Role of the ATP-Binding Cassette Transporter Abcg2 in the Phenotype and Function of Cardiac Side Population Cells
Recently, the side population (SP) phenotype has been introduced as a reliable marker to identify subpopulations of cells with stem/progenitor cell properties in various tissues. We and others have identified SP cells from postmitotic tissues, including adult myocardium, in which they have been suggested to contribute to cellular regeneration following injury. SP cells are identified and characterized by a unique efflux of Hoechst 33342 dye. Abcg2 belongs to the ATP-binding cassette (ABC) transporter superfamily and constitutes the molecular basis for the dye efflux, hence the SP phenotype, in hematopoietic stem cells. Although Abcg2 is also expressed in cardiac SP (cSP) cells, its role in regulating the SP phenotype and function of cSP cells is unknown. Herein, we demonstrate that regulation of the SP phenotype in cSP cells occurs in a dynamic, age-dependent fashion, with Abcg2 as the molecular determinant of the cSP phenotype in the neonatal heart and another ABC transporter, Mdr1, as the main contributor to the SP phenotype in the adult heart. Using loss- and gain-of-function experiments, we find that Abcg2 tightly regulates cell fate and function. Adult cSP cells isolated from mice with genetic ablation of Abcg2 exhibit blunted proliferation capacity and augmented cell death. Conversely, overexpression of Abcg2 is sufficient to enhance cell proliferation, although with a limitation of cardiomyogenic differentiation. In summary, for the first time, we reveal a functional role for Abcg2 in modulating the proliferation, differentiation, and survival of adult cSP cells that goes beyond its distinct role in Hoechst dye efflux.
Recently, the side population (SP) phenotype has been introduced as a reliable marker to identify subpopulations of cells with stem/progenitor cell properties in various tissues including the heart.1 On the molecular level, the SP phenotype is linked to the presence of ATP-binding cassette (ABC) transporters with the ability to efficiently efflux the DNA binding dye Hoechst 33342.2 This ABC transporter–dependent Hoechst efflux phenomenon confers the characteristic fluorescent-activated cell sorting (FACS) profile of SP cells as a Hoechst-low “side population” located to the periphery of the Hoechst-high main population.2
Among the various members of the ABC transporter superfamily, Abcg2 (also referred to as breast cancer resistance protein 1 [Bcrp1]) and Mdr1 (also referred to as P-glycoprotein [p-gp] or Abcb1) have been shown to efficiently efflux Hoechst 33342 and thereby confer the SP phenotype.3 Although both transporters are highly expressed in bone marrow (BM)SP cells, studies performed in mice with targeted disruption of the Mdr1a and Mdr1b genes, the murine homologs of the human Abcb1/Mdr1 gene, demonstrated that Abcg2 is the sole molecular determinant of the SP phenotype in hematopoietic stem cells.4 Moreover, Abcg2 expression is conserved in SP cells from a wide range of tissues including blood, gonad, lung, skeletal muscle and the retina, suggesting an important role of Abcg2 in stem cells.4–7
We and others have characterized SP cells isolated from adult myocardium.8–11 These cardiac (c)SP cells are phenotypically distinct from BMSP cells, in that they are not hematopoietic but exhibit the potential to differentiate into functional cardiomyocytes.10 As in SP cells from the bone marrow, Abcg2 is expressed in SP cells from the heart.9 The contribution of Abcg2 to the cSP phenotype and its biological significance in cSP progenitor cells, however, remain unknown. In this study, we find that the contribution of Abcg2 to the SP phenotype in the heart exists in an age-dependent manner, with Abcg2 as the molecular determinant of the SP phenotype in the neonatal heart and Mdr1 as the basis for the SP phenotype in the adult heart. In addition, we demonstrate that Abcg2 plays a crucial role in the maintenance of cSP progenitor cells by promoting cell proliferation and survival, while inhibiting lineage commitment. Intriguingly, for the first time, we reveal a functional role of Abcg2 in modulating proliferation, differentiation, and survival of cSP cells that goes beyond its distinct role of Hoechst dye efflux.
Materials and Methods
Male mice with genetic ablation of Abcg2 (Abcg2−/−) or Mdr1a/b (Mdr1a/b−/−) were purchased from Taconic (catalog nos. 002767-M and 001487-MM; Germantown, NY). Age-matched FVB mice were also purchased from Taconic to serve as wild-type (WT) control. Mice were studied at postnatal day (p)3, p14, and p21 and between 8 and 12 weeks of age (adult). All animal studies strictly adhered to the guidelines of the Harvard Medical School, the Institutional Animal Care and Use Committee of the Longwood Medical Area, and the National Society for Medical Research.
