugal; (M.A.); (C.M.V.); (M.R.R.); (B.C.A.)
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Lin H, Wang X, Chung M, Cai S, Pan Y. Direct fibroblast reprogramming: an emerging strategy for treating organic fibrosis. J Transl Med 2025; 23:240. [PMID: 40016790 PMCID: PMC11869441 DOI: 10.1186/s12967-024-06060-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2024] [Accepted: 12/26/2024] [Indexed: 03/01/2025] Open
Abstract
Direct reprogramming has garnered considerable attention due to its capacity to directly convert differentiated cells into desired cells. Fibroblasts are frequently employed in reprogramming studies due to their abundance and accessibility. However, they are also the key drivers in the progression of fibrosis, a pathological condition characterized by excessive extracellular matrix deposition and tissue scarring. Furthermore, the initial stage of reprogramming typically involves deactivating fibrotic pathways. Hence, direct reprogramming offers a valuable method to regenerate target cells for tissue repair while simultaneously reducing fibrotic tendencies. Understanding the link between reprogramming and fibrosis could help develop effective strategies to treat damaged tissue with a potential risk of fibrosis. This review summarizes the advances in direct reprogramming and reveals their anti-fibrosis effects in various organs such as the heart, liver, and skin. Furthermore, we dissect the mechanisms of reprogramming influenced by fibrotic molecules including TGF-β signaling, mechanical signaling, inflammation signaling, epigenetic modifiers, and metabolic regulators. Innovative methods for fibroblast reprogramming like small molecules, CRISPRa, modified mRNA, and the challenges of cellular heterogeneity and senescence faced by in vivo direct reprogramming, are also discussed.
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Affiliation(s)
- Haohui Lin
- Laboratory of Regenerative Medicine, The 2nd Affiliated Hospital, Medical School, Shenzhen University, Shenzhen, China
| | - Xia Wang
- School of Medicine, The Chinese University of Hong Kong Shenzhen, Shenzhen, China
| | - Manhon Chung
- Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Sa Cai
- Laboratory of Regenerative Medicine, The 2nd Affiliated Hospital, Medical School, Shenzhen University, Shenzhen, China.
| | - Yu Pan
- Laboratory of Regenerative Medicine, The 2nd Affiliated Hospital, Medical School, Shenzhen University, Shenzhen, China.
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Piven OO, Vaičiulevičiūtė R, Bernotiene E, Dobrzyn P. Cardiomyocyte engineering: The meeting point of transcription factors, signaling networks, metabolism and function. Acta Physiol (Oxf) 2025; 241:e14271. [PMID: 39801134 DOI: 10.1111/apha.14271] [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: 05/28/2024] [Revised: 11/22/2024] [Accepted: 01/01/2025] [Indexed: 05/02/2025]
Abstract
Direct cardiac reprogramming or transdifferentiation is a relatively new and promising area in regenerative therapy, cardiovascular disease modeling, and drug discovery. Effective reprogramming of fibroblasts is limited by their plasticity, that is, their ability to reprogram, and depends on solving several levels of tasks: inducing cardiomyocyte-like cells and obtaining functionally and metabolically mature cardiomyocytes. Currently, in addition to the use of more classical approaches such as overexpression of exogenous transcription factors, activation of endogenous cardiac transcription factors via controlled nucleases, such as CRISPR, represents another interesting way to obtain cardiomyocytes. Therefore, special attention is given to the potential of synthetic biology, in particular the CRISPR system, for the targeted conversion of only certain subpopulations of fibroblasts into cardiomyocytes. However, obtaining functionally and metabolically mature cardiomyocytes remains a challenge despite the range of recently developed approaches. In this review, we summarized current knowledge on the function and diversity of human cardiac fibroblasts and alternative cell sources for in vitro human cardiomyocyte models. We examined in detail the transcription factors that initiate cardiomyogenic reprogramming and their interactions. Additionally, we critically analyzed the strategies used for the metabolic and physiological maturation of induced cardiomyocytes.
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Affiliation(s)
- Oksana O Piven
- Laboratory of Molecular Medical Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland
- Department of Human Genetics, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv, Ukraine
| | - Raminta Vaičiulevičiūtė
- Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania
| | - Eiva Bernotiene
- Department of Regenerative Medicine, State Research Institute Centre for Innovative Medicine, Vilnius, Lithuania
- Faculty of Fundamental Sciences, VilniusTech University, Vilnius, Lithuania
| | - Pawel Dobrzyn
- Laboratory of Molecular Medical Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland
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4
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Bois A, Grandela C, Gallant J, Mummery C, Menasché P. Revitalizing the heart: strategies and tools for cardiomyocyte regeneration post-myocardial infarction. NPJ Regen Med 2025; 10:6. [PMID: 39843488 PMCID: PMC11754855 DOI: 10.1038/s41536-025-00394-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2024] [Accepted: 01/13/2025] [Indexed: 01/24/2025] Open
Abstract
Myocardial infarction (MI) causes the loss of millions of cardiomyocytes, and current treatments do not address this root issue. New therapies focus on stimulating cardiomyocyte division in the adult heart, inspired by the regenerative capacities of lower vertebrates and neonatal mice. This review explores strategies for heart regeneration, offers insights into cardiomyocyte proliferation, evaluates in vivo models, and discusses integrating in vitro human cardiac models to advance cardiac regeneration research.
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Affiliation(s)
- Axelle Bois
- Department of Anatomy and Embryology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
- Department of Cardiovascular Surgery, Université Paris Cité, INSERM U970, PARCC Hôpital Européen Georges Pompidou, 75015, Paris, France
| | - Catarina Grandela
- Department of Anatomy and Embryology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - James Gallant
- Department of Anatomy and Embryology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Christine Mummery
- Department of Anatomy and Embryology, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands.
| | - Philippe Menasché
- Department of Cardiovascular Surgery, Université Paris Cité, INSERM U970, PARCC Hôpital Européen Georges Pompidou, 75015, Paris, France
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Shi H, Spurlock BM, Liu J, Qian L. Control of cell fate upon transcription factor-driven cardiac reprogramming. Curr Opin Genet Dev 2024; 89:102226. [PMID: 39586652 PMCID: PMC11894758 DOI: 10.1016/j.gde.2024.102226] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2024] [Revised: 06/07/2024] [Accepted: 06/24/2024] [Indexed: 11/27/2024]
Abstract
Adult mammals are susceptible to substantial cardiomyocyte (CM) loss following various cardiac diseases due to the limited capacity of CM proliferation and regeneration. Recently, direct cardiac reprogramming, converting fibroblasts into induced CMs, has been achieved both in vitro and in vivo through forced expression of transcription factors (TFs). This review encapsulates the advancements made in enhancing reprogramming efficiency and underlying molecular mechanisms. It covers the optimization of TF-based reprogramming cocktails and in vivo delivery platform and recently identified regulators in enhancing reprogramming efficiency. In addition, we discuss recent insights into the molecular mechanisms of direct cardiac reprogramming from single-cell omics analyses. Finally, we briefly touch on remaining challenges and prospective direction of this field.
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Affiliation(s)
- Huitong Shi
- The McAllister Heart Institute, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Brian M Spurlock
- The McAllister Heart Institute, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. https://twitter.com/@brineshrimp2
| | - Jiandong Liu
- The McAllister Heart Institute, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Li Qian
- The McAllister Heart Institute, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
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Prieto‐Vila M, Yoshioka Y, Kuriyama N, Okamura A, Yamamoto Y, Muranaka A, Ochiya T. Adult cardiomyocytes-derived EVs for the treatment of cardiac fibrosis. J Extracell Vesicles 2024; 13:e12461. [PMID: 38940266 PMCID: PMC11211925 DOI: 10.1002/jev2.12461] [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: 01/15/2024] [Revised: 04/15/2024] [Accepted: 05/06/2024] [Indexed: 06/29/2024] Open
Abstract
Cardiac fibrosis is a common pathological feature of cardiovascular diseases that arises from the hyperactivation of fibroblasts and excessive extracellular matrix (ECM) deposition, leading to impaired cardiac function and potentially heart failure or arrhythmia. Extracellular vesicles (EVs) released by cardiomyocytes (CMs) regulate various physiological functions essential for myocardial homeostasis, which are disrupted in cardiac disease. Therefore, healthy CM-derived EVs represent a promising cell-free therapy for the treatment of cardiac fibrosis. To this end, we optimized the culture conditions of human adult CMs to obtain a large yield of EVs without compromising cellular integrity by using a defined combination of small molecules. EVs were isolated by ultracentrifugation, and their characteristics were analysed. Finally, their effect on fibrosis was tested. Treatment of TGFβ-activated human cardiac fibroblasts with EVs derived from CMs using our culture system resulted in a decrease in fibroblast activation markers and ECM accumulation. The rescued phenotype was associated with specific EV cargo, including multiple myocyte-specific and antifibrotic microRNAs, although their effect individually was not as effective as the EV treatment. Notably, pathway analysis showed that EV treatment reverted the transcription of activated fibroblasts and decreased several signalling pathways, including MAPK, mTOR, JAK/STAT, TGFβ, and PI3K/Akt, all of which are involved in fibrosis development. Intracardiac injection of CM-derived EVs in an animal model of cardiac fibrosis reduced fibrotic area and increased angiogenesis, which correlated with improved cardiac function. These findings suggest that EVs derived from human adult CMs may offer a targeted and effective treatment for cardiac fibrosis, owing to their antifibrotic properties and the specificity of cargo.
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Affiliation(s)
- Marta Prieto‐Vila
- Department of Molecular and Cellular MedicineTokyo Medical UniversityTokyoJapan
| | - Yusuke Yoshioka
- Department of Molecular and Cellular MedicineTokyo Medical UniversityTokyoJapan
| | - Naoya Kuriyama
- Department of Molecular and Cellular MedicineTokyo Medical UniversityTokyoJapan
- Department of Vascular SurgeryAsahikawa Medical UniversityAsahikawaHokkaidoJapan
| | - Akihiko Okamura
- Department of Molecular and Cellular MedicineTokyo Medical UniversityTokyoJapan
- Department of Cardiovascular MedicineNara Medical UniversityNaraJapan
| | - Yusuke Yamamoto
- Laboratory of Integrative OncologyNational Cancer Center Research InstituteTokyoJapan
| | - Asao Muranaka
- Department of Molecular and Cellular MedicineTokyo Medical UniversityTokyoJapan
| | - Takahiro Ochiya
- Department of Molecular and Cellular MedicineTokyo Medical UniversityTokyoJapan
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Ahmad W, Saleh B, Qazi REM, Muneer R, Khan I, Khan M, Salim A. Direct differentiation of rat skin fibroblasts into cardiomyocytes. Exp Cell Res 2024; 435:113934. [PMID: 38237847 DOI: 10.1016/j.yexcr.2024.113934] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2023] [Revised: 12/16/2023] [Accepted: 01/13/2024] [Indexed: 01/28/2024]
Abstract
Myocardial infarction (MI) is one of the major cardiovascular diseases caused by diminished supply of nutrients and oxygen to the heart due to obstruction of the coronary artery. Different treatment options are available for cardiac diseases, however, they do not completely repair the damage. Therefore, reprogramming terminally differentiated fibroblasts using transcription factors is a promising strategy to differentiate them into cardiac like cells in vitro and to increase functional cardiomyocytes and reduce fibrotic scar in vivo. In this study, skin fibroblasts were selected for reprogramming because they serve as a convenient source for the autologous cell therapy. Fibroblasts were isolated from skin of rat pups, propagated, and directly reprogrammed towards cardiac lineage. For reprogramming, two different approaches were adopted, i.e., cells were transfected with: (1) combination of cardiac transcription factors; GATA4, MEF2c, Nkx2.5 (GMN), and (2) combination of cardiac transcription factors; GATA4, MEF2c, Nkx2.5, and iPSC factors; Oct4, Klf4, Sox2 and cMyc (GMNO). After 72 h of transfection, cells were analyzed for the expression of cardiac markers at the mRNA and protein levels. For in vivo study, rat MI models were developed by ligating the left anterior descending coronary artery and the reprogrammed cells were transplanted in the infarcted heart. qPCR results showed that the reprogrammed cells exhibited significant upregulation of cardiac genes. Immunocytochemistry analysis further confirmed cardiomyogenic differentiation of the reprogrammed cells. For the assessment of cardiac function, animals were analyzed via echocardiography after 2 and 4 weeks of cell transplantation. Echocardiographic results showed that the hearts transplanted with the reprogrammed cells improved ejection fraction, fractional shortening, left ventricular internal systolic and diastolic dimensions, and end systolic and diastolic volumes. After 4 weeks of cell transplantation, heart tissues were harvested and processed for histology. The histological analysis showed that the reprogrammed cells improved wall thickness of left ventricle and reduced fibrosis significantly as compared to the control. It is concluded from the study that novel combination of cardiac transcription factors directly reprogrammed skin fibroblasts and differentiated them into cardiomyocytes. These differentiated cells showed cardiomyogenic characters in vitro, and reduced fibrosis and improved cardiac function in vivo. Furthermore, direct reprogramming of fibroblasts transfected with cardiac transcription factors showed better regeneration of the injured myocardium and improved cardiac function as compared to the indirect approach in which combination of cardiac and iPSC factors were used. The study after further optimization could be used as a better strategy for cell-based therapeutic approaches for cardiovascular diseases.
