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Keywords:

  • c-kit;
  • Heart;
  • Infarction;
  • Cardiac stem cell

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Cumulative evidence indicates that myocardium responds to growth or injury by recruitment of stem and/or progenitor cells that participate in repair and regenerative processes. Unequivocal identification of this population has been hampered by lack of reagents or markers specific to the recruited population, leading to controversies regarding the nature of these cells. Use of a transgenic mouse expressing green fluorescent protein driven by the c-kit promoter allows for unambiguous identification of this cell population. Green fluorescent protein (GFP) driven by the c-kit promoter labels a fraction of the c-kit+ cells recognized by antibody labeling for c-kit protein. Expression of GFP by the c-kit promoter and accumulation of GFP-positive cells in the myocardium is relatively high at birth compared with adult and declines between postnatal weeks 1 and 2, which tracks in parallel with expression of c-kit protein and c-kit-positive cells. Acute cardiomyopathic injury by infarction prompts increased expression of both GFP protein and GFP-labeled cells in the region of infarction relative to remote myocardium. Similar increases were observed for c-kit protein and cells with a slightly earlier onset and decline relative to the GFP signal. Cells coexpressing GFP, c-kit, and cardiogenic markers were apparent at 1–2 weeks postinfarction. Cardiac-resident c-kit+ cell cultures derived from the transgenic line express GFP that is diminished in parallel with c-kit by induction of differentiation. The use of genetically engineered mice validates and extends the concept of c-kit+ cells participating in the response to myocardial injury.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Throughout the life span of an organism, stem cells repair and regenerate damaged tissue. Stem cells are characterized as unspecialized, multipotent cells with the ability to self-renew [1]. Adult stem cells, such as cardiac stem cells, are few in number and are generally located in discrete regions within tissues [2]. Moreover, these cells remain in a predominantly dormant state until stimulated by injury or disease [3, 4]. The identification of pluripotent stem cells in adult tissues, including the human myocardium [5], rests with stem cell markers originally identified to mark cells of the hematopoietic lineage [6]. c-kit is a receptor tyrosine kinase found on stem cells, mast cells, germ cells, and melanocytes. The c-kit ligand, stem cell factor, is vital for normal hematopoiesis, melanogenesis, gametogenesis, and the growth and differentiation of mast cells [7, [8], [9]10]. c-kit-expressing bone marrow-derived stem cells differentiate into blood or vascular endothelial cells and play a crucial role in the amplification and mobilization of progenitor cells [11]. Thus, c-kit would seem an ideal candidate protein for identification of stem cells or precursor cells.

The recent paradigm shift in cardiac biology toward the heart as an organ capable of self-renewal and repair has created new opportunities for treatment of heart disease. However, concerns persist in relation to the nature of stem cells, their authenticity, and their role in cardiac repair, with c-kit protein as a predominant marker (along with Sca-1) to identify these putative cardiac stem cells [4, 12, [13], [14], [15], [16], [17], [18], [19], [20], [21]22]. Genetic approaches involving cell tagging or in vivo-mediated recombination in transgenic mice have been attempted to gain additional insight, but such investigations have yielded mixed findings, and the debate continues [21, 23, [24]25]. Thus, a pressing need exists for additional studies to unambiguously demonstrate the role of c-kit+ cells in cardiac biology.

The goal of this study was to clarify the role of c-kit+ cellular responses in the myocardium using genetic engineering techniques. Transgenic mice expressing green fluorescent protein (GFP) under control of the c-kit promoter were created to allow for tracking of the c-kit+ cell population. This straightforward experimental design allowed us to use conventional immunohistochemistry to validate the identity of c-kit-expressing cells using both the transgene and c-kit protein expression as markers. Evidence presented shows the association of c-kit expressing cells in postnatal development, response to myocardial infarction, and commitment of these cells to cardiogenic lineages, thereby supporting a role for c-kit+ cells in myocardial growth and repair following injury.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Transgenic Animal Models

Transgenic mouse models were housed in similar conditions in the research animal facility at San Diego State University. The c-kit-GFP reporter cDNA constructs were described previously (a generous gift from Cairns et al. [26]). Founders were injected into 0.5-day post coitum embryos, and founders from clone 2 were analyzed for GFP expression. Bone marrow cells were screened using Western blot analysis and immunocytochemical techniques to determine the best coincidence of endogenous c-kit and GFP transgene expression, and the line exhibiting the highest coincidence of c-kit and GFP expression was chosen for further analysis. All experimental procedures were designed in accordance with NIH guidelines and approved by the San Diego State University and Administration Medical Center Institutional Animal Care and Use committees.

