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1
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Cervantes-Salazar JL, Pérez-Hernández N, Calderón-Colmenero J, Rodríguez-Pérez JM, González-Pacheco MG, Villamil-Castañeda C, Rosas-Tlaque AA, Ortega-Zhindón DB. Genetic Insights into Congenital Cardiac Septal Defects-A Narrative Review. BIOLOGY 2024; 13:911. [PMID: 39596866 PMCID: PMC11592229 DOI: 10.3390/biology13110911] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/22/2024] [Revised: 11/04/2024] [Accepted: 11/04/2024] [Indexed: 11/29/2024]
Abstract
Congenital heart diseases (CHDs) are a group of complex diseases characterized by structural and functional malformations during development in the human heart; they represent an important problem for public health worldwide. Within these malformations, septal defects such as ventricular (VSD) and atrial septal defects (ASD) are the most common forms of CHDs. Studies have reported that CHDs are the result of genetic and environmental factors. Here, we review and summarize the role of genetics involved in cardiogenesis and congenital cardiac septal defects. Moreover, treatment regarding these congenital cardiac septal defects is also addressed.
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Affiliation(s)
- Jorge L. Cervantes-Salazar
- Department of Pediatric Cardiac Surgery and Congenital Heart Disease, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City 14080, Mexico; (J.L.C.-S.); (A.A.R.-T.)
| | - Nonanzit Pérez-Hernández
- Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City 14080, Mexico; (N.P.-H.); (J.M.R.-P.); (C.V.-C.)
| | - Juan Calderón-Colmenero
- Department of Pediatric Cardiology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City 14080, Mexico;
| | - José Manuel Rodríguez-Pérez
- Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City 14080, Mexico; (N.P.-H.); (J.M.R.-P.); (C.V.-C.)
| | | | - Clara Villamil-Castañeda
- Department of Molecular Biology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City 14080, Mexico; (N.P.-H.); (J.M.R.-P.); (C.V.-C.)
- Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico
| | - Angel A. Rosas-Tlaque
- Department of Pediatric Cardiac Surgery and Congenital Heart Disease, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City 14080, Mexico; (J.L.C.-S.); (A.A.R.-T.)
- Dirección General de Calidad y Educación en Salud, Secretaría de Salud, Mexico City 06600, Mexico
| | - Diego B. Ortega-Zhindón
- Department of Pediatric Cardiac Surgery and Congenital Heart Disease, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City 14080, Mexico; (J.L.C.-S.); (A.A.R.-T.)
- Programa de Maestría y Doctorado en Ciencias Médicas, Odontológicas y de la Salud, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico
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2
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Nappi F. In-Depth Genomic Analysis: The New Challenge in Congenital Heart Disease. Int J Mol Sci 2024; 25:1734. [PMID: 38339013 PMCID: PMC10855915 DOI: 10.3390/ijms25031734] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2024] [Revised: 01/25/2024] [Accepted: 01/27/2024] [Indexed: 02/12/2024] Open
Abstract
The use of next-generation sequencing has provided new insights into the causes and mechanisms of congenital heart disease (CHD). Examinations of the whole exome sequence have detected detrimental gene variations modifying single or contiguous nucleotides, which are characterised as pathogenic based on statistical assessments of families and correlations with congenital heart disease, elevated expression during heart development, and reductions in harmful protein-coding mutations in the general population. Patients with CHD and extracardiac abnormalities are enriched for gene classes meeting these criteria, supporting a common set of pathways in the organogenesis of CHDs. Single-cell transcriptomics data have revealed the expression of genes associated with CHD in specific cell types, and emerging evidence suggests that genetic mutations disrupt multicellular genes essential for cardiogenesis. Metrics and units are being tracked in whole-genome sequencing studies.
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Affiliation(s)
- Francesco Nappi
- Department of Cardiac Surgery, Centre Cardiologique du Nord, 93200 Saint-Denis, France
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3
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Selvakumar D, Clayton ZE, Prowse A, Dingwall S, Kim SK, Reyes L, George J, Shah H, Chen S, Leung HHL, Hume RD, Tjahjadi L, Igoor S, Skelton RJP, Hing A, Paterson H, Foster SL, Pearson L, Wilkie E, Marcus AD, Jeyaprakash P, Wu Z, Chiu HS, Ongtengco CFJ, Mulay O, McArthur JR, Barry T, Lu J, Tran V, Bennett R, Kotake Y, Campbell T, Turnbull S, Gupta A, Nguyen Q, Ni G, Grieve SM, Palpant NJ, Pathan F, Kizana E, Kumar S, Gray PP, Chong JJH. Cellular heterogeneity of pluripotent stem cell-derived cardiomyocyte grafts is mechanistically linked to treatable arrhythmias. NATURE CARDIOVASCULAR RESEARCH 2024; 3:145-165. [PMID: 39196193 PMCID: PMC11358004 DOI: 10.1038/s44161-023-00419-3] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/06/2022] [Accepted: 12/26/2023] [Indexed: 08/29/2024]
Abstract
Preclinical data have confirmed that human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can remuscularize the injured or diseased heart, with several clinical trials now in planning or recruitment stages. However, because ventricular arrhythmias represent a complication following engraftment of intramyocardially injected PSC-CMs, it is necessary to provide treatment strategies to control or prevent engraftment arrhythmias (EAs). Here, we show in a porcine model of myocardial infarction and PSC-CM transplantation that EAs are mechanistically linked to cellular heterogeneity in the input PSC-CM and resultant graft. Specifically, we identify atrial and pacemaker-like cardiomyocytes as culprit arrhythmogenic subpopulations. Two unique surface marker signatures, signal regulatory protein α (SIRPA)+CD90-CD200+ and SIRPA+CD90-CD200-, identify arrhythmogenic and non-arrhythmogenic cardiomyocytes, respectively. Our data suggest that modifications to current PSC-CM-production and/or PSC-CM-selection protocols could potentially prevent EAs. We further show that pharmacologic and interventional anti-arrhythmic strategies can control and potentially abolish these arrhythmias.