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Affiliation(s)
- Waqas Ahmad
- Stem Cell Research Laboratory, Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
| | - Bilal Saleh
- Stem Cell Research Laboratory, Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
| | - Rida-E-Maria Qazi
- Stem Cell Research Laboratory, Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
| | - Rabbia Muneer
- Stem Cell Research Laboratory, Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
| | - Irfan Khan
- Stem Cell Research Laboratory, Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
| | - Mohsin Khan
- Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA
| | - Asmat Salim
- Stem Cell Research Laboratory, Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan.
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Wang Q, Spurlock B, Liu J, Qian L. Fibroblast Reprogramming in Cardiac Repair. JACC Basic Transl Sci 2024; 9:145-160. [PMID: 38362341 PMCID: PMC10864899 DOI: 10.1016/j.jacbts.2023.06.012] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/04/2023] [Revised: 06/22/2023] [Accepted: 06/27/2023] [Indexed: 02/17/2024]
Abstract
Cardiovascular disease is one of the major causes of death worldwide. Limited proliferative capacity of adult mammalian cardiomyocytes has prompted researchers to exploit regenerative therapy after myocardial injury, such as myocardial infarction, to attenuate heart dysfunction caused by such injury. Direct cardiac reprogramming is a recently emerged promising approach to repair damaged myocardium by directly converting resident cardiac fibroblasts into cardiomyocyte-like cells. The achievement of in vivo direct reprogramming of fibroblasts has been shown, by multiple laboratories independently, to improve cardiac function and mitigate fibrosis post-myocardial infarction, which holds great potential for clinical application. There have been numerous pieces of valuable work in both basic and translational research to enhance our understanding and continued refinement of direct cardiac reprogramming in recent years. However, there remain many challenges to overcome before we can truly take advantage of this technique to treat patients with ischemic cardiac diseases. Here, we review recent progress of fibroblast reprogramming in cardiac repair, including the optimization of several reprogramming strategies, mechanistic exploration, and translational efforts, and we make recommendations for future research to further understand and translate direct cardiac reprogramming from bench to bedside. Challenges relating to these efforts will also be discussed.
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Affiliation(s)
- Qiaozi Wang
- Department of Pathology and Laboratory Medicine, McAllister Heart Institute, University of North Carolina, Chapel Hill, North Carolina, USA
| | - Brian Spurlock
- Department of Pathology and Laboratory Medicine, McAllister Heart Institute, University of North Carolina, Chapel Hill, North Carolina, USA
| | - Jiandong Liu
- Department of Pathology and Laboratory Medicine, McAllister Heart Institute, University of North Carolina, Chapel Hill, North Carolina, USA
| | - Li Qian
- Department of Pathology and Laboratory Medicine, McAllister Heart Institute, University of North Carolina, Chapel Hill, North Carolina, USA
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Chung S, Sung HJ. In situ Reprogramming as a Pro-Angiogenic Inducer to Rescue Ischemic Tissues. Pulse (Basel) 2024; 12:58-65. [PMID: 39022557 PMCID: PMC11249613 DOI: 10.1159/000538075] [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: 12/28/2023] [Accepted: 02/25/2024] [Indexed: 07/20/2024] Open
Abstract
Background Enhanced regenerative therapeutic strategies are required to treat intractable ischemic heart disease. Summary Since the discovery of putative endothelial progenitor cells (EPCs) in 1997, many studies have focused on their extraction, ex vivo processing, and autotransplantation under ischemic conditions. Nonetheless, numerous randomized clinical trials involving thousands of patients have yielded only marginal treatment effects, highlighting the need for advances regarding insufficient dosage and complex ex vivo processing. The prevailing paradigm of cellular differentiation highlights the potential of direct cellular reprogramming, which paves the way for in situ reprogramming. In situ reprogramming holds the promise of significantly enhancing current therapeutic strategies, yet its success hinges on the precise targeting of candidate cells for reprogramming. In this context, the spleen emerges as a pivotal "in situ reprogramming hub," owing to its dual function as both a principal site for nanoparticle distribution and a significant reservoir of putative EPCs. The in situ reprogramming of splenic EPCs offers a potential solution to overcome critical challenges, including the aforementioned insufficient dosage and complex ex vivo processing. Key Messages This review explores the latest advancements in EPC therapy and in situ reprogramming, spotlighting a pioneering study that integrates those two strategies with a specific focus on the spleen. Such an innovative approach will potentially herald a new era of regenerative therapy for ischemic heart disease.
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Affiliation(s)
- Seyong Chung
- Department of Medical Engineering, Yonsei University College of Medicine, Seoul, Republic of Korea
| | - Hak-Joon Sung
- Department of Medical Engineering, Yonsei University College of Medicine, Seoul, Republic of Korea
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Zhang J, Wang Y, Guo J, Zhang N, He J, Zhou Z, Wu F. Direct Reprogramming of Mouse Fibroblasts to Osteoblast-like Cells Using Runx2/Dlx5 Factors on Engineered Stiff Hydrogels. ACS APPLIED MATERIALS & INTERFACES 2023; 15:59209-59223. [PMID: 38102996 DOI: 10.1021/acsami.3c14777] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2023]
Abstract
Direct reprogramming of somatic cells into functional cells still faces major limitations in terms of efficiency and achieving functional maturity of the reprogramed cells. While different approaches have been developed commonly based on exploiting biochemical signals, introducing appropriate mechanical cues that stimulate the reprogramming process is rarely reported. In this study, collagen-coated polyacrylamide (PAM) hydrogels with stiffness close to that of collagenous bone (40 kPa) were adopted to augment the direct reprogramming process of mouse fibroblasts to osteoblastic-like cells. The results suggested that culturing cells on a hydrogel substrate enhanced the overexpression of osteogenic transcription factors using nonviral vectors and improved the yield of osteoblast-like cells. Particularly, a synergistic effect on achieving osteogenic functionality has been observed for the mechanical cues and overexpression of transcriptional factors, leading to enhanced osteogenic transformation and production of bone mineral matrix. Animal experiments suggested that reprogramed cells generated on matrix hydrogels accelerated bone regeneration and stimulated ectopic osteogenesis. Mechanism analysis suggested the critical involvement of actomyosin contraction and mechanical signal-mediated pathways like the RhoA-ROCK pathway, leading to a synergistic effect on the key transcriptional processes, including chromatin remodeling, nuclear translocation, and epigenetic transition. This study provides insights into the mechanical cue-enhanced direct reprogramming and cell therapy.
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Affiliation(s)
- Junwei Zhang
- National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, PR China
| | - Yao Wang
- National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, PR China
| | - Jing Guo
- National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, PR China
| | - Nihui Zhang
- National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, PR China
| | - Jing He
- National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, PR China
| | - Zongke Zhou
- Orthopedic Research Institute & Department of Orthopedics, West China Hospital of Sichuan University, Chengdu 610041, Sichuan, China
| | - Fang Wu
- National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, PR China
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Xie Y, Van Handel B, Qian L, Ardehali R. Recent advances and future prospects in direct cardiac reprogramming. NATURE CARDIOVASCULAR RESEARCH 2023; 2:1148-1158. [PMID: 39196156 DOI: 10.1038/s44161-023-00377-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/16/2023] [Accepted: 10/09/2023] [Indexed: 08/29/2024]
Abstract
Cardiovascular disease remains a leading cause of death worldwide despite important advances in modern medical and surgical therapies. As human adult cardiomyocytes have limited regenerative ability, cardiomyocytes lost after myocardial infarction are replaced by fibrotic scar tissue, leading to cardiac dysfunction and heart failure. To replace lost cardiomyocytes, a promising approach is direct cardiac reprogramming, in which cardiac fibroblasts are transdifferentiated into induced cardiomyocyte-like cells (iCMs). Here we review cardiac reprogramming cocktails (including transcription factors, microRNAs and small molecules) that mediate iCM generation. We also highlight mechanistic studies exploring the barriers to and facilitators of this process. We then review recent progress in iCM reprogramming, with a focus on single-cell '-omics' research. Finally, we discuss obstacles to clinical application.
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Affiliation(s)
- Yifang Xie
- McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Ben Van Handel
- Department of Orthopedic Surgery, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, USA
| | - Li Qian
- McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Reza Ardehali
- Section of Cardiology, Department of Internal Medicine, Baylor College of Medicine, Houston, TX, USA.
- The Texas Heart Institute, Houston, TX, USA.
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Zhou H, Yang J, Srinath C, Zeng A, Wu I, Leon EC, Qureshi TN, Reid CA, Nettesheim ER, Xu E, Duclos Z, Mohamed TMA, Farshidfar F, Fejes A, Liu J, Jones S, Feathers C, Chung TW, Jing F, Prince WS, Lin J, Yu P, Srivastava D, Hoey T, Ivey KN, Lombardi LM. Improved Cardiac Function in Postischemic Rats Using an Optimized Cardiac Reprogramming Cocktail Delivered in a Single Novel Adeno-Associated Virus. Circulation 2023; 148:1099-1112. [PMID: 37602409 DOI: 10.1161/circulationaha.122.061542] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/04/2022] [Accepted: 07/20/2023] [Indexed: 08/22/2023]
Abstract
BACKGROUND Cardiac reprogramming is a technique to directly convert nonmyocytes into myocardial cells using genes or small molecules. This intervention provides functional benefit to the rodent heart when delivered at the time of myocardial infarction or activated transgenically up to 4 weeks after myocardial infarction. Yet, several hurdles have prevented the advancement of cardiac reprogramming for clinical use. METHODS Through a combination of screening and rational design, we identified a cardiac reprogramming cocktail that can be encoded in a single adeno-associated virus. We also created a novel adeno-associated virus capsid that can transduce cardiac fibroblasts more efficiently than available parental serotypes by mutating posttranslationally modified capsid residues. Because a constitutive promoter was needed to drive high expression of these cell fate-altering reprogramming factors, we included binding sites to a cardiomyocyte-restricted microRNA within the 3' untranslated region of the expression cassette that limits expression to nonmyocytes. After optimizing this expression cassette to reprogram human cardiac fibroblasts into induced cardiomyocyte-like cells in vitro, we also tested the ability of this capsid/cassette combination to confer functional benefit in acute mouse myocardial infarction and chronic rat myocardial infarction models. RESULTS We demonstrated sustained, dose-dependent improvement in cardiac function when treating a rat model 2 weeks after myocardial infarction, showing that cardiac reprogramming, when delivered in a single, clinically relevant adeno-associated virus vector, can support functional improvement in the postremodeled heart. This benefit was not observed with GFP (green fluorescent protein) or a hepatocyte reprogramming cocktail and was achieved even in the presence of immunosuppression, supporting myocyte formation as the underlying mechanism. CONCLUSIONS Collectively, these results advance the application of cardiac reprogramming gene therapy as a viable therapeutic approach to treat chronic heart failure resulting from ischemic injury.
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Affiliation(s)
- Huanyu Zhou
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Jin Yang
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Chetan Srinath
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Aliya Zeng
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Iris Wu
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Elena C Leon
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Tawny Neal Qureshi
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Christopher A Reid
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Emily R Nettesheim
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Emma Xu
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Zoe Duclos
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Tamer M A Mohamed
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Farshad Farshidfar
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Anthony Fejes
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Jun Liu
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Samantha Jones
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Charles Feathers
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Tae Won Chung
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Frank Jing
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - William S Prince
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - JianMin Lin
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Pengzhi Yu
- Gladstone Institutes, San Francisco, CA (P.Y., D.S.)
| | - Deepak Srivastava
- Gladstone Institutes, San Francisco, CA (P.Y., D.S.)
- Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, CA (D.S.)
- Division of Cardiology, Department of Pediatrics, School of Medicine (D.S.), University of California San Francisco
- Department of Biochemistry and Biophysics (D.S.), University of California San Francisco
| | - Timothy Hoey
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Kathryn N Ivey
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
| | - Laura M Lombardi
- Tenaya Therapeutics, Inc, South San Francisco, CA (H.Z., J.Y., C.S., A.Z., I.W., E.C.L., T.N.Q., C.A.R., E.R.N., E.X., Z.D., T.M.A.M., F.F., A.F., J. Liu, S.J., C.F., T.W.C., F.J., W.S.P., J. Lin, T.H., K.N.I., L.M.L.)