Myocardial Infarctions

Surgeries were performed on 8–12-week-old transgenic mice. Myocardial infarctions were carried out under isoflurane anesthesia (Halocarbon Laboratories, River Edge, NJ, http://www.halocarbon.com) by ligating the left descending coronary artery with 8-0 suture (Ethicon, Somerville, NJ, http://www.ethicon.com). To confirm adequate ligation, cyanosis and akinesia of the affected left ventricle were observed. Sham operations were executed by opening and closing the chest. Mice were sacrificed under chloral hydrate sedation at the specific time points noted in the text. Hearts were arrested in diastole by catheterizing the abdominal aorta and flushing the heart with a high-potassium/cadmium solution. Neutral buffered formalin (NBF) fixative was perfused into the coronary arteries at systolic pressure while the left ventricle was filled with NBF at diastolic pressure. Retroperfused hearts were then removed from the chest cavity and placed in NBF overnight, followed by processing for paraffin embedding using an automated tissue processor.

Immunofluorescence Microscopy

Paraffin sections of mouse hearts cut at 5 μm were deparaffinized in xylene and rehydrated in a series of graded alcohols to distilled water. Antigen retrieval was performed in 10 mmol/l citrate, pH 6.0, using a 1,100-W microwave oven for 2 minutes at high power and 15 minutes at 50% power. The slides were allowed to cool for 10 minutes at room temperature and 15 minutes at 4°C and then washed in three changes of double-distilled water (ddiH2O) and quenched with 3% hydrogen peroxide in 1× TN buffer (150 mmol/l NaCl, 100 mmol/l Tris, pH 7.5) for 20 minutes to block endogenous perioxidase activity. Slides were then washed in several changes of ddiH2O, followed by one change in 1× TN buffer, and blocked for 1 hour in TNB (1× TN buffer containing 0.5% Blocking Buffer; proprietary formula from the Tyramide Signal Amplification kit [PerkinElmer Life and Analytical Sciences, Waltham, MA, http://las.perkinelmer.com]). Slides were incubated at 4°C overnight with primary antibodies diluted in TNB. Slides were then washed three times for 5 minutes per wash in 1× TN, incubated 2 hours at room temperature in the dark with species-specific secondary antibodies conjugated to either fluorescein isothiocyanate (FITC), CY3, or CY5 fluorophores in TNB. After secondary labeling, slides were washed three times in 1× TN for 5 minutes per wash, nuclei were stained for 20 minutes with ToPro-3 iodide (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) at 1/10,000 in 1× TN, and slides were mounted for viewing in Vectashield medium (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Detection of some antigens required signal amplification using horseradish peroxidase (HRP)-conjugated antibodies or streptavidin followed by reaction with a fluorescent tyramide substrate. When two such antigens were localized on the same slide, sequential signal amplification was required; the normal protocol for indirect immunofluorescence was followed until secondary antibodies were applied. Detection of c-kit, GFP, or GATA4 antigens was carried out using secondary antibodies to swine anti-goat HRP (1:200), donkey anti-rabbit biotin (1:3,000), and donkey anti-rabbit biotin (1:3,000), respectively. Amplification protocols followed manufacturer recommendations, with tyramide FITC and tetramethylrhodamine B isothiocyanate substrate diluted 1:50 in amplification diluent. Primary and secondary antibodies used are shown in supplemental online Tables 1 and 2, respectively. Confocal images were acquired using a Leica TCS SP2 confocal microscope (Leica, Heerbrugg, Switzerland, http://www.leica.com). All slides were treated and scanned identically using the same settings, and postacquisition processing of images was consistent for all results within each experimental set.