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Affiliation(s)
- Dinesh Selvakumar
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
- Department of Cardiology, Westmead Hospital, Westmead, New South Wales, Australia
| | - Zoe E Clayton
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
| | - Andrew Prowse
- Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, St Lucia, Queensland, Australia
| | - Steve Dingwall
- Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, St Lucia, Queensland, Australia
| | - Sul Ki Kim
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
- Department of Cardiology, Westmead Hospital, Westmead, New South Wales, Australia
| | - Leila Reyes
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
| | - Jacob George
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
| | - Haisam Shah
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
| | - Siqi Chen
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
| | - Halina H L Leung
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
| | - Robert D Hume
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
| | - Laurentius Tjahjadi
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
| | - Sindhu Igoor
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
| | - Rhys J P Skelton
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
| | - Alfred Hing
- Department of Cardiothoracic Surgery, Westmead Hospital, Westmead, New South Wales, Australia
| | - Hugh Paterson
- Sydney Imaging, Core Research Facility, the University of Sydney, Sydney, New South Wales, Australia
| | - Sheryl L Foster
- Department of Radiology, Westmead Hospital, Westmead, New South Wales, Australia
- Sydney School of Health Sciences, Faculty of Medicine and Health, the University of Sydney, Sydney, New South Wales, Australia
| | - Lachlan Pearson
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
| | - Emma Wilkie
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
| | - Alan D Marcus
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
| | - Prajith Jeyaprakash
- Department of Cardiology, Nepean Hospital, Kingswood, New South Wales, Australia
| | - Zhixuan Wu
- Institute for Molecular Bioscience, the University of Queensland, St Lucia, Queensland, Australia
| | - Han Shen Chiu
- Institute for Molecular Bioscience, the University of Queensland, St Lucia, Queensland, Australia
| | - Cherica Felize J Ongtengco
- Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, St Lucia, Queensland, Australia
| | - Onkar Mulay
- Genomics and Machine Learning Lab, Division of Genetics and Genomics, Institute for Molecular Bioscience, the University of Queensland, St Lucia, Queensland, Australia
| | - Jeffrey R McArthur
- Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia
- St. Vincent's Clinical School, UNSW, Darlinghurst, New South Wales, Australia
| | - Tony Barry
- Department of Cardiology, Westmead Hospital, Westmead, New South Wales, Australia
| | - Juntang Lu
- Department of Cardiology, Westmead Hospital, Westmead, New South Wales, Australia
| | - Vu Tran
- Department of Cardiology, Westmead Hospital, Westmead, New South Wales, Australia
| | - Richard Bennett
- Department of Cardiology, Westmead Hospital, Westmead, New South Wales, Australia
| | - Yasuhito Kotake
- Department of Cardiology, Westmead Hospital, Westmead, New South Wales, Australia
| | - Timothy Campbell
- Department of Cardiology, Westmead Hospital, Westmead, New South Wales, Australia
| | - Samual Turnbull
- Department of Cardiology, Westmead Hospital, Westmead, New South Wales, Australia
| | - Anunay Gupta
- Department of Cardiology, Westmead Hospital, Westmead, New South Wales, Australia
| | - Quan Nguyen
- Genomics and Machine Learning Lab, Division of Genetics and Genomics, Institute for Molecular Bioscience, the University of Queensland, St Lucia, Queensland, Australia
| | - Guiyan Ni
- Genomics and Machine Learning Lab, Division of Genetics and Genomics, Institute for Molecular Bioscience, the University of Queensland, St Lucia, Queensland, Australia
| | - Stuart M Grieve
- Imaging and Phenotyping Laboratory, Faculty of Medicine and Health, Charles Perkins Centre, the University of Sydney, Sydney, New South Wales, Australia
| | - Nathan J Palpant
- Institute for Molecular Bioscience, the University of Queensland, St Lucia, Queensland, Australia
| | - Faraz Pathan
- Department of Cardiology, Nepean Hospital, Kingswood, New South Wales, Australia
- Sydney Medical School, Charles Perkins Centre Nepean, Faculty of Medicine and Health, the University of Sydney, Sydney, New South Wales, Australia
| | - Eddy Kizana
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia
- Department of Cardiology, Westmead Hospital, Westmead, New South Wales, Australia
| | - Saurabh Kumar
- Department of Cardiology, Westmead Hospital, Westmead, New South Wales, Australia
| | - Peter P Gray
- Australian Institute for Bioengineering and Nanotechnology, the University of Queensland, St Lucia, Queensland, Australia
| | - James J H Chong
- Centre for Heart Research, the Westmead Institute for Medical Research, the University of Sydney, Westmead, New South Wales, Australia.
- Department of Cardiology, Westmead Hospital, Westmead, New South Wales, Australia.
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4
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Janaththani P, Tevz G, Fernando A, Malik A, Rockstroh A, Kryza T, Walpole C, Moya L, Lehman M, Nelson C, Srinivasan S, Clements J, Batra J. Unravelling the Role of Iroquois Homeobox 4 and its Interplay with Androgen Receptor in Prostate Cancer. RESEARCH SQUARE 2023:rs.3.rs-3295914. [PMID: 38076926 PMCID: PMC10705702 DOI: 10.21203/rs.3.rs-3295914/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/11/2024]
Abstract
Genome-wide association studies have linked Iroquois-Homeobox 4 (IRX4) as a robust expression quantitative-trait locus associated with prostate cancer (PCa) risk. However, the intricate mechanism and regulatory factors governing IRX4 expression in PCa remain poorly understood. Here, we unveil enrichment of androgen-responsive gene signatures in metastatic prostate tumors exhibiting heightened IRX4 expression. Furthermore, we uncover a novel interaction between IRX4 and the androgen receptor (AR) co-factor, FOXA1, suggesting that IRX4 modulates PCa cell behavior through AR cistrome alteration. Remarkably, we identified a distinctive short insertion-deletion polymorphism (INDEL), upstream of the IRX4 gene that differentially regulates IRX4 expression through the disruption of AR binding. This INDEL emerges as the most significant PCa risk-associated variant within the 5p15 locus, in a genetic analysis involving 82,591 PCa cases and 61,213 controls and was associated with PCa survival in patients undergoing androgen-deprivation therapy. These studies suggest the potential of this INDEL as a prognostic biomarker for androgen therapy in PCa and IRX4 as a potential therapeutic target in combination with current clinical management.
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Affiliation(s)
- Panchadsaram Janaththani
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
- Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Melbourne, Victoria
| | - Gregor Tevz
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
| | - Achala Fernando
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
- Centre for Genomics and Personalised Health, Queensland University of Technology, Brisbane, Queensland, Australia
| | - Adil Malik
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
| | - Anja Rockstroh
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
| | - Thomas Kryza
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
- Mater Research UQ, Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
| | - Carina Walpole
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
- Mater Research UQ, Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
| | - Leire Moya
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
| | - Melanie Lehman
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
- Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Canada
| | - Colleen Nelson
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
| | - The Australian Prostate Cancer BioResource
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
| | | | - Srilakshmi Srinivasan
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
- Centre for Genomics and Personalised Health, Queensland University of Technology, Brisbane, Queensland, Australia
| | - Judith Clements
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
| | - Jyotsna Batra
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Queensland, Australia
- Translational Research Institute, Woolloongabba, Brisbane, Queensland, Australia
- Centre for Genomics and Personalised Health, Queensland University of Technology, Brisbane, Queensland, Australia
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5
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Ahmad FS, Jin Y, Grassam-Rowe A, Zhou Y, Yuan M, Fan X, Zhou R, Mu-u-min R, O'Shea C, Ibrahim AM, Hyder W, Aguib Y, Yacoub M, Pavlovic D, Zhang Y, Tan X, Lei M, Terrar DA. Generation of cardiomyocytes from human-induced pluripotent stem cells resembling atrial cells with ability to respond to adrenoceptor agonists. Philos Trans R Soc Lond B Biol Sci 2023; 378:20220312. [PMID: 37122218 PMCID: PMC10150206 DOI: 10.1098/rstb.2022.0312] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2022] [Accepted: 12/07/2022] [Indexed: 05/02/2023] Open
Abstract
Atrial fibrillation (AF) is the most common chronic arrhythmia presenting a heavy disease burden. We report a new approach for generating cardiomyocytes (CMs) resembling atrial cells from human-induced pluripotent stem cells (hiPSCs) using a combination of Gremlin 2 and retinoic acid treatment. More than 40% of myocytes showed rod-shaped morphology, expression of CM proteins (including ryanodine receptor 2, α-actinin-2 and F-actin) and striated appearance, all of which were broadly similar to the characteristics of adult atrial myocytes (AMs). Isolated myocytes were electrically quiescent until stimulated to fire action potentials with an AM profile and an amplitude of approximately 100 mV, arising from a resting potential of approximately -70 mV. Single-cell RNA sequence analysis showed a high level of expression of several atrial-specific transcripts including NPPA, MYL7, HOXA3, SLN, KCNJ4, KCNJ5 and KCNA5. Amplitudes of calcium transients recorded from spontaneously beating cultures were increased by the stimulation of α-adrenoceptors (activated by phenylephrine and blocked by prazosin) or β-adrenoceptors (activated by isoproterenol and blocked by CGP20712A). Our new approach provides human AMs with mature characteristics from hiPSCs which will facilitate drug discovery by enabling the study of human atrial cell signalling pathways and AF. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.
