Mouse bone marrow derived progenitor cell (BPC) culture was carried out as described ^[12]. Mononuclear cells isolated from the tibias and femurs of GFP-expressing transgenic C57BL/6-TgN (ACTbEGFP) mice were cultured in EBM-2 medium supplemented with (SingleQuot Kit; Clonetics) 5% FBS on cell-culture dishes coated with 0.1% rat vitronectin/gelatin. After 4 days in culture, the adherent cells were reseeded (5×10⁴ cells/cm²) on 0.1% vitronectin/gelatin-coated 10 cm culture dishes and 4-well chamber slides and cultured for 3 additional days. Cells in 4-well slides were co-incubated with DiI-acLDL (Biomedical Technologies) for 1 hour, stained with fluorescein isothiocyanate–conjugated Banderia simplicifolia lectin 1 (FITC-BS1), and viewed under fluorescence microscope. Majority of these cells were double-stained with these markers defining them as endothelial-lineage cells ^[12]. These cells were further characterized by flow cytometry using stem and progenitor cell marker antibodies. Day-7 BPCs growing in 10 cm culture dishes were used for the chemical modification experiments using Aza and TSA described below.

Day-7 BPCs were treated with Aza (0, 10, 25, 50 and 100 nM) or TSA (0, 5, 10, 25 and 50 nM) or both drugs combined for 4 days. Total cellular RNA was obtained for qRT-PCR analysis to determine mRNA expression of pluripotency markers ^[13], ^[14]. The gene expression profiles of Primer and probe sequences specific for specific target genes are given in Table 1. Relative mRNA expression of target genes was normalized to endogenous 18S control gene (Applied Biosystems). Results were expressed as fold change in expression and values were calculated as ratio of induced expression-to-control expression.

Genomic DNA was obtained from tissue sections from un-injected (control) and BPC- and eiBPCs-derived cardiac progenitor-injected mouse hearts. Sections were stained with anti-EGFP antibody to identify areas of injected cells present in the myocardium and these sections were used for PCR analysis. Additionally, random samples of recipient spleen, lung, liver and kidney were also used for DNA extraction to compare with responses occurring in the heart. PCR was conducted for EGFP to identify DNA of EGFP-labeled transplanted cells. DNA was extracted with QIAamp DNA micro kit (Qiagen); 100 ng DNA was mixed with primers for EGFP (Table 1). Final PCR products were run on agarose gel for detection of bands corresponding to EGFP.

To determine differentiation potential of re-programmed BPCs, termed eiBPCs, into CMCs, the eiBPCs were cultured for 5 days in complete DMEM containing CMC induction medium with 5 ng/mL LIF and 3 ng/mL bone morphogenetic protein-2 (BMP-2) in 6-well culture plates (10⁶ cells per well) as well as 4-well chamber slides (10⁴ -cells per well) coated with 0.1% gelatin as in ^[13], ^[14]. Total cellular RNA was obtained from 6-well culture plates and qRT-PCR analysis of mRNA expression of CMC markers was carried out (Table 1); the markers used were GATA4, Nkx2.5, cardiotroponin T (CTT), CTI, α-sarcomeric actinin (α-SA), Mef2c, and MHC-α. For protein expression, cells were cultured for 10 days and protein expression was determined by immunochemical staining. For transplantation into myocardial infarction model, eiBPCs were cultured in CMC-conditioned medium for 24 hours to commit the cells towards CMC lineage ^[13] before transplantation.

Protein expression was evaluated by immunofluorescence staining as in ^[13], ^[14]. Briefly, cultured cells or cardiac tissue was rinsed once with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Sigma) in PBS for 30 min, then rinsed three times with PBS, permeabilized with 0.3% Triton X-100 (Sigma) in PBS for 5 min, washed twice with PBS, and incubated overnight at 4°C with primary antibodies (against Oct4, Nkx2.5, Gata 4, MAP-2, MBP, GFAP, CTT, α-SA, GFP and Ki67) diluted with 1% FBS in PBS. After 3 washes with PBS, cells were incubated with specific secondary antibodies for 1 hour at 37°C, and cells were rinsed three times with PBS, stained with DAPI to visualize cell nuclei, rinsed 3 times with PBS, dried, and mounted in Vectashield mounting medium for fluorescent imaging. All immunofluorescence staining was photographed using either confocal and immunofluorescence microscope.

Immunoprecipitation and Western blot analyses of AceH3K9, HDAC1 and LSD1 proteins were performed in BPCs and eiBPCs as in ^[13] using antibodies purchased from Cell Signaling Technology, USA.