Immunoblot Analysis

Western blot analysis was performed as described [27]. Equal amounts of protein samples were separated on a 4%–12% Bis-Tris mini-gel (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and transferred to polyvinylidene difluoride membrane. Membranes were blocked for 1 hour with 10% milk in TBST (1× Tris-buffered saline/0.1% Tween) and then probed with primary antibody overnight. The next day, the blots were washed three times, 10 minutes each time, with TBST buffer and then incubated in secondary antibody. For detection of c-kit, goat anti-c-kit (1:200; R&D Systems Inc., Minneapolis, http://www.rndsystems.com) was used as a primary antibody, followed by alkaline phosphatase-conjugated donkey anti-goat (1:5,000; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com). GFP was detected using a rabbit anti-GFP primary (1:1,000; Molecular Probes, Eugene, OR, http://probes.invitrogen.com), followed by alkaline phosphatase conjugated donkey anti-rabbit (1:5,000; Jackson Immunoresearch Laboratories) secondary antibody. Glyceraldehyde-3-phosphate dehydrogenase (1:3,000; Chemicon, Temecula, CA, http://www.chemicon.com) was used to standardize the protein loading. Blots were imaged with a Typhoon 9410 scanner, and signals were quantitated with Imagequant 5.2 software (GE Healthcare, Little Chalfont, U.K., http://www.gehealthcare.com). All quantitation was based on standardization to loading controls.

c-kit+ Cell Counts

Hearts harvested at specific developmental time points were paraffin-embedded, and three sections per block were cut and stained for c-kit and GFP. Endogenous c-kit-positive, c-kit and co-GFP-positive, and GFP-positive cells were counted throughout the left and right ventricle for each section. Area measurements for the left and right ventricles were performed using the Leica LCS Lite software.

Bone Marrow Counts

Bone marrow cell samples were collected from femurs of 10–12-week-postnatal c-kit-GFP transgenic mice by flushing with sterile phosphate-buffered saline (PBS) into a 15-ml conical tube. The bone marrow suspension was pelleted by gentle centrifugation, supernatant was aspirated, and cells were resuspended in CyGel fixing medium (Biostatus Ltd., U.K.). Resuspended cell smears were allowed to dry, fixed in 4% paraformaldehyde for 10 minutes, permeabilized in 0.1% Triton X-100/0.1 M glycine/PBS for 5 minutes, and blocked in 10% house serum/PBS. Primary antibodies against c-kit and GFP were applied in blocking buffer and allowed to incubate overnight at 4°C. CY3 and FITC-conjugated secondary antibodies were applied the following day for 2 hours at room temperature, and then ToPro-3 iodide was added to the final wash to label nuclei. Slides were coverslipped using Vectashield (Vector Laboratories). For quantitative analyses, 500 cells were counted per animal (n = 3). Cells with a distinct nucleus and either endogenous c-kit, GFP, or both c-kit and GFP were counted. Total nuclei were used to compare the number of cells in each group.

Cardiac c-kit+ Cell Isolation

Adult c-kit+ cells were isolated from 8–12-week-old mice as described by Beltrami et al. [3], with modifications. Briefly, mice were anesthetized using ketamine-xylazine solution, the chest was opened, and the aortic arch was isolated gently with sterile cotton-tipped swabs. Curved forceps were used to hold the aorta, and a 6-0 suture (Ethicon) was slid underneath. A small alligator clip was clamped to the base of the aorta distal to the aortic arch. A small incision above the alligator clip, high up on the aortic arch was created to insert a cannula. Prior to aortic cannulation, buffer was oxygenated and warmed to 37°C–40°C (all solutions made as described by Beltrami et al. [3]). The cannula was secured with suture, basic buffer was slowly perfused through the heart, and the heart was carefully removed from the mouse. The cannula with the suspended heart was attached to the Radnoti EZ Myocyte/Langendorff Isolated Heart System (Radnoti Glass Technology Inc., Monrovia, CA, http://www.radnoti.com) and perfused with basic buffer for 5 minutes, followed by perfusion digestion with collagenase II solution while the heart was submerged in the buffer for up to a maximum of 10 minutes. Upon completion of digestion, as assessed by firmness of the myocardium by touch, the heart was removed from the cannula and placed in a sterile 15-ml conical tube in buffer at 37°C. In a sterile hood, the heart was carefully minced into small pieces, and the mixture of heart and incubation buffer was carefully pipetted up and down using a sterile pipette to dissociate the cells. Larger chunks of tissue settled to the bottom of the tube while on ice followed by centrifugation (1 minute, 100g, 4°C). The supernatant was passed through a 30-μm filter (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com), and remaining cells were counted and then centrifuged (10 minutes, 600g, 4°C). The second supernatant was removed, and the pellet was resuspended in 80 μl of washing buffer and 40 μl of CD117-conjugated Miltenyi Biotec Microbeads and incubated for 20 minutes on a rocker at 4°C. One milliliter of washing buffer was then added, and the cells were resuspended. The solution was centrifuged (10 minutes, 600g, 4°C), and the supernatant was discarded, with remaining cells passed over the Miltenyi Biotec MiniMACS sorting column to select for c-kit+ cells. Cells selected by the column for c-kit+ expression were resuspended in 10% embryonic stem cells fetal bovine serum (FBS) cardiac stem cell medium (Dulbecco's modified Eagle's medium and Ham's F-12 medium [ratio, 1:1], insulin-transferrin-selenium, leukemia inhibitory factor [10 ng/ml], basic fibroblast growth factor [10 ng/ml], epidermal growth factor [20 ng/ml], 10% embryonic stem cells FBS, penicillin-streptomycin-glutamine), plated on a 35-mm cell culture-grade dish, and placed in an incubator at 37°C. Medium was changed 5–7 days later, and the resulting adherent cultures were passaged using standard trypsinization techniques.

