Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure -- Urbanek et al. 102 (24): 8692 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Urbanek et al. 10.1073/pnas.0500169102.

Supporting Information

Files in this Data Supplement:

Supporting Figure 7
Supporting Table 1
Supporting Figure 8
Supporting Table 2
Supporting Materials and Methods
Supporting Figure 9
Supporting Figure 10
Supporting Figure 11
Supporting Figure 12
Supporting Figure 13
Supporting Figure 14
Supporting Figure 15
Supporting Figure 16
Supporting Figure 17
Supporting Figure 18

Supporting Figure 7

Fig. 7. Infarct size and sampling procedures. See Materials and Methods for details.

Supporting Figure 8 Fig. 8.
Myocardial infarction and CSCs. (A–C) Three clusters of c-kit-positive CSCs (green, arrows), located in proximity of acute infarcts, are shown. The infarcted myocardium (MI) is apparent in B. Myocytes are labeled by a-sarcomeric actin (red) and nuclei by DAPI (blue). (Scale bars: 10 mm.)

Supporting Figure 9 Fig. 9.
Sca-1-like protein. (A) The identification of Sca-1-like protein in the rat, dog, and human heart (H), human cardiac endothelial cells (EC), human peripheral blood cells (PB), human bone marrow (BM), and HUVEC is shown. Mouse heart (H), kidney (K), and spleen (S) were used as positive controls. (B) The detection of Sca-1-like positive cells (yellow; arrows) in the human bone marrow is illustrated. Nuclei are stained by propidium iodide (blue). (Scale bars: 10 mm.)

Supporting Figure 10 Fig. 10.
Optical density of biochemical parameters. (A) The optical density (OD) is shown for telomerase (TERT), TERT activity, Akt, phospho-Akt, and Akt-activity on human TERT substrate. (B) The OD of TRF-1, TRF-2, DNA-PK_cs, Ku86, Ku70, and full-length and cleaved poly-ADP ribose polymerase (PARP) is shown. (C) The OD of p53, phospho-p53, p21^Cip1, p27^Kip1, p14^ARF, p16^INK4a, and Bmi-1 is shown. Results are indicated as mean ± SD. *, difference (P < 0.05) from controls (C).

Supporting Figure 11 Fig. 11.
CSC senescence. Calculated are the fraction of CSCs expressing p16^INK4a. *, difference (P < 0.05) vs. controls (C); †,difference between acute MI and chronic MI.

Supporting Figure 12 Fig. 12.
CSCs and telomere length. Telomere length in CSCs is shifted to the left to shorter telomeres in chronic MI. The filled portion of the bars corresponds to p16^INK4a and/or p53-positive CSCs; they increase in acute and chronic infarcts but more dramatically in chronic infarcts.

Supporting Figure 13 Fig. 13.
CSC death. Calculated are the fraction of CSCs undergoing apoptosis. *, difference (P < 0.05) vs. controls (C); **, difference between the border and remote myocardium in acute MI. †, difference between acute MI and chronic MI.

Supporting Figure 14 Fig. 14.
CSC in mitosis. (A) Example of a c-kit-positive CSC (green) in metaphase labeled by Ki67 (yellow) is shown at higher magnification in B–G. Metaphase chromosomes are illustrated at two optical levels of sectioning (B and C). The corresponding localization of Ki67 (yellow) on the surface of the chromosomes is depicted in D and E. The merge of metaphase chromosomes, Ki67 and c-kit, is documented in F and G. Myocytes are labeled by a -sarcomeric actin (red) and nuclei by DAPI (blue). (Scale bars: 10 m m.)

Supporting Figure 15 Fig. 15.
Lineage commitment of CSCs. (A and B) A single SMC precursor positive for c-kit (green), GATA-6 in nuclei (white dots), and a -smooth muscle actin (red in B) is shown. (C–F) An EC progenitor (C and D) and an EC precursor (E and F) are illustrated. Both cells express c-kit (green), Ets1 (yellow), and flk1 (magenta in F). A–D, acute infarcts; E and F, chronic infarcts. (Scale bars: 10 m m.)

Supporting Figure 16 Fig. 16.
Newly formed coronary arterioles. (A) A small forming arteriole with SMCs (a -smooth muscle actin, red) that contains red blood cells (yellow) is shown. Dispersed individual SMCs (red, arrows) are also seen. GATA-6 (white dots) is present in SMC nuclei. (B) Several newly formed arterioles expressing a-smooth muscle actin (red) and von Willebrand factor (green) are illustrated. Dispersed individual ECs (green, arrows) are also seen. Nuclei are labeled by DAPI (blue). (Scale bars: 10 mm.)

