Creation of MI and intracardiac injection of EPO analog
We established a mouse MI model by coronary ligation according to a novel procedure [17], and intracardiac (i.c.) injection of darbepoetin alpha (a long-acting EPO analog, EPOanlg) (30 mg/kg) was performed during MI. Detailed methods can be found in the Supplementary Material.
Echocardiography
Echocardiography was performed in each mouse to measure cardiac structure and function at the first, second and fourth week post MI using an echocardiographer for small animals (Vevo 2100, VisualSonics, Toronto, Canada). To measure the responsiveness of infarct heart to isoproterenol (ISO) challenge, left ventricular (LV) catheterization was performed to monitor hemodynamics parameters. ISO was injected into the external jugular vein after inhalation anesthesia with isoflurane at the fourteenth day post MI to check the response of MI hearts to b-adrenoceptor activation, as we reported previously [17].
Masson’s trichrome staining and determination of infarct size
Masson’s trichrome staining was used to identify cardiac infarct size. Detailed methods can be found in the Electronic Supplementary Material.
TUNEL staining
TUNEL staining was used to determine cardiomyocyte apoptosis at the seventh day post MI according to a reported method [18].
Immunofluorescent staining
Immunofluorescent staining was used to identify CD31 of endothelial cells in the myocardium and EPO receptors in Lin- Sca-1- SCs and Lin- Sca-1+ SCs. Detailed methods can be found in the Electronic Supplementary Material.
Incorporation of BrdU into post-MI hearts
Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) (5 mg/ml in 150 mM NaHCO3) was injected (50 mg/kg i.p.) twice a day for five days, beginning at the seventh day after the onset of MI, followed by two days of no injection prior to harvesting of hearts. Mice were euthanized 48 hours after the final injection and hearts were flushed with PBS, fixed with 4% paraformaldehyde and embedded in paraffin. Five μm tissue sections were cut with a microtome. To determine BrdU incorporation, tissue slides were deparaffinized, rehydrated and placed in antigen retrieval solution and kept boiling for 10 min. Tissue sections were rinsed with PBS, blocked with 5% serum, incubated with troponin I (Trop I) antibody (dilution 1:200) at 4 ºC overnight, then were incubated with BrdU primary antibody (dilution 1:10) for 2 h and rinsed again with PBS. Sections were incubated with secondary antibody (1:300 in block solution) for 1 h. Tissue slides were then rinsed with PBS for 3 × 5 min and stained with DRAQ5 for 5 min, then mounted. Trop I and Brdu-positive cells, which represented newly BrdU+/Troponin I+ cells, were counted under a fluorescence microscope. The ratio of BrdU+/Troponin I+ cells number over total nuclei was calculated.
Isolation and culture of Lin− Sca-1+ SCs
For cloning study, Lin− Sca-1+ SCs were isolated from the hearts of neonatal (1 - 3 days old) C57BL/6J mice. For other cellular experiments, Lin− Sca-1+ SCs were isolated from the hearts of adult (8-week old) male C57BL/6J mice. Detailed methods for isolating these SCs can be found in the Electronic Supplementary Material.
Above two kinds (neonatal and adult) of filtered cells were centrifuged again at 1300 rpm for 7 min at 4 ºC. Cell pellets were resuspended and cell suspension were incubated with biotin-antibody cocktail (10 μl/107 cells) at 4 ºC for 10 min. Cell pellets were then incubated with anti-biotin microbeads (20 μl/107 cells) at 4 ºC for 15 min. Cells were purified by the magnetic column and Lin− cells were obtained. Lin− cell suspension was incubated with anti-Sca-1-FITC (10 μl/107 cells) for 10 min at 4 ºC, washed with PBS, and incubated with anti-FITC microbeads (20 μl/107 cells) for 15 min at 4 ºC. Sca-1+ SCs were collected by positive magnetic sorting. After sorting, the percentage of Lin− Sca-1+ SCs was (85.03 ± 5.28)% ,which was significantly higher than the Lin− Sca-1- SCs (7.64 ± 3.31)% (p < 0.001) (Additional file 1: Fig. S1). Therefore, by magnetic sorting, high purity cardiac resident Lin− Sca-1+ SCs were obtained.