ChIp assay was performed for the Oct4 promoter in BPCs, eiBPCs and mouse ESCs as described ^[14], ^[15] to identify transcription factors binding to Oct4 following treatment with Aza and TSA.

Viability and proliferation of the modified BPCs was evaluated by Trypan-blue exclusion and MTT assays, respectively. BPCs were cultured with different concentrations of Aza and TSA in 12-well culture plates for 24 or 48 hours, cells were harvested, exposed to Trypan blue solution (final concentration 0.1%), and number of viable (unstained) and non-viable (stained) cells were counted with a hemocytometer. Effects of Aza and TSA on BPC proliferation was determined by 3, 4, 5-(dimethylthiazol-2-yl) 2, 5- diphenyl tetrazolium bromide (MTT) assay according to manufacturer's instruction (Cell Proliferation Assay kit Promega). Briefly, 10⁵ cells were cultured in 96-well culture plate (200 ul/well) in DMEM containing 10% FBS in the presence of 0, 25, 50 and 100 ng/ml 5Aza or 0, 10, 25, 50 nm/ml TSA or combination of both for 48 hours at 37°C. MTT assay results were confirmed by manual cell counting under microscope.

All procedures were performed in accordance with guidelines of Institutional Animal Care and Use Committee of University of Illinois at Chicago (ACC No: 09-061 approved dated 05/08/2009). The study involved 8-week-old male C57BL/6J mice (n = 30); Jackson Laboratories). Mice underwent surgery to induce AMI by ligation of left anterior descending coronary artery ^[16], ^[17]. Animals subdivided into 3 groups received intramyocardial injection of 5×10⁵ GFP+cardiac progenitors derived from eiBPCs (treated for 24 hours with cardiomyocyte specific medium), control BPCs, or saline, respectively, in a total volume of 10 uL at 5 sites (basal anterior, mid-anterior, mid-lateral, apical anterior and apical lateral) in the peri-infarct area immediately after surgery.

Mice underwent echocardiography just before MI (base level) and at 1, 2, and 4 weeks after AMI as described ^[16], ^[17]. Trans-thoracic echocardiography was performed with a 6- to 15-MHz transducer (SONOS 5500, Hewlett Packard). Two-dimensional images were obtained in the parasternal long and short axis and apical 4-chamber views. M-mode images of the left ventricular short axis were taken just below the level of mid-papillary muscles. Left ventricular end-diastolic and end-systolic dimensions were measured and functional shortening was determined according to modified recommendations of American Society of Echocardiography. A mean value of 3 measurements was determined for each time point. On day 28 post-AMI, mice were euthanized and aortas were perfused with saline, hearts were removed and sliced into 4 transverse sections from apex to base and fixed with 4% paraformaldehyde, methanol, or frozen in OCT compound and sectioned into 5-µm thickness slices. Immunofluorescence staining was performed to determine myocyte and endothelial cell differentiation of transplanted cells. For assessment of fibrosis, tissues sections were frozen in OCT compound and sectioned for elastic tissue/trichrome to measure average ratio of the external circumference of fibrosis area to left ventricular area.

To assess survival of transplanted cells as well as effects of cell transplantation on survival of resident myocytes, apoptosis assay was performed on heart tissue collected at different time points. Cell death was assessed using the TUNEL kit according to manufacturer's instruction (Roche Applied Science). After TUNEL assay staining, sections were examined by confocal fluorescence microscopy.

We measured mRNA levels of TNF-α, IL-1β, IL-6, MCP-1 and IL-10 in heart tissue bordering the infarcted myocardium as described ^[18].

De-paraffinized heart tissue section slides were incubated for 30 min in peroxidase suppressor to quench endogenous peroxidase activity. After washing slides two times for 3 min with a wash buffer, blocking buffer was added and then incubated for 30 min. The tissue was incubated for 30 min with anti-CD31 antibody and slides were washed two times for 3 min with wash buffer. Again the tissues were incubated for 30 min with HRP-conjugated secondary antibody and slides were washed 3 times for 3 min each with wash buffer. Finally, the metal enhanced DAB Substrate was added and incubated until the desired staining was achieved.

All experiments were made at least 3 times. Results are presented as mean+SEM. Comparisons were performed by ANOVA (GB-STAT; Dynamic Microsystems) or χ² test for percentages. All tests were 2-sided, and probability value of <0.05 was considered as statistically significant.