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Affiliation(s)
- Faizzan S. Ahmad
- Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK
- Cure8bio, Inc, 395 Fulton Street, Westbury, NY 11590, USA
| | - Yongcheng Jin
- Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK
| | | | - Yafei Zhou
- Key Laboratory of Medical Electrophysiology of the Ministry of Education and Institute of Cardiovascular Research, Southwest Medical University, Luzhou 6400, People's Republic of China
- Shaanxi Institute for Pediatric Diseases, Department of Cardiology, Xi'an Children's Hospital, Xi'an 710003, People's Republic of China
| | - Meng Yuan
- Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK
- Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA
| | - Xuehui Fan
- Key Laboratory of Medical Electrophysiology of the Ministry of Education and Institute of Cardiovascular Research, Southwest Medical University, Luzhou 6400, People's Republic of China
| | - Rui Zhou
- Key Laboratory of Medical Electrophysiology of the Ministry of Education and Institute of Cardiovascular Research, Southwest Medical University, Luzhou 6400, People's Republic of China
| | - Razik Mu-u-min
- Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK
| | - Christopher O'Shea
- Institute of Cardiovascular Sciences, College of Medicine and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK
| | - Ayman M. Ibrahim
- Aswan Heart Centre, Aswan 1242770, Egypt
- Department of Zoology, Faculty of Science, Cairo University, Cairo 12613, Egypt
| | - Wajiha Hyder
- Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK
| | - Yasmine Aguib
- Aswan Heart Centre, Aswan 1242770, Egypt
- National Heart and Lung Institute, Heart Science Centre, Imperial College London, Middlesex SW3 6LY, UK
| | - Magdi Yacoub
- Aswan Heart Centre, Aswan 1242770, Egypt
- National Heart and Lung Institute, Heart Science Centre, Imperial College London, Middlesex SW3 6LY, UK
| | - Davor Pavlovic
- Institute of Cardiovascular Sciences, College of Medicine and Dental Sciences, University of Birmingham, Birmingham B15 2TT, UK
| | - Yanmin Zhang
- Shaanxi Institute for Pediatric Diseases, Department of Cardiology, Xi'an Children's Hospital, Xi'an 710003, People's Republic of China
| | - Xiaoqiu Tan
- Key Laboratory of Medical Electrophysiology of the Ministry of Education and Institute of Cardiovascular Research, Southwest Medical University, Luzhou 6400, People's Republic of China
| | - Ming Lei
- Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK
| | - Derek A. Terrar
- Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK
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6
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Wei SJ, Du JL, Wang YB, Qu PF, Ma L, Sun ZC, Tang X, Liu K, Xi YM, Nie SJ, Jia PL, Long W, Qu YQ, Li YH, Lei PP. Whole exome sequencing with a focus on cardiac disease-associated genes in families of sudden unexplained deaths in Yunnan, southwest of China. BMC Genomics 2023; 24:57. [PMID: 36721086 PMCID: PMC9890689 DOI: 10.1186/s12864-022-09097-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2022] [Accepted: 12/22/2022] [Indexed: 02/02/2023] Open
Abstract
OBJECTIVES To explore the causes of sudden unexpected death (SUD) and to search for high-risk people, whole exome sequencing (WES) was performed in families with SUDs. METHODS: Whole exome sequencing of 25 people from 14 SUD families were screened based on cardiac disease-associated gene variants, and their echocardiograms and electrocardiograms (ECG) were also examined. The protein function of mutated genes was predicted by SIFT, PolyPhen2 and Mutation Assessor. RESULTS In the group of 25 people from 14 SUD families, 49 single nucleotide variants (SNVs) of cardiac disease-associated genes were found and verified by Sanger sequencing. 29 SNVs of 14 cardiac disorder-related genes were predicted as pathogens by software. Among them, 7 SNVs carried by two or more members were found in 5 families, including SCN5A (c.3577C > T), IRX4 (c.230A > G), LDB3 (c.2104 T > G), MYH6 (c.3G > A), MYH6 (c.3928 T > C), TTN (c.80987C > T) and TTN (c.8069C > T). 25 ECGs were collected. In summary, 4 people had J-point elevation, 2 people had long QT syndrome (LQTS), 4 people had prolonged QT interval, 3 people had T-wave changes, 3 people had sinus tachycardia, 4 people had sinus bradycardia, 4 people had left side of QRS electrical axis, and 3 people had P wave broadening. Echocardiographic results showed that 1 person had atrial septal defect, 1 person had tricuspid regurgitation, and 2 people had left ventricular diastolic dysfunction. CONCLUSIONS Of the 14 heart disease-associated genes in 14 SUDs families, there are 7 possible pathological SNVS may be associated with SUDs. Our results indicate that people with ECG abnormalities, such as prolonged QT interval, ST segment changes, T-wave change and carrying the above 7 SNVs, should be the focus of prevention of sudden death.
