Recent studies have shown that cardiac stem cells (CSCs) from the adult mammalian heart can give rise to functional cardiomyocytes; however, the definite surface markers to identify a definitive single entity of CSCs and the molecular mechanisms regulating their growth are so far unknown. Here, we demonstrate a single-cell deposition analysis to isolate individually selected CSCs from adult murine hearts and investigate the signals required for their proliferation and survival. Clonally proliferated CSCs express stem cell antigen-1 (Sca-1) with embryonic stem (ES) cell-like and mesenchymal cell-like characteristics and are associated with telomerase reverse transcriptase (TERT). Using a transgene that expresses a GFP reporter under the control of the TERT promoter, we demonstrated that TERTGFP-positive fractions from the heart were enriched for cells expressing Sca-1. Knockdown of Sca-1 transcripts in CSCs led to retarded ex vivo expansion and apoptosis through Akt inactivation. We also show that ongoing CSC proliferation and survival after direct cell-grafting into ischemic myocardium require Sca-1 to upregulate the secreted paracrine effectors that augment neoangiogenesis and limit cardiac apoptosis. Thus, Sca-1 might be an essential component to promote CSC proliferation and survival to directly facilitate early engraftment, and might indirectly exert the effects on late cardiovascular differentiation after CSC transplantation.

Introduction

The adult mammalian heart harbors a population of mitotically competent cardiac stem cells (CSCs) that can be isolated by using FACS to recognize the cells expressing surface antigens KIT and stem cell antigen-1 (Sca-1) or by targeting a reporter gene driven by the promoter for islet-1, a LIM-homeodomain transcription factor (Beltrami et al., 2003; Laugwitz et al., 2005; Matsuura et al., 2004; Moretti et al., 2006; Oh et al., 2003; Pfister et al., 2005). These cells express essential cardiac transcriptional factors but do not express more mature markers of structural genes; however, the exact contribution of cell fusion in the process of adopting cardiac muscle phenotype after cell transfer into ischemic myocardium remains controversial (Beltrami et al., 2003; Oh et al., 2003). Within the adult heart, CSCs often reside in cardiac niches with supporting cells that provide a specialized environment to replenish and maintain a balance of survival, proliferation and self-renewal of CSCs through symmetric or asymmetric division in order to replace the mature cells that are lost during injury or turnover (Urbanek et al., 2006).

The general lack of definitive molecular markers to identify cardiac stem cells raises the fundamental question of whether these cardiac stem cells are derived from a single entity. CSCs in the mammalian heart share several cell-surface markers with hematopoietic and endothelial progenitor cells (Linke et al., 2005; Messina et al., 2004; Urbanek et al., 2003). Although the hierarchies of hematopoietic stem cells have been well characterized, evidence supporting the role of bone marrow-derived Lin-Kit+ cells in cardiac-lineage induction has been controversial (Kawada et al., 2004; Murry et al., 2004; Orlic et al., 2001). Recent reports have demonstrated that genetic disruption of Kit in mice mainly affects marrow-derived hematopoietic and endothelial cell development for cardiac repair, that could be rescued by bone marrow replacement with wild-type cells, through the failure of progenitor-cell mobilization from marrow and reduced release of cytokines and chemokines that may participate in the cardioprotective paracrine signaling (Ayach et al., 2006; Fazel et al., 2006). These studies do not exclude the possible functional role of KIT in resident CSCs as the principal mediator in the regenerating process during cardiac injury, but suggest that defining CSCs using specific cell-surface markers may not be optimal to address the identity of these cells, as indicated by their partially overlapping expression in human hearts (Urbanek et al., 2005b).

Decline of CSC function may be a major cause of the decrease in regenerative capacity in aging and disease (Rota et al., 2006). Although some of the growth factors involved in Kit+ CSC proliferation and survival have been identified, factors regulating Kit CSCs have yet to be defined (Gude et al., 2006; Limana et al., 2005; Urbanek et al., 2005a). In this study, we sought to identify single proliferative cells from the adult heart without progenitor selection using particular surface markers. Using this unbiased approach, we have established clonal CSC lines and demonstrated that the majority of the telomerase-active progenitor-cell colonies expressed Sca-1 and showed mesenchymal-cell-like character. We also show that targeting the Sca-1 transcripts in CSCs used for cell grafting leads to failure of their ability to prevent cardiac remodeling after myocardial infarction. The antiapoptotic and angiogenetic paracrine activities of intrinsic Sca-1 signaling in CSCs promote direct CSC proliferation and survival, and contribute to neovascularization in the host myocardium for efficient cardiovascular regeneration.

