International Journal of Cardiology 138 (2010) 40 – 49 www.elsevier.com/locate/ijcard
Differentiation and migration of Sca1+/CD31− cardiac side population cells in a murine myocardial ischemic model Simon X. Liang a,1 , Terence Y.L. Tan a,1 , Leonie Gaudry b , Beng Chong a,⁎ a
b
Center for Vascular Research, Department of Medicine and Hematology, St George Hospital, St George Clinical School, University of New South Wales, Sydney, 2052, Australia Centre for Vascular Research, Department of Hematology, The Prince of Wales Hospital, University of New South Wales, Sydney, Australia Received 9 December 2007; received in revised form 10 June 2008; accepted 8 August 2008 Available online 28 February 2009
Abstract Background: Side population cells are a rare subset of cells found in the adult heart that are highly enriched for stem and progenitor cell activity. Recent studies have suggested that Sca1+/CD31− cardiac side population cells are capable of differentiation into cardiomyocytes in vitro. However, the response of these cells to myocardial injury remains unknown in vivo. Methods: Sca1+/CD31− cardiac side population cells were isolated from mouse (C57BL6/J) hearts by FACS. These cells were labeled and delivered via an intramyocardial injection into an infracted mouse heart. The differentiation potential of these cells was determined by immunohistochemistry two weeks later. We further tested the migration potential and the relationship of SDF-1α/CXCR4 to these cells. Results: The transplanted cells were found to express cardiomyocyte or endothelial cell specific markers. Furthermore, when these cells were transplanted into non-infarct myocardium after myocardial infarction, they were found in the damaged myocardium. Consistent with their homing property, we found that SDF-1α and CXCR4 were up-regulated in the damaged myocardium and on Sca1+/CD31− cardiac side population cells respectively following myocardial infarction. We also show that SDF-1α induced migration of Sca1+/CD31− cardiac side population cells in vitro. Conclusions: Our results have suggested that Sca1+/CD31− cardiac side population cells are able to migrate into damaged myocardium from non-ischemic area of the heart and differentiate into both cardiomyocyte- and endothelial-like cells following acute ischemic injury. The SDF-1α/CXCR4 system might play an important role in the migration of these cells. © 2009 Elsevier Ireland Ltd. All rights reserved. Keywords: Stem cells; Side population cells; Myocardial ischemia; Migration; SDF-1α/CXCR4
1. Introduction The traditional view on the heart as a terminally differentiated organ has been challenged by recent studies. Accumulated evidence has suggested that there are different populations of cardiac stem or progenitor cells that reside within the adult heart. Cells expressing c-kit, Sca1, islet-1, cardiospheres and a subpopulation of cardiac side population (CSP) cells, Sca1+/CD31− CSP cells have been shown to ⁎ Corresponding author. Tel.: +61 2 91132010; fax: +61 2 91133998. E-mail address:
[email protected] (B. Chong). 1 These authors contributed equally to this publication. 0167-5273/$ - see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2008.08.032
differentiate into cardiomyocytes [1–6]. SP cells are defined by their ability to actively efflux Hoechst 33342 dye. This unique property is mediated through the action of cell surface localized p-glycoprotein multidrug/ATP-binding cassette transporter protein [7,8]. SP cells have been identified in various adult mammalian tissues and organs, including bone marrow, heart, liver, skeletal muscle, brain, kidney and lung [5,7,9–11]. Notably, SP cell populations identified in these organs, though rare, are highly enriched for stem or progenitor cell activity. Similarly, a recent study has shown that cardiac SP cells that lacked the structural cardiac genes were able to differentiate into α-actinin positive, cardiomyocyte-like cells when they were co-cultured with cardiac main population cells