On day 7, BPCs obtained from C57BL/6J mouse bone marrow as described ^[12] were analyzed by immunofluorescence and flowcytometry (Figure 1A, B). Most cells were positive for Dil-acLDL and showed binding to fluorescein isothicyanate-conjugated Banderia simplicifolia lectin 1 (FITC-BS1). FACS analysis also showed BPCs were a heterogeneous population. They were positive for progenitor cell markers such as CD34 (17%), CD133 (10%), Sca1 (100%), and c-kit (12%) and some were also positive for endothelial cell markers CD31 (10%) and VE-cadherin (16%). BPCs were treated for 4 days with Aza alone, TSA alone, or both Aza and TSA at various concentrations. BPCs treated with Aza or TSA alone (Figure 1C) or with both agents in combination induced expression of Oct4, Nanog and Sox2 (Figure 1D, p<0.001 control vs. Aza+TSA treatment group). Maximum expression of these factors occurred at combined concentrations of 50 nM Aza and 25 nM TSA. Immunofluorescence analysis of Oct4 expression showed that BPCs treated with Aza+TSA showed significantly up-regulated expression of pluripotent genes at passage 0 (Figure 1E) but expression decreased markedly by passage 6 (data not shown). The expression of Oct4 gene transcript was supported by Oct4 Western blot protein expression data (Figure 1F). The results shown in Figure 1A-E demonstrate that pluripotent genes in BPCs were activated by co-treatment with Aza and TSA in a concentration-dependent manner. We also observed by qRT-PCR that BPCs treated with 50 nM Aza and 25 nM TSA resulted in silencing of eNOS and VE-cadherin genes compared to control cells (Figure 1G), suggesting that these drugs promoted de-differentiation of endothelial cells present in the BPC population.

BPCs were cultured with different concentrations of Aza and TSA in 12-well culture plates for 24 or 48 hours, cells were harvested, exposed to Trypan blue solution (final concentration 0.1%), and number of viable (unstained) and non-viable (stained) cells were counted with a hemocytometer. Treatment with up to 50 nM Aza and 25 nM TSA had no effect on cell viability (Figure 2A, p = 0.09). The modified BPCs also showed normal ability to proliferate (Figure 2B, C p<0.05)

We determined whether Aza and TSA treatment of BPCs altered acetylation and methylation status of AceH3K9, HDAC1, and LSD1 proteins involved in induction of multipotency ^[19]. BPCs were treated with 5Aza and TSA, either alone or in combination for 2 days. As shown in Figure 3A, expression of acetylated H3 lysine9 (AceH3K9) protein was significantly increased in BPCs treated with TSA alone (p<0.01) or with Aza plus TSA (p<0.005) compared to controls. HDAC1 protein expression decreased after treatment of BPCs with either TSA alone (p<0.005) or combination of Aza+TSA (p<0.005) compared to control cells (Figure 3B). Compared to control BPCs, the treated BPCs displayed decreased LSD1 expression (p<0.05) (Figure 3C).

We also determined whether Aza and TSA induced changes in AceH3K9 protein interaction with Oct4 promoter ^[14] to assess whether these agents can epigenetically modify BPCs. As shown in Figure 3D, Oct4 promoter was highly acetylated in positive control mouse ESCs, whereas acetylation of H3 region in Oct4 promoter was not detected in untreated BPCs but it was much evident in the treated BPC (p<0.05).

To address whether Aza and TSA modified BPCs (eiBPCs) could give rise into myocyte progenitor lineage cells and cardiomyocytes, eiBPC were cultured for 5 or 10 days in CMC induction medium. We observed that cardiac progenitor cell markers Flk1 and c-kit were up-regulated as early as 24 hr after combined treatment with Aza and TSA compared to control or BPC treated with Aza or TSA alone (Figure 4A, B). Cardiomyocyte specific markers, GATA4, Nkx2.5, CTT, α-sarcomeric actinin, Mef2c, MHC-α and CTI were up-regulated after combined treatment of Aza and TSA at day 5 compared to BPCs treated with Aza or TSA alone (Figure 4C, D; P<0.001 control vs. cells treated with both Aza and TSA). eiBPCs or cardiac progenitor cells derived from eiBPCs failed to differentiate into endodermal lineage of cells and did not form teratomas in SCID mice (data not shown) suggesting that eiBPCs or cardiac progenitors derived from eiBPCs had been partially re-programmed but were not pluripotent.