Results

Clonal isolation of cardiac stem cells in the adult heart

To identify the single entity of CSCs in the adult heart, we employed an unbiased approach using a single-cell clonogenic isolation technique to isolate a proliferative cell population. Singly dissociated GFP-labelled transgenic cells derived from the hearts of GFP transgenic mice were plated at a density of one cell per well in serum-free medium (Fig. 1A,B). Altogether, 11,520 single cells were deposited, and from 9541 single cells determined by inspection on day 1 to be present as one individual cell per well, a total of 11 clones arose within 7 days. Eight out 11 clones failed to grow in serum-free medium after 7 days in culture, and 3 clones (∼0.03%) could proliferate to form spherical clusters and were continuously expanded after 14 days (Fig. 1C). The three independent colonies were re-dissociated and re-plated in low-serum for expansion, and individual CSC colonies were used for the following experiments to characterize clonal CSCs.

Characterization of clonally amplified CSCs

Immunophenotyping revealed that the clonal CSCs strongly expressed Sca-1, which is used as a marker to identify cardiac progenitor cells from the adult heart (Matsuura et al., 2004; Oh et al., 2003), whereas KIT-positive cells were rarely detected (Fazel et al., 2006; Gude et al., 2006; Pfister et al., 2005) (Fig. 1D). Notably, CSCs did not express the hematopoietic and endothelial progenitor-cell-specific surface antigens CD45, CD34 and CD31, but did express the typical mesenchymal stem-cell surface antigens CD90, CD105, CD29, CD44, CD106, CD73 and CD13 (Pittenger and Martin, 2004). The three individual CSCs exhibited an identical immunophenotyping for the surface marker analysis. The cell membrane antigens Sca-1, KIT, CD45, and CD34 were not destroyed by collagenase treatment as tested in bone marrow (data not shown).

Gene expression was then examined in CSC clones using reverse transcriptase (RT)-PCR (Fig. 1E). Three individual colonies were analyzed and most of the clones expressed Bcrp1, polycomb group protein Bmi1 and also telomerase reverse transcriptase (TERT), which has been reported to be absent in cardiac fibroblasts (Leri et al., 2001). Although Nanog was detectable in all of the colonies examined, none of the colonies – unlike embryonic stem (ES) cells – were positive for OCT4 or UTF1. Some but not all of the colonies expressed HNF3β, brachyury and SOX2, which are endodermal, mesodermal and ectodermal precursor markers, respectively. These results distinguished clonal CSCs from mouse fibroblasts, which are negative for all of the ES cell markers described above (Takahashi and Yamanaka, 2006). In addition, all of the colonies analyzed expressed nestin, a marker of immature neural progenitor cells (Joannides et al., 2004).

TERT-expressing cells in postnatal heart are associated with Sca-1 expression

TERT has been identified as a key factor controlling telomerase activity, telomere length, and cell growth (Blackburn, 2001). We measured the telomerase activity in clonal CSCs. The three individual CSCs displayed significantly elevated telomerase activity (Fig. 2A). To directly characterize TERT-expressing cells in the adult heart, we engineered transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the mouse Tert promoter (Fig. 2B). We identified two transgenic founders by genomic DNA screening and established two independent lines (Fig. 2C). In order to examine the efficacy of mouse Tert promoter in the heart in vivo, cells isolated from transgenic hearts were sorted by EGFP signal and the EGFP-positive cells were found to be TERT-expressing cells, which were not detectable in EGFP populations (Fig. 2D). As expected from high telomerase activities of clonal CSCs shown in Fig. 2A, three individual CSC clones showed TERT expression (Fig. 2D). To further characterize the TERT-positive cells in the heart, FACS analysis was performed. FACS of the cells prepared from the heart of adult mice expressing TERT-EGFP indicated that TERTGFP-positive cells constitute a population that is positive for Sca-1 but rarely expressing detectable levels of c-kit, CD45, CD34, or CD31 (Fig. 2E).