Statistical Analyses

Comparisons of the results of time course analyses were assessed for significance by analysis of variance with Tukey's post hoc analyses. Significance determinations for single time points or individual sample sets were analyzed by Student's two-tailed t tests.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

GFP Colocalizes with a Subpopulation of c-kit-Positive Cells in the c-kit+-GFP Transgenic Mouse Line

GFP expression in the cells of c-kit-GFP transgenics was verified by analyses of bone marrow cell preparations harvested from 10–12-week-old mice. Bone marrow cells were stained with c-kit and GFP, and the number cells expressing c-kit only, GFP only, or coincident c-kit and GFP was counted by confocal microscopy (Fig. 1). c-kit was expressed on 6.3% of all the cells, of which 32% (2.02%) were also GFP+ (Fig. 1, top). An even smaller subpopulation of 0.83% of total bone marrow cells were GFP+, which we attribute to cells that may have lost c-kit expression but retain the GFP transgene for a prolonged time period. Subsequently, we stained the bone marrow for another hematopoietic marker, Sca-1, and colocalized it with c-kit and GFP. The results indicate that approximately half of the bone marrow cells that have the phenotype of GFP+/c-kit− are GFP+/c-kit−/Sca-1+ (supplemental online Fig. 2). There were significantly more c-kit+ cells than dual c-kit+/GFP+ or the relatively rare c-kit−/GFP+ cells (Fig. 1, top; p < .001). Thus, the GFP transgene is expressed predominantly in the appropriate c-kit+ population, although only a subset of c-kit+ cells exhibit transgene protein accumulation.

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Figure Figure 1.. GFP colocalizes with a subpopulation of c-kit-positive bone marrow cells in c-kit-GFP transgenic mice. Top: Histogram representing the number of c-kit-positive, c-kit + GFP-positive, and GFP-positive bone marrow cells (*, p < .05; **, p < .001) (n = 4). Bottom: Confocal scans show representative fields of adult bone marrow cells labeled with antibodies to c-kit (red), GFP (green), and ToPro nuclear stain (blue), as shown in single-channel scans along the left of each color overlay. Abbreviation: GFP, green fluorescent protein.

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Expression of c-kit Is Elevated in Postnatal Myocardial Development

The myocardium undergoes rapid postnatal growth and development within the first 1–2 weeks after birth [28, [29], [30], [31]32], so the role of c-kit+ cells during this time period relative to young adult samples at 10 weeks of age was assessed by confocal microscopy. c-kit+ cells localized by immunohistochemistry were counted relative to area measured in hearts at 2 days, 1 week, 2 weeks, and 10 weeks (postnatal age) (Fig. 2). As previously performed for bone marrow quantitations, cells groups expressing c-kit only, GFP only, and c-kit coincident with GFP were counted versus area measurements. c-kit+/GFP− cells were significantly more numerous at 2 days (11.57 ± 1.9 cells per mm2) or 1 week (16.97 ± 1.9 cells per mm2) relative to the 2-week (0.3 ± 1.9 cells per mm2) or 10-week (0.0 ± 0.0 cells per mm2) time points (Fig. 2B; p < .05). Similarly, c-kit+/GFP+ cells were significantly more numerous at 2 days (7.16 ± 2.0 cells per mm2) or 1 week (7.12 ± 1.2 cells per mm2) relative to the 2-week (0.3 ± 1.9 cells per mm2) or 10-week (0.0 ± 0.0 cells per mm2) time points (Fig. 2B; p < .05). No significant difference was observed in any of the groups between 2 days and 1 week. Persistence of rare c-kit−/GFP+ cells at early time points (0.77 ± 0.3 cells per mm2 at 2 days or 0.20 ± 0.2 cells per mm2 at 1 week) corroborates findings from bone marrow analyses (Fig. 1), suggesting that this population results from GFP that is retained after c-kit protein expression is downregulated. Microscopy results were corroborated by immunoblot analyses for both c-kit and GFP protein performed on lysates of myocardial samples prepared from hearts of c-kit-GFP transgenic mice at 2 days, 1 week, 2 weeks, and 10 weeks post-birth. c-kit protein levels were highest in 2-day-old and 1-week-old hearts and then significantly decreased in 2- and 10-week-old hearts, with a similar trend of expression for GFP (Fig. 3). The decrease in c-kit protein expression from 1 to 10 weeks after birth was significant (p < .05). Collectively, these results demonstrate an increased frequency of the c-kit+ cell population in early postnatal growth up to 1 week after birth that declines by 2 weeks of age.