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[12]. Pfister et al. have further demonstrated that a subpopulation of CSP cells, Sca1+/CD31− CSP cells are capable of differentiation into functional cardiomyocytes following co-culture with adult cardiomyocytes [5]. To date, the role of Sca1+/CD31− CSP cells in repairing injured myocardium has been yet to be established. It has been shown that CSP cells within the damaged site die after acute myocardial infarction (MI) [13]. Thus we decided to investigate whether CSP cells can mobilize from non-damaged myocardium into the damaged site and repair the damaged tissue. A recent study has shown that cardiac Sca1+ cells home to the damaged site after acute MI through the circulation system [14]. Another study, however, suggested that cardiac c-kit+ cells were able to mobilize within the myocardium [15]. In contrast, very little is known about the migration of CSP cells. In this work, we showed that Sca1+/CD31− CSP cells are capable of differentiation into cardiomyocyte- and endothelial-like cells in MI region, supporting the idea that they represent a specific cardiac stem/ progenitor cell subpopulation. Stem cells migration during inflammation and tissue damage is a complicated process and regulated by a number of growth factors, cytokines and chemokines. Chemokines and their receptors have been shown to play important roles in hematopoietic stem cells mobilization, providing directional signals for stem cell migration [16]. Most chemokine receptors bind more than one chemokine ligand and many chemokines bind more than one chemokine receptor. However, unlike others chemokines, stromal cell-derived factor-1α (SDF-1α) uniquely binds to its receptor, CXCR4 and is its only ligand [17]. The importance of SDF-1α/CXCR4 system in hematopoiesis, cardiac and neuronal development is furthermore underscored by the fetal lethality of either SDF-1α or CXCR4 knockout mice [18]. Recent studies have suggested that SDF1α/CXCR4 system is a key factor in transmigration of circulating hematopoietic stem cells [19]. Notably, SDF-1α was shown to be up-regulated in the damaged zone after myocardial infarction [20,21]. Over expression of SDF-1α in the infarcted heart could induce haematopoietic stem cell homing to infarcted myocardium [20]. However it is unclear whether the SDF-1α/CXCR4 system mediates migration of Sca1+/ CD31− CSP cells after MI. In this study, we investigated the possible role of the SDF-1α/CXCR4 system in recruitment of Sca1+/CD31− CSP cells from non-infarcted area into the infarcted myocardium after MI. 2. Materials and methods 2.1. Animals For all experiments, wild-type C57BL/6J mice (female, 8–10 weeks) were purchased from ARC (Animal Resources Centre, Perth, Western Australia). The general health status of the animals was monitored daily. The studies described herein were approved by the institutional animal ethical committee.
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2.2. Myocardial infarction murine model and cells labeling For the identification and tracking of the Sca1+/CD31− CSP cells in vivo, the cells were labeled with PKH26, a red fluorescence cell tracker dye (Sigma-Aldrich, USA) before intramyocardial injection, according to the manufacturer's instructions. This dye has been similarly used to identify and track donor cells in the MI heart [22,23]. Experimental MI was induced by left anterior descending (LAD) coronary artery ligation as previously described [24,25]. Briefly, mice were anesthetized with 0.1 ml/10 g body weight of a cocktail consisting of 1 part Hypnorm, 1 part Midazolom and 2 parts distilled water, and ventilated with a standard rodent ventilator (Harvard Ventilator). The thoracic cavity was opened by incision between the second and the third intercostals space and a rodent rib spreader was introduced to allow for visualization of the heart. MI was induced by a permanent ligation of the LAD coronary artery. PKH26 labeled Sca1+/CD31− CSP cells (approximately 150,000) in PBS were injected into the peri-infarct zone immediately following LAD coronary artery ligation. At various time points (24 h, 3, 6 and 14 days) after MI, the mice were sacrificed by cervical dislocation and the whole heart was removed. Sham-operated animals underwent an identical procedure without ligation of the LAD coronary artery. 