Generation of Sca-1 knockdown (KD) mice

To functionally characterize clonal CSCs, majority of which could be marked by Sca-1 expression, we generated Sca-1 KD mice in which double-stranded (ds)-Sca-1 RNA was expressed under the control of an RNA polymerase II promoter (Fig. 3A) (Shinagawa and Ishii, 2003). The vector pDECAP-Sca-1 expressing ds-Sca-1 RNA with a small loop for transcript pausing and full-length Sca-1 were co-transfected into HEK 293 cells at various concentrations, and the reduction in Sca-1 expression was examined by both RT-PCR and FACS (Fig. 3B,C). Two lines of Sca-1 KD mice were obtained, in which endogenous Sca-1 protein levels (Fig. 3D) in the heart were apparently reduced.

Targeting Sca-1 transcripts affects proliferation and survival but not differentiation of CSCs

To test the function of Sca-1 in CSC development, we examined the ability to clonally proliferate in vitro of single cells from the adult heart of Sca-1 KD and non-transgenic (NTG) littermate mice using a single-cell deposition analysis. This revealed that the percentage of colony-forming cells from Sca-1 KD hearts was significantly lower than that from NTG hearts (Sca-1 KD ∼0.007% vs NTG ∼0.03%, Fig. 3E). We isolated 11 clones from NTG hearts and four clones from Sca-1 KD hearts, of which eight NTG- and two Sca-1 KD-clones exhibited features of mesenchymal phenotype (data not shown), showed Nanog and Bcrp1 expression by RT-PCR, and could proliferate for more than 14 days (Fig. 3F). Of the clones obtained, four clones expressed brachyury, which is a primitive streak marker (Gadue et al., 2006). Sca-1 expression in CSCs isolated from Sca-1 KD mice was markedly inhibited compared with NTG controls (Fig. 3G). Therefore, we investigated whether Sca-1 expression affects the replicative growth of clonal CSCs in independent cell-culture (Fig. 3H). As shown in Fig. 3I, Sca-1 KD CSCs showed significantly impaired growth kinetics compared with those of NTG CSCs. We determined the molecular mechanisms by which Sca-1 KD mice showed retarded CSC growth. As shown in Fig. 3J, BrdU incorporation and phosphorylation of histone H3 were clearly reduced in the Sca-1 KD CSCs compared with NTG controls, whereas p53 expression levels were significantly increased in the Sca-1 KD CSCs. Telomerase activities were also significantly impaired in the Sca-1 KD CSCs (Fig. 3K).

Of the CSC clones isolated, clones 2, 3 and 6 from NTG and clone 1 from Sca-1 KD mice, all of which expressed brachyury, were chosen for subsequent series of experiments. We asked whether the decrease in clonal CSC growth mediated by Sca-1 KD is associated with an increase in apoptosis. CSCs were isolated from the hearts of NTG and Sca-1 KD mice and were incubated with 100 and 200 μM H2O2 for 18 hours, and the surviving cells were analyzed by TUNEL staining. As shown in Fig. 4A, H2O2 induced apoptosis in a dose-dependent manner, and the extent of apoptosis was significantly higher in CSCs isolated from Sca-1 KD hearts than that in NTG-CSCs.

Activation of EGF and bFGF signaling in endothelial cells leads to the phosphorylation of a number of downstream effectors, including Akt and MAPKs (Sulpice et al., 2002). To test the role of these kinases in Sca-1-mediated CSC growth, the activation of Akt and MAPKs in response to EGF and bFGF was examined (Fig. 4B). Incubation of CSCs with EGF and bFGF resulted in a rapid enhancement of Akt, ERK1/2, and JNK1/2, but not in phosphorylation of p38. Although activation of Akt could be abolished by inhibition of Sca-1 transcripts, phosphorylation of three MAPKs was unaffected. These results raise the issue of whether Sca-1-mediated signaling regulates CSC differentiation in vitro. The potential of CSCs to give rise to cardiovascular lineages was not affected by targeting Sca-1 transcripts – as shown by immunostaining (Fig. 5A) and by RT-PCR to assess gene profiles typical of cardiac muscle, smooth muscle and endothelial cell differentiation after specific inductions for 14 days (Fig. 5B), and a study investigating the Ca2+ transient in beating cardiomyocytes (Fig. 5C).