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Figure Figure 2.. Developmental expression of c-kit and GFP in mouse myocardium assessed by confocal microscopy in c-kit-GFP transgenic mice. (A): Representative scans of myocardial sections at the time points indicated in upper right corner of each micrograph. Stem cells are indicated (yellow arrows), with c-kit (red), GFP (green), ToPro (magenta), and tropomyosin (red) as shown in single-channel scans along the left of each color overlay. (B): Histogram derived from microscopy shows developmental expression of c-kit+/GFP− (red), c-kit+/GFP+ (yellow), and c-kit−/GFP+ (green) at the time points indicated along the x-axis (n = 3). Abbreviation: GFP, green fluorescent protein.

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Figure Figure 3.. Developmental expression of c-kit and GFP in mouse myocardium of c-kit-GFP transgenic mice. (A): Immunoblot of whole heart lysates of 2-day-old, 1-week-old, 2-week-old, and 10-week-old mice labeled for c-kit or GFP, with GAPDH used to control for minor variation in sample loading. c-kit+ control is a sample of cultured c-kit+ cells derived from the heart. (B): Quantitation of c-kit and GFP protein levels normalized to those of 2-day old mice. Results are expressed as the mean ± SEM of 2-day old mice. n = 3 for all mice. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein.

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Expression of c-kit Is Elevated in Response to Pathological Challenge

The c-kit-GFP transgenic line was used to determine the upregulation of c-kit+ cells activity at 1, 4, 7, 10, and 14 days following myocardial infarction (MI) induced by permanent coronary artery occlusion. c-kit protein levels were not elevated in the myocardium of sham versus 1-day-post-MI samples, but significant increases were observed by 4, 7, 10, and 14 days post-MI in lysates prepared from tissue excised from the infarct and border zone regions (Fig. 4, infarct region; p < .05, with p < .01 for day 10). GFP expression levels followed a trend similar to that observed for c-kit, with significantly increased expression between sham and 7-, 10-, and 14-day-post-MI samples (Fig. 4, infarct region; p < .05 for day 10, with p < .01 for days 7 or 14). GFP expression increases slightly after c-kit expression, which could reflect the inability of the transgene promoter to recapitulate the complex spatiotemporal regulation of the endogenous c-kit gene with 100% efficiency. In comparison, no demonstrable changes in expression of c-kit or GFP markers were observed in the remote myocardium, with undetectable signal throughout the time course (Fig. 4, remote). Paraffin sections immunolabeled to detect both c-kit and GFP showed c-kit+ cell recruitment to the area of injury. Coincident labeling for both GFP and c-kit was observed in all cells within border zone and infarct regions of myocardium at both early and late stages of the time course (Fig. 4). At 10–14 days following infarction, colocalization of c-kit, GFP, and tropomyosin was detected (Figs. 4, 5), as well as the cardiogenic lineage marker GATA4 (Fig. 5).