2.3. Sca1+/CD31− CSP cell isolation, fluorescence-activated cell sorting and analysis, and magnetic enrichment of CD45− cells Cell suspension were obtained from enzyme-digested heart as previously described [5,9]. Briefly, minced cardiac tissue was digested with 0.1% collagenase B (Roche Diagnostics, Germany), 2.4 U/ml dispase II (Roche Diagnostics, Germany), and 2.5 mmol/l CaCl2 at 37 °C for 45 min and washed with HBSS (Invitrogen, USA) containing 2% fetal calf serum (FCS) (Invitrogen, USA) and 10 mmol/l HEPES (Invitrogen, USA). Removal of erythrocytes and debris was accomplished by sequential filtration through 70 µm and 40 µm filters (BD Biosciences, USA), and then a Percoll gradient (Amersham Bioscience, Sweden) [26]. Cell suspensions were incubated with Hoechst 33342 (5 µg/ml) (SigmaAldrich, USA) at 37 °C for 90 min in DMEM supplemented with 2% FCS, 10 mmol/l HEPES at a concentration of 106 nucleated cells/ml in the presence or absence of verapamil (50 mM) (Sigma-Aldrich, USA) [7]. At the completion of Hoechst staining, cells were placed immediately on ice. Cells were subsequently labeled with monoclonal rat anti-mouse antibodies, included Sca1-FITC, CD31− PE, CD45− APC, CXCR4-biotin and Sca1-biotin detected with streptavidinPE or APC (BD Pharmingen, USA) and rabbit anti-mouse polyclonal antibody vWF (Chemicon, USA) detected with a secondary FITC-conjugated donkey anti-rabbit polyclonal antibody (Chemicon, USA). For all studies, cells stained with an isotype control antibody were employed as negative
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controls, and to establish gating parameters for positive cells. All stains were performed at 4 °C for 30 min in the dark. Fluorescence-activated cell sorting and analysis were performed using FACStar Plus flow cytometer (Becton Dickinson and Co.). The Hoechst dye was excited at 350 nm. Fluorescence emission was collected with a 405/30 nm band pass filter (Hoechst blue) and a 675 nm long pass edge filter (Hoechst red). SP cells were identified as described previously [7]. A 488-nm argon laser for exciting PE and FITC, and a 633-nm HeNe laser for APC were used. Data were analyzed using BD FACSDiva™ software v4.12 (Becton Dickinson and Co.). For magnetic enrichment, cells labeled with CD45− biotin were incubated with streptavidin (STV)-conjugated microbeads (Miltenyi Biotech, Germany) and purified by two cycles of magnetic selection. Sorted cells were reanalyzed by flow cytometry. 2.4. Immunostaining The heart tissue was harvested 3 or 14 days post-MI and injection, frozen in OCT (Tissue-Tek, USA) and sectioned at 5 µm. Hematoxylin and Eosin (HE) staining was performed according to the standard method. Immunostaining was performed as previously described [11,27]. Primary antibodies used included: polyclonal rabbit anti-mouse vWF (Chemicon, USA), polyclonal rabbit anti-mouse SDF-1 (eBioscience, USA), monoclonal rat anti-mouse antibody CD31− biotin (BD Pharmingen, USA), monoclonal mouse anti-mouse antibodies; α-actinin (Sigma-Aldrich, USA) Troponin T (Labvision, UK). Visualization was achieved using conjugated secondary antibodies including goat anti-rabbit biotin (Vector Lab, USA), STV-FITC (BD Pharmingen, USA) or -Cy3 (Sigma-Aldrich, USA), Goat anti-mouse Alexa fluor 488 (Invitrogen, USA). Nuclei were counter-stained with 4, 6-diamidino-2-phenylindol dihydrochloride (DAPI) before evaluation by fluorescent microscopy. In this study, all cells stained with an isotype control antibody were employed as negative controls. All stains were performed in the dark. Slides were examined with an Olympus DP70 microscope and pictures taken with a Spot digital camera BX51. 2.5. In vivo migration study To test the migration of Sca1+/CD31− CSP cells from the non-ischemic region to the ischemic area of the infarct heart, PKH26 labeled Sca1+/CD31− CSP cells were injected into the non-ischemic right ventricle of a ligated LAD artery heart. Briefly, labeled cells suspended in PBS were first loaded into a syringe before being fitted with a 30 gauge needle pre filled with PKH26 dye to mark the site of injection in the right ventricle. The injection site was into the right ventricle with the needle directed towards the right auricle to ensure that the cells were delivered away from the ischemic area (Fig. 1). The ventricular wall surrounding the injection site was first observed to be well perfused with no pallor and contracting
Fig. 1. Schematic diagram of migration in vivo assay. Labeled Sca1+/CD31− CSP cells were transplanted into the right ventricle (non-ischemic area) of a LAD ligated infarcted mouse heart.