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Affiliation(s)
- Si-Jie Wei
- grid.285847.40000 0000 9588 0960Department of Forensic Medicine, Kunming Medical University, Kunming, 650500 Yunnan Province China
| | - Jin-Liang Du
- grid.449428.70000 0004 1797 7280Forensic Science Center of Jining Medical University, Jining, Shandong 272000 People’s Republic of China
| | - Yue-Bing Wang
- Yunnan Institute of Endemic Disease Control and Prevention, Dali, 671000 Yunnan Province China
| | - Peng-Fei Qu
- grid.285847.40000 0000 9588 0960Department of Forensic Medicine, Kunming Medical University, Kunming, 650500 Yunnan Province China ,grid.11135.370000 0001 2256 9319School of Basic Medicine, Peking University Health Science Center, Beijing, 100191 China
| | - Lin Ma
- Yunnan Institute of Endemic Disease Control and Prevention, Dali, 671000 Yunnan Province China
| | - Zhong-Chun Sun
- grid.285847.40000 0000 9588 0960Department of Forensic Medicine, Kunming Medical University, Kunming, 650500 Yunnan Province China
| | - Xue Tang
- Yunnan Institute of Endemic Disease Control and Prevention, Dali, 671000 Yunnan Province China
| | - Kai Liu
- grid.285847.40000 0000 9588 0960Department of Forensic Medicine, Kunming Medical University, Kunming, 650500 Yunnan Province China
| | - Yan-Mei Xi
- Yunnan Institute of Endemic Disease Control and Prevention, Dali, 671000 Yunnan Province China
| | - Sheng-Jie Nie
- grid.285847.40000 0000 9588 0960Department of Forensic Medicine, Kunming Medical University, Kunming, 650500 Yunnan Province China
| | - Peng-Lin Jia
- grid.285847.40000 0000 9588 0960Department of Forensic Medicine, Kunming Medical University, Kunming, 650500 Yunnan Province China
| | - Wu Long
- grid.285847.40000 0000 9588 0960Department of Forensic Medicine, Kunming Medical University, Kunming, 650500 Yunnan Province China
| | - Yong-Qiang Qu
- grid.285847.40000 0000 9588 0960Department of Forensic Medicine, Kunming Medical University, Kunming, 650500 Yunnan Province China
| | - Yu-Hua Li
- grid.285847.40000 0000 9588 0960Department of Forensic Medicine, Kunming Medical University, Kunming, 650500 Yunnan Province China
| | - Pu-Ping Lei
- grid.285847.40000 0000 9588 0960Department of Forensic Medicine, Kunming Medical University, Kunming, 650500 Yunnan Province China
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7
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Myosin light chain 2 marks differentiating ventricular cardiomyocytes derived from human embryonic stem cells. Pflugers Arch 2021; 473:991-1007. [PMID: 34031754 DOI: 10.1007/s00424-021-02578-3] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2021] [Revised: 05/01/2021] [Accepted: 05/05/2021] [Indexed: 12/13/2022]
Abstract
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great value for studies of human cardiac development, drug discovery, disease modeling, and cell therapy. However, the mixed cardiomyocyte subtypes (ventricular-, atrial-, and nodal-like myocytes) and the maturation heterogeneity of hPSC-CMs restrain their application in vitro and in vivo. Myosin light chain 2 (MYL2, encoding the ventricular/cardiac muscle isoform MLC2v protein) is regarded as a ventricular-specific marker of cardiac myocardium; however, its restricted localization to ventricles during human heart development has been questioned. Consequently, it is currently unclear whether MYL2 definitively marks ventricular hESC-CMs. Here, by using a MYL2-Venus hESC reporter line, we characterized a time-dependent increase of the MYL2-Venus positive (MLC2v-Venus+) hESC-CMs during differentiation. We also compared the molecular, cellular, and functional properties between the MLC2v-Venus+ and MYL2-Venus negative (MLC2v-Venus-) hESC-CMs. At early differentiation stages of hESC-CMs, we reported that both MLC2v-Venus- and MLC2v-Venus+ CMs displayed ventricular-like traits but the ventricular-like cells from MLC2v-Venus+ hESC-CMs displayed more developed action potential (AP) properties than that from MLC2v-Venus- hESC-CMs. Meanwhile, about a half MLC2v-Venus- hESC-CM population displayed atrial-like AP properties, and a half showed ventricular-like AP properties, whereas only ~ 20% of the MLC2v-Venus- hESC-CMs expressed the atrial marker nuclear receptor subfamily 2 group F member 2 (NR2F2, also named as COUPTFII). At late time points, almost all MLC2v-Venus+ hESC-CMs exhibited ventricular-like AP properties. Further analysis demonstrates that the MLC2v-Venus+ hESC-CMs had enhanced Ca2+ transients upon increase of the MLC2v level during cultivation. Concomitantly, the MLC2v-Venus+ hESC-CMs showed more defined sarcomeric structures and better mitochondrial function than those in the MLC2v-Venus- hESC-CMs. Moreover, the MLC2v-Venus+ hESC-CMs were more sensitive to hypoxic stimulus than the MLC2v-Venus- hESC-CMs. These results provide new insights into the development of human ventricular myocytes and reveal a direct correlation between the expression profile of MLC2v and ventricular hESC-CM development. Our findings that MLC2v is predominantly a ventricular marker in developmentally immature hESC-CMs have implications for human development, drug screening, and disease modeling, and this marker should prove useful in overcoming issues associated with hESC-CM heterogeneity.
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8
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Mantri M, Scuderi GJ, Abedini-Nassab R, Wang MFZ, McKellar D, Shi H, Grodner B, Butcher JT, De Vlaminck I. Spatiotemporal single-cell RNA sequencing of developing chicken hearts identifies interplay between cellular differentiation and morphogenesis. Nat Commun 2021; 12:1771. [PMID: 33741943 PMCID: PMC7979764 DOI: 10.1038/s41467-021-21892-z] [Citation(s) in RCA: 120] [Impact Index Per Article: 30.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2020] [Accepted: 02/17/2021] [Indexed: 01/01/2023] Open
Abstract
Single-cell RNA sequencing is a powerful tool to study developmental biology but does not preserve spatial information about tissue morphology and cellular interactions. Here, we combine single-cell and spatial transcriptomics with algorithms for data integration to study the development of the chicken heart from the early to late four-chambered heart stage. We create a census of the diverse cellular lineages in developing hearts, their spatial organization, and their interactions during development. Spatial mapping of differentiation transitions in cardiac lineages defines transcriptional differences between epithelial and mesenchymal cells within the epicardial lineage. Using spatially resolved expression analysis, we identify anatomically restricted expression programs, including expression of genes implicated in congenital heart disease. Last, we discover a persistent enrichment of the small, secreted peptide, thymosin beta-4, throughout coronary vascular development. Overall, our study identifies an intricate interplay between cellular differentiation and morphogenesis.
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Affiliation(s)
- Madhav Mantri
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
- Computational Biology Ph.D. Program, Cornell University, Ithaca, NY, USA
| | - Gaetano J Scuderi
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
| | - Roozbeh Abedini-Nassab
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
- Department of Engineering, University of Neyshabur, Neyshabur, Iran
| | - Michael F Z Wang
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
- Computational Biology Ph.D. Program, Cornell University, Ithaca, NY, USA
| | - David McKellar
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
| | - Hao Shi
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
| | - Benjamin Grodner
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA
| | - Jonathan T Butcher
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.
| | - Iwijn De Vlaminck
- Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.
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9
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Wang YM, Khederzadeh S, Li SR, Otecko NO, Irwin DM, Thakur M, Ren XD, Wang MS, Wu DD, Zhang YP. Integrating Genomic and Transcriptomic Data to Reveal Genetic Mechanisms Underlying Piao Chicken Rumpless Trait. GENOMICS PROTEOMICS & BIOINFORMATICS 2021; 19:787-799. [PMID: 33631431 PMCID: PMC9170765 DOI: 10.1016/j.gpb.2020.06.019] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/13/2019] [Revised: 04/14/2020] [Accepted: 06/10/2020] [Indexed: 11/19/2022]
Abstract
Piao chicken, a rare Chinese native poultry breed, lacks primary tail structures, such as pygostyle, caudal vertebra, uropygial gland, and tail feathers. So far, the molecular mechanisms underlying tail absence in this breed remain unclear. In this study, we comprehensively employed comparative transcriptomic and genomic analyses to unravel potential genetic underpinnings of rumplessness in Piao chicken. Our results reveal many biological factors involved in tail development and several genomic regions under strong positive selection in this breed. These regions contain candidate genes associated with rumplessness, including Irx4, Il18, Hspb2, and Cryab. Retrieval of quantitative trait loci (QTL) and gene functions implies that rumplessness might be consciously or unconsciously selected along with the high-yield traits in Piao chicken. We hypothesize that strong selection pressures on regulatory elements might lead to changes in gene activity in mesenchymal stem cells of the tail bud. The ectopic activity could eventually result in tail truncation by impeding differentiation and proliferation of the stem cells. Our study provides fundamental insights into early initiation and genetic basis of the rumpless phenotype in Piao chicken.