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13
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Sweat ME, Cao Y, Zhang X, Burnicka-Turek O, Perez-Cervantes C, Arulsamy K, Lu F, Keating EM, Akerberg BN, Ma Q, Wakimoto H, Gorham JM, Hill LD, Kyoung Song M, Trembley MA, Wang P, Gianeselli M, Prondzynski M, Bortolin RH, Bezzerides VJ, Chen K, Seidman JG, Seidman CE, Moskowitz IP, Pu WT. Tbx5 maintains atrial identity in post-natal cardiomyocytes by regulating an atrial-specific enhancer network. NATURE CARDIOVASCULAR RESEARCH 2023; 2:881-898. [PMID: 38344303 PMCID: PMC10854392 DOI: 10.1038/s44161-023-00334-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/18/2023] [Accepted: 08/21/2023] [Indexed: 02/15/2024]
Abstract
Understanding how the atrial and ventricular heart chambers maintain distinct identities is a prerequisite for treating chamber-specific diseases. Here, we selectively knocked out (KO) the transcription factor Tbx5 in the atrial working myocardium to evaluate its requirement for atrial identity. Atrial Tbx5 inactivation downregulated atrial cardiomyocyte (aCM) selective gene expression. Using concurrent single nucleus transcriptome and open chromatin profiling, genomic accessibility differences were identified between control and Tbx5 KO aCMs, revealing that 69% of the control-enriched ATAC regions were bound by TBX5. Genes associated with these regions were downregulated in KO aCMs, suggesting they function as TBX5-dependent enhancers. Comparing enhancer chromatin looping using H3K27ac HiChIP identified 510 chromatin loops sensitive to TBX5 dosage, and 74.8% of control-enriched loops contained anchors in control-enriched ATAC regions. Together, these data demonstrate TBX5 maintains the atrial gene expression program by binding to and preserving the tissue-specific chromatin architecture of atrial enhancers.
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Affiliation(s)
- Mason E. Sweat
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
| | - Yangpo Cao
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
- Department of Pharmacology, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China
| | - Xiaoran Zhang
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
| | - Ozanna Burnicka-Turek
- Department of Pediatrics, Pathology, and Human Genetics, The University of Chicago, Chicago, IL
| | - Carlos Perez-Cervantes
- Department of Pediatrics, Pathology, and Human Genetics, The University of Chicago, Chicago, IL
| | - Kulandai Arulsamy
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
| | - Fujian Lu
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
| | - Erin M. Keating
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
| | - Brynn N. Akerberg
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
| | - Qing Ma
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
| | - Hiroko Wakimoto
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Joshua M. Gorham
- Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
| | - Lauren D. Hill
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
| | - Mi Kyoung Song
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
- Department of Pediatrics, Seoul National University College of Medicine, Seoul, Korea
| | - Michael A. Trembley
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
| | - Peizhe Wang
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
| | - Matteo Gianeselli
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
| | | | - Raul H. Bortolin
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
| | | | - Kaifu Chen
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
| | - Jonathan G. Seidman
- Department of Pediatrics, Pathology, and Human Genetics, The University of Chicago, Chicago, IL
| | - Christine E. Seidman
- Department of Pediatrics, Pathology, and Human Genetics, The University of Chicago, Chicago, IL
| | - Ivan P. Moskowitz
- Department of Pharmacology, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China
| | - William T. Pu
- Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115
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14
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Romero-Tejeda M, Fonoudi H, Weddle CJ, DeKeyser JM, Lenny B, Fetterman KA, Magdy T, Sapkota Y, Epting CL, Burridge PW. A novel transcription factor combination for direct reprogramming to a spontaneously contracting human cardiomyocyte-like state. J Mol Cell Cardiol 2023; 182:30-43. [PMID: 37421991 PMCID: PMC10495191 DOI: 10.1016/j.yjmcc.2023.06.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/14/2022] [Revised: 06/06/2023] [Accepted: 06/27/2023] [Indexed: 07/10/2023]
Abstract
The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2+ cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach.
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Affiliation(s)
- Marisol Romero-Tejeda
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Hananeh Fonoudi
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Carly J Weddle
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Jean-Marc DeKeyser
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Brian Lenny
- Department of Epidemiology and Cancer Control, St. Jude Children's Hospital, Memphis, TN, USA
| | - K Ashley Fetterman
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Tarek Magdy
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Yadav Sapkota
- Department of Epidemiology and Cancer Control, St. Jude Children's Hospital, Memphis, TN, USA
| | - Conrad L Epting
- Departments of Pediatrics and Pathology, Northwestern University and Ann & Robert H.Lurie Children's Hospital of Chicago, Chicago, IL 60611, USA
| | - Paul W Burridge
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
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15
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Sun J, Wang L, Matthews RC, Walcott GP, Yu-An L, Wei Y, Zhou Y, Zangi L, Zhang J. CCND2 Modified mRNA Activates Cell Cycle of Cardiomyocytes in Hearts With Myocardial Infarction in Mice and Pigs. Circ Res 2023; 133:484-504. [PMID: 37565345 PMCID: PMC10529295 DOI: 10.1161/circresaha.123.322929] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/13/2023] [Accepted: 08/01/2023] [Indexed: 08/12/2023]
Abstract
BACKGROUND Experiments in mammalian models of cardiac injury suggest that the cardiomyocyte-specific overexpression of CCND2 (cyclin D2, in humans) improves recovery from myocardial infarction (MI). The primary objective of this investigation was to demonstrate that our specific modified mRNA translation system (SMRTs) can induce CCND2 expression in cardiomyocytes and replicate the benefits observed in other studies of cardiomyocyte-specific CCND2 overexpression for myocardial repair. METHODS The CCND2-cardiomyocyte-specific modified mRNA translation system (cardiomyocyte SMRTs) consists of 2 modRNA constructs: one codes for CCND2 and contains a binding site for L7Ae, and the other codes for L7Ae and contains recognition elements for the cardiomyocyte-specific microRNAs miR-1 and miR-208. Thus, L7Ae suppresses CCND2 translation in noncardiomyocytes but is itself suppressed by endogenous miR-1 and -208 in cardiomyocytes, thereby facilitating cardiomyocyte-specific CCND2 expression. Experiments were conducted in both mouse and pig models of MI, and control assessments were performed in animals treated with an SMRTs coding for the cardiomyocyte-specific expression of luciferase or green fluorescent protein (GFP), in animals treated with L7Ae modRNA alone or with the delivery vehicle, and in Sham-operated animals. RESULTS CCND2 was abundantly expressed in cultured, postmitotic cardiomyocytes 2 days after transfection with the CCND2-cardiomyocyte SMRTs, and the increase was accompanied by the upregulation of markers for cell-cycle activation and proliferation (eg, Ki67 and Aurora B kinase). When the GFP-cardiomyocyte SMRTs were intramyocardially injected into infarcted mouse hearts, the GFP signal was observed in cardiomyocytes but no other cell type. In both MI models, cardiomyocyte proliferation (on day 7 and day 3 after treatment administration in mice and pigs, respectively) was significantly greater, left-ventricular ejection fractions (days 7 and 28 in mice, days 10 and 28 in pigs) were significantly higher, and infarcts (day 28 in both species) were significantly smaller in animals treated with the CCND2-cardiomyocyte SMRTs than in any other group that underwent MI induction. CONCLUSIONS Intramyocardial injections of the CCND2-cardiomyocyte SMRTs promoted cardiomyocyte proliferation, reduced infarct size, and improved cardiac performance in small and large mammalian hearts with MI.
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Affiliation(s)
- Jiacheng Sun
- Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham
- These authors contributed equally to this work
| | - Lu Wang
- Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham
- These authors contributed equally to this work
| | - Rachel C. Matthews
- Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham
| | - Gregory P. Walcott
- Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham
- Department of Medicine, Division of Cardiovascular Disease, School of Medicine, University of Alabama at Birmingham
| | - Lu Yu-An
- Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham
| | - Yuhua Wei
- Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham
| | - Yang Zhou
- Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham
| | - Lior Zangi
- Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029
- Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029
| | - Jianyi Zhang
- Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham
- Department of Medicine, Division of Cardiovascular Disease, School of Medicine, University of Alabama at Birmingham
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16
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Kabłak-Ziembicka A, Badacz R, Okarski M, Wawak M, Przewłocki T, Podolec J. Cardiac microRNAs: diagnostic and therapeutic potential. Arch Med Sci 2023; 19:1360-1381. [PMID: 37732050 PMCID: PMC10507763 DOI: 10.5114/aoms/169775] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/20/2023] [Accepted: 07/18/2023] [Indexed: 09/22/2023] Open
Abstract
MicroRNAs are small non-coding post-translational biomolecules which, when expressed, modify their target genes. It is estimated that microRNAs regulate production of approximately 60% of all human proteins and enzymes that are responsible for major physiological processes. In cardiovascular disease pathophysiology, there are several cells that produce microRNAs, including endothelial cells, vascular smooth muscle cells, macrophages, platelets, and cardiomyocytes. There is a constant crosstalk between microRNAs derived from various cell sources. Atherosclerosis initiation and progression are driven by many pro-inflammatory and pro-thrombotic microRNAs. Atherosclerotic plaque rupture is the leading cause of cardiovascular death resulting from acute coronary syndrome (ACS) and leads to cardiac remodeling and fibrosis following ACS. MicroRNAs are powerful modulators of plaque progression and transformation into a vulnerable state, which can eventually lead to plaque rupture. There is a growing body of evidence which demonstrates that following ACS, microRNAs might inhibit fibroblast proliferation and scarring, as well as harmful apoptosis of cardiomyocytes, and stimulate fibroblast reprogramming into induced cardiac progenitor cells. In this review, we focus on the role of cardiomyocyte-derived and cardiac fibroblast-derived microRNAs that are involved in the regulation of genes associated with cardiomyocyte and fibroblast function and in atherosclerosis-related cardiac ischemia. Understanding their mechanisms may lead to the development of microRNA cocktails that can potentially be used in regenerative cardiology.
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Affiliation(s)
- Anna Kabłak-Ziembicka
- Department of Interventional Cardiology, Institute of Cardiology, Jagiellonian University Medical College, Krakow, Poland
- Noninvasive Cardiovascular Laboratory, the John Paul II Hospital, Krakow, Poland
| | - Rafał Badacz
- Department of Interventional Cardiology, Institute of Cardiology, Jagiellonian University Medical College, Krakow, Poland
- Department of Interventional Cardiology, the John Paul II Hospital, Krakow, Poland
| | - Michał Okarski
- Student Scientific Group of Modern Cardiac Therapy at the Department of Interventional Cardiology, Jagiellonian University Medical College, Krakow, Poland
| | - Magdalena Wawak
- Department of Interventional Cardiology, the John Paul II Hospital, Krakow, Poland
| | - Tadeusz Przewłocki
- Noninvasive Cardiovascular Laboratory, the John Paul II Hospital, Krakow, Poland
- Department of Cardiac and Vascular Diseases Institute of Cardiology, Jagiellonian University Medical College, Krakow, Poland
| | - Jakub Podolec
- Department of Interventional Cardiology, Institute of Cardiology, Jagiellonian University Medical College, Krakow, Poland
- Department of Interventional Cardiology, the John Paul II Hospital, Krakow, Poland
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17
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Chi C, Song K. Cellular reprogramming of fibroblasts in heart regeneration. J Mol Cell Cardiol 2023; 180:84-93. [PMID: 36965699 PMCID: PMC10347886 DOI: 10.1016/j.yjmcc.2023.03.009] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/31/2022] [Revised: 03/10/2023] [Accepted: 03/21/2023] [Indexed: 03/27/2023]
Abstract
Myocardial infarction causes the loss of cardiomyocytes and the formation of cardiac fibrosis due to the activation of cardiac fibroblasts, leading to cardiac dysfunction and heart failure. Unfortunately, current therapeutic interventions can only slow the disease progression. Furthermore, they cannot fully restore cardiac function, likely because the adult human heart lacks sufficient capacity to regenerate cardiomyocytes. Therefore, intensive efforts have focused on developing therapeutics to regenerate the damaged heart. Several strategies have been intensively investigated, including stimulation of cardiomyocyte proliferation, transplantation of stem cell-derived cardiomyocytes, and conversion of fibroblasts into cardiac cells. Resident cardiac fibroblasts are critical in the maintenance of the structure and contractility of the heart. Fibroblast plasticity makes this type of cells be reprogrammed into many cell types, including but not limited to induced pluripotent stem cells, induced cardiac progenitor cells, and induced cardiomyocytes. Fibroblasts have become a therapeutic target due to their critical roles in cardiac pathogenesis. This review summarizes the reprogramming of fibroblasts into induced pluripotent stem cell-derived cardiomyocytes, induced cardiac progenitor cells, and induced cardiomyocytes to repair a damaged heart, outlines recent findings in utilizing fibroblast-derived cells for heart regeneration, and discusses the limitations and challenges.