normally before the cells were transplanted. The heart was harvested three days post-injection and was cut at 5 µm sections commencing at the injection site in the right ventricle and ending at the left ventricle. Each section was subsequently examined for the presence of PKH26 labeled cells by fluorescent microscopy. 2.6. Chemotaxis assay The in vitro migration of Sca1+/CD31− CPS cells was assessed using transwells polycarbonate membrane (Millipore, USA) with 8 µm pores size as previously described [28]. Briefly, 50,000 cells in 50 µl serum-free DMEM were added to the upper surface of membranes in different chemotaxis chambers. SDF-1α (R and D Systems, USA) at concentrations 0 ng/ml, 50 ng/ml, 100 ng/ml and 500 ng/ml was placed in the lower compartment of each chemotaxis chamber. After 4 h incubation at 37 °C in 5% CO2 and 95% humidity, the upper surface of membranes was scraped free of cells and debris, and gently washed with phosphate-buffered saline (PBS). Membranes were then fixed and stained using DiffQuick cell fixation and staining kit (Fronine, Australia). Cell migration was measured by counting the number of cells that had migrated through pores and adhered to the lower surface of the membrane in five adjacent high-power fields (40×). To block SDF-1α signal, the cells were incubated with CXCR4 antibody (10 µg/ml) (eBioscience, USA) for 30 min before being added to the upper surface of membranes in the chambers that contained SDF-1α at concentration 500 ng/ml. Experiments were performed in triplicate. Cell numbers of experimental groups were expressed as fold increase compared to control groups (SDF-1α 0 ng/ml), respectively.
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2.7. Statistics analysis
Table 1 Surface markers expressed on CSP cells.
Data are presented as mean ± SD. Experimental data were compared using Student's t-test. Results were considered statistically significant when p b 0.05.
Surface markers
CSP cells (%)
Sca1+/CD31+ Sca1+/CD31− Sca1−/CD31− Sca1−/CD31+ CD45+
66.3 ± 2.1 11.2 ± 1.3 21.5 ± 2.4 ≤1 1.7 ± 0.9
3. Results 3.1. Sca1+/CD31− CSP cells differentiation into cardiomyocyte-like and endothelial-like cells after acute MI
N = 3.
A recent study has shown that Sca1+/CD31− CSP cells that lacked cardiac contractile proteins, such as α-actinin and Troponin T, are capable of differentiation into mature and functional cardiomyocytes in vitro [5]. In this study we wanted to determine whether this same population of cells could undergo differentiation into cardiomyocytes and other types of cells in vivo after acute MI by using a mouse MI model. Using Hoechst 33342 staining, a Hoechst-low cell population was detected in the adult mouse heart (Fig. 2A). Hoechst efflux in this side population of cells was completely inhibited by treatment with a specific inhibitor verapamil (Fig. 2A). CSP cells were largely negative for CD45 (Fig. 2B, C). But they widely expressed the stem cell antigen Sca1 (Fig. 2D). Surface markers expressed on CSP cells were shown in Table 1 and Fig. 1D. These results were consistent with
previous studies by others [5,8]. Notably, expression of vWF was very rare on the Sca1+/CD31− CSP cells (1.1 +0.3%, Fig. 2E, F). Based on their immunophenotype, Sca1+/CD31− CSP cells were isolated from cell suspension of adult mouse hearts by FACS. The purity of Sca1+/CD31− SP cells isolated from a single sort was more than 95% (data not shown). Following their isolation, Sca1+/CD31− CSP cells (100,000– 150,000) were labeled with PKH26 and injected into the perinfarct region immediately after coronary artery ligation. After two weeks, the heart was harvested and sections were taken from the peri-infarct and infarct region. Implanted cells labeled with PKH26 were observed to be distributed in the peri-infarct and infarct region (Fig. 3A–D). Some of the implanted cells located at the peri-infarct region were closely aligned with existing surviving cardiomyocytes and showed similar staining with the neighboring, surviving cardiomyocyte by HE staining (Fig. 3D), indicating that some of the implanted Sca1+/CD31− CSP cells might differentiate into cardiomyocyte-like