We used a standardized mouse AMI model ^[16], ^[17] to test the efficacy of cardiac progenitors derived from eiBPCs (as described Methods) in improving LV function. Physiological assessment of LV function was made before AMI and on days 7, 14 and 28 post-AMI. The day 28 post-AMI echocardiography showed significantly improved LV diastolic dimensions (LVDd) and percentage of LV fractional shortening (%LVFS) in mice transplanted with eiBPCs and BPCs compared to saline control (Figure 5A, B). Mouse hearts treated cardiac progenitors derived from eiBPCs had better functional outcome post-AMI than BPC treatment alone. Myocardial regeneration was only seen in the infarcted myocardium in hearts treated with Aza- plus TSA-modified BPCs. Masson's trichrome-stained sections showed markedly enlarged LV in saline-injected AMI mice and LVs were less enlarged in mouse hearts injected with Aza- plus TSA-modified BPCs (Figure 5C). Heart sections were also quantified for % LV fibrosis as the ratio of length of fibrosis scar-to-LV circumference. We observed marked reduction in LV fibrosis in hearts treated with eiBPC-derived cardiac progenitors compared with hearts treated with BPCs or saline (Figure 5D). We also examined the capillary density in ischemic area by DAB staining using anti-CD31 antibody marking for capillaries (Figure 5E). Five randomly chosen sections were quantified in microscopic visual fields on each slide and mean was calculated. We observed a closely similar number of capillaries in BPC-transplanted hearts (28±3) and eiBPC-derived cardiac progenitor-transplanted hearts (25±2) (Figure 5E, P = 0.06), suggesting that propensity of BPCs to differentiate into endothelial cells was similar to cardiac progenitors from eiBPCs.

To determine effects of cardiac progenitors derived from eiBPCs on expression of pro-inflammatory and anti-inflammatory cytokines and chemokines (TNF-α, IL-1β, IL-6, MCP-1 and IL-10) in the myocardium at 3 days post-AMI was assessed by qRT-PCR. Expression of pro-inflammatory cytokines and chemokines was increased in myocardium of saline-treated and was decreased in BPC-treated (Figure 6A-D, p<0.01 vs. saline) and eiBPC-derived cardiac progenitor-treated myocardium (Figure 6A-D, p<0.005 vs. saline, P<0.05 vs. BPC). The anti-inflammatory cytokine IL-10 mRNA expression was increased in eiBPC-derived cardiac progenitor group compared to saline- or BPC-treated group (Figure 6E, p<0.01 vs. saline, p<0.05 vs. BPC) treated myocardium. Even in non-injected eiBPC-derived cardiac progenitors, there was increased IL-10 and IL-10R mRNA expression vs. BPCs (Figure 6F).

Immunofluorescence staining was performed to determine in situ differentiation of transplanted GFP+eiBPC-derived cardiac progenitors in the myocardium. The 28 days post-cell injected heart tissue sections were co-stained with CMC tissue markers cardiotroponin T (CTT) (Figure 7A) and α-SA (Figure 7B). The GPF+ cells (green) expressing the two CMC proteins (red) were double positive (yellow) merged images indicating the co-localization and differentiation of injected cells. Double positive cells were counted at 5 randomly chosen fields on each slide, and the mean is presented as double positive cells/mm2 (Figure 7C). As shown in Figure 7, transplantation of cardiac progenitors derived from eiBPCs induced differentiation of progenitor cells to CMCs in situ; transplantation of eiBPC-derived cardiac progenitors also showed greater number of cells co-expressing the CMC markers than transplantation of BPCs alone (p<0.01). Genomic DNA obtained from un-injected hearts and hearts from BPC-injected hearts and eiBPC-derived cardiac progenitor-injected hearts was also used for PCR analysis. Additionally, samples of spleen, lung, liver and kidney were used for DNA extraction. PCR for EGFP used to identify DNA of GFP-labeled transplanted cells in tissue. The bands on agarose gel corresponding to eGFP showed that green cells were present in heart tissue (Figure 7D) and bands from the other organs did not express GFP DNA (data not shown).

As shown in Figure 8, differentiation into GFP+CD31+ double positive cells was marginally increased in myocardial sections obtained from mouse hearts transplanted with BPCs (Figure 8A) compared to larger increase in mouse hearts transplanted with eiBPC-derived cardiac progenitors (Figure 8B). Endothelial differentiation in myocardial tissue was quantified by counting CD31+GFP+ cells (Figure 8C). Number of GFP+CD31+ cells similarly increased in both BPC-transplanted hearts (12±2) and eiBPC-derived cardiac progenitor transplanted hearts (10±1, P = 0.09).

Transplanted eiBPC-derived cardiac progenitors also showed evidence of proliferation in vivo, as determined by co-localization of GFP+ cells with nuclear proliferation antigen, Ki67, seen at day 28 post-transplantation (Figure 9A, C), whereas fewer GFP+Ki67+ cells were seen in BPC-transplanted hearts. This proliferative effect is consistent with the in vitro data above (Figure 2). In addition the number of apoptotic cells was less in eiBPC-derived cardiac progenitor transplanted group than in BPC-transplanted group (Figure 9 B, D).