Skip to main content
SpringerLink
Log in

Cardiac cell proliferation assessed by EdU, a novel analysis of cardiac regeneration

  • Original Research
  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

Emerging evidence suggests that mammalian hearts maintain the capacity for cardiac regeneration. Rapid and sensitive identification of cardiac cellular proliferation is prerequisite for understanding the underlying mechanisms and strategies of cardiac regeneration. The following immunologically related markers of cardiac cells were analyzed: cardiac transcription factors Nkx2.5 and Gata 4; specific marker of cardiomyocytes TnT; endothelial cell marker CD31; vascular smooth muscle marker smooth muscle myosin IgG; cardiac resident stem cells markers IsL1, Tbx18, and Wt1. Markers were co-localized in cardiac tissues of embryonic, neonatal, adult, and pathological samples by 5-ethynyl-2′-deoxyuridine (EdU) staining. EdU was also used to label isolated neonatal cardiomyocytes in vitro. EdU robustly labeled proliferating cells in vitro and in vivo, co-immunostaining with different cardiac cells markers. EdU can rapidly and sensitively label proliferating cardiac cells in developmental and pathological states. Cardiac cell proliferation assessed by EdU is a novel analytical tool for investigating the mechanism and strategies of cardiac regeneration in response to injury.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Anversa P, Kajstura J (1998) Ventricular myocytes are not terminally differentiated in the adult mammalian heart. Circ Res 13:1–14

    Article  Google Scholar 

  • Beltrami AP, Urbanek K, Kajstura J, Yan SM, Finato N, Bussani R, Nadal-Ginard B, Silvestri F, Leri A, Beltrami CA, Anversa P (2001) Evidence that human cardiac myocytes divided after myocardial infarction. N Eng J Med 344:1750–1757

    Article  CAS  Google Scholar 

  • Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabé-Heider F, Walsh S, Zupicich J, Alkass K, Buchholz BA, Druid H, Jovinge S, Frisén J (2009) Evidence for cardiomyocyte renewal in humans. Science 324:98–102

    Article  CAS  Google Scholar 

  • Buck SB, Bradford J, Gee KR, Agnew BJ, Clarke ST, Salic A (2008) Detection of S-phase cell cycle progression using 5-ethynyl-2′-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2′-deoxyuridine antibodies. Biotechniques 44:927–929

    Article  CAS  Google Scholar 

  • Cappella P, Gasparri F, Pulici M, Moll J (2008) A novel method based on click chemistry, which overcomes limitations of cell cycle analysis byclassical determination of BrdU incorporation, allowing multiplex antibody staining. Cytometry A 73:626–636

    Article  Google Scholar 

  • Chehrehasa F, Meedeniya AC, Dwyer P, Abrahamsen G, Mackay-Sim A (2009) EdU, a new thymidine analogue for labelling proliferating cells in the nervous system. J Neurosci Methods 15:122–130

    Article  Google Scholar 

  • Chen K, Bai H, Arzigian M, Gao YX, Bao J, Wu WS, Shen WF, Wu L, Wang ZZ (2010) Endothelial cells regulate cardiomyocyte development from embryonic stem cells. J Cell Biochem 111:29–39

    Article  CAS  Google Scholar 

  • Dodou E, Verzi MP, Anderson JP, Xu SM, Black BL (2004) Mef2c is a direct transcriptional target of ISL1 and GATA factors in the anterior heart field during mouse embryonic development. Development 131:3931–3942

    Article  CAS  Google Scholar 

  • Formigli L, Francini F, Nistri S, Margheri M, Luciani G, Naro F, Silvertown JD, Orlandini SZ, Meacci E, Bani D (2009) Skeletal myoblasts overexpressing relaxin improve differentiation and communication of primary murine cardiomyocyte cell cultures. J Mol Cell Cardiol 47:335–345

    Article  CAS  Google Scholar 

  • Guner-Ataman B, Paffett-Lugassy N, Adams MS, Nevis KR, Jahangiri L, Obregon P, Kikuchi K, Poss KD, Burns CE, Burns CG (2013) Zebrafish second heart field development relies on progenitor specification in anterior lateral plate mesoderm and nkx2.5 function. Development 140:1353–1363

    Article  CAS  Google Scholar 

  • Harvey RP (1996) NK-2 homeobox genes and heart development. Dev Biol 178:203–216

    Article  CAS  Google Scholar 

  • Hsieh PC, Segers VF, Davis ME, MacGillivray C, Gannon J, Molkentin JD, Robbins J, Lee RT (2007) Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med 13:970–974

    Article  CAS  Google Scholar 

  • Hsu TL, Hanson SR, Kishikawa K, Wang SK, Sawa M, Wong CH (2007) Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells. Proc Natl Acad Sci USA 20:2614–2619

    Article  Google Scholar 

  • Jopling C, Sleep E, Raya M, Martí M, Raya A, Izpisúa Belmonte JC (2010) Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature 25:606–609

    Article  Google Scholar 

  • Kikuchi K, Holdway JE, Werdich AA, Anderson RM, Fang Y, Egnaczyk GF, Evans T, Macrae CA, Stainier DY, Poss KD (2010) Primary contribution to zebrafish heart regeneration by gata4(+) cardiomyocytes. Nature 25:601–605

    Article  Google Scholar 

  • Lagrue E, Abe H, Lavanya M, Touhami J, Bodard S, Chalon S, Battini JL, Sitbon M, Castelnau P (2010) Regional characterization of energy metabolism in the brain of normal and MPTP-intoxicated mice using new markers of glucose and phosphate transport. J Biomed Sci 17:91