cells in an ischemic environment. To further confirm this, cardiac-specific markers including Troponin T and cardiac α-actinin were used to identify those implanted cells. Our results have shown that some of the transplanted Sca1+/ CD31− CSP cell expressed Troponin T (Fig. 4A–D) and cardiac α-actinin (Fig. 4E–H) after acute MI. However the fraction of the transplanted Sca1+/CD31− CSP cells that appeared to differentiate into cardiomyocyte-like cells (αactinin positive) was less than 4%. Interestingly, we also found that some of the transplanted Sca1+/CD31− CSP cells expressed endothelial-specific markers including vWF (Fig. 4I–L) and CD31 (Fig. 4M–P) as well in the ischemic area. The fraction of the implanted Sca1+/CD31− CSP cells that differentiated into endothelial-like cells (vWF positive) were approximately 20%. Taken together, these observations, therefore have suggested that Sca1+/CD31− CSP cells are able to differentiate into cardiomyocyte- and endothelial-like cells in an ischemic environment after acute MI. The percentages of cardiomyocyte- and endothelial-like cell differentiation were thought to be an underestimation due to the limitations of the PKH26 cell tracker dye. The PKH26 dye might be diluted or lost in the cell cytoplasma when cells undergo cell division and differentiation. Thus, transplanted cells that had undergone mitotic division or have differentiated into larger differentiated cardiomyocytes might have lost their ability to be identified due to the dilution of the fluorescent dye.
Fig. 2. Isolation of CSP cells from the adult mouse heart. (A) CSP (left lower box) cells were identified based on Hoechst staining that was blocked by verapamil (right upper box). CSP cells represented approximately 1.0% of the total heart cells. (B) Isotype control of CD45 for CSP cells. (C) CD45+ CSP cell represented approximately 1.7% of the total CSP cells. (D) Sca1+/CD31− CSP cells represented approximately 11% of the total CSP cells. (E) Isotype control of vWF for CSP cells. (F) Sca1+/CD31− CSP cells rarely expressed vWF (1.1± 0.3%). Data represent means ± SD. (N = 3).
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Fig. 3. Transplanted Sca1+/CD31− CSP cells were found in both MI and the border of MI region. (A) Transplanted labeled Sca1+/CD31− CSP cells (red) were found in infarcted mouse heart. (B) Phase contrast image of the same section. (C) Overlay of A and B images showed that labeled CSP cells (green) located in both MI and the border of MI region (encircled). (D) HE staining of the same section. (Scale bar, 100 µm).
3.2. Migration of Sca1+/CD31− CSP cells from non-ischemic region to ischemic area within myocardium after MI A previous study has showed that cardiac Sca1+ cells were able to home to peri-infarct zone of the infarcted heart but not the non-ischemic area and the right ventricle after they were injected intravenously [14]. To determine whether Sca1+/CD31− CSP cells are able to migrate from non-MI area to MI region within myocardium after acute MI, we developed a migration assay as described in the method section. Labeled Sca1+/CD31− CSP cells were injected into the remote area of the right ventricle of a LAD artery ligated heart. Three days later, transplanted PKH26 labeled cells were found in the ischemic region of the left ventricle that
was characterized by the presence of intensive infiltrate of inflammatory cells and the disruption of cardiac fibers (Fig. 5A–D). The injection site was identified with the visible needle tract by the dye staining the cardiac fibers both at the tip of the injection site and at the point of entry into the myocardium due to the earlier priming of the needle (Fig. 5E–H). In comparison with the ischemic region, the injection site was surrounded by regular and intact cardiac fibers. The absence of obvious infiltrate of inflammatory cells in the injection site further conformed that the site was in the non-ischemic area of the right ventricle (Fig. 5H, I). This result suggests that Sca1+/CD31− CSP cells are able to mobilize from the non-ischemic region to the ischemic region within myocardium following acute MI.