Cardiac and BMSP Cell Preparation
Cardiomyocyte-depleted mononuclear cell suspensions were prepared as previously described to obtain cSP cells.10 (For details, see the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.) BMSP cells were isolated form the tibia and femur as previously described.1
Cell Viability Assay
Cell death was determined by an annexin V kit (Abcam) using FACS analysis, and cell viability was determined by CellTiter-Glo and CellTiter-Blue (Promega) using a luminescence/fluorescence plate reader, respectively, according to the instructions of the manufacturer.
cSP Expansion and Lentiviral Infection
Freshly isolated cSP cells were cultured in expansion medium (α-MEM culture medium supplemented with 20% FBS, 2 mmol/L l-glutamine, and 1% penicillin/streptomycin). Vector pSPORT1 (American Type Culture Collection vector no. 10471063) was enzymatically digested with BamHI, and the resulting Abcg2 cDNA was blunted and subsequently cloned into the HPV-422 lentivirus vector (kindly provided by Dr P. Allen, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass). The bicistronic vector HPV-422 encodes the Abcg2 and IRES-GFP under the promoter EF1a. WT cSP cells from passage 4 to 6 were infected with the Abcg2-IRES-GFP lentivirus, and 48 hours postinfection green fluorescent protein–positive (GFP+) cSP cells were sorted and further expanded. Culture medium was replaced every 72 hours.
The proliferative capacity of cSP cells was determined in expanded passage 4 to 6 cSP cells using total cell number, expression of Ki67 and phospho-histone H3, total protein, and total DNA. For detailed protocols, see the online data supplement.
Cardiomyogenic Differentiation Capacity
The role of Abcg2 in regulating the ability of cSP cells to undergo cardiomyogenic differentiation was determined in our established coculture system.10 cSP cells were transfected with GFP-expressing lentivirus, and cocultures were stained for α-sarcomeric actinin (Sigma). (For details, see the online data supplement.)
Statistical differences between groups were evaluated using Student’s unpaired t test or ANOVA, as appropriate. All data are presented as means±SEM. A probability value of <0.05 was considered statistically significant.
Abcg2 Regulates the SP Phenotype in cSP Cells in an Age-Dependent Manner
Given published data suggesting that Abcg2 is the sole molecular determinant of the SP cell phenotype in bone marrow cells, we first sought to determine whether Abcg2 also regulates the SP phenotype in cardiac cells. To establish the role of Abcg2 in mediating the SP cell phenotype, bone marrow and cardiac cell suspensions were isolated from 8- to 12-week-old age-matched mice with genetic ablation of Abcg2 (Abcg2−/−) and WT counterparts. FACS analysis of bone marrow cell suspensions from Abcg2−/− mice demonstrated a complete lack of BMSP cells (WT: 0.20±0.05%; Abcg2−/−: 0.02±0.02%) compared to WT mice, suggesting that Abcg2 is indeed required for conferring the SP phenotype in bone marrow cells (Figure 1A). In contrast, cSP cells from Abcg2−/− hearts revealed a clearly detectable, although significantly reduced, SP population (WT: 0.8±0.1%; Abcg2−/−: 0.46±0.1%) (Figure 1B).
To confirm the correct FACS gating of SP cells (Figure 2A and 2B), we used 2 potent inhibitors of ABC transporters, verapamil and fumitremorgin C (FTC). Treatment of cells with verapamil or FTC completely abolished the SP cell band in both WT and Abcg2−/− cardiac cell suspensions (Figure 2C and 2F). To further verify that Hoechst dye efflux in Abcg2−/− hearts was mediated via ABC transport, cardiac cell suspensions were preincubated with 2-deoxyglucose to deplete ATP and thereby inactivate ATP-dependent transporter function. Depletion of ATP also resulted in a complete loss of SP cells in both WT and Abcg2−/− cell suspensions, indicating that dye efflux in Abcg2−/− cell suspensions is indeed mediated in an ATP-dependent manner (Figure 2G and 2H).