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Affiliation(s)
- Congwu Chi
- Division of Cardiology, Department of Medicine, The University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA
| | - Kunhua Song
- Division of Cardiology, Department of Medicine, The University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Gates Center for Regenerative Medicine and Stem Cell Biology, The University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
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18
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Brlecic PE, Bonham CA, Rosengart TK, Mathison M. Direct cardiac reprogramming: A new technology for cardiac repair. J Mol Cell Cardiol 2023; 178:51-58. [PMID: 36965701 PMCID: PMC10124164 DOI: 10.1016/j.yjmcc.2023.03.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/03/2022] [Revised: 03/03/2023] [Accepted: 03/21/2023] [Indexed: 03/27/2023]
Abstract
Cardiovascular disease is one of the leading causes of morbidity and mortality worldwide, with myocardial infarctions being amongst the deadliest manifestations. Reduced blood flow to the heart can result in the death of cardiac tissue, leaving affected patients susceptible to further complications and recurrent disease. Further, contemporary management typically involves a pharmacopeia to manage the metabolic conditions contributing to atherosclerotic and hypertensive heart disease, rather than regeneration of the damaged myocardium. With modern healthcare extending lifespan, a larger demographic will be at risk for heart disease, driving the need for novel therapeutics that surpass those currently available in efficacy. Transdifferentiation and cellular reprogramming have been looked to as potential methods for the treatment of diseases throughout the body. Specifically targeting the fibrotic cells in cardiac scar tissue as a source to be reprogrammed into induced cardiomyocytes remains an appealing option. This review aims to highlight the history of and advances in cardiac reprogramming and describe its translational potential as a treatment for cardiovascular disease.
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Affiliation(s)
- Paige E Brlecic
- Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA
| | - Clark A Bonham
- Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA
| | - Todd K Rosengart
- Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA
| | - Megumi Mathison
- Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX, USA.
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19
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Guo QY, Yang JQ, Feng XX, Zhou YJ. Regeneration of the heart: from molecular mechanisms to clinical therapeutics. Mil Med Res 2023; 10:18. [PMID: 37098604 PMCID: PMC10131330 DOI: 10.1186/s40779-023-00452-0] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/09/2021] [Accepted: 03/22/2023] [Indexed: 04/27/2023] Open
Abstract
Heart injury such as myocardial infarction leads to cardiomyocyte loss, fibrotic tissue deposition, and scar formation. These changes reduce cardiac contractility, resulting in heart failure, which causes a huge public health burden. Military personnel, compared with civilians, is exposed to more stress, a risk factor for heart diseases, making cardiovascular health management and treatment innovation an important topic for military medicine. So far, medical intervention can slow down cardiovascular disease progression, but not yet induce heart regeneration. In the past decades, studies have focused on mechanisms underlying the regenerative capability of the heart and applicable approaches to reverse heart injury. Insights have emerged from studies in animal models and early clinical trials. Clinical interventions show the potential to reduce scar formation and enhance cardiomyocyte proliferation that counteracts the pathogenesis of heart disease. In this review, we discuss the signaling events controlling the regeneration of heart tissue and summarize current therapeutic approaches to promote heart regeneration after injury.
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Affiliation(s)
- Qian-Yun Guo
- Beijing Key Laboratory of Precision Medicine of Coronary Atherosclerotic Disease, Beijing Institute of Heart Lung and Blood Vessel Disease, Clinical Center for Coronary Heart Disease, Department of Cardiology, Beijing Anzhen Hospital, Capital Medical University, Beijing, 100029, China
| | - Jia-Qi Yang
- Beijing Key Laboratory of Precision Medicine of Coronary Atherosclerotic Disease, Beijing Institute of Heart Lung and Blood Vessel Disease, Clinical Center for Coronary Heart Disease, Department of Cardiology, Beijing Anzhen Hospital, Capital Medical University, Beijing, 100029, China
| | - Xun-Xun Feng
- Beijing Key Laboratory of Precision Medicine of Coronary Atherosclerotic Disease, Beijing Institute of Heart Lung and Blood Vessel Disease, Clinical Center for Coronary Heart Disease, Department of Cardiology, Beijing Anzhen Hospital, Capital Medical University, Beijing, 100029, China
| | - Yu-Jie Zhou
- Beijing Key Laboratory of Precision Medicine of Coronary Atherosclerotic Disease, Beijing Institute of Heart Lung and Blood Vessel Disease, Clinical Center for Coronary Heart Disease, Department of Cardiology, Beijing Anzhen Hospital, Capital Medical University, Beijing, 100029, China.
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20
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Sweat ME, Cao Y, Zhang X, Burnicka-Turek O, Perez-Cervantes C, Akerberg BN, Ma Q, Wakimoto H, Gorham JM, Song MK, Trembley MA, Wang P, Lu F, Gianeselli M, Prondzynski M, Bortolin RH, Seidman JG, Seidman CE, Moskowitz IP, Pu WT. Tbx5 maintains atrial identity by regulating an atrial enhancer network. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.04.21.537535. [PMID: 37131696 PMCID: PMC10153240 DOI: 10.1101/2023.04.21.537535] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/04/2023]
Abstract
Understanding how the atrial and ventricular chambers of the heart maintain their distinct identity is a prerequisite for treating chamber-specific diseases. Here, we selectively inactivated the transcription factor Tbx5 in the atrial working myocardium of the neonatal mouse heart to show that it is required to maintain atrial identity. Atrial Tbx5 inactivation downregulated highly chamber specific genes such as Myl7 and Nppa , and conversely, increased the expression of ventricular identity genes including Myl2 . Using combined single nucleus transcriptome and open chromatin profiling, we assessed genomic accessibility changes underlying the altered atrial identity expression program, identifying 1846 genomic loci with greater accessibility in control atrial cardiomyocytes compared to KO aCMs. 69% of the control-enriched ATAC regions were bound by TBX5, demonstrating a role for TBX5 in maintaining atrial genomic accessibility. These regions were associated with genes that had higher expression in control aCMs compared to KO aCMs, suggesting they act as TBX5-dependent enhancers. We tested this hypothesis by analyzing enhancer chromatin looping using HiChIP and found 510 chromatin loops that were sensitive to TBX5 dosage. Of the loops enriched in control aCMs, 73.7% contained anchors in control-enriched ATAC regions. Together, these data demonstrate a genomic role for TBX5 in maintaining the atrial gene expression program by binding to atrial enhancers and preserving tissue-specific chromatin architecture of atrial enhancers.
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21
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Perveen S, Vanni R, Lo Iacono M, Rastaldo R, Giachino C. Direct Reprogramming of Resident Non-Myocyte Cells and Its Potential for In Vivo Cardiac Regeneration. Cells 2023; 12:1166. [PMID: 37190075 PMCID: PMC10136631 DOI: 10.3390/cells12081166] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2023] [Revised: 04/10/2023] [Accepted: 04/12/2023] [Indexed: 05/17/2023] Open
Abstract
Cardiac diseases are the foremost cause of morbidity and mortality worldwide. The heart has limited regenerative potential; therefore, lost cardiac tissue cannot be replenished after cardiac injury. Conventional therapies are unable to restore functional cardiac tissue. In recent decades, much attention has been paid to regenerative medicine to overcome this issue. Direct reprogramming is a promising therapeutic approach in regenerative cardiac medicine that has the potential to provide in situ cardiac regeneration. It consists of direct cell fate conversion of one cell type into another, avoiding transition through an intermediary pluripotent state. In injured cardiac tissue, this strategy directs transdifferentiation of resident non-myocyte cells (NMCs) into mature functional cardiac cells that help to restore the native tissue. Over the years, developments in reprogramming methods have suggested that regulation of several intrinsic factors in NMCs can help to achieve in situ direct cardiac reprogramming. Among NMCs, endogenous cardiac fibroblasts have been studied for their potential to be directly reprogrammed into both induced cardiomyocytes and induced cardiac progenitor cells, while pericytes can transdifferentiate towards endothelial cells and smooth muscle cells. This strategy has been indicated to improve heart function and reduce fibrosis after cardiac injury in preclinical models. This review summarizes the recent updates and progress in direct cardiac reprogramming of resident NMCs for in situ cardiac regeneration.
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Affiliation(s)
| | - Roberto Vanni
- Department of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, Italy
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22
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Garry GA, Olson EN. Reprogramming of cardiac cell fate as a therapeutic strategy for ischemic heart disease. J Mol Cell Cardiol 2023; 179:2-6. [PMID: 36997058 DOI: 10.1016/j.yjmcc.2023.03.013] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/26/2023] [Revised: 03/19/2023] [Accepted: 03/27/2023] [Indexed: 04/01/2023]
Abstract
Direct reprogramming of resident cardiac fibroblasts to induced cardiomyocytes is an attractive therapeutic strategy to restore function and remuscularize the injured heart. The cardiac transcription factors Gata4, Mef2c, and Tbx5 have been the mainstay of direct cardiac reprogramming strategies for the past decade. Yet, recent discoveries have identified alternative epigenetic factors capable of reprogramming human cells in the absence of these canonical factors. Further, single-cell genomics evaluating cellular maturation and epigenetics in the setting of injury and heart failure models following reprogramming have continued to inform the mechanistic underpinnings of this process and point toward future areas of discovery for the field. These discoveries and others covered in this review have provided complementary approaches that further enhance the effectiveness of reprogramming as a means of promoting cardiac regeneration following myocardial infarction and heart failure.
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Affiliation(s)
- Glynnis A Garry
- Department of Molecular Biology, the Hamon Center for Regenerative Science and Medicine, and Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
| | - Eric N Olson
- Department of Molecular Biology, the Hamon Center for Regenerative Science and Medicine, and Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA
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23
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Romero-Tejeda M, Fonoudi H, Weddle CJ, DeKeyser JM, Lenny B, Fetterman KA, Magdy T, Sapkota Y, Epting C, Burridge PW. A Novel Transcription Factor Combination for Direct Reprogramming to a Spontaneously Contracting Human Cardiomyocyte-like State. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.03.14.532629. [PMID: 36993577 PMCID: PMC10055062 DOI: 10.1101/2023.03.14.532629] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/19/2023]
Abstract
UNLABELLED The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4,960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD , SMAD6 , and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2 + cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach. HIGHLIGHTS Using network-based algorithm Mogrify, acoustic liquid handling, and high-content kinetic imaging cytometry we screened the effect of 4,960 unique transcription factor combinations. Using 24 patient-specific human fibroblast samples we identified the combination of MYOCD , SMAD6 , and TBX20 (MST) as the most successful direct reprogramming combination. MST cocktail results in reprogrammed cells with spontaneous contraction, cardiomyocyte-like calcium transients, and expression of cardiomyocyte associated genes.
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24
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Yamada Y, Sadahiro T, Ieda M. Development of direct cardiac reprogramming for clinical applications. J Mol Cell Cardiol 2023; 178:1-8. [PMID: 36918145 DOI: 10.1016/j.yjmcc.2023.03.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/28/2022] [Revised: 02/21/2023] [Accepted: 03/06/2023] [Indexed: 03/16/2023]
Abstract
The incidence of cardiovascular diseases is increasing worldwide, and cardiac regenerative therapy has great potential as a new treatment strategy, especially for ischemic heart disease. Direct cardiac reprogramming is a promising new cardiac regenerative therapy that uses defined factors to induce transdifferentiation of endogenous cardiac fibroblasts (CFs) into induced cardiomyocyte-like cells (iCMs). In vivo reprogramming is expected to restore lost cardiac function without necessitating cardiac transplantation by converting endogenous CFs that exist abundantly in cardiac tissues directly into iCMs. Indeed, we and other groups have demonstrated that in vivo cardiac reprogramming improves cardiac contractile function and reduces scar area after acute myocardial infarction (MI). Recently, we demonstrated that in vivo cardiac reprogramming is an innovative cardiac regenerative therapy that not only regenerates the myocardium, but also reverses fibrosis by inducing the quiescence of pro-fibrotic fibroblasts, thereby improving heart failure in chronic MI. In this review, we summarize the recent progresses in in vivo cardiac reprogramming, and discuss its prospects for future clinical applications and the challenges of direct human reprogramming, which has been a longstanding issue.
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Affiliation(s)
- Yu Yamada
- Department of Cardiology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba City, Ibaraki 305-8575, Japan
| | - Taketaro Sadahiro
- Department of Cardiology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba City, Ibaraki 305-8575, 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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25
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Haridhasapavalan KK, Borthakur A, Thummer RP. Direct Cardiac Reprogramming: Current Status and Future Prospects. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2023; 1436:1-18. [PMID: 36662416 DOI: 10.1007/5584_2022_760] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Advances in cellular reprogramming articulated the path for direct cardiac lineage conversion, bypassing the pluripotent state. Direct cardiac reprogramming attracts major attention because of the low or nil regenerative ability of cardiomyocytes, resulting in permanent cell loss in various heart diseases. In the field of cardiology, balancing this loss of cardiomyocytes was highly challenging, even in the modern medical world. Soon after the discovery of cell reprogramming, direct cardiac reprogramming also became a promising alternative for heart regeneration. This review mainly focused on the various direct cardiac reprogramming approaches (integrative and non-integrative) for the derivation of induced autologous cardiomyocytes. It also explains the advancements in cardiac reprogramming over the decade with the pros and cons of each approach. Further, the review highlights the importance of clinically relevant (non-integrative) approaches and their challenges for the prospective applications for personalized medicine. Apart from direct cardiac reprogramming, it also discusses the other strategies for generating cardiomyocytes from different sources. The understanding of these strategies could pave the way for the efficient generation of integration-free functional autologous cardiomyocytes through direct cardiac reprogramming for various biomedical applications.