Fig. 4. Transplanted Sca1+/CD31− CSP cells differentiate into cardiomyocyte- and endothelial-like cells after 14 days in MI region. Immuno-fluorescence staining of cardiac-specific markers α-actinin (A) and Troponin (E), and enodthelial cell markers vWF (I) and CD31 (M). They were green. (B), (F), (J) and (N) Transplanted Sca1+/CD31− CSP cells (red). (C), (G) (K) and (O) Nuclei (blue) were counter-stained by DAPI. (D), (H), (L) and (P) were merged images of A, B, C, and E, F, G and I, J, K and M, N, O respectively. (Scale bars, 10 µm).
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Fig. 5. Migration of Sca1+/CD31− CSP cells from the non-ischemic region into the infarcted area. (A) Migrated Sca1+/CD31− CSP cells (red) into the infarcted area. (B) Phase contrast of section A. (C) Merged image of A and B. (D) HE staining of same section shown in C showing migrated CSP cells into the infarcted region as seen by the disruption of cardiac fibers and inflammatory cell infiltration. Two labeled intact cells (boxed), as an example, have been indicated on the left lower corner. (Scale bar, 50 µm for A, B, C and D). (E) PKH26 dye (red) staining the myocardium at the tip of the injection site (arrow with dot) and at the point of entry into the myocardium (arrow). (F) Phase contrast of section E. (G) Merged image of E and F. (H) HE staining of the same section showing the preservation of cardiomyocyte architecture and little inflammatory cells at the injection site. (Scale bar, 100 µm for E, F, G and H). (I) Magnified view of boxed area showing the tip of the injection site. (Scale bar, 25 µm).
3.3. Up-regulation of the expression of CXCR4 on Sca1+/ CD31− CSP cells in post-ischemic hearts Studies have reported that CXCR4 plays an important role in cell migration in various cell types that includes hematopoietic and non-hematopoietic cells [29,30]. Recently it was shown that cardiac progenitor cells derived from the bone marrow expressed CXCR4 and these cells were chemo attracted to the infarcted heart in an SDF-1α/CXCR4 dependent manner [31]. We therefore wanted to investigate whether there was an up-regulation of CXCR4 on Sca1+/CD31− CSP cells in the infarcted heart as they were able to mobilize from the non-ischemic area to the ischemic region within the myocardium following acute MI. Due to the numbers of CD45+ SP cells and the expression of CXCR4 on these cells were increased following acute MI (3 days) (Fig. 6A,B,C,D), six colour analyses (Hoechst red, Hoechst blue, Sca1, CD31, CXCR4 and CD45) was required. However, there was technical difficulty for six colour analyses due to rare numbers of CSP cells. In order to enrich CD45− cells in post-MI heart suspension, CD45+ cells were depleted by magnetic separation. The purity of CD45− CSP cells was 98% in post-MI heart suspension, which was confirmed by flow cytometry
analyses (Fig. 6E, F). Based on multiple colour flow cytometry analyses, our results showed that the expression of CXCR4 was significantly increased on Sca1+/CD31− CSP cells after acute MI. Comparing to the non-ischemic heart (Fig. 7A–D), there was more than 3-fold increase of the expression of CXCR4 on Sca1+/CD31− CSP cells in 3 days MI heart (Fig. 7E–I). Notably, as with non-CD45 depleted CSP cells and other SP populations, the detection of CD45− depleted CSP cells was blocked by verapamil (50 mM), confirming that CD45 depletion did not change the SP phenotype (Fig. 7G). In addition, there was little change seen in the proportion of Sca1+/CD31− CSP cells in CSP cells between normal and damaged hearts (Fig. 7B, F). 3.4. Up-regulation of expression of SDF-1α in ischemic myocardium Recent studies have shown that SDF-1α gene expression was up-regulated after acute MI [20,21]. In this study, we investigated whether the expression of SDF-1α was increased in ischemic myocardium. We found that the expression of SDF-1α was able to be detected only in ischemic myocardium by immunostaining (Fig. 8A–D). This result
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4. Discussion
Fig. 6. Increase of CD45+ SP cell numbers and increase of expression of CXCR4 on CD45+ SP and CSP (CD45−) cells after acute MI (3 days), and enrichment of CSP cells. (A) and (B) CSP (left lower box) cells were gated for analysis in normal and MI heart. (C) CD45+ SP cell were less 4%, and only 6% and less 1% of CSP and CD45+ SP cells were CXCR4+ respectively in normal heart. (D) Numbers of CD45+ SP cells were increased to approximately 34%, and the expression of CXCR4 on CSP and CD45+ SP cells was increased to approximately 23% and 22% respectively in 3 days MI heart. (E) CD45− CSP cells were 66.3% before magnetic sorting. (F) Purification of CD45− CSP cells reached 98% after magnetic sorting, confirmed by flow cytometry analysis.
