Reassessment of c-Kit + Cells for Cardiomyocyte Contribution in Adult Heart

HomeCirculationVol. 140, No. 2Reassessment of c-Kit+ Cells for Cardiomyocyte Contribution in Adult Heart Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBReassessment of c-Kit+ Cells for Cardiomyocyte Contribution in Adult Heart Lingjuan He, PhD, Maoying Han, BS, Zhenqian Zhang, BS, Yan Li, PhD, Xiuzhen Huang, BS, Xiuxiu Liu, BS, Wenjuan Pu, PhD, Huan Zhao, BS, Qing-Dong Wang, MD, PhD, Yu Nie, PhD and Bin Zhou, MD, PhD Lingjuan HeLingjuan He Lingjuan He, PhD, 320 Yueyang Road A2110, Shanghai 200031, China. Email E-mail Address: [email protected] The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, China (L.H.). State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence on Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, China (L.H., M.H., Z.Z., Y.L., X.H., X.L., W.P., H.Z., B.Z.). Search for more papers by this author , Maoying HanMaoying Han State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence on Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, China (L.H., M.H., Z.Z., Y.L., X.H., X.L., W.P., H.Z., B.Z.). ShanghaiTech University, China (M.H., B.Z.). Search for more papers by this author , Zhenqian ZhangZhenqian Zhang State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence on Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, China (L.H., M.H., Z.Z., Y.L., X.H., X.L., W.P., H.Z., B.Z.). Search for more papers by this author , Yan LiYan Li State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence on Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, China (L.H., M.H., Z.Z., Y.L., X.H., X.L., W.P., H.Z., B.Z.). Search for more papers by this author , Xiuzhen HuangXiuzhen Huang State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence on Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, China (L.H., M.H., Z.Z., Y.L., X.H., X.L., W.P., H.Z., B.Z.). Search for more papers by this author , Xiuxiu LiuXiuxiu Liu State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence on Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, China (L.H., M.H., Z.Z., Y.L., X.H., X.L., W.P., H.Z., B.Z.). Search for more papers by this author , Wenjuan PuWenjuan Pu State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence on Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, China (L.H., M.H., Z.Z., Y.L., X.H., X.L., W.P., H.Z., B.Z.). Search for more papers by this author , Huan ZhaoHuan Zhao State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence on Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, China (L.H., M.H., Z.Z., Y.L., X.H., X.L., W.P., H.Z., B.Z.). Search for more papers by this author , Qing-Dong WangQing-Dong Wang Bioscience Heart Failure, Cardiovascular, Renal, and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden (Q.-D.W.). Search for more papers by this author , Yu NieYu Nie Yu Nie, PhD, Fuwai Hospital, Beijing, 100037, China. Email E-mail Address: [email protected] State Key Laboratory of Cardiovascular Disease, Fuwai Hospital, National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (Y.N.). Search for more papers by this author and Bin ZhouBin Zhou Bin Zhou, MD, PhD, 320 Yueyang Road A2110, Shanghai 200031, China. Email E-mail Address: [email protected] State Key Laboratory of Cell Biology, Chinese Academy of Sciences Center for Excellence on Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, China (L.H., M.H., Z.Z., Y.L., X.H., X.L., W.P., H.Z., B.Z.). ShanghaiTech University, China (M.H., B.Z.). Key Laboratory of Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, China (B.Z.). Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China (B.Z.). Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, China (B.Z.). Search for more papers by this author Originally published8 Jul 2019https://doi.org/10.1161/CIRCULATIONAHA.119.039909Circulation. 2019;140:164–166The myogenic potential of Kit+ cardiac stem cells (CSCs) remains controversial.1–3 Recent communications arising in Nature argued that all previous Kit–Cre knock-in drivers fail to fate-map cardiac stem cells.3 Two major caveats on Kit–Cre tools for the study of Kit+ CSCs have been proposed.3 First, all reported Kit–Cre knock-in mice are haploinsufficient for Kit, because the knock-in cDNA encoding for recombinase replaces endogenous Kit expression.2,4,5 Second, Kit–Cre mice were insufficient to label low–Kit-expressing cells.3 Here we generated 2 new Kit–Cre drivers that label all Kit+ cells efficiently without removing the endogenous Kit gene. New fate-mapping data showed that Kit+ CSCs do not contribute to de novo cardiomyocytes during cardiac homeostasis