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. 2011 Jun 8;474(7353):640-4.
doi: 10.1038/nature10188.

De novo cardiomyocytes from within the activated adult heart after injury

Nicola Smart  1 , Sveva Bollini, Karina N Dubé, Joaquim M Vieira, Bin Zhou, Sean Davidson, Derek Yellon, Johannes Riegler, Anthony N Price, Mark F Lythgoe, William T Pu, Paul R Riley
Affiliations

De novo cardiomyocytes from within the activated adult heart after injury

Nicola Smart et al. Nature. .

Abstract

A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction. A therapeutic ideal--relative to cell transplantation--would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm's tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.

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Figures

Figure 1
Figure 1. Activated Wt1+ cells give rise to cardiac progenitors in the injured adult heart
a, Schematic of constitutive or pulse-chase labelling of Wt1+ cells. b–e, FACS analyses of whole hearts at day 7 (d7) after myocardial infarction (MI) revealed a significant increase in GFP+ (b, c) and YFP+ (d, e) cells following priming with Tβ4, as compared to PBS-treated controls (Co); x-axes represent either GFP (b, c) or YFP (d, e) fluorescent wavelengths on a logarithmic scale and y-axes represent total cell numbers isolated by FACs (b–e). f, g, Multi-photon imaging at day 7 after myocardial infarction revealed YFP+ cells within the epicardium and subepicardial region migrating towards underlying myocardium. Scale bars in f, 20 μm; g, 10 μm. h, Three-dimensional Imaris reconstruction of migrating YFP+ cells (green) amidst non-labelled cells (red). ep, epicardium; my, myocardium. i–l, YFP+ cells that co-stained for Isl1 (highlighted by white arrowheads in k) resided in the epicardium proximal to areas of scarred myocardium 2 days (d2) after myocardial infarction. sc, scar region. Scale bar in l (also applies to i–l), 50 μm. m, Significant increase in Isl1 expression in primed hearts at days 2, 4 and 7 after myocardial infarction relative to sham-operated controls *P ≤ 0.05, *P ≤ 0.01, ***P ≤ 0.001; MI group 1 and group 2 versus sham; myocardial infarction categories: purple, mild injury; cream, severe injury; n=6 hearts per sham and MI groups. n, Significant increases in Isl1+/YFP+ cells at days 2 (*P ≤ 0.05), 4 (**P ≤ 0.01) and 7 (***P ≤ 0.001) after myocardial infarction and Nkx2-5+/YFP+ cells by day 7 (***P ≤ 0.001) alongside phospho-histone H3+ (P-HH3+) proliferating YFP+ progenitors at day 7 (***P ≤ 0.001), compared to sham-operated controls. P values were calculated by Student’s t-test (m) and paired ANOVA (n). Error bars represent mean±s.e.m. N values are numbers of hearts analysed for each group: N=3 (m); N=4 (d2 and d4) and N=7 (d7) (n).
Figure 2
Figure 2. Activated adult Wt1+ progenitors differentiate into structurally coupled cardiomyocytes
a–c, At day 14, YFP+ cells that co-expressed cTnT resided in the left ventricular wall within the border zone (white arrowheads in c; dashed white line demarcates extent of peri-infarct region). bz, border zone, sc, scar region. d, e, YFP+ cells expressing SαA as determined by epifluorescence (d) and confocal microscopy (e; white arrowhead highlights mature YFP+ cardiomyocyte; white asterisks highlight less mature YFP+ cardiomyocytes). f, g, Mature YFP+ cardiomyocytes stained positive for cTnT with sarcomeric banding (white arrowheads). f–k, Evidence of structural coupling between YFP+ and resident YFP− cardiomyocytes through Ncad+ adherens junction (white arrowhead in i) and Cx43+ gap junction formation (white arrowhead/arrows in j, k). l, YFP+/cTnT+ cardiomyocytes were located adjacent to necrotic myocardium within the scar (white asterisks). m, n, Rhod-2 loading of distal YFP− cardiomyocytes (m) was compared against YFP+ cardiomyocytes within the peri-infarct region (n). o, p, Calcium transients across clustered YFP− and YFP+ cardiomyocytes as evidence of functional coupling. q, r, Distal YFP− spontaneous calcium transients (q) were compared against YFP+ transients (r). s, Representative traces plotted per cardiac cycle (AU, arbitrary units). All scale bars are 20 μm, except for a, 100 μm; b, 150 μm; and c, 150 μm. Scale bar in m applies to m–r.