Flow cytometry to detect cell cycle
Lin− Sca-1+ SCs isolated from adult mice were used. Purified Lin− Sca-1+ SCs suspension was centrifuged and 2 ml pre-cooled 70% ethanol was added to the pallet overnight at 4 ºC. The immobilized cells were centrifuged at 2000 rpm for 4 min. Cells pellets were rinsed with PBS for 3 times, and then incubated with 100 μl RNase A and 400 μl propyl iodide (PI) for 15 min at RT. Flow cytometry was used to detect fluorescence signal at the excitation wavelength of 488 nm. The percentages of Lin− Sca-1+ SCs at G0/G1, S and G2/M phases were calculated.
Measurement of cell proliferation
The third passage of Lin− Sca-1+ SCs isolated from adult mice were seeded to 24-well plates (5 × 103 per well). The Lin− Sca-1+ SCs were divided into EPO group and PBS (vehicle) group (6 wells per group) randomly. EPO (0.5 μmol/l) was added to each well of EPO group every 48 hours. Meanwhile, the same volume of PBS was added to the wells of vehicle group. Cells were cultured in Cytation 5 cell imaging microtiter plate and cell numbers were counted every 4 h.
Assessment of SCs clonality
The third passage of Lin− Sca-1+ SCs isolated from neonatal mice were seeded to 6-well plates (1 × 105 per well). EPO (0.5 μmol/l) was added to each well of EPO group every 48 hours, and the same volume of PBS was added to each well of vehicle group. At the seventh day of treatment, cloning numbers were counted under a microscope.
Transwell assay to measure SCs migration
The third passage of Lin− Sca-1+ SCs isolated from adult mice were recovered and seeded into the upper chamber of a transwell (200 μl, 5 × 104 cells/ml). Stem cell culture medium (500 μl) was added into the bottom chamber. After cells were adhered, serum-free medium was used to starve cells overnight. Then the culture media with different concentrations of EPO (0, 0.05, 0.1, 0.5, 1, 5 μmol/l) were added to the bottom chamber. After 24 h, cells in the upper chamber removed to the bottom surface. The transwell chamber was immersed in 4% paraformaldehyde for 30 min at RT and rinsed with PBS for 3 × 5 min. The transwell chamber was placed in 0.1% crystal violet solution for 20 min at RT. After rinsing with water for 3 × 5 min, transwell chamber was placed under a microscope for data collection. Finally, the transwell chamber was decolored with 1 ml of 33% glacial acetic acid for 10 min and then the optical density (O.D.) values representing migrated Lin− Sca-1+ SCs were determined in different groups with a microplate reader at the wavelength of 570 nm.
Western blotting
Western Blotting was used to evaluate the expression levels of EPO receptor in Lin− Sca-1- and Lin− Sca-1+ SCs, and the expression levels of p38, p-p38, Stat-5, p-Stat-5, Erk-1/2 and p-Erk-1/2 in Lin− Sca-1+ SCs. Detailed methods for Western blotting can be found in the Electronic Supplementary Material.
Reagents
Reagents used in this study were purchased from the following suppliers: EPO, Sigma; darbepoetin alpha, Amgen, Thousand Oaks, CA; mouse lineage depletion kit, Miltenyi Biotec; anti-Sca-1 MicroBead Kit, MACS; LS column, Miltenyi Biotec; Masson’s trichrome stain kit, propyl iodide (PI), ribonuclease A, BrdU, DAPI and anti-p-Stat-5A/B (pSer725/730), Sigma; transwell, Corning; anti-CD31, BD; anti-Stat-5 (c-17) and secondary antibodies, Santa Cruz; anti-phospho-p38 MAPK, anti-p38 MAPK (Thr180/Tyr182), anti-phosphoPlus® p44/42 MAPK (Erk-1/2) (Thr202/Tyr204), anti-p44/42 MAPK (Erk-1/2) and anti-troponin I, Cell Signaling. All other chemicals were purchased from Sigma (St. Louis, MO).
Statistical analysis
All values are presented as mean ± standard error (S.E.M.) of independent experiments from given n-sizes. Statistical significance was determined by student’s t-test or one-way ANOVA followed by the Bonferroni post-hoc test where appropriate. The survival rate was analyzed by Gehan Breslow-Wilcoxon test. p value of < 0.05 were considered statistically significant.