or after injury. All animal procedures followed were in accordance with the institutional guidelines.To circumvent the knockout of c-Kit gene when Cre is inserted into ATG of endogenous gene locus, we designed an alternative strategy to insert Cre after the last coding exon of Kit gene (Figure [A]). We used a self-cleaving 2A peptide (2A) or an internal ribosomal entry site (IRES) to permit Cre expression after endogenous Kit expression (Figure [A]). To circumvent the labeling inefficiency by induced recombinases such as CreER5 or MerCreMer,2,4 we used constitutively active Cre recombinase to mark all Kit+ cells and their lineages (Figure [A]). All mouse experiments were used in accordance with the guidelines from the Institutional Animal Care and Use Committee. All data were presented as means±SEM, and the statistical analysis was done by 1-way ANOVA with Turkey’s multiple comparison tests. Although homozygous Kit knockout (knock-in of reporter or Cre at ATG locus) are lethal,5Kit2A-Cre/2A-Cre or KitIRES-Cre/IRES-Cre homozygous mice survive normally to adult stage at the expected Mendelian frequency (22.2% and 26.1%, respectively, Figure [B and C]). In addition, we did not detect any difference in body size and weight, ratio of heart weight to body weight and heart function, tissue fibrosis, cardiomyocyte size, and capillary density between wild-type, heterozygous, and homozygous littermate groups (Figure [B through H]). We confirmed that Kit expression remains intact in the homozygous mice (Figure [I]). We found that both Kit–2A–Cre and Kit–IRES–Cre labeled all hematopoietic cells from bone marrow and all coronary endothelial cells and also a subset of cardiomyocytes (data not shown). We therefore generated 2 new Kit–Cre lines efficiently label Kit+ cells without loss of Kit gene allele and expression.Download figureDownload PowerPointFigure. Generation of efficient Kit–Cre knock-in lines without removing endogenous Kit gene and their fate mapping studies. A, Schematic figure showing knock-in strategies for Kit-2A-Cre or Kit-IRES-Cre alleles by homologous recombination. B, Quantification of the heart-to-body weight percentage in wild-type, heterozygous, and homozygous mice. C, Gross view of littermate mice of different genotypes. Ratio on mouse denotes the number of this genotype to all mice studied. D, Hematoxylin and eosin (H.E.) staining on heart sections of different genotyped mice. E, Echocardiographic (Echo) images of different genotyped mice. F, Sirius Red staining on heart sections. G, Wheat germ agglutinin (WGA) staining on heart sections and quantification of relative cardiomyocyte size in each field. H, Immunostaining for VE-cadherin (CDH5) on heart sections and quantification of vessel density in each field. I, Immunostaining for c-Kit on heart sections show its expression in tissues of all groups (12–16W). J, Strategy for labeling of Kit+ cells by dual recombinases-based system. K, Schematic figure showing experimental strategy. L and M, Immunostaining for green fluorescent protein (GFP), tdTomato, and Troponin I (TNNI3) on myocardial infarction (MI) heart sections. Magnification of boxed regions in M shows all GFP+ cardiomyocytes were tdTomato+TNNI3+ (yellow arrowheads). N, Quantification of the percentage of GFP+tdTomato+ or GFP+tdTomato– cardiomyocytes in different regions of MI heart or sham heart tissue. O, Immunostaining for GFP, tdTomato, and TNNI3 on MI heart sections. Arrowheads indicate tdTomato+GFP+ cardiomyocytes. The data are means±SEM; n = 5–6. Statistical analysis was done by 1-way ANOVA with Turkey’s multiple comparisons for B, G, and H. Black scale bars, 1 mm; white scale bars, 100 µm. Each image is representative of at least 5 individual mouse samples. CAG indicates CMV immediate enhancer/β-actin promoter; DAPI, 4´,6´-diamino-2-phenylindole; IRES, internal ribosomal entry site; MI, myocardial infarction; n.s., not significant; Tam, tamoxifen; Tnnt2, Troponin T2 promoter; and UTR, untranslated region.Using new genetic tools, we next re-examined whether Kit+ cells contribute to new cardiomyocytes in the adult heart during homeostasis. In the adult mouse heart (8–10 weeks), Kit–2A–Cre and Kit–IRES–Cre labeled both TNNI3+ cardiomyocytes and TNNI3– noncardiomyocytes. It is known that pre-existing cardiomyocytes self-renew slowly during homeostasis. Whether noncardiomyocytes labeled by new Kit–Cre tools contain stem cells that are able to generate new cardiomyocytes remains to be examined. To distinguish putative new cardiomyocytes derived from Kit+ cardiomyocytes or Kit+ noncardiomyocytes, we first marked them with 2 distinct genetic markers based