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Affiliation(s)
- Yun-Mei Wang
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; Kunming College of Life Science, University of the Chinese Academy of Sciences, Kunming 650223, China; Center for Neurobiology and Brain Restoration, Skolkovo Institute of Science and Technology, Moscow 143026, Russia
| | - Saber Khederzadeh
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; Kunming College of Life Science, University of the Chinese Academy of Sciences, Kunming 650223, China
| | - Shi-Rong Li
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; Kunming College of Life Science, University of the Chinese Academy of Sciences, Kunming 650223, China
| | - Newton Otieno Otecko
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; Kunming College of Life Science, University of the Chinese Academy of Sciences, Kunming 650223, China
| | - David M Irwin
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5S 1A8, Canada
| | - Mukesh Thakur
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; Zoological Survey of India, Kolkata 700053, India
| | - Xiao-Die Ren
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; Kunming College of Life Science, University of the Chinese Academy of Sciences, Kunming 650223, China
| | - Ming-Shan Wang
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; Kunming College of Life Science, University of the Chinese Academy of Sciences, Kunming 650223, China.
| | - Dong-Dong Wu
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; Kunming College of Life Science, University of the Chinese Academy of Sciences, Kunming 650223, China.
| | - Ya-Ping Zhang
- State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; Kunming College of Life Science, University of the Chinese Academy of Sciences, Kunming 650223, China.
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10
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Assi E, D'Addio F, Mandò C, Maestroni A, Loretelli C, Ben Nasr M, Usuelli V, Abdelsalam A, Seelam AJ, Pastore I, Magagnotti C, Abdi R, El Essawy B, Folli F, Corradi D, Zuccotti G, Cetin I, Fiorina P. Placental proteome abnormalities in women with gestational diabetes and large-for-gestational-age newborns. BMJ Open Diabetes Res Care 2020; 8:8/2/e001586. [PMID: 33188009 PMCID: PMC7668299 DOI: 10.1136/bmjdrc-2020-001586] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/18/2020] [Revised: 09/24/2020] [Accepted: 10/12/2020] [Indexed: 01/06/2023] Open
Abstract
INTRODUCTION Gestational diabetes mellitus (GDM) is the most frequent metabolic complication during pregnancy and is associated with development of short-term and long-term complications for newborns, with large-for-gestational-age (LGA) being particularly common. Interestingly, the mechanism behind altered fetal growth in GDM is only partially understood. RESEARCH DESIGN AND METHODS A proteomic approach was used to analyze placental samples obtained from healthy pregnant women (n=5), patients with GDM (n=12) and with GDM and LGA (n=5). Effects of altered proteins on fetal development were tested in vitro in human embryonic stem cells (hESCs). RESULTS Here, we demonstrate that the placental proteome is altered in pregnant women affected by GDM with LGA, with at least 37 proteins differentially expressed to a higher degree (p<0.05) as compared with those with GDM but without LGA. Among these proteins, 10 are involved in regulating tissue differentiation and/or fetal growth and development, with bone marrow proteoglycan (PRG2) and dipeptidyl peptidase-4 (DPP-4) being highly expressed. Both PRG2 and DPP-4 altered the transcriptome profile of stem cells differentiation markers when tested in vitro in hESCs, suggesting a potential role in the onset of fetal abnormalities. CONCLUSIONS Our findings suggest that placental dysfunction may be directly responsible for abnormal fetal growth/development during GDM. Once established on a larger population, inhibitors of the pathways involving those altered factors may be tested in conditions such as GDM and LGA, in which therapeutic approaches are still lacking.
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Affiliation(s)
- Emma Assi
- International Center for T1D Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, Department of Biomedical and Clinical Sciences, Università di Milano, Milano, Italy
| | - Francesca D'Addio
- International Center for T1D Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, Department of Biomedical and Clinical Sciences, Università di Milano, Milano, Italy
| | - Chiara Mandò
- "G. Pardi" Laboratory of Maternal-Fetal Translational Research, Department of Biomedical and Clinical Sciences "L. Sacco", Università di Milano, Milano, Lombardia, Italy
| | - Anna Maestroni
- International Center for T1D Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, Department of Biomedical and Clinical Sciences, Università di Milano, Milano, Italy
| | - Cristian Loretelli
- International Center for T1D Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, Department of Biomedical and Clinical Sciences, Università di Milano, Milano, Italy
| | - Moufida Ben Nasr
- International Center for T1D Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, Department of Biomedical and Clinical Sciences, Università di Milano, Milano, Italy
- Nephrology Division, Boston Children's Hospital, Boston, Massachusetts, USA
| | - Vera Usuelli
- International Center for T1D Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, Department of Biomedical and Clinical Sciences, Università di Milano, Milano, Italy
- Nephrology Division, Boston Children's Hospital, Boston, Massachusetts, USA
| | - Ahmed Abdelsalam
- International Center for T1D Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, Department of Biomedical and Clinical Sciences, Università di Milano, Milano, Italy
| | - Andy Joe Seelam
- International Center for T1D Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, Department of Biomedical and Clinical Sciences, Università di Milano, Milano, Italy
| | - Ida Pastore
- Department of Endocrinology, ASST Fatebenefratelli-Sacco, Milano, Lombardia, Italy
| | - Cinzia Magagnotti
- ProMiFa, Protein Microsequencing Facility, Ospedale San Raffaele, Milano, Lombardia, Italy
| | - Reza Abdi
- Transplantation Research Center, Renal Division, Brigham & Women's, Harvard University, Boston, Massachusetts, USA
| | - Basset El Essawy
- Transplantation Research Center, Renal Division, Brigham & Women's, Harvard University, Boston, Massachusetts, USA
- Faculty of Medicine, Al-Azhar University, Cairo, Egypt
| | - Franco Folli
- Endocrinologia e Metabolismo, Dipartimento di Scienze della Salute, Universita di Milano, Milano, Italy
| | - Domenico Corradi
- Department of Biomedical, Biotechnological and Translational Sciences, Unit of Pathology, University of Parma, Parma, Emilia-Romagna, Italy
| | - Gianvincenzo Zuccotti
- International Center for T1D Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, Department of Biomedical and Clinical Sciences, Università di Milano, Milano, Italy
- Department of Pediatrics, Ospedale dei Bambini V Buzzi, Milano, Lombardia, Italy
| | - Irene Cetin
- "G. Pardi" Laboratory of Maternal-Fetal Translational Research, Department of Biomedical and Clinical Sciences "L. Sacco", Università di Milano, Milano, Lombardia, Italy
| | - Paolo Fiorina
- International Center for T1D Pediatric Clinical Research Center Romeo ed Enrica Invernizzi, Department of Biomedical and Clinical Sciences, Università di Milano, Milano, Italy
- Nephrology Division, Boston Children's Hospital, Boston, Massachusetts, USA
- Department of Endocrinology, ASST Fatebenefratelli-Sacco, Milano, Lombardia, Italy
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11
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Overton IM, Sims AH, Owen JA, Heale BSE, Ford MJ, Lubbock ALR, Pairo-Castineira E, Essafi A. Functional Transcription Factor Target Networks Illuminate Control of Epithelial Remodelling. Cancers (Basel) 2020; 12:cancers12102823. [PMID: 33007944 PMCID: PMC7652213 DOI: 10.3390/cancers12102823] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2020] [Revised: 09/16/2020] [Accepted: 09/24/2020] [Indexed: 12/15/2022] Open
Abstract
Cell identity is governed by gene expression, regulated by transcription factor (TF) binding at cis-regulatory modules. Decoding the relationship between TF binding patterns and gene regulation is nontrivial, remaining a fundamental limitation in understanding cell decision-making. We developed the NetNC software to predict functionally active regulation of TF targets; demonstrated on nine datasets for the TFs Snail, Twist, and modENCODE Highly Occupied Target (HOT) regions. Snail and Twist are canonical drivers of epithelial to mesenchymal transition (EMT), a cell programme important in development, tumour progression and fibrosis. Predicted "neutral" (non-functional) TF binding always accounted for the majority (50% to 95%) of candidate target genes from statistically significant peaks and HOT regions had higher functional binding than most of the Snail and Twist datasets examined. Our results illuminated conserved gene networks that control epithelial plasticity in development and disease. We identified new gene functions and network modules including crosstalk with notch signalling and regulation of chromatin organisation, evidencing networks that reshape Waddington's epigenetic landscape during epithelial remodelling. Expression of orthologous functional TF targets discriminated breast cancer molecular subtypes and predicted novel tumour biology, with implications for precision medicine. Predicted invasion roles were validated using a tractable cell model, supporting our approach.