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Affiliation(s)
- Krishna Kumar Haridhasapavalan
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India
| | - Atreyee Borthakur
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India
| | - Rajkumar P Thummer
- Laboratory for Stem Cell Engineering and Regenerative Medicine, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India.
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26
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Direct cardiac reprogramming: basics and future challenges. Mol Biol Rep 2023; 50:865-871. [PMID: 36308583 DOI: 10.1007/s11033-022-07913-0] [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: 04/29/2022] [Revised: 08/29/2022] [Accepted: 09/01/2022] [Indexed: 02/01/2023]
Abstract
BACKGROUND Heart failure is the leading cause of morbidity and mortality worldwide and is characterized by reduced cardiac function. Currently, cardiac transplantation therapy is applied for end-stage heart failure, but it is limited by the number of available donors. METHODS AND RESULTS Following an assessment of available literature, a narrative review was conducted to summarizes the current status and challenges of cardiac reprogramming for clinical application. Scientists have developed different regenerative treatment strategies for curing heart failure, including progenitor cell delivery and pluripotent cell delivery. Recently, a novel strategy has emerged that directly reprograms cardiac fibroblast into a functional cardiomyocyte. In this treatment, transcription factors are first identified to reprogram fibroblast into a cardiomyocyte. After that, microRNA and small molecules show great potential to optimize the reprogramming process. Some challenges regarding cell reprogramming in humans are conversion efficiency, virus utilization, immature and heterogenous induced cardiomyocytes, technical reproducibility issues, and physiological effects of depleted fibroblasts on myocardial tissue. CONCLUSION Several strategies have shown positive results in direct cardiac reprogramming. However, direct cardiac reprogramming still needs improvement if it is used as a mainstay therapy in humans, and challenges need to be overcome before cardiac reprogramming can be considered a viable therapeutic strategy. Further advances in cardiac reprogramming studies are needed in cardiac regenerative therapy.
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27
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Liu M, Liu J, Zhang T, Wang L. Direct cardiac reprogramming: Toward the era of multi-omics analysis. CELL INSIGHT 2022; 1:100058. [PMID: 37193352 PMCID: PMC10120284 DOI: 10.1016/j.cellin.2022.100058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/03/2022] [Revised: 09/29/2022] [Accepted: 09/29/2022] [Indexed: 05/18/2023]
Abstract
Limited regenerative capacity of adult cardiomyocytes precludes heart repair and regeneration after cardiac injury. Direct cardiac reprograming that converts scar-forming cardiac fibroblasts (CFs) into functional induced-cardiomyocytes (iCMs) offers promising potential to restore heart structure and heart function. Significant advances have been achieved in iCM reprogramming using genetic and epigenetic regulators, small molecules, and delivery strategies. Recent researches on the heterogeneity and reprogramming trajectories elucidated novel mechanisms of iCM reprogramming at single cell level. Here, we review recent progress in iCM reprogramming with a focus on multi-omics (transcriptomic, epigenomic and proteomic) researches to investigate the cellular and molecular machinery governing cell fate conversion. We also highlight the future potential using multi-omics approaches to dissect iCMs conversion for clinal applications.
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Affiliation(s)
- Mengxin Liu
- Department of Cardiology, Institute of Myocardial Injury and Repair, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China
- Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, 430071, China
| | - Jie Liu
- Department of Cardiology, Institute of Myocardial Injury and Repair, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China
- Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, 430071, China
| | - Tong Zhang
- Department of Cardiology, Institute of Myocardial Injury and Repair, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China
- Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, 430071, China
- State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan, 430062, China
| | - Li Wang
- Department of Cardiology, Institute of Myocardial Injury and Repair, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China
- Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Wuhan University, Wuhan, 430071, China
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28
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Tang Y, Aryal S, Geng X, Zhou X, Fast VG, Zhang J, Lu R, Zhou Y. TBX20 Improves Contractility and Mitochondrial Function During Direct Human Cardiac Reprogramming. Circulation 2022; 146:1518-1536. [PMID: 36102189 PMCID: PMC9662826 DOI: 10.1161/circulationaha.122.059713] [Citation(s) in RCA: 36] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
BACKGROUND Direct cardiac reprogramming of fibroblasts into cardiomyocytes has emerged as a promising strategy to remuscularize injured myocardium. However, it is insufficient to generate functional induced cardiomyocytes from human fibroblasts using conventional reprogramming cocktails, and the underlying molecular mechanisms are not well studied. METHODS To discover potential missing factors for human direct reprogramming, we performed transcriptomic comparison between human induced cardiomyocytes and functional cardiomyocytes. RESULTS We identified TBX20 (T-box transcription factor 20) as the top cardiac gene that is unable to be activated by the MGT133 reprogramming cocktail (MEF2C, GATA4, TBX5, and miR-133). TBX20 is required for normal heart development and cardiac function in adult cardiomyocytes, yet its role in cardiac reprogramming remains undefined. We show that the addition of TBX20 to the MGT133 cocktail (MGT+TBX20) promotes cardiac reprogramming and activates genes associated with cardiac contractility, maturation, and ventricular heart. Human induced cardiomyocytes produced with MGT+TBX20 demonstrated more frequent beating, calcium oscillation, and higher energy metabolism as evidenced by increased mitochondria numbers and mitochondrial respiration. Mechanistically, comprehensive transcriptomic, chromatin occupancy, and epigenomic studies revealed that TBX20 colocalizes with MGT reprogramming factors at cardiac gene enhancers associated with heart contraction, promotes chromatin binding and co-occupancy of MGT factors at these loci, and synergizes with MGT for more robust activation of target gene transcription. CONCLUSIONS TBX20 consolidates MGT cardiac reprogramming factors to activate cardiac enhancers to promote cardiac cell fate conversion. Human induced cardiomyocytes generated with TBX20 showed enhanced cardiac function in contractility and mitochondrial respiration.
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Affiliation(s)
- Yawen Tang
- Department of Biomedical Engineering (Y.T., X.G., V.G.F., J.Z., Y.Z.), Heersink School of Medicine, School of Engineering, University of Alabama at Birmingham
| | - Sajesan Aryal
- Department of Medicine, Division of Hematology and Oncology (S.A., X.Z., R.L.), Heersink School of Medicine, School of Engineering, University of Alabama at Birmingham.,O’Neal Comprehensive Cancer Center (S.A., X.Z., R.L.), Heersink School of Medicine, School of Engineering, University of Alabama at Birmingham
| | - Xiaoxiao Geng
- Department of Biomedical Engineering (Y.T., X.G., V.G.F., J.Z., Y.Z.), Heersink School of Medicine, School of Engineering, University of Alabama at Birmingham
| | - Xinyue Zhou
- Department of Medicine, Division of Hematology and Oncology (S.A., X.Z., R.L.), Heersink School of Medicine, School of Engineering, University of Alabama at Birmingham.,O’Neal Comprehensive Cancer Center (S.A., X.Z., R.L.), Heersink School of Medicine, School of Engineering, University of Alabama at Birmingham
| | - Vladimir G. Fast
- Department of Biomedical Engineering (Y.T., X.G., V.G.F., J.Z., Y.Z.), Heersink School of Medicine, School of Engineering, University of Alabama at Birmingham
| | - Jianyi Zhang
- Department of Biomedical Engineering (Y.T., X.G., V.G.F., J.Z., Y.Z.), Heersink School of Medicine, School of Engineering, University of Alabama at Birmingham
| | - Rui Lu
- Department of Medicine, Division of Hematology and Oncology (S.A., X.Z., R.L.), Heersink School of Medicine, School of Engineering, University of Alabama at Birmingham.,O’Neal Comprehensive Cancer Center (S.A., X.Z., R.L.), Heersink School of Medicine, School of Engineering, University of Alabama at Birmingham
| | - Yang Zhou
- Department of Biomedical Engineering (Y.T., X.G., V.G.F., J.Z., Y.Z.), Heersink School of Medicine, School of Engineering, University of Alabama at Birmingham
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29
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Ricketts SN, Qian L. The heart of cardiac reprogramming: The cardiac fibroblasts. J Mol Cell Cardiol 2022; 172:90-99. [PMID: 36007393 DOI: 10.1016/j.yjmcc.2022.08.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/14/2022] [Revised: 07/29/2022] [Accepted: 08/13/2022] [Indexed: 12/14/2022]
Abstract
Cardiovascular disease is the leading cause of death worldwide, outpacing pulmonary disease, infectious disease, and all forms of cancer. Myocardial infarction (MI) dominates cardiovascular disease, contributing to four out of five cardiovascular related deaths. Following MI, patients suffer adverse and irreversible myocardial remodeling associated with cardiomyocyte loss and infiltration of fibrotic scar tissue. Current therapies following MI only mitigate the cardiac physiological decline rather than restore damaged myocardium function. Direct cardiac reprogramming is one strategy that has promise in repairing injured cardiac tissue by generating new, functional cardiomyocytes from cardiac fibroblasts (CFs). With the ectopic expression of transcription factors, microRNAs, and small molecules, CFs can be reprogrammed into cardiomyocyte-like cells (iCMs) that display molecular signatures, structures, and contraction abilities similar to endogenous cardiomyocytes. The in vivo induction of iCMs following MI leads to significant reduction in fibrotic cardiac remodeling and improved heart function, indicating reprogramming is a viable option for repairing damaged heart tissue. Recent work has illustrated different methods to understand the mechanisms driving reprogramming, in an effort to improve the efficiency of iCM generation and create an approach translational into clinic. This review will provide an overview of CFs and describe different in vivo reprogramming methods.
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Affiliation(s)
- Shea N Ricketts
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Li Qian
- Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA; McAllister Heart Institute, University of North Carolina, Chapel Hill, NC 27599, USA.
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30
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Drouin-Ouellet J, Li D, Lu YR, Echegaray CV. The 2022 International Society for Stem Cell Research (ISSCR) Annual Meeting: Celebrating 20 Years of Achievements. Cell Reprogram 2022; 24:212-222. [DOI: 10.1089/cell.2022.0105] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Affiliation(s)
| | - Dan Li
- Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Massachusetts General Hospital, Harvard University, Boston, Massachusetts, USA
| | - Yuancheng Ryan Lu
- Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
| | - Camila Vazquez Echegaray
- Department of Molecular Medicine and Gene Therapy, Lund Stem Cell Centre, Wallenberg Centre for Molecular Medicine, Lund University, Lund, Sweden
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Razzaq SS, Khan I, Naeem N, Salim A, Begum S, Haneef K. Overexpression of GATA binding protein 4 and myocyte enhancer factor 2C induces differentiation of mesenchymal stem cells into cardiac-like cells. World J Stem Cells 2022; 14:700-713. [PMID: 36188117 PMCID: PMC9516467 DOI: 10.4252/wjsc.v14.i9.700] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/28/2022] [Revised: 06/20/2022] [Accepted: 08/30/2022] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Heart diseases are the primary cause of death all over the world. Following myocardial infarction, billions of cells die, resulting in a huge loss of cardiac function. Stem cell-based therapies have appeared as a new area to support heart regeneration. The transcription factors GATA binding protein 4 (GATA-4) and myocyte enhancer factor 2C (MEF2C) are considered prominent factors in the development of the cardiovascular system. AIM To explore the potential of GATA-4 and MEF2C for the cardiac differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs). METHODS hUC-MSCs were characterized morphologically and immunologically by the presence of specific markers of MSCs via immunocytochemistry and flow cytometry, and by their potential to differentiate into osteocytes and adipocytes. hUC-MSCs were transfected with GATA-4, MEF2C, and their combination to direct the differentiation. Cardiac differentiation was confirmed by semiquantitative real-time polymerase chain reaction and immunocytochemistry. RESULTS hUC-MSCs expressed specific cell surface markers CD105, CD90, CD44, and vimentin but lack the expression of CD45. The transcription factors GATA-4 and MEF2C, and their combination induced differentiation in hUC-MSCs with significant expression of cardiac genes i.e., GATA-4, MEF2C, NK2 homeobox 5 (NKX2.5), MHC, and connexin-43, and cardiac proteins GATA-4, NKX2.5, cardiac troponin T, and connexin-43. CONCLUSION Transfection with GATA-4, MEF2C, and their combination effectively induces cardiac differentiation in hUC-MSCs. These genetically modified MSCs could be a promising treatment option for heart diseases in the future.