on cardiomyocyte-specific marker cardiac troponin T2 (Tnnt2). We generated a new Tnnt2–DreER knock-in line to add a tdTomato marker on pre-existing cardiomyocytes that includes Kit+ cardiomyocytes (Figure [J]). Through serial tissue sections and cardiomyocyte isolation Tnnt2–DreER;R26–rox–tdTomato mice, we confirmed that all tdTomato+ cells were indeed Tnnt2+ cardiomyocytes that express mature sarcomere in the adult heart (data not shown). By combining Kit–Cre;R26–GFP and Tnnt2–DreER;R26–rox–tdTomato lines, we could distinguish GFP+tdTomato– noncardiomyocytes from GFP+tdTomato+ cardiomyocytes in Kit+ cell population (Figure [J]). We examined whether GFP+tdTomato– noncardiomyocytes give rise to new cardiomyocytes, which would maintain as GFP+tdTomato– (Figure [J]). We induced mice with tamoxifen at 2 weeks before myocardial infarction (MI) and analyzed tissue samples at 4 weeks after MI (Figure [K]). We could readily detect GFP+tdTomato+ cardiomyocytes in the infarct and border regions (Figure [L through N]). However, no GFP+tdTomato– cardiomyocytes were detected in all tissue sections (1258 sections from 5 mouse hearts). We found that most of the Kit–2A–Cre–labeled noncardiomyocytes were endothelial cells. We confirmed the above results by Kit–IRES–Cre mouse line using same strategy (Figure [O]). The lack of GFP+tdTomato– cardiomyocytes in the infarcted myocardium demonstrated that Kit+ nonmyocytes did not contribute to any new cardiomyocyte after MI.This study combined the 2 newly generated Kit–Cre drivers and dual recombinase–mediated lineage tracing strategy to circumvent the Kit–Cre lineage tracing issues3 and then address their fates in the adult heart. Different from previous Kit–Cre tools2,4,5, the 2 new Kit–Cre drivers do not have the issue of Kit haploinsufficiency and at the same time label all Kit+ cells and their descendants. This study resolved the concerns of Kit–Cre lineage tracing issues, providing a compelling genetic evidence of absence of endogenous Kit+ CSCs for myocardial renewal and repair in the adult heart.Sources of FundingThis work was supported byNational Key Research & Development Program of China Grants 2017YFC1001303 and 2018YFA0108100; Strategic Priority Research Program of the Chinese Academy of Sciences Grants XDA16010507 and XDB19000000; National Science Foundation of China Grants 31730112, 91639302, 31625019, 91849202, 81761138040, 81872241, and 31701292; Young Elite Scientists Sponsorship Program Grant 2017QNRC001; Youth Innovation Promotion Association Grant 2060299; a Sanofi Fellowship; funds from Astrazeneca; and a Royal Society–Newton Advanced Fellowship.DisclosuresNone.FootnotesData sharing: The data that support the findings of this study and research materials, as well as experimental procedures and protocols, are available from the corresponding author upon reasonable request.Lingjuan He, PhD, 320 Yueyang Road A2110, Shanghai 200031, China. Email [email protected]ac.cnBin Zhou, MD, PhD, 320 Yueyang Road A2110, Shanghai 200031, China. Email [email protected]ac.cnYu Nie, PhD, Fuwai Hospital, Beijing, 100037, China. Email [email protected]orgReferences1. Ellison GM, Vicinanza C, Smith AJ, Aquila I, Leone A, Waring CD, Henning BJ, Stirparo GG, Papait R, Scarfò M, Agosti V, Viglietto G, Condorelli G, Indolfi C, Ottolenghi S, Torella D, Nadal-Ginard B. Adult c-kit+ cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair.Cell. 2013; 154:827–842. doi: 10.1016/j.cell.2013.07.039CrossrefMedlineGoogle Scholar2. van Berlo JH, Kanisicak O, Maillet M, Vagnozzi RJ, Karch J, Lin SC, Middleton RC, Marbán E, Molkentin JD. c-kit+ cells minimally contribute cardiomyocytes to the heart.Nature. 2014; 509:337–341. doi: 10.1038/nature13309CrossrefMedlineGoogle Scholar3. 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Jennbacken K, Wågberg F, Karlsson U, Eriksson J, Magnusson L, Chimienti M, Ricchiuto P, Bernström J, Ding M, Ross-Thriepland D, Xue Y, Peiris D, Aastrup T, Tegel H, Hober S, Sivertsson Å, Uhlén M, Strömstedt P, Davies R and Holmberg Schiavone L (2019) Phenotypic Screen with the Human Secretome Identifies FGF16 as Inducing Proliferation of iPSC-Derived Cardiac Progenitor Cells, International Journal of Molecular Sciences, 10.3390/ijms20236037, 20:23, (6037) July 9, 2019Vol 140, Issue 2 Advertisement Article InformationMetrics © 2019 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.119.039909PMID: 31283370 Originally publishedJuly 8, 2019 Keywordsc-Kit+ cellscardiomyocytesgenetic lineage tracingdual recombinasePDF download Advertisement SubjectsBasic Science ResearchCell Biology/Structural BiologyMyocardial RegenerationStem Cells