Loss of Sca-1 transcripts in CSCs fails to improve cardiac function due to diminished donor-cell proliferation, survival and engraftment after cell transplantation

The data described above support the hypothesis that loss of Sca-1 results in a retarded regenerative capacity of CSCs in vivo. To further examine this possibility, we performed cell transfer experiments into ischemic myocardium. 5×105 CSCs that had been clonally isolated and expanded from the hearts of Sca-1 KD (C1) and NTG (C6) mice were transplanted into wild-type (WT) mice 1 hour after myocardial infarction. Cardiac MRI was performed 4 weeks after cell grafting and showed that transplantation of Sca-1-KD CSCs resulted in significantly larger left ventricular volume and an increased infarct rate as compared with NTG-CSC implantation (Fig. 6A,B).

We examined the in-vivo effects of Sca-1-mediated CSC regulation we observed in vitro. At day 3 after CSC transplantation into ischemic myocardium, Sca-1 KD CSCs showed significantly fewer engraftments than NTG-CSCs, as verified by measurement of lacZ activity (Fig. 7A). This observation was confirmed by the lower Ki67 expression in Sca-1 KD CSCs, indicating that the proliferative potential was significantly impaired in Sca-1 KD CSCs (Fig. 7A). To assess whether Sca-1-mediated control of CSC survival may also be applied to the process of donor-cell engraftment, we analyzed the viability of grafted CSCs, labeled by β-galactosidase (β-gal) staining, on day 3 after the cell grafting. As shown in Fig. 7B, grafted Sca-1 KD CSCs in the ischemic myocardium resulted in more apoptotic cells than NTG-CSCs, suggesting that the transplanted Sca-1 KD CSCs were also susceptible to cell death in vivo.

To further test whether these effects of Sca-1 during the early phase of CSC transplantation may contribute to the early CSC-engraftment and late regeneration process of cardiovascular-lineage cells, we investigated the presence of lacZ+ donor cells at day 7 and characterized their individual phenotypes 4 weeks after transplantation. As shown in Fig. 8A, the frequency of lacZ+ cells observed 7 days post cell transfer was significantly lower in Sca-1 KD CSC grafts compared with NTG-CSC transplantation, resulting in substantially insufficient cardiovascular regeneration within the ischemic regions 4 weeks after CSC transplantation (Fig. 8B-D).

Sca-1 KD CSC transplantation fails to prevent myocardial apoptosis and limits angiogenesis, partially due to the failure of paracrine effector secretion

Last, we assessed whether the loss of Sca-1 in transplanted CSCs affects myocardial apoptosis and angiogenesis. At day 3, transplantation of Sca-1 KD CSCs resulted in a high level of myocardial apoptosis in the ungrafted area of the infarcted border zone, whereas fewer TUNEL-positive cells were observed in NTG-CSC-injected hearts (Fig. 9A). Furthermore, transplantation of Sca-1 KD CSCs failed to improve capillary density 2 weeks after infarction in the ischemic region as compared with NTG-CSC injection (Fig. 9B). To explore the molecular mechanisms of Sca-1-mediated myocardial apoptosis and neoangiogenesis, we then oxygen-starved CSCs for 8 hours and measured the levels of mRNA for secreted paracrine factors by RT-PCR. As shown in Fig. 9C, downregulation of hepatocyte growth factor (HGF) in Sca-1 KD CSCs was evident at baseline normoxia. After hypoxia, a greater increase in the expression of VEGF and HGF was observed in NTG-CSCs compared with that in Sca-1 KD CSCs. The expression pattern of insulin-like growth factor-1 (IGF1) under normoxic and hypoxic conditions was comparable in CSCs from NTG and Sca-1 KD hearts.

