Isolation and Culture of Cardiac Stem Cells (CSCs). CSCs were isolated from the ventricle of adult Fischer 344 rats. Clonogenic cells obtained by cell sorting and single cell cloning were infected with a retrovirus carrying EGFP (1). The viral titer was 10⁶ cfu/ml. A single clonogenic c-kit^POS cell expressing EGFP was used to develop a c-kit^POS-EGFP^POS clone, which was used in these studies.

Ischemia/Reperfusion Injury. Female Fischer 344 rats (age, 3 months; weight, 175 ± 20 g) were anesthetized with ketamine (37 mg/kg) and xylazine (5 mg/kg) and ventilated with a rodent respirator (Harvard Apparatus). Anesthesia was maintained with isoflurane inhalation and body temperature was kept at 37°C with a heating pad. After administration of antibiotics, the chest was opened and the heart exposed. All animals underwent a 90-min occlusion of the left anterior descending coronary artery followed by reperfusion, and the chest was closed. Four hours after reperfusion, rats were reanesthetized, the chest reopened, and a thin catheter (Intracath, 22G, Becton Dickinson) was advanced into the aortic root via the left ventricular (LV) apex (2). The aorta and the pulmonary artery were occluded briefly with a snare for two 20-s intervals, 10 min apart, and vehicle or CSCs were injected into the aortic root during the occlusion (2). CSCs were suspended in normal saline (1 × 10⁶ CSCs diluted in 1 ml). In sham-operated rats, the chest was opened but no injection was done. Rats were killed 5 weeks later for histological studies; the number of rats was n = 17 in the untreated group, n = 24 in the treated group, and n = 12 in the sham-operated group.

Locomotion of CSCs. After the coronary occlusion/reperfusion protocol and the injection of EGFP^POS-clonogenic CSCs 4 h after reperfusion, the heart was excised, perfused retrogradely through the aorta with Tyrode solution containing 30 mM KCl, and placed in a bath mounted on top of the microscope stage of a two-photon microscope (Bio-Rad Radiance 2100MP). The heart was continuously perfused and superfused at 37°C with oxygenated Tyrode solution containing rhodamine-labeled dextran. The two-photon microscope was positioned to view the left ventricular free wall. EGFP and rhodamine were excited at 960 and 840 nm, respectively, with mode-locked Ti:Sapphire femtosecond laser (Tsunami, Spectra-Physics); 4D image stacks were acquired at emission wavelengths of 515 and 600 nm, respectively. Hearts were studied at three intervals (20 min, 8 h, and 12 h after the injection of cells); at each interval, three to five hearts were used.

Echocardiography and Hemodynamics. Serial echocardiograms were obtained at baseline (2 days before coronary occlusion) and 2 and 35 days after reperfusion by using an HDI 5000 echocardiography machine (Philips Medical Systems, Besl, The Netherlands) equipped with 15-7 MHz linear broadband and 12-5 MHz phased array transducers. Before echocardiography, rats were weighed and anesthetized with i.p. injection of pentobarbital (25 mg/kg). The anterior chest was shaved, and rats were placed in the left lateral decubitus position. A rectal temperature probe was placed, and the body temperature was carefully maintained between 37.0°C and 37.5°C with a heating pad throughout the study. The parasternal long-axis, parasternal short-axis, and apical four-chamber views were used to obtain 2D, M-mode, and spectral Doppler images (3-5). Systolic and diastolic anatomic parameters were obtained from M-mode tracings at the midpapillary level. The ejection fraction (EF) was calculated by the area-length method. The stroke volume (SV) was calculated from the aortic area and the time-velocity integral of the aortic flow. The mean velocity of circumferential fiber shortening (Vcf) was determined from the LV fractional shortening (LVFS) and the rate-corrected ejection time (ET) by the following formula: Vcf = LVFS/ET (6).

The hemodynamic studies were performed at 35 days after surgery, just before death. Rats were anesthetized with pentobarbital and ventilated. A 2-French catheter (Millar Instruments, Houston) connected to a chart recorder was introduced via the apex into the LV for the evaluation of pressures in the open-chest preparation.