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Affiliation(s)
- Syeda Saima Razzaq
- Dr. Zafar H. Zaidi Center for Proteomics, University of Karachi, Karachi 75270, Pakistan
| | - Irfan Khan
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
| | - Nadia Naeem
- Dow Research Institute of Biotechnology & Biomedical Sciences (DRIBBS), Dow University of Health Sciences (DUHS), Ojha Campus, Karachi 75200, Pakistan
| | - Asmat Salim
- Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
| | - Sumreen Begum
- Stem Cells Research Laboratory (SCRL), Sindh Institute of Urology and Transplantation (SIUT), Karachi 74200, Pakistan
| | - Kanwal Haneef
- Dr. Zafar H. Zaidi Center for Proteomics, University of Karachi, Karachi 75270, Pakistan.
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Lyra-Leite DM, Gutiérrez-Gutiérrez Ó, Wang M, Zhou Y, Cyganek L, Burridge PW. A review of protocols for human iPSC culture, cardiac differentiation, subtype-specification, maturation, and direct reprogramming. STAR Protoc 2022; 3:101560. [PMID: 36035804 PMCID: PMC9405110 DOI: 10.1016/j.xpro.2022.101560] [Citation(s) in RCA: 54] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
The methods for the culture and cardiomyocyte differentiation of human embryonic stem cells, and later human induced pluripotent stem cells (hiPSC), have moved from a complex and uncontrolled systems to simplified and relatively robust protocols, using the knowledge and cues gathered at each step. HiPSC-derived cardiomyocytes have proven to be a useful tool in human disease modelling, drug discovery, developmental biology, and regenerative medicine. In this protocol review, we will highlight the evolution of protocols associated with hPSC culture, cardiomyocyte differentiation, sub-type specification, and cardiomyocyte maturation. We also discuss protocols for somatic cell direct reprogramming to cardiomyocyte-like cells.
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Affiliation(s)
- Davi M Lyra-Leite
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
| | - Óscar Gutiérrez-Gutiérrez
- Stem Cell Unit, Clinic for Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Göttingen, Germany
| | - Meimei Wang
- Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Yang Zhou
- Department of Biomedical Engineering, University of Alabama at Birmingham, Birmingham, AL, USA
| | - Lukas Cyganek
- Stem Cell Unit, Clinic for Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Göttingen, Göttingen, Germany
| | - Paul W Burridge
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
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33
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Conservation of miR combo based direct cardiac reprogramming. Biochem Biophys Rep 2022; 31:101310. [DOI: 10.1016/j.bbrep.2022.101310] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2022] [Revised: 07/01/2022] [Accepted: 07/05/2022] [Indexed: 11/18/2022] Open
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Sierra-Pagan JE, Garry DJ. The regulatory role of pioneer factors during cardiovascular lineage specification – A mini review. Front Cardiovasc Med 2022; 9:972591. [PMID: 36082116 PMCID: PMC9445115 DOI: 10.3389/fcvm.2022.972591] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2022] [Accepted: 08/03/2022] [Indexed: 11/15/2022] Open
Abstract
Cardiovascular disease (CVD) remains the number one cause of death worldwide. Ischemic heart disease contributes to heart failure and has considerable morbidity and mortality. Therefore, alternative therapeutic strategies are urgently needed. One class of epigenetic regulators known as pioneer factors has emerged as an important tool for the development of regenerative therapies for the treatment of CVD. Pioneer factors bind closed chromatin and remodel it to drive lineage specification. Here, we review pioneer factors within the cardiovascular lineage, particularly during development and reprogramming and highlight the implications this field of research has for the future development of cardiac specific regenerative therapies.
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Affiliation(s)
- Javier E. Sierra-Pagan
- Cardiovascular Division, Department of Medicine, University of Minnesota, Minneapolis, MN, United States
| | - Daniel J. Garry
- Cardiovascular Division, Department of Medicine, University of Minnesota, Minneapolis, MN, United States
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, United States
- Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota, Minneapolis, MN, United States
- *Correspondence: Daniel J. Garry
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35
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Soltani Dehnavi S, Eivazi Zadeh Z, Harvey AR, Voelcker NH, Parish CL, Williams RJ, Elnathan R, Nisbet DR. Changing Fate: Reprogramming Cells via Engineered Nanoscale Delivery Materials. ADVANCED MATERIALS (DEERFIELD BEACH, FLA.) 2022; 34:e2108757. [PMID: 35396884 DOI: 10.1002/adma.202108757] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/31/2021] [Revised: 04/02/2022] [Indexed: 06/14/2023]
Abstract
The incorporation of nanotechnology in regenerative medicine is at the nexus of fundamental innovations and early-stage breakthroughs, enabling exciting biomedical advances. One of the most exciting recent developments is the use of nanoscale constructs to influence the fate of cells, which are the basic building blocks of healthy function. Appropriate cell types can be effectively manipulated by direct cell reprogramming; a robust technique to manipulate cellular function and fate, underpinning burgeoning advances in drug delivery systems, regenerative medicine, and disease remodeling. Individual transcription factors, or combinations thereof, can be introduced into cells using both viral and nonviral delivery systems. Existing approaches have inherent limitations. Viral-based tools include issues of viral integration into the genome of the cells, the propensity for uncontrollable silencing, reduced copy potential and cell specificity, and neutralization via the immune response. Current nonviral cell reprogramming tools generally suffer from inferior expression efficiency. Nanomaterials are increasingly being explored to address these challenges and improve the efficacy of both viral and nonviral delivery because of their unique properties such as small size and high surface area. This review presents the state-of-the-art research in cell reprogramming, focused on recent breakthroughs in the deployment of nanomaterials as cell reprogramming delivery tools.
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Affiliation(s)
- Shiva Soltani Dehnavi
- ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, ANU College of Health & Medicine, Canberra, ACT, 2601, Australia
- Research School of Chemistry, ANU College of Science, Canberra, ACT, 2601, Australia
- ANU College of Engineering & Computer Science, Canberra, ACT, 2601, Australia
| | - Zahra Eivazi Zadeh
- Biomedical Engineering Department, Amirkabir University of Technology, Tehran, 15875-4413, Iran
- The Graeme Clark Institute, The University of Melbourne, Melbourne, VIC, 3010, Australia
- Department of Biomedical Engineering, Faculty of Engineering and Information Technology, The University of Melbourne, Melbourne, VIC, 3010, Australia
| | - Alan R Harvey
- School of Human Sciences, The University of Western Australia, and Perron Institute for Neurological and Translational Science, Perth, WA, 6009, Australia
| | - Nicolas H Voelcker
- Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia
- Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility, 151 Wellington Road, Clayton, VIC, 3168, Australia
- CSIRO Manufacturing, Bayview Avenue, Clayton, VIC, 3168, Australia
| | - Clare L Parish
- The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Melbourne, VIC, 3010, Australia
| | - Richard J Williams
- iMPACT, School of Medicine, Deakin University, Waurn Ponds, VIC, 3216, Australia
| | - Roey Elnathan
- Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, Parkville, VIC, 3052, Australia
- Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility, 151 Wellington Road, Clayton, VIC, 3168, Australia
- CSIRO Manufacturing, Bayview Avenue, Clayton, VIC, 3168, Australia
- iMPACT, School of Medicine, Deakin University, Waurn Ponds, VIC, 3216, Australia
| | - David R Nisbet
- ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, ANU College of Health & Medicine, Canberra, ACT, 2601, Australia
- Research School of Chemistry, ANU College of Science, Canberra, ACT, 2601, Australia
- The Graeme Clark Institute, The University of Melbourne, Melbourne, VIC, 3010, Australia
- Department of Biomedical Engineering, Faculty of Engineering and Information Technology, The University of Melbourne, Melbourne, VIC, 3010, Australia
- Melbourne Medical School, Faculty of Medicine, Dentistry and Health Science, The University of Melbourne, Melbourne, VIC, 3010, Australia
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36
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Ko T, Nomura S. Manipulating Cardiomyocyte Plasticity for Heart Regeneration. Front Cell Dev Biol 2022; 10:929256. [PMID: 35898398 PMCID: PMC9309349 DOI: 10.3389/fcell.2022.929256] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2022] [Accepted: 06/14/2022] [Indexed: 01/14/2023] Open
Abstract
Pathological heart injuries such as myocardial infarction induce adverse ventricular remodeling and progression to heart failure owing to widespread cardiomyocyte death. The adult mammalian heart is terminally differentiated unlike those of lower vertebrates. Therefore, the proliferative capacity of adult cardiomyocytes is limited and insufficient to restore an injured heart. Although current therapeutic approaches can delay progressive remodeling and heart failure, difficulties with the direct replenishment of lost cardiomyocytes results in a poor long-term prognosis for patients with heart failure. However, it has been revealed that cardiac function can be improved by regulating the cell cycle or changing the cell state of cardiomyocytes by delivering specific genes or small molecules. Therefore, manipulation of cardiomyocyte plasticity can be an effective treatment for heart disease. This review summarizes the recent studies that control heart regeneration by manipulating cardiomyocyte plasticity with various approaches including differentiating pluripotent stem cells into cardiomyocytes, reprogramming cardiac fibroblasts into cardiomyocytes, and reactivating the proliferation of cardiomyocytes.
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37
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Kuang J, Huang T, Pei D. The Art of Reprogramming for Regenerative Medicine. Front Cell Dev Biol 2022; 10:927555. [PMID: 35846373 PMCID: PMC9280648 DOI: 10.3389/fcell.2022.927555] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2022] [Accepted: 06/08/2022] [Indexed: 11/13/2022] Open
Abstract
Traditional pharmaceuticals in the forms of small chemical compounds or macromolecules such as proteins or RNAs have provided lifesaving solutions to many acute and chronic conditions to date. However, there are still many unmet medical needs, especially those of degenerative nature. The advent of cell-based therapy holds the promise to meet these challenges. In this review, we highlight a relatively new paradigm for generating or regenerating functional cells for replacement therapy against conditions such as type I diabetes, myocardial infarction, neurodegenerative diseases and liver fibrosis. We focus on the latest progresses in cellular reprogramming for generating diverse functional cell types. We will also discuss the mechanisms involved and conclude with likely general principles underlying reprogramming.
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Affiliation(s)
- Junqi Kuang
- Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, China
- Laboratory of Cell Fate Control, School of Life Sciences, Westlake University, Hangzhou, China
| | - Tao Huang
- Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, China
- Laboratory of Cell Fate Control, School of Life Sciences, Westlake University, Hangzhou, China
- College of Life Sciences, Zhejiang University, Hangzhou, China
| | - Duanqing Pei
- Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, China
- Laboratory of Cell Fate Control, School of Life Sciences, Westlake University, Hangzhou, China
- *Correspondence: Duanqing Pei,
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38
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Zhang X, Zhang Q, Chen L, Cai B, Zeng M, Ou S, Chen Y, Feng Z, Chen H, Cao S, Kang K. Appropriate Exogenous Expression Stoichiometry of GATA4 as an Important Factor for Cardiac Reprogramming of Human Dermal Fibroblasts. Cell Reprogram 2022; 24:283-293. [PMID: 35762944 DOI: 10.1089/cell.2022.0014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022] Open
Abstract
Reprogramming of human dermal fibroblasts (HDFs) into induced cardiomyocyte-like cells (iCMs) represents a promising strategy for human cardiac regeneration. Different cocktails of cardiac transcription factors can convert HDFs into iCMs, although with low efficiency and immature phenotype. Here, GATA4, MEF2C, TBX5, MESP1, and MYOCD (GMTMeMy for short) were used to reprogram HDFs by retrovirus infection. We found that the exogenous expression stoichiometry of GATA4 (GATA4 stoichiometry) significantly affected reprogramming efficiency. When 1/8 dosage of GATA4 virus (GATA4 dosage) plus MTMeMy was used, the reprogramming efficiency was obviously improved compared with average pooled virus encoding each factor, which measured, by the expression level of cardiac genes, the percentage of cardiac troponin T and alpha-cardiac myosin heavy-chain immunopositive cells and the numbers of iCMs showing calcium oscillation or beating synchronously in co-culture with mouse CMs. In addition, we prepared conditioned maintenance medium (CMM) by CM differentiation of H9 human embryonic stem cell line. We found that compared with traditional maintenance medium (TMM), CMM made iCMs show well-organized sarcomere formation and characteristic calcium oscillation wave earlier. These findings demonstrated that appropriate GATA4 stoichiometry was essential for cardiac reprogramming and some components in CMM were important for maturation of iCMs.
