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To the Heart of the Matter: De novo cardiomyocytes from within the activated adult heart after injury

From Nature
By Stuart P. Atkinson

The potential for cell replacement through the differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) is a promising means of therapeutic intervention in many disease states, but may be limited by several problems, such as limited graft survival, restricted homing to the site of injury and host immune rejection. An alternative to this is the possibility of stimulating resident adult stem cells within the tissue to aid repair. It is generally assumed that the cells of the adult epicardium (the outer layer of heart tissue) are quiescent, incapable of migration or differentiation, while cells of the embryonic epicardium possess an innate ability to proliferate, migrate and differentiate into a number of mature cardiovascular cell types. However, data now suggests that resident stem/progenitor cells in the adult heart may produce de novo cardiomyocytes following injury.

Previous studies using Wt1GFPCre/+, Wt1CreERT2/+; or Tbx18Cre mice transgenic mice have definitively shown that embryonic epicardial progenitor cells (EDPCs) contribute to the cardiomyocyte lineage (Zhou et al and Cai et al) but translating this to the potential study of adult EDPCs is very difficult due to the embryonic restriction or relative unspecificity of these markers. However, other previous studies have shown that treatment with thymosin B4 (TB4) (“priming”) leads to the activation of adult EDPCs, subsequent induction of vascular precursors for neovascularisation and re-expression of Wt1 (Smart et al (2008) and Bock-Marquette et al) hence allowing for the study of potential adult cardiomyocyte precursors. Now, a new study in Nature from the laboratory of Paul R. Riley at the Molecular Medicine Unit, UCL Institute of Child Health, London, utilises this system to identify de novo cardiomyocytes in the mouse adult heart (Smart et al (2011)) by establishing both constitutive (GFP+) and pulse (YFP+) labelling of Wt1+ progenitors.

Initial experiments with epicardial explants from Tβ4-primed Wt1GFPCre/+ adult hearts without myocardial infarction, led to the outgrowth of cells with a differentiated cardiac muscle phenotype and GFP-expression at 14 days of in vitro culture. GFP+ cells expressed Isl1 (post-natal cardioblast marker) and Nkx2-5 (early cardiomyocyte progenitor marker) while progenitor-like GFP+ cells which expressed mature markers for cardiomyocyte differentiation (cTnT, SaA and MyBPC) were also prevalent, demonstrating the presence of a progenitor cell maturing towards a cardiomyocyte fate.

In vivo analysis in the Tβ4-primed mouse heart showed that Wt1 and Tbx18 gene expression was increased at 2 days post-injury and persisted in GFP+ cells found at 4 days compared to non-primed injury only mice which only show an increase in Wt1 at 7 days. Further detailed analysis found increased Wt1 in small round progenitor-like cells within the epicardium, sub-epicardial region and underlying myocardium. Proliferative Ki67+ progenitor cells were observed in epicardial and sub-epicardial regions alongside YFP+ cells, which expanded in number from day 2–14. An epicardial subpopulation was positive for Isl1 and Nkx2-5 at day 2 post-injury and these proposed cardiac progenitors significantly increased by day 7. By day 14, BrdU+/YFP+ cells were located within the area of the injury and co-expressed SαA and cTnT and their size, gross morphology and inherent ultrastructure resembled mature cardiomyocytes. Similar to the in vitro analysis, these results suggest the presence of an activated progenitor cell giving rise to mature cardiomyocytes.

These de novo cardiomyocytes appeared appropriately integrated with the myocardium and formed connections with each other, as determined by the presence of both Ncad-adherens and Cx43-gap junctions. Further, YFP+ and YFP- cardiomyocytes showed synchronous calcium transients and very similar kinetics, suggesting appropriate integration with resident myocardium and, importantly, functionality. The study failed to locate any mature GFP+/YFP+ cardiomyocytes in the absence of injury or Tβ4 priming and further control experiments ruled out that traced cardiomyocytes were labelled by virtue of ectopic activation of the fluorophore from the Wt1 knock-in alleles and the possibility of the GFP+ progenitor population arising from existing vasculature. Further functional analysis into the effects of priming on cardiac function and myocardial regeneration, suggested a significant improvement in functional parameters, alongside beneficial changes in infarct/scar volume with increased left ventricular mass over time were recorded with Tβ4 treatment.

Overall the study suggests that myocardial infarction in the Tβ4-primed mice leads to the appearance of Wt1+ progenitor/stem cells at the site of the injury which give rise to functional mature de novo cardiomyocytes that improve cardiac function and myocardial regeneration. Both these findings represent an important step forward in the study of heart disease and the role of resident adult stem cells in injury-response.



Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart.
Zhou B, Ma Q, Rajagopal S, Wu SM, Domian I, Rivera-Feliciano J, Jiang D, von Gise A, Ikeda S, Chien KR, Pu WT.
Nature. 2008 Jul 3;454(7200):109-13.

A myocardial lineage derives from Tbx18 epicardial cells.
Cai CL, Martin JC, Sun Y, Cui L, Wang L, Ouyang K, Yang L, Bu L, Liang X, Zhang X, Stallcup WB, Denton CP, McCulloch A, Chen J, Evans SM.
Nature. 2008 Jul 3;454(7200):104-8.

De novo cardiomyocytes from within the activated adult heart after injury.
Smart N, Bollini S, Dubé KN, Vieira JM, Zhou B, Davidson S, Yellon D, Riegler J, Price AN, Lythgoe MF, Pu WT, Riley PR.
Nature. 2011 Jun 8. doi: 10.1038/nature10188.

Thymosin beta4 mediated PKC activation is essential to initiate the embryonic coronary developmental program and epicardial progenitor cell activation in adult mice in vivo.
Bock-Marquette I, Shrivastava S, Pipes GC, Thatcher JE, Blystone A, Shelton JM, Galindo CL, Melegh B, Srivastava D, Olson EN, DiMaio JM.
J Mol Cell Cardiol. 2009 May;46(5):728-38.

Thymosin beta4 induces adult epicardial progenitor mobilization and neovascularization.
Smart N, Risebro CA, Melville AA, Moses K, Schwartz RJ, Chien KR, Riley PR.
Nature. 2007 Jan 11;445(7124):177-82. Epub 2006 Nov 15.