Discussion

Recent reports have shown that clonogenic CSCs reside in mammalian hearts, judging on the basis of specific cell-surface markers which are also expressed by hematopoietic and endothelial progenitor cells (Beltrami et al., 2003; Linke et al., 2005). Using an unbiased approach, our study has demonstrated that clonally proliferated CSCs express Sca-1 with ES cell-like and mesenchymal-cell-like characteristics, and are associated with TERT expression.

Our results, showing low expression of KIT in clonal Kit+ CSCs, differ from the findings of some previous studies (Linke et al., 2005; Messina et al., 2004) but are consistent with those of recent reports about the adult heart (Fazel et al., 2006; Gude et al., 2006; Matsuura et al., 2004; Oh et al., 2003; Pfister et al., 2005; Tateishi et al., 2007). The reasons for this discrepancy are unclear. However, retrospective analysis data that directly sorted TERT-expressing cells from TERT-EGFP hearts (that were genetically isolated without the modification by cell culture) have shown that the majority of heart-resident TERT-positive cells could be identified via the expression of Sca-1. This implies that our findings were neither the result of intra-clonal variability nor due to contamination by cardiac fibroblasts during cell expansion. Recent report demonstrated that the expression levels of Sca-1 and KIT appear to be changed during myocardial maturation in ES cells (Wu et al., 2006).

Mesenchymal stem cells have been isolated from many tissues, including human heart (da Silva Meirelles et al., 2006; Tateishi et al., 2007). It is notable that CSCs expressed general characteristics of mesenchymal stem cells according to the analysis of cell-surface markers and partially showed the embryonic factors, as previously reported in clonal amniotic fluid-derived mesenchymal stem cells (Tsai et al., 2006). These observations indicated that the epithelial-mesenchymal transition (EMT) may occur in adult CSCs to produce proliferative precursors, which may undergo a reversible commitment into the directions of either mesenchymal or cardiac lineage, depending on the inductive conditions (Wessels and Perez-Pomares, 2004). Several reports suggest that the source of CSCs may include the neural crest (Tomita et al., 2005), primitive epicardial cells (Hay, 2005) or perivascular cells (da Silva Meirelles et al., 2006) through the EMT.

Regulating stem cell self-renewal is an essential feature of the niche where stem cells must be exposed to sufficient intrinsic-factors to maintain the proper stem cell number for the demands of tissue repair. We focused on Sca-1-mediated regulation in CSCs for the first time and found that normal Sca-1 function is associated with CSC proliferation and survival, contributing to early donor-cell engraftment and late cardiovascular differentiation, which is consistent with the prevailing view of the role of Sca-1 in the ability of hematopoietic stem cells and bone-marrow-derived mesenchymal stem cells to self-replicate (Bonyadi et al., 2003; Ito et al., 2003). Although the function of Sca-1 in skeletal muscle progenitors was not consistent with our observations in CSCs, the cell fate decision might be cell-type specific and/or age dependent (Mitchell et al., 2005). The mode of action of Lin-Kit+ cells in the heart or bone marrow has been intensively investigated in gain- (Dawn et al., 2006; Urbanek et al., 2005a) and loss-of-function experiments, and the function of these cells was validated by bone marrow reconstitution studies to completely rescue the defective cardiac repair in c-kit mutant mice after infarction (Ayach et al., 2006; Fazel et al., 2006), consistent with the lack of cardiac decompensation after pressure-overload in c-kit mutant mice (Hara et al., 2002) and our present observation indicating the rarity of c-kit+ cells in TERT-expressing CSCs.