was similar to another study recently reported [32]. In contrast, the expression of SDF-1α was undetectable in remote and non-ischemic myocardium of the same heart (Fig. 8E–H). 3.5. SDF-1α induces chemotaxis of Sca1+/CD31− CSP cells via CXCR4 SDF-1α induced chemotaxis has been reported in various types of cells via its receptor CXCR4. We, therefore, tested the ability of SDF-1α to induce chemotaxis in this cell population in vitro. Utilizing a chemotaxis assay, we detected a significant migratory response towards the SDF-1α gradient (50 ng/ml, 100 ng/ml and 500 ng/ml) in this cell population when compared to the control (SDF-1α 0 ng/ml) (Fig. 9). The chemotactic response to SDF-1α was abolished by the blockage of CXCR4 with an anti-CXCR4 antibody (Fig. 9), which further confirmed the role of SDF-1α in this migratory response.
There have recently been several reports of the existence of endogenous cardiac stem cell like population of cells [3,4,33–35]. The differentiation of Sca1+/CD31− CSP cells into functional cardiomyocytes in vitro has been demonstrated by Pfister et al. [5]. However the response of these cells to myocardial injury remains unknown. In our present study we found that Sca1+/CD31− CSP cells were able to differentiate into cardiomyocyte-like and endothelial-like cells in the infarcted region. Despite this, Sca1+/CD31− CSP cells are able to differentiate into cardiomyocyte- and endothelial-like cells, however, it is possible that the majority of these cells in the infarcted region might not all survive and die by necrosis and apoptosis. Stem and progenitor cells migrating from the surrounding and non-ischemic area, therefore, may more efficiently repair the damaged tissue. In our present study, one of the important finding is that Sca1+/CD31− CSP cells are able to migrate to ischemic myocardium from nonischemic myocardium following acute MI. However, the mechanism of Sca1+/CD31− CSP cell migration is still unclear. The cell migration could be through micro-circulation or the myocardial interstitium or both. Studies in rat and mouse models have showed that cardiac derived c-kit+ cells in rat and Sca1+ cells in mouse are able to home to damaged myocardium through circulation [4,36] as well as through the myocardial interstitium within the myocardium [15]. These studies indicated that the migration of stem cells to the damaged myocardium could be dependent and independent from the circulation. Recent studies have suggested that the interaction between SDF-1α and its receptor CXCR4 plays an important role in mobilization and homing of hematopoietic and non-hematopoietic cells [19,37–39]. Several studies have shown that the gene expression of SDF-1α was significantly increased in the damaged myocardium after MI. The up-regulation of SDF-1α led to homing of BMC to the infarcted area of the heart [20,21]. Consistent with these studies, the expression of SDF-1α was detected in some damaged cardiomyocytes by immunofluorescence staining in MI heart in our current study. The expression of CXCR4 was also markedly increased on Sca1+/ CD31− CSP cells after acute. Importantly, our results have demonstrated that SDF-1α significantly induced migration of Sca1+/CD31− CSP cells in vitro, indicating that SDF-1α/ CXCR4 system may play an important role in migration of Sca1+/CD31− CSP cells after acute MI. After showing migration of Sca-l+/CD31− CSP cells into the infarcted area of heart and differentiation of these cells into cardiomyocyte-like and endothelial-like cells, improvement of myocardial function would be expected. However, we have not carried out echocardiographical and other studies to demonstrate improved ventricular function. This is a weakness of our present study. In conclusion, in this paper we describe the use of a mouse MI model to further demonstrate that Sca-l+/CD31−
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Fig. 7. Expression of CXCR4 was increased on Sca1+/CD31− CSP cells in CD45+ depleted heart suspension cells after MI (3 days). (A) CSP cells were gated for analysis in normal heart. (B) Sca1+/CD31− CSP cells (10.7%) were further gated for analysis of expression of CXCR4 in normal heart. (C) Isotype control for CXCR4. (D) Expression of CXCR4 was approximately 6% in normal heart. (E) CSP cells were gated for analysis in 3 days Ml heart. (F) Sca1+/CD31− CSP cells (10.1%) were further gated for analysis of expression of CXCR4 in 3 days MI heart. (G) CSP cells (left box) were blocked by verapamil after CD45+ cell depleted in 3 day MI heart. (H) The expression of CXCR4 was 20.3% in MI heart. (I) The expression of CXCR4 on Sca1+/CD31− CSP cells of MI heart was at least 3-fold higher than on Sca1+/CD31− CSP cells of normal heart. The increase of expression of CXCR4 on Sca1+/CD31− CSP cells was significant after MI. Data represent means ± SD. (N = 4).