Supporting Figure 17 Fig. 17.
Vascular progenitors and precursors. *, difference (P < 0.05) vs. controls (C); **, difference between the border and remote myocardium in acute MI. †, difference between acute and chronic MI.

Supporting Figure 18 Fig. 18.
Myocyte regeneration within the infarcted myocardium. (A and B) Small developing myocytes within the infarct are positive for telomerase (magenta in A), MCM5 (white in A) and MEF2C (yellow in B) in the nuclei, and connexin 43 (green, arrowheads in B) in the plasmamembrane. (Scale bars: 10 mm.)

Table 1. Patient population

	No. of patients	Age, years	Sex (male/ female)	Duration of the disease	Infarct size, %
Controls	12	60 ± 20	5/6	—	—
Acute myocardial infarction	20	62 ± 13	13/7	6 ± 3 days	34 ± 6
End-stage postinfarction ischemic cardiomyopathy	20	56 ± 7	15/5	69 ± 45 months	23 ± 7

4, 6, and 6 control, acute, and chronic samples, respectively, used in previous studies were included (1, 2). Control hearts and hearts with acute infarcts were collected 10–20 hours after death. Samples from the heart of patients who underwent cardiac transplantation were processed immediately after cardiectomy. Specimens were fixed in formalin or frozen in liquid nitrogen.

1. Kajstura, J., Leri, A., Finato, N., Di Loreto, C., Beltrami, C. A. & Anversa, P. (1998) Proc. Natl. Acad. Sci. USA 95, 8801–8805.

2. Beltrami, A. P., Urbanek, K., Kajstura, J., Yan, S. M., Finato, N., Bussani, R., Nadal-Ginard, B., Silvestri, F., Leri, A., Beltrami, C. A., et al.. (2001) N. Engl. J. Med. 344, 1750–1757.

Table 2. Cell markers, function, and antibody labeling

Cell markers	Function	Antibody labeling
1. CSC epitopes
c-kit	Stem cell factor receptor	Conjugated primary Ab
MDR1	ABC cassette transporter	Conjugated primary Ab
Sca-1	Adhesion molecule	Conjugated primary Ab
2. Transcription factors of cardiac cell lineages
GATA-4	Differentiation of cardiac cells	Conjugated primary Ab
MEF2C	Differentiation of cardiomyocytes	Conjugated primary Ab
GATA-5	Regulator of Nkx2.5	Conjugated primary Ab
GATA-6	Differentiation of VSMCs	Conjugated primary Ab
Ets1	Differentiation of ECs	Conjugated primary Ab
Erg1	Differentiation of ECs	Conjugated primary Ab
3. Structural proteins of cardiac cell lineages
Nestin	Intermediate filament of immature cells	Primary/secondary Ab
Desmin	Intermediate filament of muscle cells	Primary/secondary Ab
Α -sarcomeric actin	Contractile protein of cardiomyocytes	Primary/secondary Ab
Cardiac myosin	Contractile protein of cardiomyocytes	Primary/secondary Ab
Connexin 43	Electrical coupling	Primary/secondary Ab
N-cadherin	Mechanical coupling	Primary/secondary Ab
Α -SM actin	Contractile protein of VSMCs	Primary/secondary Ab
TGF β1 receptor	VSMC receptor	Primary/secondary Ab
Flk1	VEGF2 receptor in ECs	Primary/secondary Ab
VE-cadherin	Adhesion molecule of ECs	Primary/secondary Ab
Von Willebrand Factor	Factor VIII receptor in ECs	Primary/secondary Ab
Vimentin	Intermediate filament in ECs and Fs	Primary/secondary Ab
4. Hematopoietic markers
Gata-1	Differentiation of blood cells	Conjugated primary Ab
Gata-2	Differentiation of blood cells	Conjugated primary Ab
Cd34	HSC/EC antigen	Conjugated primary Ab
Cd45	Pan-myeloid marker	Conjugated primary Ab
Cd45ro	T lymphocyte subset marker	Conjugated primary Ab
Cd8	T lymphocyte subset marker	Conjugated primary Ab
Cd20	B lymphocyte marker	Conjugated primary Ab
Glycophorin A	Erythroid marker	Conjugated primary Ab

CSC, cardiac stem cells; VSMCs, vascular smooth muscle cells; ECs, endothelial cells; Fs, fibroblasts; HSC, hematopoietic stem cells; Ab, antibody.