Sca-1 was originally identified as an antigen upregulated in activated lymphocytes, and was shown to be linked to the lipid bilayer as a glycosyl phosphatidylinositol (PtdIns)-anchored protein that activates cell signaling via mediators such as Akt (Reiser et al., 1986). The proliferation of CSCs appears to be dependent on the capacity of the cells to undergo cell cycle progression through the phosphorylation of Akt in response to EGF and bFGF stimulation, as observed in neural stem cells (Groszer et al., 2006). Our observations are supported by two independent gain-of-function studies demonstrating that the nuclear-targeting of Akt leads to the rapid expansion of comparatively rare Kit+ CSCs in the postnatal heart (Gude et al., 2006), and ex-vivo transduction of Akt to bone marrow-derived MSCs can functionally repair the ischemic myocardium through the upregulation of secreted paracrine effectors (Gnecchi et al., 2006; Jiang et al., 2006). Consistent with these studies, our present study also demonstrated that the functional improvement of damaged myocardium after CSCs transplantation was attenuated by Sca-1 KD, in which new vessel formation and inhibition of myocardial apoptosis by release of angiogenic growth factors and myocyte regeneration by grafted CSCs were severely impaired.

Taken together, our results suggest that Sca-1 is expressed in the majority of intrinsic CSCs in the adult heart, which have characteristics of ES-like and mesenchymal-like cells, and implicate the role of Sca-1 in CSC maintenance and function. Sca-1-mediated signaling is important in CSC development in normal circumstances and its beneficial effect might be involved the responses to hypoxic and ischemic conditions. The cardioprotective effect of CSC transplantation that we have shown here indicates that Sca-1-mediated ligand responses may participate in the production of angiogenic and antiapoptotic paracrine effectors, consistent with recent observations demonstrating that induction of VEGF and HGF activates bone marrow-derived mesenchymal stem cells through PI 3-kinase–Akt pathway (Forte et al., 2006; Okuyama et al., 2006). It will be of interest to assess the gene expression profile in CSCs by targeting Sca-1 transcripts to identify the factors responsible for optimizing CSC therapy in heart failure.

Materials and Methods

Clonal isolation and culture of CSCs

Hearts from 6-week-old to-12-week-old GFP transgenic mice (provided by M. Okabe, Osaka University Medical School) (Okabe et al., 1997), Sca-1 KD mice or NTG mice were excised and were perfused with cold PBS to remove the blood cells. The tissues were washed twice, and aortic and pulmonary vessels were removed from the hearts. The dissected hearts were minced, and digested twice for 20 minutes at 37°C with 0.2% type II collagenase and 0.01% DNAse I (Worthington Biochemical Corp, NJ). The cells were passed through a 40-μm filter to remove the debris and were plated into 25-cm2 dishes in DMEM (Invitrogen) for 30 minutes to allow fibroblasts to adhere. The non-adherent cells were collected and size-fractionated with a 30-70% Percoll gradient to obtain single-cell suspensions by removal of mature cardiomyocytes. For clonal analysis, the resulting cell suspensions were plated in 96-well plates at 1 cell per 100 μl by the limiting dilution technique (Yoon et al., 2005) with serum-free growth medium: DMEM/F12 containing B27 supplement, 20 ng/ml EGF (Sigma), and 40 ng/ml bFGF (Promega). Wells were visually inspected 24 hours after plating to exclude those containing more than one cell per well; then, clones derived from a single cell were further cultivated. On day 14, clonally expanded CSCs from single cells were cultured in low-serum medium consisting of growth medium supplemented with 1×B27 supplement, 2% FBS, and 10 ng/ml leukemia inhibitory factor (CHEMICON). At 60-70% confluence, cells from individual clones were serially reseeded in six-well plates, 25-cm2, 75-cm2 and 175-cm2 flasks for further expansion. Hypoxic conditions were created by incubating cells at 37°C in a CO2 multi-gas incubator (ASTEC) with an atmosphere of 5% CO2 and 95% N2 for 8 hours.

CSC differentiation

For cardiac differentiation analysis, single-cell-derived CSCs were cultured in differentiation medium containing 10% FBS, insulin-transferrin-selenium, and 10 nM dexamethasone (Sigma) for 14 days. Differentiation medium consisting of DMEM/F12 supplemented with 10 ng/ml VEGF or 50 ng/ml PDGF-BB (both from R&D Systems) and 10% FBS was used to induce endothelial and smooth muscle cell differentiation for 14 days, respectively.