We and others have previously shown that cSP cells express high levels of stem cell antigen 1 (Sca-1) and moderate levels of CD31 but lack the pan-hematopoietic marker CD45.10 To further immunophenotypically characterize Abcg2−/− cSP cells, we compared the expression of cell surface markers, Sca-1, CD31 and CD45, in Abcg2−/− and WT cSP cells using FACS analysis. As shown in the Table, deficiency of Abcg2 did not alter the expression pattern of Sca-1, CD31, and CD45, as compared to WT cSP cells, further suggesting that lacking Abcg2 does not alter the expression pattern of major cell surface markers in adult cSP cells.
In addition to Abcg2, another member of the ABC transporter superfamily, the P-glycoprotein Mdr1, has been demonstrated to exhibit the capacity to efflux the Hoechst 33342 dye.12 We therefore hypothesized that Mdr1 may be involved in the regulation of the cSP phenotype in the adult heart. Quantitative RT-PCR analysis (Figure I in the online data supplement) and immunocytochemistry (Figure 2I through 2Q) in WT and Abcg2−/− cSP cells confirmed the expression of Mdr1a and Mdr1b genes and proteins independent of the expression of Abcg2.
To determine the contribution of Mdr1 to the Hoechst 33342 efflux phenotype in the adult heart, cSP cells were isolated from hearts of adult mice with targeted disruption of Mdr1a/b genes. Strikingly, Mdr1a/b−/− hearts exhibited a severe depletion of cSP (Figure 3A). In contrast, bone marrow from Mdr1a/b−/− animals exhibited normal BMSP cell numbers (data not shown). To ensure that the limited number of cSP cells observed in Mdr1a/b−/− hearts was not attributable to experimental variables, in particular Hoechst concentration, cell ratio, and staining duration, we used an internal control by mixing 1 part of cardiomyocyte-depleted mononuclear cells ubiquitously expressing enhanced green fluorescent protein (GFP+ control cells) with 3 parts of non-GFP, Mdr1a/b−/−, or WT cardiomyocyte-depleted mononuclear cells (Figure 3B). Indeed, analysis of these cell mixtures revealed almost exclusively GFP+ cells among the cSP cells (>98%), with no significant contribution from Mdr1a/b−/− cells to the SP band, thereby confirming the severely impaired Hoechst efflux capacity in Mdr1a/b−/− cSP cells (Figure 3B). In contrast, 1:3 cell mixtures of GFP+ control cells and WT cardiac cells revealed only ≈25% GFP+ control cells within the SP cell population, confirming the dye efflux competence of WT cardiac cells (Figure 3B).
Whereas Abcg2 is enriched in early neonatal cSP cells, its expression level is markedly decreased postnatally.9 To determine whether ABC-transporter expression in the heart is age-dependent, quantitative RT-PCR analysis for Abcg2 and Mdr1a/b was performed in neonatal and adult mouse hearts. These analyses confirmed profound downregulation of Abcg2 gene expression in the adult cSP cells as compared to the neonatal cSP cells. In contrast, Mdr1a/b expression levels demonstrated a reverse pattern with low expression in the neonatal cSP cells and high expression in the adult cSP cells (supplemental Figure II). To determine whether Abcg2 and Mdr1a/b mediate the SP phenotype in cSP cells in an age-dependent manner, cSP cells were isolated from Abcg2−/−, Mdr1a/b−/−, and age-matched WT mice at p3, p14, and p21 and 8 to 12 weeks (adult) of age (Figure 4A through 4D). In contrast to adult hearts, early postnatal (p3) Abcg2−/− hearts demonstrated almost no detectable cSP cells, whereas a similar number of SP cells was observed in Mdr1a/b−/− and WT hearts (Figure 4A). The lack of Hoechst dye efflux was confirmed in cSP cells from Abcg2−/− hearts at p3 using GFP-mixing studies, similar to those described above (data not shown). A gradual decrease in cSP cells from p3 (9.4±1.2%) to adulthood (0.8±0.1%) was noted in WT hearts. Abcg2−/− hearts, however, demonstrated a gradual increase in cSP cells from day 3 postnatally (0.18±0.1%) to adult (0.46±0.1%) and thereafter maintained cSP cell levels into adulthood. The opposite profile was observed in Mdr1a/b−/− hearts, with cSP cell numbers dropping dramatically within the first 3 weeks of postnatal life and being barely detectable in adulthood. Taken together, these data demonstrate that the cSP cell phenotype is mediated by Abcg2 and Mdr1a/b in an age-dependent fashion.