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Affiliation(s)
- Xiangyu Zhang
- Department of Cardiovascular Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China
| | - Qi Zhang
- Department of Neurology, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China
| | - Lijun Chen
- Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academic and Sciences, Guangzhou Medical University, Guangzhou, China
| | - Baomei Cai
- Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Center for Cell Lineage and Atlas, Guangzhou, China
| | - Mengying Zeng
- Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Center for Cell Lineage and Atlas, Guangzhou, China
| | - Sihua Ou
- Department of Neurology, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China
| | - Yating Chen
- Department of Neurology, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China
| | - Ziyu Feng
- Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Center for Cell Lineage and Atlas, Guangzhou, China
| | - Huan Chen
- Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Center for Cell Lineage and Atlas, Guangzhou, China
| | - Shangtao Cao
- Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Center for Cell Lineage and Atlas, Guangzhou, China
| | - Kai Kang
- Department of Cardiovascular Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China
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Metabolic Determinants in Cardiomyocyte Function and Heart Regenerative Strategies. Metabolites 2022; 12:metabo12060500. [PMID: 35736435 PMCID: PMC9227827 DOI: 10.3390/metabo12060500] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2022] [Revised: 05/25/2022] [Accepted: 05/26/2022] [Indexed: 02/04/2023] Open
Abstract
Heart disease is the leading cause of mortality in developed countries. The associated pathology is characterized by a loss of cardiomyocytes that leads, eventually, to heart failure. In this context, several cardiac regenerative strategies have been developed, but they still lack clinical effectiveness. The mammalian neonatal heart is capable of substantial regeneration following injury, but this capacity is lost at postnatal stages when cardiomyocytes become terminally differentiated and transit to the fetal metabolic switch. Cardiomyocytes are metabolically versatile cells capable of using an array of fuel sources, and the metabolism of cardiomyocytes suffers extended reprogramming after injury. Apart from energetic sources, metabolites are emerging regulators of epigenetic programs driving cell pluripotency and differentiation. Thus, understanding the metabolic determinants that regulate cardiomyocyte maturation and function is key for unlocking future metabolic interventions for cardiac regeneration. In this review, we will discuss the emerging role of metabolism and nutrient signaling in cardiomyocyte function and repair, as well as whether exploiting this axis could potentiate current cellular regenerative strategies for the mammalian heart.
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40
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Afouda BA. Towards Understanding the Gene-Specific Roles of GATA Factors in Heart Development: Does GATA4 Lead the Way? Int J Mol Sci 2022; 23:5255. [PMID: 35563646 PMCID: PMC9099915 DOI: 10.3390/ijms23095255] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Revised: 04/29/2022] [Accepted: 05/03/2022] [Indexed: 02/04/2023] Open
Abstract
Transcription factors play crucial roles in the regulation of heart induction, formation, growth and morphogenesis. Zinc finger GATA transcription factors are among the critical regulators of these processes. GATA4, 5 and 6 genes are expressed in a partially overlapping manner in developing hearts, and GATA4 and 6 continue their expression in adult cardiac myocytes. Using different experimental models, GATA4, 5 and 6 were shown to work together not only to ensure specification of cardiac cells but also during subsequent heart development. The complex involvement of these related gene family members in those processes is demonstrated through the redundancy among them and crossregulation of each other. Our recent identification at the genome-wide level of genes specifically regulated by each of the three family members and our earlier discovery that gata4 and gata6 function upstream, while gata5 functions downstream of noncanonical Wnt signalling during cardiac differentiation, clearly demonstrate the functional differences among the cardiogenic GATA factors. Such suspected functional differences are worth exploring more widely. It appears that in the past few years, significant advances have indeed been made in providing a deeper understanding of the mechanisms by which each of these molecules function during heart development. In this review, I will therefore discuss current evidence of the role of individual cardiogenic GATA factors in the process of heart development and emphasize the emerging central role of GATA4.
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Affiliation(s)
- Boni A Afouda
- Institute of Medical Sciences, Foresterhill Health Campus, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK
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41
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Deciphering Cardiac Biology and Disease by Single-Cell Transcriptomic Profiling. Biomolecules 2022; 12:biom12040566. [PMID: 35454155 PMCID: PMC9032111 DOI: 10.3390/biom12040566] [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: 03/11/2022] [Revised: 04/02/2022] [Accepted: 04/04/2022] [Indexed: 11/29/2022] Open
Abstract
By detecting minute molecular changes in hundreds to millions of single cells, single-cell RNA sequencing allows for the comprehensive characterization of the diversity and dynamics of cells in the heart. Our understanding of the heart has been transformed through the recognition of cellular heterogeneity, the construction of regulatory networks, the building of lineage trajectories, and the mapping of intercellular crosstalk. In this review, we introduce cardiac progenitors and their transcriptional regulation during embryonic development, highlight cellular heterogeneity and cell subtype functions in cardiac health and disease, and discuss insights gained from the study of pluripotent stem-cell-derived cardiomyocytes.
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42
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Pascale E, Caiazza C, Paladino M, Parisi S, Passaro F, Caiazzo M. MicroRNA Roles in Cell Reprogramming Mechanisms. Cells 2022; 11:940. [PMID: 35326391 PMCID: PMC8946776 DOI: 10.3390/cells11060940] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2022] [Revised: 02/28/2022] [Accepted: 03/08/2022] [Indexed: 02/01/2023] Open
Abstract
Cell reprogramming is a groundbreaking technology that, in few decades, generated a new paradigm in biomedical science. To date we can use cell reprogramming to potentially generate every cell type by converting somatic cells and suitably modulating the expression of key transcription factors. This approach can be used to convert skin fibroblasts into pluripotent stem cells as well as into a variety of differentiated and medically relevant cell types, including cardiomyocytes and neural cells. The molecular mechanisms underlying such striking cell phenotypes are still largely unknown, but in the last decade it has been proven that cell reprogramming approaches are significantly influenced by non-coding RNAs. Specifically, this review will focus on the role of microRNAs in the reprogramming processes that lead to the generation of pluripotent stem cells, neurons, and cardiomyocytes. As highlighted here, non-coding RNA-forced expression can be sufficient to support some cell reprogramming processes, and, therefore, we will also discuss how these molecular determinants could be used in the future for biomedical purposes.
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Affiliation(s)
- Emilia Pascale
- Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II”, Via Pansini 5, 80131 Naples, Italy; (E.P.); (C.C.); (M.P.); (S.P.)
| | - Carmen Caiazza
- Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II”, Via Pansini 5, 80131 Naples, Italy; (E.P.); (C.C.); (M.P.); (S.P.)
| | - Martina Paladino
- Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II”, Via Pansini 5, 80131 Naples, Italy; (E.P.); (C.C.); (M.P.); (S.P.)
| | - Silvia Parisi
- Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II”, Via Pansini 5, 80131 Naples, Italy; (E.P.); (C.C.); (M.P.); (S.P.)
| | - Fabiana Passaro
- Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II”, Via Pansini 5, 80131 Naples, Italy; (E.P.); (C.C.); (M.P.); (S.P.)
| | - Massimiliano Caiazzo
- Department of Molecular Medicine and Medical Biotechnology, University of Naples “Federico II”, Via Pansini 5, 80131 Naples, Italy; (E.P.); (C.C.); (M.P.); (S.P.)
- Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands
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43
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Zhang X, Chen L, Huang X, Chen H, Cai B, Qin Y, Chen Y, Ou S, Li X, Wu Z, Feng Z, Zeng M, Guo W, Li H, Zhou C, Yu S, Pan M, Liu J, Kang K, Cao S, Pei D. MYOCD is Required for Cardiomyocyte-like Cells Induction from Human Urine Cells and Fibroblasts Through Remodeling Chromatin. Stem Cell Rev Rep 2022; 18:2414-2430. [PMID: 35246800 DOI: 10.1007/s12015-022-10339-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 01/20/2022] [Indexed: 01/14/2023]
Abstract
Despite direct reprogramming of human cardiac fibroblasts into induced cardiomyocytes (iCM) holds great potential for heart regeneration, the mechanisms are poorly understood. Whether other human somatic cells could be reprogrammed into cardiomyocytes is also unknown. Here, we report human urine cells (hUCs) could be converted into CM-like cells from different donors and the related chromatin accessibility dynamics (CAD) by assay for transposase accessible chromatin(ATAC)-seq. hUCs transduced by MEF2C, TBX5, MESP1 and MYOCD but without GATA4 expressed multiple cardiac specific genes, exhibited Ca2+ oscillation potential and sarcomeric structures, and contracted synchronously in coculture with mouse CM. Additionally, we found that MYOCD is required for both closing and opening critical loci, mainly by hindering the opening of loci enriched with motifs for the TEAD and AP1 family and promoting the closing of loci enriched with ETS motifs. These changes differ partially from CAD observed during iCM induction from human fibroblasts. Collectively, our study offers one practical platform for iCM generation and insights into mechanisms for iCM fate determination.
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Affiliation(s)
- Xiangyu Zhang
- Department of Cardiovascular Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, Heilongjiang, China
| | - Lijun Chen
- CAS Key Laboratory of Regenerative Biology, South China Institutes for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China.,Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academic and Sciences, Guangzhou Medical University, Guangzhou, 511436, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China
| | - Xingnan Huang
- Laboratory of Cell Fate Control, School of Life Sciences, Westlake University, Hangzhou, 310024, China
| | - Huan Chen
- Center for Cell Lineage and Atlas, Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, 510005, China
| | - Baomei Cai
- Center for Cell Lineage and Atlas, Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, 510005, China
| | - Yue Qin
- Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academic and Sciences, Guangzhou Medical University, Guangzhou, 511436, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China.,University of Chinese Academy of Science, Beijing, 100049, China
| | - Yating Chen
- CAS Key Laboratory of Regenerative Biology, South China Institutes for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China.,Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academic and Sciences, Guangzhou Medical University, Guangzhou, 511436, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China
| | - Sihua Ou
- CAS Key Laboratory of Regenerative Biology, South China Institutes for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China.,Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academic and Sciences, Guangzhou Medical University, Guangzhou, 511436, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China
| | - Xiaoxi Li
- Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academic and Sciences, Guangzhou Medical University, Guangzhou, 511436, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China
| | - Zichao Wu
- CAS Key Laboratory of Regenerative Biology, South China Institutes for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China.,Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academic and Sciences, Guangzhou Medical University, Guangzhou, 511436, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China
| | - Ziyu Feng
- Center for Cell Lineage and Atlas, Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, 510005, China
| | - Mengying Zeng
- Center for Cell Lineage and Atlas, Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, 510005, China
| | - Wenjing Guo
- Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academic and Sciences, Guangzhou Medical University, Guangzhou, 511436, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China
| | - Heying Li
- Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academic and Sciences, Guangzhou Medical University, Guangzhou, 511436, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China
| | - Chunhua Zhou
- Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academic and Sciences, Guangzhou Medical University, Guangzhou, 511436, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China
| | - Shengyong Yu
- Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academic and Sciences, Guangzhou Medical University, Guangzhou, 511436, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China
| | - Mengjie Pan
- CAS Key Laboratory of Regenerative Biology, South China Institutes for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China.,Center for Cell Lineage and Atlas, Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, 510005, China.,University of Chinese Academy of Science, Beijing, 100049, China
| | - Jing Liu
- Joint School of Life Science, Guangzhou Institutes of Biomedicine and Health, Chinese Academic and Sciences, Guangzhou Medical University, Guangzhou, 511436, China.,Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Science, Chinese Academic and Sciences, Guangzhou, 510530, China.,Center for Cell Lineage and Atlas, Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, 510005, China
| | - Kai Kang
- Department of Cardiovascular Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, Heilongjiang, China.
| | - Shangtao Cao
- Center for Cell Lineage and Atlas, Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, 510005, China.
| | - Duanqing Pei
- Laboratory of Cell Fate Control, School of Life Sciences, Westlake University, Hangzhou, 310024, China.
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44
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Garbern JC, Lee RT. Heart regeneration: 20 years of progress and renewed optimism. Dev Cell 2022; 57:424-439. [PMID: 35231426 PMCID: PMC8896288 DOI: 10.1016/j.devcel.2022.01.012] [Citation(s) in RCA: 43] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2021] [Revised: 01/10/2022] [Accepted: 01/18/2022] [Indexed: 02/06/2023]
Abstract
Cardiovascular disease is a leading cause of death worldwide, and thus there remains great interest in regenerative approaches to treat heart failure. In the past 20 years, the field of heart regeneration has entered a renaissance period with remarkable progress in the understanding of endogenous heart regeneration, stem cell differentiation for exogenous cell therapy, and cell-delivery methods. In this review, we highlight how this new understanding can lead to viable strategies for human therapy. For the near term, drugs, electrical and mechanical devices, and heart transplantation will remain mainstays of cardiac therapies, but eventually regenerative therapies based on fundamental regenerative biology may offer more permanent solutions for patients with heart failure.