Morphometry and Histology. After the hemodynamic measurements, the thorax was opened, the abdominal aorta was cannulated, the heart was arrested in diastole with 0.15 ml of CdCl₂ (100 mM), and the myocardium was perfused retrogradely through the aorta with 10% buffered formalin. The right atrium was cut to allow drainage. The perfusion pressure was adjusted to match the mean arterial pressure. The LV chamber was filled with fixative from a pressure reservoir set at height equivalent to the in vivo measured LV end-diastolic pressure (LVEDP) (3). After measuring the major longitudinal intracavitary axis, the LV was sectioned serially into five rings perpendicular to the longitudinal axis of the heart, and the thickness of the free wall, infarcted region, and septum as well as the transverse LV chamber diameter were determined by an image analyzer. The minimal and maximal luminal diameters at midregion were used with the longitudinal axis to compute chamber volume (7). Measurements of wall thickness, chamber radius, and LVEDP were used to calculate diastolic wall stress at each site examined. Slices were then embedded in paraffin, and infarct size was determined as the number of myocytes lost from the LV (7). An average sarcomere length of 2.1 m m was used in all cases to correct the raw measurements of LV anatomical parameters (7). The anatomical data were obtained in 17 untreated and 18 treated infarcted rats.

Immunohistochemistry was performed in formalin-fixed 4-m m-thick histological sections. Primitive cells were identified by the c-kit antibody; myocytes were recognized with GATA-4, MEF2C, nestin, desmin, cardiac myosin heavy chain, a -sarcomeric actin, N-cadherin, and connexin 43 antibodies. Endothelial cells were recognized with Ets-1 and von Willebrand factor antibodies and smooth muscle cells with GATA-6 and a -smooth muscle actin antibodies. Scar tissue was detected with a mixture of collagen type III and type I antibodies. Cycling cells were detected with BrdUrd, Ki67, and MCM5 antibodies. Colocalization of cell-specific markers with EGFP was used to identify cells that originated from CSCs. Nuclei were identified with propidium iodide (PI) (1, 8, 9). All of the primary and secondary antibodies used in these studies are specified in Table 2. The quantitative analysis was performed in 10 treated rats. In each animal, for each parameter examined, the sampling area ranged between 3 and 17 mm² of regenerating myocardium.

Cell Volume of EGFP^POS and EGFP^NEG Myocytes. LV myocytes were enzymatically dissociated from CSC-treated and untreated hearts and subsequently fixed with 4% paraformaldehyde. Cells were then stained in suspension, and the average volumes of new small and large EGFP^POS myocytes and EGFP^NEG myocytes were measured by 3D optical sectioning by confocal microscopy (5, 7). The total number of myocytes measured was 1,200 in sham-operated rats, 1,400 in infarcted untreated rats and 1,300 in infarcted treated rats. In addition, 600 EGFP^POS myocytes were measured in infarcted treated rats.

Calculation of the Infarct Size and the Extent of Regeneration by the Number of Cardiomyocytes Lost and Regenerated. To obtain this information, we determined the volume of the LV in treated and untreated infarcted rats and in sham-operated controls by dividing LV weight by the specific gravity of muscle tissue, 1.06 mg/ml (7). These values and the measurements of the volume fraction of scarred and viable myocardium in the LV were used to calculate the total volume of infarcted and noninfarcted myocardium in the LV (Fig. 6). Subsequently, the total volume of myocytes in the ventricle was obtained by multiplying the noninfarcted LV volume by the volume fraction of myocytes in the myocardium. The quotient between the total myocyte volume and the myocyte cell volume allowed the computation of the total number of LV myocytes in each group of animals.

The comparison between the number of myocytes present in the LV of sham-operated animals and the number of myocytes found in the LV of untreated or CSC-treated infarcted animals enabled us to calculate the percentage of myocytes lost and remaining in each of the two infarcted animal groups. The fraction of myocytes lost was 27% (-6.0 × 10⁶ myocytes) and 33% (-7.2 × 10⁶ myocytes) in treated and untreated infarcted hearts, respectively (Fig. 6). In the treated group, this value reflects the size of the original infarct because the contribution of EGFP^POS myocytes was not included in these measurements (this was done to assess infarct size independently of the myocyte regeneration associated with the injection of CSCs). These determinations were performed in 17 infarcted untreated, 18 infarcted treated, and 12 sham-operated rats.