Abcg2 Regulates cSP Cell Proliferation
Expression of Abcg2 has been associated with cellular proliferation in cancer cell lines.13 Using gain- and loss-of-function approaches, we investigated the role of Abcg2 in the regulation of cSP cell proliferation. cSP cells isolated from adult age-matched Abcg2−/− and WT mouse hearts were subjected to proliferation assays. As shown in Figure 5A, the proliferation capacity, as determined by total cell number, was markedly decreased in cSP cells lacking Abcg2. Consistent with the decrease in cell proliferation, the expression of the cell cycle markers Ki67 that identifies cells in G1, S, G2, and M phases (Figure 5B) and phospho-histone H3 (Figure 5C and 5D) that identifies cells in M phase were decreased by ≈50% and ≈70%, respectively, in Abcg2−/− cSP cells when compared to WT cSP cells. Likewise, total protein and DNA content were significantly decreased in Abcg2−/− cSP cells cultured in the expansion media, thus further supporting impaired cell proliferation seen in Abcg2−/− cSP cells (Figure 5E and 5F). Conversely, the proliferation capacity was significantly enhanced in cSP cells following overexpression of Abcg2 via lentiviral-mediated gene transfer, with an increase in both total cell number (Figure 5G) and expression of Ki67 (data not shown) as compared to WT cSP cells. Taken together, these data demonstrate a functional role of Abcg2 in facilitating proliferation of cSP cells.
Regulation of cSP Cell Survival by Abcg2
Emerging evidence also suggests that Abcg2 may play a critical role in protecting primitive cells from cellular injury.14,15 To date, it is unclear whether Abcg2 also exerts cell protective effects on cSP cells. To determine whether Abcg2 is implicated in cSP cell survival, we first assessed apoptosis and necrosis in cSP cells under normal culture conditions using annexin V and propidium iodide staining, respectively. As illustrated in Figure 6A and 6B, even under normal culture conditions, significantly elevated numbers of both apoptotic and necrotic cells were observed in cSP cells lacking Abcg2 as compared to WT cells. In addition, cell viability assays measuring cellular metabolic capacity by means of ATP quantitation (CellTiter-Glo, Promega) or conversion of the redox dye resazurin to the fluorescent end product resorufin (CellTiter-Blue, Promega) demonstrated significantly decreased metabolic capacity in Abcg2−/− cSP cells (Figure 6C and 6D), thus further confirming impaired viability.
Because oxidative stress is a common mediator of cell death in myocardial injury of various causes, we further investigated oxidative stress–induced cell death in WT and Abcg2−/− cSP cells after exposing cSP cell cultures to 200 μmol/L H2O2. In this model, oxidative stress–induced cell death was significantly higher in cSP cells lacking Abcg2 as compared to WT cSP cells (Figure 6E). Taken together, our data indicate a protective role of Abcg2 in cSP cells under normal culture (21% O2) and H2O2-induced oxidative stress conditions.
Overexpression of Abcg2 Impairs the Ability of cSP Cells to Undergo Cardiomyogenic Differentiation
Regulation of proliferation and differentiation maintains progenitor cell homeostasis. We have found that Abcg2 is an essential regulator of cSP cell proliferation. We next sought to determine whether Abcg2 mediates cardiomyogenic differentiation of cSP cells. Using a previously described coculture system with adult rat cardiomyocytes, cardiomyogenic differentiation was assessed in WT and Abcg2−/− cSP cells, as well as in Abcg2-overexpressing cSP cells (Figure 7A through 7I). To track the cell fate of cells in coculture, cSP cells were infected with lentivirus expressing GFP. Genetic deficiency of Abcg2 did not limit the cardiomyogenic differentiation of cSP cells (Figure 7D through 7F). Overexpression of Abcg2 via lentiviral-mediated gene transfer significantly decreased cardiomyogenic differentiation of cSP cells (Figure 7G through 7I). Moreover, overexpression of Abcg2 maintained cSP cells in a proliferative state even under conditions promoting cardiomyogenic differentiation.
Since the first isolation of BMSP cells more than a decade ago,2 the SP phenotype has been widely used to identify stem/progenitor cells in various tissues.2,7,16–19 More recently, the ABC transporter Abcg2 has been identified as the sole molecular determinant of the SP phenotype in bone marrow cells.4 However, the role of ABC transporters in the regulation of SP phenotype and function of cSP cells remains unknown. Herein, we demonstrate not only a dynamic, age-dependent regulation of the cSP phenotype by Abcg2 but also a functional role of Abcg2 in modulating proliferation, differentiation, and survival of cSP cells that goes beyond its distinct role in Hoechst dye efflux.
