Stem cells in the dog heart are self-renewing, clonogenic, and multipotent and regenerate infarcted myocardium, improving cardiac function

Linke et al. 10.1073/pnas.0502678102.

Supporting Information

Files in this Data Supplement:

Fig. 7. Detection of Sca-1-like protein in cardiac progenitor cells. Sca-1-like protein was identified by immunoprecipitation and Western blot of progenitor cells sorted for c-kit, MDR1, and Sca-1-like epitopes. Samples of myocardium from the mouse, rat, and human hearts were used for comparison. An Sca-1-like band was detected at 18.4 kDa. The mouse thymus (Th), bone marrow (BM), and kidney (K) were used as positive controls.

Fig. 8. FACS analysis of cardiac progenitor cells. The separation of c-kit (A and B), Sca-1-like (C and D), and c-kit-MDR1-Sca-1-like (E--H) positive cardiac progenitor cells is shown in these bivariate distribution plots.

Fig. 9. Identification and characterization of cardiac progenitor cells. (A) Isolated and sorted cardiac progenitor cells express c-kit (green), MDR1 (magenta), and Sca-1-like (yellow) alone or together (white). GATA-4 (green), MEF2C (magenta), Ets-1 (yellow), and GATA-6 (white) are seen in some of their nuclei (blue). (B) The distribution of cardiac progenitor cells with different epitopes is illustrated as mean ± SD. (C) Individual progenitor cells were sorted to place single sorted cells one in each well of a 96-well Terasaki plate. (Scale bars, 10 mm.)

Fig. 10. Progenitor cells and clone formation. A mixture of undifferentiated and committed cells positive for GATA-4, MEF2C, or Ets-1 is present in these two clones. (Scale bars, 10 mm.)

Fig. 11. Cardiac cell lineages generated by the expansion of different clones. The relative proportion of myocytes, SMCs, and ECs derived from these clones is shown as mean ± SD.

Fig. 12. Number of cells generated by expansion of clones formed by single cells with distinct stem cell surface antigens. Values are mean ± SD. *, significant difference from expanded c-kit clones; **, significant difference from expanded MDR1 clones.

Fig. 13. Effects of HGF, IGF-1, and other growth factors on the migration and invasive properties of cardiac progenitor cells. The greater impact of HGF on the migration and invasion capacity of cardiac progenitor cells is apparent. The role of HGF on these assays is followed by SCF. Inhibitors of MMP-2 and MMP-9 markedly attenuate the invasive ability of progenitor cells in the presence of HGF and SCF. Values are mean ± SD.

Fig. 14. Effects of HGF and IGF-1 on the growth and death of cardiac progenitor cells. HGF and IGF-1 attenuate H₂O₂-induced apoptosis (A) and enhance proliferation (B) of cardiac progenitor cells in SFM. Values are mean ± SD. *, significant difference from SFM; **, significant difference from HGF.

Fig. 15. Cardiac progenitor cells form HGF and IGF-1. Cardiac progenitor cells synthesize and secrete HGF and IGF-1 in response to stimulation by each growth factor: HGF (A) and IGF-1 (B). Values are mean ± SD. *, significant difference from SFM.

Fig. 16. Number of progenitor cells in the myocardium. Values are mean ± SD. *, significant difference from control myocardium (SO); **, significant difference from nontreated infarcts. Inf, infarcted region; Bor, border zone; Rem, remote myocardium.

Fig. 17. Number of replicating progenitor cells in the myocardium. Values are mean ± SD. *, significant difference from control myocardium (SO); **, significant difference from nontreated infarcts. Inf, infarcted region; Bor, border zone; Rem, distant myocardium.

Fig. 18. Number of dying progenitor cells in the myocardium. Values are mean ± SD. *, significant difference from control myocardium (SO); **, significant difference from nontreated infarcts. Inf, infarcted region; Bor, border zone; Rem, distant myocardium.

Fig. 19. Effects of growth factors on regional and global ventricular function. Negative values of fractional shortening reflect paradoxical motion. Values are mean ± SD. *, significant difference from 2 days; **, significant difference from nontreated infarcts.

Fig. 20. Growth factors induce an improvement of regional cardiac performance. Myocardial contraction measured by sonomicrometer crystals. (Left) Baseline conditions before coronary artery occlusion. (Center) Recordings at 2 days after infarction. (Right) Recordings at 28 days after infarction. A--C represent hypokinetic infarcted segments (Bottom Center in each composite) from GF-treated hearts; in these cases contractile function returned to baseline values (Bottom Right in each composite). In all examples, end diastolic segment length decreases from 2 to 28 days because of the shrinkage of the necrotic myocardium and scar formation. LVP, left ventricular pressure; SL, segment length.

Fig. 21. Expression of specific proteins in newly formed myocytes within the infarcted myocardium. (A--F) Developing myocytes are positive for a-sarcomeric actin (A, E, and F), cardiac myosin heavy chain (B), troponin I (C), and a-actinin (D). The maturing myocytes express connexin 43 (E) and N-cadherin (F). Myocyte nuclei are positive for BrdUrd (A and C, white dots) and MEF2C (B and D, green dots). (Scale bars in A--F, 10 mm.) (G) Size distribution of newly formed myocytes.

Fig. 22. Growth factors improve ventricular remodeling after infarction. BrdUrd labeling of myocyte nuclei (white, arrowheads) in the viable myocardium of the border zone (A and C) and distant portion of the left ventricular wall (B and D) in GF-treated (A and B) and nontreated (C and D) infarcted hearts. (Scale bars, 10 mm.)

Fig. 23. Myocyte replication in the surviving myocardium. Values are mean ± SD. *, significant difference from nontreated infarcts.

Fig. 24. Myocyte hypertrophy in the surviving myocardium. (A--C) Myocyte cross-sectional area is increased more in the border zone of nontreated (B) than in GF-treated (C) infarcted hearts. (A) Control myocardium. Myocyte surface is defined by laminin (green). (Scale bars, 10 mm.) (D) Values are mean ± SD. *, significant difference from control myocardium; **, significant difference from nontreated infarcts.

Fig. 25. Ventricular arrhythmias. Number of dissociated ventricular beats over time is shown. The incidence of rhythm disturbances was lower in treated infarcted dogs (*).

Table 1. List of markers employed for the identification of lineage-negative CSCs and committed cells

CSC, cardiac stem cell; VSMCs, vascular smooth muscle cells; ECs, endothelial cells; HSC, hematopoietic stem cell. Direct immunolabeling, primary antibody conjugated with fluorochrome; indirect immunolabeling, secondary antibody conjugated with fluorochrome.

• Early Edition
• Archives
• Online Submission

• Feature Articles

  (Expanded research articles of exceptional breadth)

• Commentaries

  (Expert commentary on leading research)

• Letters

  (Online-only letters to the editor)

• Inaugural Articles

  (Articles by recently elected NAS members)

• PNAS Profiles

  (Biographical profiles of Academy members)

• Sustainability Science

  (Interactions of human and environmental systems)

• Collected Papers

  (Editorials, Perspectives, Reviews, Colloquia, etc.)

• Special Features

  (Solicited articles on exceptional research topics)

• Sackler Colloquia

  (Scientific reports held under NAS auspices)