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Figure Figure 4.. Increases in c-kit+ and GFP+ cells in the myocardium of c-kit-GFP transgenic mice following infarction injury. (A): Immunoblot of c-kit and GFP protein levels in infarct and remote regions of myocardium, with GAPDH as loading control to control for minor variations in protein loading. Positive controls to show antibody recognition include cultured stem cells (+c-kit control) and α-myosin heavy chain driving GFP whole heart lysate (+GFP control). (B): Quantitation of c-kit and GFP protein levels in myocardial lysates of tissue excised from the infarct region 1, 4, 7, 10, and 14 days post-MI relative to sham-operated control tissue (n = 3). (C, D): Evolution of c-kit+ and GFP+ cellular response to MI in c-kit-GFP transgenic myocardium at early (C) or late (D) time points, as indicated in upper right corner of each micrograph. (C): Representative confocal scans of infarcted myocardium labeled at 1 and 4 days post-MI together with sham-operated control, with coincidence of c-kit+ (blue) and GFP (green) shown (yellow arrows), as shown in single-channel scans along the left of each color overlay. Viable myocardium is labeled by sarcomeric tropomyosin (red), whereas nuclei are stained with ToPro dye (magenta). (D): Representative confocal scans of infarcted myocardium labeled at 7, 10, and 14 days post-MI, with coincidence of c-kit+ (red) and GFP (green) shown (yellow arrows), as shown in single-channel scans along the left of each color overlay. Infarct and border zone are labeled as appropriate. Viable myocardium was labeled by sarcomeric tropomyosin (blue), whereas nuclei were stained with ToPro dye (magenta). Changes in color designations between (A) and (B) series scans were made to highlight the relevant cell population in scan sets. Abbreviations: d, day; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; MI, myocardial infarction.

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Figure Figure 5.. Colocalization of c-kit+/GATA4+ cells in the infarct region at late stages of time course. (A): Representative confocal scans of infarcted myocardium labeled at 10 days post-MI with coincidence of c-kit+ (red) and GATA4 (green) as indicated (yellow arrows), as shown in single-channel scans along the left of each color overlay. (B): Representative confocal scan of infarcted myocardium labeled at 14 days post-MI, with coincidence of c-kit+ (red) and GATA4 (green) as indicated (yellow arrows). Sarcomeric tropomyosin (magenta) and nuclei (blue) are also shown. Boxed inset regions in the upper right of each panel show higher magnification of the cell from the scan, as indicated. Abbreviations: d, days; MI, myocardial infarction.

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Cardiac Progenitor Cells Express Markers of Cardiovascular Lineages Following Infarction Injury

The c-kit-GFP transgenic line was used to clarify the relationship between c-kit+ progenitor cells and markers of cardiovascular lineage commitment by confocal microscopy performed upon sections at 7, 10, and 14 days post-MI. Cells can be operationally defined as cardiac progenitor cells as characterized by coexpression of cardiac-specific protein markers, such as sarcomeric tropomyosin, along with the stem cell marker c-kit. Commitment of cells to progenitor status was evaluated by coincident labeling for c-kit or GFP, together with GATA4 (cardiogenic potential), sarcomeric tropomyosin (cardiomyocytes), α-smooth muscle actin (vasculature), or von Willebrand's factor (endothelial). Coincidence between GATA4 and c-kit was detected at 10–14 days post-MI, as well as less frequently observed triple colocalization among c-kit, GATA4, and tropomyosin (Fig. 5, inset). In addition, GFP+ cells in the infarct region were also coincident for expression of connexin43 (supplemental online Fig. 1). GFP+ cells coincident with either α-smooth muscle actin or von Willebrand's factor in the infarct zone were present by 7 days and persisted through 14 days post-MI (Fig. 6). These results indicate the presence of cells in the myocardium following infarction injury with cardiac progenitor cell phenotypic characteristics for three major lineages of tissue types found in the heart: cardiomyocytes, vasculature, and endothelium.

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Figure Figure 6.. Coincidence of GFP+ cells with markers of vascular and endothelial lineages in the c-kit-GFP transgenic mouse. Shown are representative confocal scans of myocardium at 7 (A, B) and 14 (C, D) days postinfarction. Sections were labeled for either α-SMA or von Willebrand's (both shown in yellow) in conjunction with GFP (green), ToPro for nuclei (magenta), and sarcomeric tropomyosin (red). Coincidence of GFP with either α-SMA or von Willebrand's is indicated (arrows in overlay scans; arrowheads in the single-channel scans along the left of each color overlay). Boxed regions (C1, D1) show detail of cells indicated by asterisks in low-power scans. Abbreviations: d, days; GFP, green fluorescent protein; MI, myocardial infarction; α-SMA, α-smooth muscle actin.