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Affiliation(s)
- Jessica C. Garbern
- Department of Stem Cell and Regenerative Biology and the Harvard Stem Cell Institute, Harvard University, 7 Divinity Ave, Cambridge, MA 02138, USA,Department of Cardiology, Boston Children’s Hospital, 300 Longwood Ave, Boston, MA 02115, USA
| | - Richard T. Lee
- Department of Stem Cell and Regenerative Biology and the Harvard Stem Cell Institute, Harvard University, 7 Divinity Ave, Cambridge, MA 02138, USA,Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, 75 Francis St, Boston, MA 02115, USA,Corresponding author and lead contact: Richard T. Lee, Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Ave, Cambridge, MA 02138, Phone: 617-496-5394, Fax: 617-496-8351,
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45
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Nam YJ. Translational perspectives on cardiac reprogramming. Semin Cell Dev Biol 2022; 122:14-20. [PMID: 34210578 PMCID: PMC8712611 DOI: 10.1016/j.semcdb.2021.06.020] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Revised: 05/21/2021] [Accepted: 06/23/2021] [Indexed: 02/03/2023]
Abstract
Loss of cardiac muscle after cardiac injury is replaced by cardiac fibrosis, due to very limited regenerative capacity of the heart. Although initially beneficial, persistent cardiac fibrosis leads to pump failure and conduction abnormalities, common modes of death following cardiac injury. Thus, directly reprogramming cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs) by forced expression of cardiogenic factors (referred to as cardiac reprogramming) is particularly attractive in that it targets cardiac fibroblasts, a major source of cardiac fibrosis, to induce new cardiac muscle. Over the last decade, remarkable progresses have been made on cardiac reprogramming, particularly focusing on how to enhance conversion of fibroblasts to iCMs in vitro. However, it still remains elusive whether this new regenerative approach can be translated into clinical practice. This review discusses progresses and challenges of cardiac reprogramming in the translational context.
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Affiliation(s)
- Young-Jae Nam
- Department of Medicine, Division of Cardiovascular Medicine, Vanderbilt University Medical Center, Nashville, TN, USA,Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA,Vanderbilt Center for Stem Cell Biology, Vanderbilt University, Nashville, TN, USA,To whom correspondence may be addressed: Young-Jae Nam, M.D., Ph.D., Department of Medicine, Division of Cardiovascular Medicine, Vanderbilt University Medical Center, Nashville , Tennessee 37232, USA, Phone: 615-936-5436,
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46
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Xie Y, Liu J, Qian L. Direct cardiac reprogramming comes of age: Recent advance and remaining challenges. Semin Cell Dev Biol 2022; 122:37-43. [PMID: 34304993 PMCID: PMC8782931 DOI: 10.1016/j.semcdb.2021.07.010] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2021] [Revised: 06/30/2021] [Accepted: 07/12/2021] [Indexed: 02/03/2023]
Abstract
The adult human heart has limited regenerative capacity. As such, the massive cardiomyocyte loss due to myocardial infarction leads to scar formation and adverse cardiac remodeling, which ultimately results in chronic heart failure. Direct cardiac reprogramming that converts cardiac fibroblast into functional cardiomyocyte-like cells (also called iCMs) holds great promise for heart regeneration. Cardiac reprogramming has been achieved both in vitro and in vivo by using a variety of cocktails that comprise transcription factors, microRNAs, or small molecules. During the past several years, great progress has been made in improving reprogramming efficiency and understanding the underlying molecular mechanisms. Here, we summarize the direct cardiac reprogramming methods, review the current advances in understanding the molecular mechanisms of cardiac reprogramming, and highlight the novel insights gained from single-cell omics studies. Finally, we discuss the remaining challenges and future directions for the field.
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47
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Garry GA, Bassel-Duby R, Olson EN. Direct reprogramming as a route to cardiac repair. Semin Cell Dev Biol 2022; 122:3-13. [PMID: 34246567 PMCID: PMC8738780 DOI: 10.1016/j.semcdb.2021.05.019] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2021] [Accepted: 05/14/2021] [Indexed: 02/03/2023]
Abstract
Ischemic heart disease is the leading cause of morbidity, mortality, and healthcare expenditure worldwide due to an inability of the heart to regenerate following injury. Thus, novel heart failure therapies aimed at promoting cardiomyocyte regeneration are desperately needed. In recent years, direct reprogramming of resident cardiac fibroblasts to induced cardiac-like myocytes (iCMs) has emerged as a promising therapeutic strategy to repurpose the fibrotic response of the injured heart toward a functional myocardium. Direct cardiac reprogramming was initially achieved through the overexpression of the transcription factors (TFs) Gata4, Mef2c, and Tbx5 (GMT). However, this combination of TFs and other subsequent cocktails demonstrated limited success in reprogramming adult human and mouse fibroblasts, constraining the clinical translation of this therapy. Over the past decade, significant effort has been dedicated to optimizing reprogramming cocktails comprised of cardiac TFs, epigenetic factors, microRNAs, or small molecules to yield efficient cardiac cell fate conversion. Yet, efficient reprogramming of adult human fibroblasts remains a significant challenge. Underlying mechanisms identified to accelerate this process have been centered on epigenetic remodeling at cardiac gene regulatory regions. Further studies to achieve a refined understanding and directed means of overcoming epigenetic barriers are merited to more rapidly translate these promising therapies to the clinic.
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Affiliation(s)
- Glynnis A. Garry
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX,The Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX,Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, University of Texas Southwestern Medical Center, Dallas, TX
| | - Rhonda Bassel-Duby
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX,The Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX,Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, University of Texas Southwestern Medical Center, Dallas, TX
| | - Eric N. Olson
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX,The Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX,Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, University of Texas Southwestern Medical Center, Dallas, TX,Correspondence: Eric N. Olson, Ph.D. 5323 Harry Hines Boulevard, Dallas, Texas, 75390-9148, Tel: 214-648-1187,
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48
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Mehanna RA, Essawy MM, Barkat MA, Awaad AK, Thabet EH, Hamed HA, Elkafrawy H, Khalil NA, Sallam A, Kholief MA, Ibrahim SS, Mourad GM. Cardiac stem cells: Current knowledge and future prospects. World J Stem Cells 2022; 14:1-40. [PMID: 35126826 PMCID: PMC8788183 DOI: 10.4252/wjsc.v14.i1.1] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/26/2021] [Revised: 07/02/2021] [Accepted: 01/06/2022] [Indexed: 02/06/2023] Open
Abstract
Regenerative medicine is the field concerned with the repair and restoration of the integrity of damaged human tissues as well as whole organs. Since the inception of the field several decades ago, regenerative medicine therapies, namely stem cells, have received significant attention in preclinical studies and clinical trials. Apart from their known potential for differentiation into the various body cells, stem cells enhance the organ's intrinsic regenerative capacity by altering its environment, whether by exogenous injection or introducing their products that modulate endogenous stem cell function and fate for the sake of regeneration. Recently, research in cardiology has highlighted the evidence for the existence of cardiac stem and progenitor cells (CSCs/CPCs). The global burden of cardiovascular diseases' morbidity and mortality has demanded an in-depth understanding of the biology of CSCs/CPCs aiming at improving the outcome for an innovative therapeutic strategy. This review will discuss the nature of each of the CSCs/CPCs, their environment, their interplay with other cells, and their metabolism. In addition, important issues are tackled concerning the potency of CSCs/CPCs in relation to their secretome for mediating the ability to influence other cells. Moreover, the review will throw the light on the clinical trials and the preclinical studies using CSCs/CPCs and combined therapy for cardiac regeneration. Finally, the novel role of nanotechnology in cardiac regeneration will be explored.
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Affiliation(s)
- Radwa A Mehanna
- Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Marwa M Essawy
- Oral Pathology Department, Faculty of Dentistry/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Mona A Barkat
- Human Anatomy and Embryology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Ashraf K Awaad
- Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Eman H Thabet
- Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Heba A Hamed
- Histology and Cell Biology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Hagar Elkafrawy
- Medical Biochemistry Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Nehal A Khalil
- Medical Biochemistry Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Abeer Sallam
- Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Marwa A Kholief
- Forensic Medicine and Clinical toxicology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Samar S Ibrahim
- Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Ghada M Mourad
- Histology and Cell Biology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt.
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49
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Mehanna RA, Essawy MM, Barkat MA, Awaad AK, Thabet EH, Hamed HA, Elkafrawy H, Khalil NA, Sallam A, Kholief MA, Ibrahim SS, Mourad GM. Cardiac stem cells: Current knowledge and future prospects. World J Stem Cells 2022. [PMID: 35126826 DOI: 10.4252/wjsc.v14.i1.1]] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Regenerative medicine is the field concerned with the repair and restoration of the integrity of damaged human tissues as well as whole organs. Since the inception of the field several decades ago, regenerative medicine therapies, namely stem cells, have received significant attention in preclinical studies and clinical trials. Apart from their known potential for differentiation into the various body cells, stem cells enhance the organ's intrinsic regenerative capacity by altering its environment, whether by exogenous injection or introducing their products that modulate endogenous stem cell function and fate for the sake of regeneration. Recently, research in cardiology has highlighted the evidence for the existence of cardiac stem and progenitor cells (CSCs/CPCs). The global burden of cardiovascular diseases' morbidity and mortality has demanded an in-depth understanding of the biology of CSCs/CPCs aiming at improving the outcome for an innovative therapeutic strategy. This review will discuss the nature of each of the CSCs/CPCs, their environment, their interplay with other cells, and their metabolism. In addition, important issues are tackled concerning the potency of CSCs/CPCs in relation to their secretome for mediating the ability to influence other cells. Moreover, the review will throw the light on the clinical trials and the preclinical studies using CSCs/CPCs and combined therapy for cardiac regeneration. Finally, the novel role of nanotechnology in cardiac regeneration will be explored.
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Affiliation(s)
- Radwa A Mehanna
- Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Marwa M Essawy
- Oral Pathology Department, Faculty of Dentistry/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Mona A Barkat
- Human Anatomy and Embryology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Ashraf K Awaad
- Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Eman H Thabet
- Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Heba A Hamed
- Histology and Cell Biology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Hagar Elkafrawy
- Medical Biochemistry Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Nehal A Khalil
- Medical Biochemistry Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Abeer Sallam
- Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Marwa A Kholief
- Forensic Medicine and Clinical toxicology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Samar S Ibrahim
- Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt
| | - Ghada M Mourad
- Histology and Cell Biology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt.
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50
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López-Muneta L, Linares J, Casis O, Martínez-Ibáñez L, González Miqueo A, Bezunartea J, Sanchez de la Nava AM, Gallego M, Fernández-Santos ME, Rodriguez-Madoz JR, Aranguren XL, Fernández-Avilés F, Segovia JC, Prósper F, Carvajal-Vergara X. Generation of NKX2.5GFP Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts. Front Cell Dev Biol 2022; 9:797927. [PMID: 35127713 PMCID: PMC8815860 DOI: 10.3389/fcell.2021.797927] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2021] [Accepted: 12/06/2021] [Indexed: 01/14/2023] Open
Abstract
Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5GFP reporter cardiac fibroblasts. We first established a new NKX2.5GFP reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells. The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5GFP hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5GFP cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.
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Affiliation(s)
- Leyre López-Muneta
- Regenerative Medicine Program, Foundation for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, Pamplona, Spain
| | - Javier Linares
- Regenerative Medicine Program, Foundation for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, Pamplona, Spain
| | - Oscar Casis
- Departament of Physiology, Faculty of Pharmacy, University of the Basque Country UPV/EHU, Vitoria-Gasteiz, Spain
| | - Laura Martínez-Ibáñez
- Program of Cardiovascular Diseases, Foundation for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, Pamplona, Spain
| | - Arantxa González Miqueo
- Program of Cardiovascular Diseases, Foundation for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, Pamplona, Spain
- Centro de Investigación Biomédica en Red Cardiovascular (CIBERCV), Instituto de Salud Carlos III, Madrid, Spain
| | - Jaione Bezunartea
- Retinal Pathologies and New Therapies Group, Experimental Ophthalmology Laboratory, Department of Ophthalmology, University of Navarra Clinic, Pamplona, Spain
| | - Ana Maria Sanchez de la Nava
- Department of Cardiology, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón (IISGM), Madrid, Spain
- Centro de Investigación Biomedica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain
| | - Mónica Gallego
- Departament of Physiology, Faculty of Pharmacy, University of the Basque Country UPV/EHU, Vitoria-Gasteiz, Spain
| | - María Eugenia Fernández-Santos
- Department of Cardiology, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón (IISGM), Madrid, Spain
- Centro de Investigación Biomedica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain
| | - Juan Roberto Rodriguez-Madoz
- Hemato-oncology Program, CIMA Universidad de Navarra, Instituto de Investigación Sanitaria de Navarra (IdiSNA), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Pamplona, Spain
| | - Xabier L. Aranguren
- Regenerative Medicine Program, Foundation for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, Pamplona, Spain
| | - Francisco Fernández-Avilés
- Department of Cardiology, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón (IISGM), Madrid, Spain
- Centro de Investigación Biomedica en Red de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain
- Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain
| | - José Carlos Segovia
- Cell Technology Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Unidad Mixta de Terapias Avanzadas, Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain
| | - Felipe Prósper
- Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Instituto de Investigación Sanitaria de Navarra (IdiSNA), Department of Hematology and Cell Therapy, University of Navarra Clinic, Pamplona, Spain
| | - Xonia Carvajal-Vergara
- Regenerative Medicine Program, Foundation for Applied Medical Research (CIMA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, Pamplona, Spain
- *Correspondence: Xonia Carvajal-Vergara,
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