CSP cells are able to differentiate into cardiomyocyte-like and endothelial-like cells within the damaged myocardium. These cells, therefore, are highly enriched for stem and progenitor cell activity. Importantly, our data have shown that a salient feature in these cells is their ability of migration from non-ischemic area into the ischemic region within myocardium after acute MI. Our results have further suggested that SDF-1α expressed by damaged myocardium might be one of the important factors that induced the migration of Sca1+/CD31− CSP cells by the specific SDF-
1α/CXCR4 interaction towards the damaged region of the infarcted heart. This provides a means for further study of the mechanism of migration of these cells within myocardium after acute MI. Taken together, our results have suggested that Sca1+/CD31− CSP cells are able to migrate into damaged myocardium from non-damaged and remote myocardium, and differentiate into cardiomyocyte-like and endothelial-like cells following acute MI. Thus, these cells may play important roles in repairing and healing the damaged cardiac tissue after MI. Understanding and enhancing such processes may hold
Fig. 8. Expression of SDF-1α was detected by immuno-fluorescence staining at the border area of MI hearts. (A) α-actinin staining marking the border of the infarcted heart. (B) SDF-1α staining (C) Nuclei were counter-stained with Dapi. (D) Merged image of A, B and C showed that there is increased expression of SDF at the border of the infarcted heart (yellow). (Scale bar, 50 µm). (E) α-actinin staining of non-ischemic area of MI heart. (F) Absence of SDF-1α staining in non-ischemic area. (G) Nuclei were counter-stained with Dapi. (H) Merged image of E, F and G showed that the expression of SDF-1α was undetectable in nonischemic area of MI heart. (Scale bars, 50 µm).
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Fig. 9. SDF-1α induced chemotactic response in Sca1+/CD31− CSP cells. Chemotaxis assay showed that chemotactic responses of Sca1+/CD31− CSP cells to SDF-1α at every concentration (50 ng/ml, 100 ng/ml and 500 ng/ml, shown as black columns) were significantly above control (SDF-1α 0 ng/ml, shown as white column). The chemotactic response of Sca1+/CD31− CSP cells to SDF-1α at concentration 500 ng/ml was blocked by a CXCR4 antibody (shown as grey column). (N = 3).
enormous potential possibilities for therapeutic myocardial regeneration in ischemic heart disease. Role of funding source This work was supported by Australia Research Council (DP0558687 to B.C.) and Hematology Research Fellowship, University of New South Wales (T.YL.Tan). Conflict of interests None declared. Acknowledgments We are grateful to Susan Wan and Gavin Mackenzie for their excellent technical work. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [40]. References [1] Hierlihy AM, Seale P, Lobe CG, Rudnicki MA, Megeney LA. The post-natal heart contains a myocardial stem cell population. FEBS Lett 2002;530(1–3):239–43. [2] Urbanek K, Torella D, Sheikh F, et al. Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure. PNAS 2005;102(24):8692–7. [3] Beltrami AP, Barlucchi L, Torella D, et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114 (6):763–76. [4] Oh H, Bradfute SB, Gallardo TD, et al. Cardiac progenitor cells from adult myocardium: Homing, differentiation, and fusion after infarction. PNAS 2003;100(21):12313–8.
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