Construction of targeting vector and generation of transgenic mice

Full-length Sca-1 cDNA was cloned using the following primers: forward: 5′-CTCTGAGGATGGACACTTCT-3′, reverse: 5′-GGTCTGCAGGAGGACTGAGC-3′. The 404-bp ds-RNA fragment targeting the N-terminus of Sca-1 was selectively amplified and subcloned into the pDECAP vector (Shinagawa and Ishii, 2003). The plasmid encoding EGFP driven by the mouse Tert promoter was provided by N. Hole (University of Durham) (Armstrong et al., 2000) and subcloned into the human growth hormone polyadenylation sequence. Each expression cassette was released and microinjected into the pronuclei of fertilized C57BL/6 oocytes. PCR analysis of tail DNA was used to identify founder transgenic mice.

Retroviral transduction of CSCs

To track cells after injection into the infarcted myocardium, CSCs were engineered to express the bacterial lacZ reporter gene. This was done by retroviral infection with a vector (pMSCV-LacZ) encoding the lacZ gene and a puromycin resistance gene. After selection with puromycin, the transduction efficiency was evaluated by X-gal staining.

FACS analysis and cell sorting

Single-cell suspensions were stained with the following antibodies: phycoerythrin (PE)-conjugated antibodies against Sca-1, KIT, CD45, CD44, CD90, CD31, CD73, CD106, CD34, CD13, CD29, and isotype control IgG (all from BD Biosciences). Allophycocyanin (APC)-conjugated goat anti-rat IgG was used to detect rat anti-mouse CD105 (Southern Biotech). Dead cells were eliminated using propidium iodide (Sigma) and 10,000 to 50,000 events were collected per sample using a FACS Calibur flow cytometer (BD Biosciences). Bone marrow cells were flushed from the tibiae and femurs of 6-week-old to 12-week-old C57BL/6 mice and compared (with or without collagenase and filtration) (Oh et al., 2003). Single-cell suspensions were harvested from TERT-EGFP transgenic and NTG hearts as the method for CSC preparation, and the EGFP-positive cells were analyzed and sorted on BD FACSAria (Becton Dickinson).

RT-PCR and telomerase activity

Total RNA was prepared from cultured cells using TRIzol (Invitrogen) and cDNA was generated with the SuperScript III First-Strand Synthesis System (Invitrogen). PCR reactions were performed with gene-specific primers. Primer sequences are available on request. To evaluate VEGF, HGF, and IGF1 expression, cDNA was subjected to 40 rounds of amplification (ABI PRISM 7700, Applied Biosystems) with Assay-on-Demand™ primer-probes sets (Applied Biosystems). The mRNA levels were expressed relative to an endogenous control (18S RNA) and the fold-increase in the respective groups versus normoxia NTG-CSCs was calculated. Telomerase activity of single-cell-derived CSCs was measured using a TRAP assay kit, TRAPEZE (CHEMICON), as previously described (Oh et al., 2001).

Calcium transient

Cells were washed three times with 1 mM Ca2+ Tyrode's solution as previously described (Kaneko et al., 2000), additional 15 minutes incubation with 1 mM Ca2+ Tyrode's containing 1 mM probenecid at 37°C was performed to allow hydrolysis of acetoxymethyl esters within the cells. Fluorescence imaging was performed at 24°C using a fixed-stage microscope (BX50WI, Olympus, Japan) equipped with a multi-pinhole-type confocal scanning system (CSU-21, Yokogawa, Japan). Digitized fluorescence signals were analyzed with Image J software.

Western blotting

Whole protein lysates were extracted with lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.25% sodium deoxycholate, 1 mM EDTA, 1% Nonidet P-40, 1 mM PMSF and protease inhibitor (PIERCE). For phosphorylation of Akt, ERK1/2, JNK1/2 and p38, 1 mM Na3VO4 and 1 mM NaF were added. Transferred membranes were incubated with rat anti-mouse Sca-1 monoclonal antibody (clone D7, BD Biosciences), antibodies against phosphorylated Akt (S473), Akt, phosphorylated ERK1/2 (T202/Y204), ERK1/2, phosphorylated SAPK/JNK (Thr 183/Tyr185), SAPK/JNK, phosphorylated p38 MAPK (Thr180/Tyr182), p38 MAPK (all from Cell Signaling), or mouse monoclonal anti-GAPDH (Chemicon). Horseradish peroxidase (HRP)-conjugated goat anti-rat IgG, HRP-conjugated sheep anti-mouse IgG and HRP-conjugated donkey anti-rabbit IgG (Amersham Biosciences) were used as secondary antibodies.