Figure 3
Figure 3. Prospective donor Wt1+/GFP+ cells at day 4 after myocardial infarction seem to be derived from epicardium
a, b, Immunostaining for Wt1 and anti-podoplanin (PDPN) revealed that the day 4 donor Wt1+ cells were restricted to the epicardium and subepicardial space. ep, epicardium; my, myocardium; sc, scar; ses, subepicardial space. c, e, Anti-GFP and anti-cTnI co-staining revealed spatial restriction to the equivalent epicardial regions as for Wt1 staining with exclusion from the myocardium. d, cTnT, MyBPC and Actn2 were not expressed in the FACS GFP+ population at day 4 after myocardial infarction, whereas expression of all three myocardial markers was significantly upregulated in GFP+ cells at day 14 consistent with contribution of de novo cardiomyocytes. f–h, Wt1+ cells were restricted to the epicardium and excluded from MLC2v–YFP+ ventricular cardiomyocytes in regions of healthy (g) and scarred (h) myocardium. i–k, Expression of Wt1/GFP was excluded from within the Pecam+/Flk1+ coronary vasculature (i, j; cv, coronary vein; ca, coronary artery) and confirmed by a lack of expression of Pecam and Tie2 in the day 4 GFP+ FACS population (k). l, m, Cytospin/immunostaining revealed a homogeneous GFP+ fraction at day 4, without contamination from GFP− cells (l), which lacked vascular (Pecam and SMA) and myocardial (SαA, cTnT) markers (m) as compared to the GFP− fraction (n). *P ≤ 0.05, **P ≤ 0.01, all statistics by Students t-test. Error bars represent mean ± s.e.m., N values are numbers of hearts analysed for each group: N=6 (day 4) and N=7 (day 14) (d); N=6 (k). All scale bars are 50 μm, except a, e, 500 μm; f, 200 μm; m, n, 20 μm.
Figure 4
Figure 4. Transplanted donor Wt1+ progenitors differentiate into cardiomyocytes within host myocardium in the absence of cell fusion
a, Schematic of cell transplantation regimen. b, After 24 h post-transplantation GFP+ cells within the epicardium and subepicardial region at the injection site were absent in remote regions. ep, epicardium; my, myocardium. c, Transplanted GFP+ cells expressed Nkx2-5, indicative of a myocardial progenitor phenotype (white arrowhead highlights a GFP+/Nkx2-5+ progenitor; white arrows highlight GFP−/Nkx2-5+ progenitors and asterisks highlight epicardium cells negative for both GFP and Nkx2-5). d–f, Donor GFP+ cells with an intermediate differentiated phenotype (highlighted by white arrowhead, d), alongside those with evidence of sarcomeric banding that co-expressed cTnT were observed within host myocardium (e, f). sc, scar. g–j, GFP+/cTnT+ cardiomyocytes (g–i) with sarcomeric banding (highlighted by white arrowheads in h) were traced for FISH analyses to reveal a single XY karyotype (j). X, X chromosome; Y, Y chromosome. k–o, Reciprocal transplantation (XX into XY) followed by confocal microscopy (k) revealed GFP+ cardiomyocytes (l) that had the donor XX karyotype (m) relative to the XY karyotype of host GFP− cardiomyocytes (highlighted by white arrowhead; n, o). p–s, In female Wt1CreERT2/+;R26REYFP/+ mice, previously tracked YFP+ cTnT+ (p) and SαA+ cardiomyocytes (r and examples shown in Fig. 3), had an XX karyotype (white box insets) supporting transdifferentiation in the absence of cell fusion with resident cardiomyocytes (q, s). Scale bars: c, 25μm; d–f, 20 μm; g (applies to g–j), k (applies to k–o), p (applies to p–q), r (applies to r–s), 10 μm.
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References

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