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Affiliation(s)
- Ian M. Overton
- MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK; (A.H.S.); (B.S.E.H.); (M.J.F.); (A.L.R.L.); (E.P.-C.); (A.E.)
- Department of Systems Biology, Harvard University, Boston, MA 02115, USA;
- Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh, Edinburgh EH9 3BF, UK
- Patrick G Johnston Centre for Cancer Research, Queen’s University Belfast, Belfast BT9 7AE, UK
- Correspondence:
| | - Andrew H. Sims
- MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK; (A.H.S.); (B.S.E.H.); (M.J.F.); (A.L.R.L.); (E.P.-C.); (A.E.)
| | - Jeremy A. Owen
- Department of Systems Biology, Harvard University, Boston, MA 02115, USA;
- Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Bret S. E. Heale
- MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK; (A.H.S.); (B.S.E.H.); (M.J.F.); (A.L.R.L.); (E.P.-C.); (A.E.)
| | - Matthew J. Ford
- MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK; (A.H.S.); (B.S.E.H.); (M.J.F.); (A.L.R.L.); (E.P.-C.); (A.E.)
| | - Alexander L. R. Lubbock
- MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK; (A.H.S.); (B.S.E.H.); (M.J.F.); (A.L.R.L.); (E.P.-C.); (A.E.)
| | - Erola Pairo-Castineira
- MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK; (A.H.S.); (B.S.E.H.); (M.J.F.); (A.L.R.L.); (E.P.-C.); (A.E.)
| | - Abdelkader Essafi
- MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK; (A.H.S.); (B.S.E.H.); (M.J.F.); (A.L.R.L.); (E.P.-C.); (A.E.)
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12
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Jia Z, Zhang Y, Yan A, Wang M, Han Q, Wang K, Wang J, Qiao C, Pan Z, Chen C, Hu D, Ding X. 1,25-dihydroxyvitamin D3 signaling-induced decreases in IRX4 inhibits NANOG-mediated cancer stem-like properties and gefitinib resistance in NSCLC cells. Cell Death Dis 2020; 11:670. [PMID: 32820157 PMCID: PMC7441324 DOI: 10.1038/s41419-020-02908-w] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2020] [Revised: 07/29/2020] [Accepted: 07/29/2020] [Indexed: 11/24/2022]
Abstract
Recent studies have demonstrated that acquisition of cancer stem-like properties plays an essential role in promoting epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) resistance in non-small cell lung cancer (NSCLC); however, how to regulate cancer stem-like properties and EGFR-TKI resistance is largely unclear. In this study, we discovered that increased iroquois-class homeodomain protein 4 (IRX4) was related to gefitinib resistance in NSCLC cells. Knockdown of IRX4 inhibited cell proliferation, sphere formation, and the expression of CD133, ALDH1A1, NANOG, Sox2 and Notch1, and the transcriptional activity of NANOG promoter. IRX4 overexpression increased the protein level of NANOG and CD133 in PC-9 cells. Combination of knocking-down IRX4 with gefitinib increased cell apoptosis and decreased cell viability and the expression of p-EGFR and NANOG in PC-9/GR cells. IRX4 knockdown in a PC-9/GR xenograft tumor model inhibited tumor progression and the expression of NANOG and CD133 more effectively than single treatment alone. Knockdown of NANOG inhibited the expression of CD133 and restored gefitinib cytotoxicity, and NANOG overexpression-induced cancer stem-like properties and gefitinib resistance could be obviously reversed by knocking-down IRX4. Further, we found that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) reduced obviously the expression of IRX4 and NANOG by inhibiting the activation of TGF-β1/Smad3 signaling pathway; moreover, combination of 1,25(OH)2D3 and gefitinib decreased cell viability and proliferation or tumor progression and the expression of IRX4 and NANOG compared with single treatment alone both in PC-9/GR cells and in a PC-9/GR xenograft tumor model. These results reveal that inhibition of IRX4-mediated cancer stem-like properties by regulating 1,25(OH)2D3 signaling may increase gefitinib cytotoxicity. Combination therapy of gefitinib and 1,25(OH)2D3 by targeting IRX4 and NANOG, could provide a promising strategy to improve gefitinib cytotoxicity.
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Affiliation(s)
- Zhirong Jia
- School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China
| | - Yameng Zhang
- School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China
| | - Aiwen Yan
- School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China
| | - Meisa Wang
- School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China
| | - Qiushuang Han
- School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China
| | - Kaiwei Wang
- School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China
| | - Jie Wang
- School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China.,Department of Pharmacy, the First Affiliated Hospital of Xinjiang Medical University, 830054, Urumqi, China
| | - Chen Qiao
- School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China.,Precision Medicine Laboratory, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China
| | - Zhenzhen Pan
- School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China
| | - Chuansheng Chen
- School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China
| | - Dong Hu
- Key Laboratory of Industrial Dust Prevention and Control & Occupational Safety and Health of the Ministry of Education, Medical School, Anhui University of Science and Technology, 232001, Huainan, China.
| | - Xuansheng Ding
- School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China. .,Precision Medicine Laboratory, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, 211198, Nanjing, China.
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13
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Peng Y, Pang J, Hu J, Jia Z, Xi H, Ma N, Yang S, Liu J, Huang X, Tang C, Wang H. Clinical and molecular characterization of 12 prenatal cases of Cri-du-chat syndrome. Mol Genet Genomic Med 2020; 8:e1312. [PMID: 32500674 PMCID: PMC7434726 DOI: 10.1002/mgg3.1312] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2020] [Revised: 04/20/2020] [Accepted: 04/27/2020] [Indexed: 12/12/2022] Open
Abstract
Background This study aimed to define the molecular basis for 12 prenatal cases of Cri‐du‐chat syndrome (CdCS) and the potential genotyping‐phenotyping association. Methods Karyotyping and single nucleotide polymorphism array analyses for copy number variants were performed. Results Nine cases had 5p terminal deletions and three had 5p interstitial deletions, and these cases had variable deletion sizes with partial overlapping. Phenotypically, besides intrauterine growth restriction (IUGR) and brain as well as heart abnormalities, hypospadias, and lung dysplasia were observed. Potential genetic causes for specific phenotypes in these cases were identified. Conclusion This study defined the molecular bases for the patients of CdCS, which is important for genetic counseling for these families. The findings of present study expand the clinical features of CdCS in the fetal period, and provided important information for further refining the genotypic–phenotypic correlations for this syndrome.