    Article  CAS  Google Scholar 

  • Lepilina A, Coon AN, Kikuchi K, Holdway JE, Roberts RW, Burns CG, Poss KD (2006) A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell 127:607–619

    Article  CAS  Google Scholar 

  • Linask KK, Lash JW (1993) Early heart development: dynamics of endocardial cell sorting suggests a common origin with cardiomyocytes. Dev Dyn 196:62–69

    Article  CAS  Google Scholar 

  • Narmoneva DA, Vukmirovic R, Davis ME, Kamm RD, Lee RT (2004) Endothelial cells promote cardiac myocyte survival and spatial reorganization: implications for cardiac regeneration. Circulation 110:962–968

    Article  Google Scholar 

  • Nassiri SM, Khaki Z, Soleimani M, Ahmadi SH, Jahanzad I, Rabbani S, Sahebjam M, Ardalan FA, Fathollahi MS (2007) The similar effect of transplantation of marrow-derived mesenchymal stem cells with or without prior differentiation induction in experimental myocardial infarction. J Biomed Sci 14:745–755

    Article  Google Scholar 

  • Porrello ER, Mahmoud AI, Simpson E, Hill JA, Richardson JA, Olson EN, Sadek HA (2011) Transient regenerative potential of the neonatal mouse heart. Science 25:1078–1080

    Article  Google Scholar 

  • Qu D, Wang G, Wang Z, Zhou L, Chi W, Cong S, Ren X, Liang P, Zhang B (2011) 5-Ethynyl-2′-deoxycytidine as a new agent for DNA labeling: detection of proliferating cells. Anal Biochem 417:112–121

    Article  CAS  Google Scholar 

  • Salic A, Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc Natl Acad Sci USA 19:2415–2420

    Article  Google Scholar 

  • Saravanakumar M, Devaraj H (2012) Notch signalling in cardiovasculogenesis: insight into their role in early cardiovasculardevelopment. Mol Biol Rep 40:3537–3547

    Article  Google Scholar 

  • Shu T, Zeng B, Ren X, Li Y (2010) HO-1 modified mesenchymal stem cells modulate MMPs/TIMPs system and adverse remodeling in infarcted myocardium. Tissue Cell 42:217–222

    Article  CAS  Google Scholar 

  • Studzinski GP, Harrison LE (1999) Differentiation-related changes in the cell cycle traverse. Int Rev Cytol 189:1–58

    Article  CAS  Google Scholar 

  • Wada R, Muraoka N, Inagawa K, Yamakawa H, Miyamoto K, Sadahiro T, Umei T, Kaneda R, Suzuki T, Kamiya K, Tohyama S, Yuasa S, Kokaji K, Aeba R, Yozu R, Yamagishi H, Kitamura T, Fukuda K, Ieda M (2013) Induction of human cardiomyocyte-like cells from fibroblasts by defined factors. Proc Natl Acad Sci USA 110:12667–12672

    Article  CAS  Google Scholar 

  • Yang J, Xia J, He Y, Zhao J, Zhang G (2013) MSCs transplantation with application of G-CSF reduces apoptosis or increases VEGF in rabbit model of myocardial infarction. Cytotechnology. doi:10.1007/s10616-013-9655-2

    Google Scholar 

  • Zeng B, Chen H, Zhu C, Ren X, Lin G, Cao F (2008) Effects of combined mesenchymal stem cells and heme oxygenase-1 therapy on cardiac performance. Eur J Cardiothorac Surg 34:850–856

    Article  Google Scholar 

  • Zeng B, Lin G, Ren X, Zhang Y, Chong H (2010a) Over-expression of HO-1 on mesenchymal stem cells promotes angiogenesis and improves myocardial function in infarcted myocardium. J Biomed Sci 17:80

    Article  Google Scholar 

  • Zeng C, Pan F, Jones LA, Lim MM, Griffin EA, Sheline YI, Mintun MA, Holtzman DM, Mach RH (2010b) Evaluation of 5-ethynyl-2′-deoxyuridine staining as a sensitive and reliable method for studying cell proliferation in the adult nervous system. Brain Res 1319:21–32

    Article  CAS  Google Scholar 

  • Zeng B, Ren XF, Cao F, Zhou XY, Zhang J (2011) Developmental patterns and characteristics of epicardial cell markers Tbx18 and Wt1 in murine embryonic heart. J Biomed Sci 18:67

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Chinese National Nature Science Foundation (30900609, 81270271).

Conflict of interest

The authors do not have any possible conflicts of interest.

Author information

Authors and Affiliations

  1. Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei, People’s Republic of China

    Bin Zeng, Suiyang Tong & Hao Xia

  2. Department of Developmental and Regenerative Biology, Mount sinai school of medicine, New York, NY, 10029, USA

    Bin Zeng

  3. College of Veterinary Medicine, Northeast Agricultural University, Harbin, Hei Longjiang, People’s Republic of China

    Xiaofeng Ren

Authors
  1. Bin Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Suiyang Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiaofeng Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hao Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Bin Zeng or Hao Xia.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10616_2014_9827_MOESM1_ESM.ppt

Supplementary Fig. 1: EdU-labeled cardiac proliferating cells in adult hearts at E10.5, E17.5, and neonatal mice at 7 days (×40). The highest red fluorescence in the atrial and ventricular walls and ventricular septum (VS) in the fetus; staining was noticeably reduced after birth (PPT 1000 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zeng, B., Tong, S., Ren, X. et al. Cardiac cell proliferation assessed by EdU, a novel analysis of cardiac regeneration. Cytotechnology 68, 763–770 (2016). https://doi.org/10.1007/s10616-014-9827-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10616-014-9827-8

Keywords

Search

Navigation