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Adult Cardiac c-kit+ Cells Express Stem Markers In Vitro and Inducible Cardiogenic Lineage Marker upon Differentiation

Adult cardiac c-kit+ cells were isolated from hearts of c-kit-GFP transgenic mice to confirm expression of phenotypic markers in vitro. These cultured cells are c-kit+, GFP+, Ki67+ (a marker of cell cycling; [33, [34]35]), nucleostemin+ (a marker of pluripotent stem cells; [36, 37]), and GATA4− under normal culture conditions (Fig. 7A–7D). Following dexamethasone treatment (10 nM for 1 week), morphological differences in the cultures were observed upon differentiation: cells increased in size and lost their spindle shape. Moreover, expression levels of cellular markers were altered as c-kit and GFP expression decreased in parallel, whereas expression of Mef2c (a cardiomyocyte-specific transcription factor [38, 39]) was markedly increased in selected cells (Fig. 7E–7H).

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Figure Figure 7.. Cardiac stem cell (CSC) cultures from hearts of c-kit-GFP transgenic mice show markers of cardiogenic lineage. Undifferentiated CSCs were c-kit+ (A–D), GFP+ (B), nucleostemin+ (C), and Ki67+ (D). Colors for the labels of each protein are shown in the names of each single-channel scan along the left of the corresponding color overlay, with nuclei of cells indicated in blue. Upon induction of differentiation, CSC cultures switched from c-kit+/GFP+ (E) to c-kit−/GFP− (F) and showed increased immunoreactivity for MEF2C in the differentiated (H) versus undifferentiated (G) state. Abbreviation: GFP, green fluorescent protein.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Studies of hematopoiesis have yielded important insights regarding the biology of self-renewing cell populations, including the association of the cell surface marker c-kit with stem cells [6, [7]8, 40]. Recognizing that c-kit expression represents a generic marker of stem cells rather than being restricted to cells destined for a hematopoietic fate, researchers from the cardiovascular community use c-kit as a preeminent identifier of stem cells within the myocardium [3, 41, 42]. There is evidence that hematopoietic stem cells can also migrate into and become resident in damaged myocardium [43, 44]. Use of c-kit alone cannot definitively address the origin of the cell population as cardiac or circulating bone marrow. However, the presence of c-kit+ cells in the myocardium and their dynamic responses to myocardial development and injury, as demonstrated in this study, are powerful evidence for growth and repair of the heart. In particular, the significantly increased frequency of c-kit+ cells in postnatal myocardium relative to adult tissue supports a role for this cell population in the growth of myocardium (Fig. 2). Use of c-kit+ cells for myocardial regeneration and inhibition of damage demonstrates the multifaceted potential of this cell population for facilitating reparative processes in the myocardium [5, 41, 42, 45, 46]. Despite this rapid accumulation of intervention-oriented experimental literature for cardiac repair, basic biological aspects of the resident c-kit+ cell population within the myocardium remain relatively obscure and require additional scrutiny to take full advantage of these cells for therapeutic purposes.

Involvement of c-kit+ cells in response to pathologic injury is well documented, but evolution of this response and understanding of the role that stem cells play in myocardial injury remains obscure. The c-kit-GFP transgenic mouse line described in this study uses genetic engineering to authenticate the identity of c-kit+ cells in postnatal growth and response of the myocardium to infarction injury. To demonstrate that localization of endogenous c-kit protein as a stem cell marker in the myocardium is not an experimental artifact, stem cells were genetically tagged using GFP. The c-kit-GFP transgenic mouse expresses GFP under the regulatory elements of the c-kit promoter that tags a subpopulation of endogenous c-kit+ cells with GFP in bone marrow (Fig. 1). The basis for GFP tagging a subset of the total c-kit+ cell population is unknown, but it could be due to the complexity of the c-kit promoter and additional regulatory elements not entirely captured in the cDNA constructs [26]. However, GFP expression is highly specific and restricted to appropriate cell types on the basis of the high coincidence of GFP and c-kit colocalization in myocardial sections; in addition, (a) GFP+ differentiated cell types were never observed, and (b) and confocal microscopy of testes sections revealed classic GFP expression coincident with c-kit, which plays important roles in spermatogenesis (data not shown; [9, 10]). The comparatively low level of c-kit−/GFP+ cells found in the bone marrow could be due to a temporal disconnect between turnover of c-kit versus turnover of GFP protein, with prolonged GFP retention before clearance from the cytoplasm.