Sample fixation and X-gal staining

Hearts were fixed in 1% paraformaldehyde, 0.2% glutaraldehyde, and 0.2% Nonidet P-40. X-gal staining was performed with the following reagents: 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.2% Nonidet P-40, and 1 mg/ml X-gal (Invitrogen). After staining, samples were post-fixed with 4% paraformadehyde and embedded in frozen OCT compound or paraffin.

Histology and immunofluorescence

Fixed cells and frozen sections were stained using the following primary antibodies: mouse anti-cardiac troponin-T (Ab1, Neo Markers), rat anti-mouse CD31 (BD Biosciences), Cy3-conjugated anti-α-SMA (Sigma), rabbit anti-p53 (FL-393, Santa Cruz) and rabbit anti-phosphorylated histone H3 (Ser10, Upstate). Secondary antibodies were conjugated to Alexa Fluor 555 or Alexa Fluor 568, and nuclei were visualized using DAPI (Molecular Probes). BrdU incorporation was examined by incubation with 10 μM BrdU for 1 hour using a detection kit (Roche). For Ki67 immunohistochemistry, we used a Vectastain ABC Elite kit (Vector Laboratories). After antigen retrieval using citrate buffer (pH 6.0) and blockage of endogenous peroxidase activity using 0.3% hydrogen peroxide, the sections were incubated with rat anti-mouse Ki67 antibody (DAKO) for 1 hour at room temperature. Then, the sections were treated with biotinylated secondary antibody followed by incubation with avidin horseradish peroxidase complex. Finally, the sections were counterstained with hematoxylin or H&E staining. Capillary density was estimated by CD31 immunostaining with a Vectastain ABC Elite kit. Apoptotic CSCs or cardiomyocytes were evaluated by the TUNEL assay in fixed cells and paraffin-embedded sections with an ApopTag kit (Chemicon). H2O2 was purchased from Wako. Images were captured with a BZ-8000 (Keyence, Japan) and IX 71 (Olympus Corporation, Japan).

Myocardial infarction and cell grafting

Ligation of the left anterior descending (LAD) coronary artery was performed in 12-week-old to 24-week-old C57BL/6 mice (Shimizu Laboratory Supplies, Japan) in accordance with the animal care and use guidelines at Kyoto University Hospital. One hour after the LAD ligation, 5×105 cells were suspended in 20 μl of PBS and injected into two sites of the infarcted border zone. In the control group, mice were sham-operated by receiving a left thoracotomy without coronary artery ligation.

Cardiac function and infarct size

Cardiac MRI studies were performed using a 7 T MR scanner, Unity Inova (Varian Inc., Palo Alto, CA) with a 25-mm home-built solenoid-type volume coil. Analysis of end-systolic and end-diastolic LV volumes and LV mass was done using an operator-interactive threshold technique, and stroke volume and cardiac output were calculated. All measurements were performed and analyzed by an individual blinded to the animal group. For in vivo determination of infarct size, end-diastolic epicardial and endocardial contours were traced on the MRI short-axis slices; only akinetic and dyskinetic segments were considered to be infarcted areas (Yang et al., 2004).

Statistics

Data are expressed as the mean ± s.e. Two-tailed Student's t test was used to compare the clonality of Sca-1 KD- and NTG-CSCs. Comparison of groups in remaining experiments was unpaired analyses using two-tailed Student's t test. Significance level was set at P<0.05 (StatView).

Acknowledgements

We thank the following investigators for their kind gifts of mice or plasmids: M. Okabe, N. Hole, S. Ishii and Y. Yoshida, A. Kosugi. We also thank M. Nishikawa for technical assistance. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, and by Grants-in-Aid from the Ministry of Health, Labor and Welfare.

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