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Affiliation(s)
- Ying Peng
- Department of Medical Genetics, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, Hunan, China.,National Health Commission Key Laboratory of Birth Defects Research, Prevention and Treatment, Changsha, Hunan, China
| | - Jialun Pang
- Department of Medical Genetics, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, Hunan, China.,National Health Commission Key Laboratory of Birth Defects Research, Prevention and Treatment, Changsha, Hunan, China
| | - Jiancheng Hu
- Department of Medical Genetics, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, Hunan, China.,National Health Commission Key Laboratory of Birth Defects Research, Prevention and Treatment, Changsha, Hunan, China
| | - Zhengjun Jia
- Department of Medical Genetics, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, Hunan, China.,National Health Commission Key Laboratory of Birth Defects Research, Prevention and Treatment, Changsha, Hunan, China
| | - Hui Xi
- Department of Medical Genetics, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, Hunan, China.,National Health Commission Key Laboratory of Birth Defects Research, Prevention and Treatment, Changsha, Hunan, China
| | - Na Ma
- Department of Medical Genetics, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, Hunan, China.,National Health Commission Key Laboratory of Birth Defects Research, Prevention and Treatment, Changsha, Hunan, China
| | - Shuting Yang
- Department of Medical Genetics, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, Hunan, China.,National Health Commission Key Laboratory of Birth Defects Research, Prevention and Treatment, Changsha, Hunan, China
| | - Jing Liu
- Department of Medical Genetics, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, Hunan, China.,National Health Commission Key Laboratory of Birth Defects Research, Prevention and Treatment, Changsha, Hunan, China
| | - Xiaoliang Huang
- Department of Medical Genetics, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, Hunan, China.,National Health Commission Key Laboratory of Birth Defects Research, Prevention and Treatment, Changsha, Hunan, China
| | - Chengyuan Tang
- Department of Nephrology, Hunan Key Laboratory of Kidney Disease and Blood Purification, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Hua Wang
- Department of Medical Genetics, Hunan Provincial Maternal and Child Health Care Hospital, Changsha, Hunan, China.,National Health Commission Key Laboratory of Birth Defects Research, Prevention and Treatment, Changsha, Hunan, China
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14
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miR-128a Acts as a Regulator in Cardiac Development by Modulating Differentiation of Cardiac Progenitor Cell Populations. Int J Mol Sci 2020; 21:ijms21031158. [PMID: 32050579 PMCID: PMC7038042 DOI: 10.3390/ijms21031158] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2020] [Revised: 02/04/2020] [Accepted: 02/06/2020] [Indexed: 11/18/2022] Open
Abstract
MicroRNAs (miRs) appear to be major, yet poorly understood players in regulatory networks guiding cardiogenesis. We sought to identify miRs with unknown functions during cardiogenesis analyzing the miR-profile of multipotent Nkx2.5 enhancer cardiac progenitor cells (NkxCE-CPCs). Besides well-known candidates such as miR-1, we found about 40 miRs that were highly enriched in NkxCE-CPCs, four of which were chosen for further analysis. Knockdown in zebrafish revealed that only miR-128a affected cardiac development and function robustly. For a detailed analysis, loss-of-function and gain-of-function experiments were performed during in vitro differentiations of transgenic murine pluripotent stem cells. MiR-128a knockdown (1) increased Isl1, Sfrp5, and Hcn4 (cardiac transcription factors) but reduced Irx4 at the onset of cardiogenesis, (2) upregulated Isl1-positive CPCs, whereas NkxCE-positive CPCs were downregulated, and (3) increased the expression of the ventricular cardiomyocyte marker Myl2 accompanied by a reduced beating frequency of early cardiomyocytes. Overexpression of miR-128a (4) diminished the expression of Isl1, Sfrp5, Nkx2.5, and Mef2c, but increased Irx4, (5) enhanced NkxCE-positive CPCs, and (6) favored nodal-like cardiomyocytes (Tnnt2+, Myh6+, Shox2+) accompanied by increased beating frequencies. In summary, we demonstrated that miR-128a plays a so-far unknown role in early heart development by affecting the timing of CPC differentiation into various cardiomyocyte subtypes.
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Atkinson SP. Previews. Stem Cells Transl Med 2019. [PMCID: PMC6766597 DOI: 10.1002/sctm.19-0270] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022] Open
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Hoff K, Lemme M, Kahlert AK, Runde K, Audain E, Schuster D, Scheewe J, Attmann T, Pickardt T, Caliebe A, Siebert R, Kramer HH, Milting H, Hansen A, Ammerpohl O, Hitz MP. DNA methylation profiling allows for characterization of atrial and ventricular cardiac tissues and hiPSC-CMs. Clin Epigenetics 2019; 11:89. [PMID: 31186048 PMCID: PMC6560887 DOI: 10.1186/s13148-019-0679-0] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2018] [Accepted: 05/03/2019] [Indexed: 02/07/2023] Open
Abstract
Background Cardiac disease modelling using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) requires thorough insight into cardiac cell type differentiation processes. However, current methods to discriminate different cardiac cell types are mostly time-consuming, are costly and often provide imprecise phenotypic evaluation. DNA methylation plays a critical role during early heart development and cardiac cellular specification. We therefore investigated the DNA methylation pattern in different cardiac tissues to identify CpG loci for further cardiac cell type characterization. Results An array-based genome-wide DNA methylation analysis using Illumina Infinium HumanMethylation450 BeadChips led to the identification of 168 differentially methylated CpG loci in atrial and ventricular human heart tissue samples (n = 49) from different patients with congenital heart defects (CHD). Systematic evaluation of atrial-ventricular DNA methylation pattern in cardiac tissues in an independent sample cohort of non-failing donor hearts and cardiac patients using bisulfite pyrosequencing helped us to define a subset of 16 differentially methylated CpG loci enabling precise characterization of human atrial and ventricular cardiac tissue samples. This defined set of reproducible cardiac tissue-specific DNA methylation sites allowed us to consistently detect the cellular identity of hiPSC-CM subtypes. Conclusion Testing DNA methylation of only a small set of defined CpG sites thus makes it possible to distinguish atrial and ventricular cardiac tissues and cardiac atrial and ventricular subtypes of hiPSC-CMs. This method represents a rapid and reliable system for phenotypic characterization of in vitro-generated cardiomyocytes and opens new opportunities for cardiovascular research and patient-specific therapy. Electronic supplementary material The online version of this article (10.1186/s13148-019-0679-0) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Kirstin Hoff
- Department of Congenital Heart Disease and Pediatric Cardiology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.,DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany
| | - Marta Lemme
- DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany.,Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Anne-Karin Kahlert
- Department of Congenital Heart Disease and Pediatric Cardiology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.,DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany.,Institute for Clinical Genetics, Carl Gustav Carus Faculty of Medicine, Dresden, Germany
| | - Kerstin Runde
- Department of Congenital Heart Disease and Pediatric Cardiology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany
| | - Enrique Audain
- Department of Congenital Heart Disease and Pediatric Cardiology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.,DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany
| | - Dorit Schuster
- Institute of Human Genetics, Christian-Albrechts-University Kiel & University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany
| | - Jens Scheewe
- Department of Congenital Heart Disease and Pediatric Cardiology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany
| | - Tim Attmann
- Department of Congenital Heart Disease and Pediatric Cardiology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany
| | - Thomas Pickardt
- National Register for Congenital Heart Defects, DZHK (German Centre for Cardiovascular Research), Berlin, Germany.,Competence Network for Congenital Heart Defects, DZHK (German Centre for Cardiovascular Research), Berlin, Germany
| | - Almuth Caliebe
- Institute of Human Genetics, Christian-Albrechts-University Kiel & University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany
| | - Reiner Siebert
- Institute of Human Genetics, University Hospital Ulm, Ulm, Germany
| | - Hans-Heiner Kramer
- Department of Congenital Heart Disease and Pediatric Cardiology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.,DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany
| | - Hendrik Milting
- Erich and Hanna Klessmann Institute for Cardiovascular Research & Development (EHKI), Heart and Diabetes Center NRW, Ruhr University Bochum, Bad Oeynhausen, Germany
| | - Arne Hansen
- DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany.,Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Ole Ammerpohl
- Institute of Human Genetics, University Hospital Ulm, Ulm, Germany
| | - Marc-Phillip Hitz
- Department of Congenital Heart Disease and Pediatric Cardiology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany. .,DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany. .,Institute of Human Genetics, Christian-Albrechts-University Kiel & University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany. .,Wellcome Trust Sanger Institute, Cambridge, UK.
