Improved cardiac function in infarcted mice after treatment with pluripotent embryonic stem cells

Abstract

Because pluripotent embryonic stem cells (ESCs) are able to differentiate into any tissue, they are attractive agents for tissue regeneration. Although improvement of cardiac function has been observed after transplantation of pluripotent ESCs, the extent to which these effects reflect ESC-mediated remuscularization, revascularization, or paracrine mechanisms is unknown. Moreover, because ESCs may generate teratomas, the ability to predict the outcome of cellular differentiation, especially when transplanting pluripotent ESCs, is essential; conversely, a requirement to use predifferentiated ESCs would limit their application to highly characterized subsets that are available in limited numbers. In the experiments reported here, we transplanted low numbers of two murine ESC lines, respectively engineered to express a beta-galactosidase gene from either a constitutive (elongation factor) or a cardiac-specific (alpha-myosin heavy chain) promoter, into infarcted mouse myocardium. Although ESC-derived tumors formed within the pericardial space in 21% of injected hearts, lacZ histochemistry revealed that engraftment of ESC was restricted to the ischemic myocardium. Echocardiographic monitoring of ESC-injected hearts that did not form tumors revealed functional improvements by 4 weeks postinfarction, including significant increases in ejection fraction, circumferential fiber shortening velocity, and peak mitral blood flow velocity. These experiments indicate that the infarcted myocardial environment can support engraftment and cardiomyogenic differentiation of pluripotent ESCs, concomitant with partial functional recovery.

Figure 1. Constitutive expression of EF-lacZ, and cardiac-specific expression of α-MHC-lacZ, enables respective identification of donor cells and cardiomyocytes

(A) Scheme of transgene insertion into the HPRT locus of F3-ESCs, enabling constitutive expression of lacZ from the elongation factor (EF) promoter or cardiac-specific expression from the α-MHC promoter. F3-ESCs have a Neo gene replacing the native HPRT promoter (P) and Exon 1. The HPRT targeting vector, pMP8NEB-lacZ, removes the Neo gene and reconstitutes the HPRT promoter (P) and exon 1, while introducing the transgene. (B) Transgenic ESCs stained with X-gal, showing expression of lacZ in cultured EF-lacZ cells and (C) in the hearts of embryos derived solely from α-MHC-lacZ cells via tetraploid aggregation. (D) To induce infarction, ligatures were placed around the left anterior descending (LAD) and first branch of the circumflex (LCX) arteries. EF-lacZ or α-MHC-lacZ cells were transplanted 60 minutes later, at the inferior border of the infarction site (red circle). (D) ECG changes were noted in all animals after placing the two ligatures; after 60 minutes, a Q-wave and blanching of the infarcted myocardium was observed. (E) Photograph showing that this protocol produced infarction of the anterolateral free wall of the left ventricle, resulting in significant thinning of the myocardial wall between the ligatures.

Figure 2. Allogeneic ESCs survive after transplantation into acutely infarcted myocardium

(A) After 9 weeks, whole hearts stained with X-gal revealed transplanted EF-lacZ cells throughout the infarcted tissue, boundaries of which are denoted by the broken line; red circle = site of injection. (B) Top Panels: whole mount of X-gal stained heart showing the presence of EF-lacZ donor cells in infarcted area and the absence of staining in control hearts; bar = 750 µm. Middle Panels: Cross-sections of the X-gal stained hearts shown in the top panel, showing homogeneous distribution of EF-lacZ cells; bar = 200 µm. Bottom Panels: Comparison of collagen deposition, assessed by Masson’s Trichrome staining, between hearts injected with EF-lacZ cells or with medium-only (controls); bar = 200 µm. Note: β-gal+ cells were present only in the scar tissue observed in the anterior/lateral free wall. (C) Heart injected with EF-lacZ cells containing a β-gal+ tumor located between epicardium and pericardium. This mass, which is magnified in the lower part of panel C, was not incorporated into the myocardium. The blue color indicates the origin of cells in this tumor from the EF-lacZ cells; however, whether they are cardiomyocytes cannot be discerned. Bar = 500 µm. (D) Cross-section of X-gal reacted whole mount heart that had been infarcted and injected with α-MHC-lacZ cells; the blue color denotes de novo cardiogenesis. Bar = 200 µm.

Figure 3. Echocardiography demonstrates significant functional improvements in animals transplanted with EF-lacZ or α-MHC-lacZ cells

Panels A and B are representative M-mode echocardiographic tracings of parasternal short axis views of (A) infarcted hearts injected with medium only, which exhibit a significantly thin, akinetic anterior LV wall and abnormal ventricular diameters during diastole and systole. Yellow arrow = luminal edge of anterior wall; red arrow = luminal edge of posterior wall; purple bar = distance between anterior wall and posterior wall during systole. (B) Infarcted hearts injected with EF-lacZ cells, which displayed improved anterior wall motion and thickness, as well as normalized ventricular diameters. (C) LEFT: Ejection fractions of non-infarcted sham mice, compared to medium controls and α-MHC-lacZ-injected hearts 4 and 8 weeks after infarction/transplantation, indicating significant improvement after ESC treatment. RIGHT: The PeakE/PeakA ratio, which assesses diastolic function/ventricular filling, shows complete normalization 4 weeks after ESC transplantation. Error bars in panel C indicate the standard error of the mean (SEM). Panel D shows the extent of improvement in selected functional parameters conferred by either ESC line: LVDs, fractional shortening, and shortening velocity; by contrast, neither ESC line improved PWd or LVDd. The P-values of <0.05 above each pair of bars denote the probability that the extent of improvement is different from medium-injected controls.