Myocardial aspects of GFP expression also support authenticity of c-kit population tagging in the c-kit-GFP transgenic line. Developmental expression of c-kit in mouse heart decreases with age, as was found for expression of GFP in c-kit-GFP mice (Fig. 2). Similarly, c-kit+ cells are recruited to the site of an infarct and aid in regenerating the injured heart, as was proportionally represented by the demonstration of GFP accumulation following MI (Figs. 3, Figure 4., Figure 5., Figure 6.7). Furthermore, expression of GFP parallels that of c-kit in response over time and was restricted to the area of damage in the myocardium. The evolution of c-kit+ cell presence in response to pathological challenge shows maximal infiltration at approximately 10 days postinfarction (Fig. 4), which is delayed relative to the inflammatory infiltrates that arrive comparatively soon after injury [47]. The response was localized to the region of damage, as the remote myocardium showed no evidence of c-kit or GFP throughout the time course. In the later stages of the time course, the c-kit+ cell population was found to coexpress markers of commitment to the cardiac lineage, including proteins typical of cardiomyocytes, vascular smooth muscle, and endothelium. In vitro data indicate that upon differentiation, c-kit expression will diminish (Fig. 7). Therefore, markers of differentiation and stem cell markers, such as c-kit, will be coexpressed only very transiently, and although these cells are very rare, they can be observed [28]. Only endogenous c-kit+ cellular responses to myocardial insult were examined in this study and not adoptively transferred populations as in previous reports alluding to cell fusion [24, 48], and to our knowledge, fusion between endogenous c-kit+ cells and other myocardial cells has not been observed. Nevertheless, the ultimate fate of these progenitor cell populations is the subject of ongoing and future studies, however there is tantalizing evidence in published reports of stem cells possessing the capacity to regenerate myocardial tissue [49, [50], [51]52].

The isolation of resident cardiac stem cells, which were originally selected on the basis of c-kit surface antigen expression, and subsequent in vitro culturing and differentiation provide further validation of the GFP tag, as well as the cardiogenicity of these cells. Undifferentiated cultured cells expressed markers consistent with their capacity for self-renewal (Ki67), as well as multipotentiality (nucleostemin). Furthermore, expression of c-kit as well as the GFP transgene was readily observed, consistent with previous reports of cardiac stem cell isolation and characterization [3]. A subpopulation of these cultured cells also expressed relatively low levels of Mef2c, a cardiomyocyte transcription factor, indicating that a fraction of the culture had undergone commitment to cardiac progenitors destined for a cardiomyocyte lineage. Immunoreactivity of the cells for Mef2c was markedly enhanced by induction of differentiation by treatment with dexamethasone. Immunocytochemistry also showed a loss of endogenous c-kit as well as GFP transgene expression following stimulation to differentiate, further supporting the validity of the c-kit-GFP transgene as a marker of c-kit-positive cells. Taken together, these results demonstrate that cardiac c-kit+ cells isolated from the c-kit-GFP transgenic line express and appropriately downregulate their transgene in response to commitment to a cardiogenic lineage.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

With increasing acceptance of c-kit+ cells as an authentic source for cellular-based myocardial repair, the challenge is to enhance the potential of these cells to mediate regenerative processes in the damaged heart. GFP tagging of the c-kit+ population will be a valuable approach for in vivo tracking of cells following injury [53]. The evolution of the c-kit+ cell response described in this study indicates that 1–2 weeks after injury is a critical time for expansion, engraftment, and commitment. Focusing future studies upon this window, with manipulations to enhance the survival and growth of stem cells with cytokines and paracrine factors, may augment cellular repair processes in the damaged heart.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

We thank all members of the Sussman laboratory for helpful discussion and comments. M.A.S. is supported by NIH Grants 5R01-HL067245, 1R01-HL091102, 1P01-HL085577, and 1P01-AG023071 (Anversa Principal Investigator). J.A.M. and N.A.G. are Fellows of the Rees-Stealy Research Foundation and the San Diego State University Heart Institute. J.F. and B.B. contributed equally to this work.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of Potential Conflicts of Interest
  9. Acknowledgements
  10. References
  11. Supporting Information
FilenameFormatSizeDescription
SC-07-0751_Supplemental_Data.pdf45KSupplemental Data
SC-07-0751_Supplemental_Figure_1.pdf213KSupplemental Figure 1
SC-07-0751_Supplemental_Figure_2.pdf292KSupplemental Figure 2

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