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Hong SP, Song S, Lee S, Jo H, Kim HK, Han J, Park JH, Cho SW. Regenerative potential of mouse embryonic stem cell-derived PDGFRα + cardiac lineage committed cells in infarcted myocardium. World J Stem Cells 2019; 11:44-54. [PMID: 30705714 PMCID: PMC6354102 DOI: 10.4252/wjsc.v11.i1.44] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/27/2018] [Revised: 12/06/2018] [Accepted: 01/06/2019] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Pluripotent stem cell-derived cardiomyocytes (CMs) have become one of the most attractive cellular resources for cell-based therapy to rescue damaged cardiac tissue.
AIM We investigated the regenerative potential of mouse embryonic stem cell (ESC)-derived platelet-derived growth factor receptor-α (PDGFRα)+ cardiac lineage-committed cells (CLCs), which have a proliferative capacity but are in a morphologically and functionally immature state compared with differentiated CMs.
METHODS We induced mouse ESCs into PDGFRα+ CLCs and αMHC+ CMs using a combination of the small molecule cyclosporin A, the rho-associated coiled-coil kinase inhibitor Y27632, the antioxidant Trolox, and the ALK5 inhibitor EW7197. We implanted PDGFRα+ CLCs and differentiated αMHC+ CMs into a myocardial infarction (MI) murine model and performed functional analysis using transthoracic echocardiography (TTE) and histologic analysis.
RESULTS Compared with the untreated MI hearts, the anterior and septal regional wall motion and systolic functional parameters were notably and similarly improved in the MI hearts implanted with PDGFRα+ CLCs and αMHC+ CMs based on TTE. In histologic analysis, the untreated MI hearts contained a thinner ventricular wall than did the controls, while the ventricular walls of MI hearts implanted with PDGFRα+ CLCs and αMHC+ CMs were similarly thicker compared with that of the untreated MI hearts. Furthermore, implanted PDGFRα+ CLCs aligned and integrated with host CMs and were mostly differentiated into α-actinin+ CMs, and they did not convert into CD31+ endothelial cells or αSMA+ mural cells.
CONCLUSION PDGFRα+ CLCs from mouse ESCs exhibiting proliferative capacity showed a regenerative effect in infarcted myocardium. Therefore, mouse ESC-derived PDGFRα+ CLCs may represent a potential cellular resource for cardiac regeneration.
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Affiliation(s)
- Seon Pyo Hong
- Center for Vascular Research, Institute of Basic Science (IBS), Daejeon 34141, South Korea
| | - Sukhyun Song
- Center for Vascular Research, Institute of Basic Science (IBS), Daejeon 34141, South Korea
| | - Seungjoo Lee
- Department of Neurosurgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505, South Korea
| | - Hyeonju Jo
- Cardiovascular and Metabolic Disease Center, Department of Physiology, Department of Health Sciences and Technology, BK21 plus Project Team, Inje University College of Medicine, Busan 47392, South Korea
| | - Hyoung Kyu Kim
- Cardiovascular and Metabolic Disease Center, Department of Physiology, Department of Health Sciences and Technology, BK21 plus Project Team, Inje University College of Medicine, Busan 47392, South Korea
| | - Jin Han
- Cardiovascular and Metabolic Disease Center, Department of Physiology, Department of Health Sciences and Technology, BK21 plus Project Team, Inje University College of Medicine, Busan 47392, South Korea
| | - Jae-Hyeong Park
- Department of Cardiology in Internal Medicine, School of Medicine, Chungnam National University Hospital, Chungnam National University, Daejeon 35015, South Korea
| | - Sung Woo Cho
- Division of Cardiology, Department of Internal Medicine, Inje University College of Medicine, Seoul Paik Hospital, Seoul 04551, South Korea
- Cardiovascular and Metabolic Disease Center, Inje University College of Medicine, Busan 47392, South Korea
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Hong SP, Song S, Lee S, Jo H, Kim HK, Han J, Park JH, Cho SW. Regenerative potential of mouse embryonic stem cell-derived PDGFRα + cardiac lineage committed cells in infarcted myocardium. World J Stem Cells 2019. [DOI: 10.4252/wjsc.v11.i1.45] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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Lalit PA, Rodriguez AM, Downs KM, Kamp TJ. Generation of multipotent induced cardiac progenitor cells from mouse fibroblasts and potency testing in ex vivo mouse embryos. Nat Protoc 2017; 12:1029-1054. [PMID: 28426026 DOI: 10.1038/nprot.2017.021] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Here we describe a protocol to generate expandable and multipotent induced cardiac progenitor cells (iCPCs) from mouse adult fibroblasts using forced expression of Mesp1, Tbx5, Gata4, Nkx2.5 and Baf60c (MTGNB) along with activation of Wnt and JAK/STAT signaling. This method does not use iPS cell factors and thus differs from cell activation and signaling-directed (CASD) reprogramming to cardiac progenitors. Our method is specific to direct CPC reprogramming, whereas CASD reprogramming can generate various cell types depending on culture conditions and raises the possibility of transitioning through a pluripotent cell state. The protocol describes how to isolate and infect primary fibroblasts; induce reprogramming and observe iCPC colonies; expand and characterize reprogrammed iCPCs by immunostaining, flow cytometry and gene expression; differentiate iCPCs in vitro into cardiac-lineage cells; and test the embryonic potency of iCPCs via injection into the cardiac crescent of mouse embryos. A scientist experienced in molecular cell biology and embryology can reproduce this protocol in 12-16 weeks. iCPCs can be used for studying cardiac biology, drug discovery and regenerative medicine.
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Affiliation(s)
- Pratik A Lalit
- Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.,Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Adriana M Rodriguez
- Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.,Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Karen M Downs
- Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.,Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA
| | - Timothy J Kamp
- Department of Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.,Stem Cell and Regenerative Medicine Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.,Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin, USA
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