Nuclear DNA Content in New Myocytes and in Situ Hybridization. Myocytes and lymphocytes were stained with Ki67 to measure PI fluorescence intensity by confocal microscopy in noncycling and cycling EGFP^POS myocyte nuclei. These values were compared with the PI intensity of noncycling and cycling lymphocyte nuclei from the peripheral blood. Together, 120 nuclei of EGFP^POS myocytes and 150 lymphocyte nuclei were analyzed. For the detection of chromosome 12, sections were exposed to a denaturating solution containing 70% formamide. After dehydration with ethanol, sections were hybridized with Rat Chromosome 12/Y Paint probe (Cambio) for 3 h (8, 10). Nuclei were stained with PI. In each animal, an average of 10 mm² of regenerating tissue was analyzed by confocal microscopy.

Western Immunoblotting. EGFP^POS/c-kit^POS CSCs were obtained from four different clones and lysed with RIPA buffer. The equivalents of 60 m g of protein were separated by electrophoresis on a 10% SDS/PAGE. Proteins were transferred onto nitrocellulose filters and exposed to a rabbit polyclonal anti-CXCR4 antibody (Santa Cruz Biotechnology). After repeated washing, blots were incubated with a specific secondary antibody. The CXCR4 receptor was detected as a 43-kDa band. Equal loading was determined by the expression of b -tubulin (mouse monoclonal antitubulin, Santa Cruz Biotechnology).The HL-60 and A-10 whole cell lysates (Santa Cruz Biotechnology) were used as controls with high and low expression of CXCR4, respectively.

Statistical Analysis. Data are reported as means ± SEM. Morphometric, histological, echocardiographic, and hemodynamic data were analyzed with unpaired or paired Student’s t tests, as appropriate (11). Comparisons involving three groups (sham-operated, untreated infarcted, and treated infarcted) were performed by one- or two-way (time and group) ANOVA as appropriate followed by Student’s t tests with the Bonferroni correction (11).

1. Beltrami, A. P., Barlucchi, L., Torella, D., Baker, M., Limana, F., Chimenti, S., Kasahara, H., Rota, M., Musso, E., Urbanek, K., et al.. (2003) Cell 114, 763-776.

2. Hajjar, R. J., Schmidt, U., Matsui, T., Guerrero, J. L., Lee, K. H., Gwathmey, J. K., Dec, G. W., Semigran, M. J. & Rosenzweig, A. (1998) Proc. Natl. Acad. Sci. USA 95, 5251-5256.

3. Orlic, D., Kajstura, J., Chimenti, S., Limana, F., Jakoniuk, I., Quaini, F., Nadal-Ginard, B., Bodine, D. M., Leri, A. & Anversa, P. (2001) Proc. Natl. Acad. Sci. USA 98, 10344-10349.

4. Hoit, B. D. (2001) J. Mol. Cell Cardiol. 33, 27-35.

5. Limana, F., Urbanek, K., Chimenti, S., Quaini, F., Leri, A., Kajstura, J., Nadal-Ginard, B., Izumo, S. & Anversa, P. (2002) Proc. Natl. Acad. Sci. USA 99, 6257-6262.

6. Carabello, B. A., Zile, M. R., Tanaka, R. & Cooper, G., 4th (1992) Am. J. Physiol. 263, H1137-H1144.

7. Anversa, P. & Olivetti, G. (2002) in The Heart, eds. Page, E., Fozzard, H. A. & Solaro, R. J. (Oxford Univ. Press, New York), Vol. I, pp. 75-144.

8. Quaini, F., Urbanek, K., Beltrami, A. P., Finato, N., Beltrami, C. A., Nadal-Ginard, B., Kajstura, J., Leri, A. & Anversa, P. (2002) N. Engl. J. Med. 346, 5-15.

9. Urbanek, K., Quaini, F., Tasca, G., Torella, D., Castaldo, C., Nadal-Ginard, B., Leri, A., Kajstura, J., Quaini, E. & Anversa, P. (2003) Proc. Natl. Acad. Sci. USA 101, 10440-10445.

10. Orlic, D., Kajstura, J., Chimenti, S., Jakoniuk, I., Anderson, S. M., Li, B., Pickel, J., McKay, R., Nadal-Ginard, B., Bodine, D. M., et al.. (2001) Nature 410, 701-705.

11. Wallenstein, S., Zucker, C. L. & Fleiss, J. L. (1980) Circ. Res. 47, 1-9.