Role of Abcg2 in Conferring the SP Phenotype in the Heart
Although Abcg2 and Mdr1 are both expressed in hematopoietic stem cells identified by the SP phenotype, Abcg2 was shown to be the sole molecular determinant of the SP phenotype in bone marrow cells.4 These findings in the bone marrow led to the assumption that Abcg2 may also be responsible for the dye efflux observed in SP cells isolated from other tissues, particularly given the preferential expression pattern of Abcg2 in SP cells as compared to main population cells. Similar to their expression in bone marrow, Abcg2 and Mdr1 were shown to be expressed in the heart, although their contribution to the cSP phenotype remained to be elucidated. Using mouse models with targeted gene ablation of either Abcg2 or Mdr1, we demonstrate that the cSP cell phenotype is not governed by a single ABC transporter but rather is regulated in an age-dependent manner by both Abcg2 and Mdr1. Our findings show that during early postnatal development, Abcg2 represents the main transporter responsible for dye efflux in cardiac cells and thus constitutes the molecular basis for the SP phenotype in the neonatal and early postnatal heart. This is in accordance with the role of Abcg2 in bone marrow–derived cells and may indicate that cSP cells of the early postnatal heart share phenotypic characteristics of bone marrow–derived SP cells. Whether cSP cells developmentally originate from blood-borne cells, however, remains to be determined. It is important to point out that our present data provide no evidence to either support or dispute such notion. Prior data from our laboratory using labeled bone marrow transplantation, however, suggest that extracardiac stem cells only contribute to the maintenance of resident cSP cell pools following injury, with little role in the maintenance of cSP cell numbers under normal physiological conditions.20
Although our results agree with previous reports of persistent Abcg2 expression in cSP cells throughout adulthood,9 we find that the contribution of Abcg2 to the SP phenotype diminishes in the adult heart. Our results show that very limited cSP cells can be detected in early neonatal Abcg2−/− hearts, with clearly detectable cSP cells in adult Abcg2−/− hearts, albeit at a lower total number as compared to WT hearts. These Abcg2−/− cSP cells are sensitive to verapamil, FTC, and 2-deoxyglucose treatment, thus suggesting that their Hoechst-extruding ability is mediated through another ABC transporter. Analyses of mice completely lacking Mdr1 identified the P-glycoprotein as the essential ABC transporter for Hoechst efflux in adult cSP cells. It is important to note that the putative Abcg2 inhibitor FTC, which was previously shown to have no effect on Mdr1-mediated mitoxantrone efflux in mitoxantrone-resistance-selected human colon carcinoma cell lines,21 did inhibit Mdr1-mediated Hoechst efflux in cSP cells, suggesting that the specificity of FTC may depend on the cell type and the substrate.
Interestingly, this regulatory role of Mdr1 in the cSP phenotype is limited to cSP cells from mice older than 3 weeks of age, with limited contribution of Mdr1 to the SP phenotype in early neonatal mouse hearts. Although the origin of cardiac stem/progenitor cells and their relationship with bone marrow–derived stem cells remains speculative at this time, our data suggest that BMSP cells do not significantly contribute to the maintenance of cSP cells under physiological conditions, as evidenced by the lack of SP cells in adult Mdr1a/b−/− hearts, whereas Mdr1a/b−/− bone marrow contains normal SP cells. Thus, the present data are consistent with our previous findings demonstrating that BMSP cells only contribute to the maintenance of cSP cells following cardiac injury such as myocardial infarction.20
Our data dispute the perception that a single universal ABC transporter is responsible for the SP phenotype and suggest that developmental status and local microenvironment dictate the relative contribution of ABC transporters to the SP phenotype at the given tissue. In line with this observation is the recent demonstration of contribution of both Mdr1 and Abcg2 transporters to the SP phenotype in mammary glands.22
Role of Abcg2 in Regulating the Function of cSP Cells
Abcg2 has been found to be highly expressed in various proliferating stem/progenitor cells and tumor cell lines, although its role in the regulation of SP cell proliferation and differentiation remains unknown. Using gain- and loss-of function approaches, we demonstrate that overexpression of Abcg2 is sufficient to increase the proliferative capacity of cSP cells, whereas lack of Abcg2 expression markedly impairs their expandability in vitro. The marked decrease in cells being in M phase of the cell cycle, as measured by phospho-histone H3 expression, suggests that the absence of Abcg2 may hamper cell cycle progression. This association of Abcg2 expression with cell proliferation is in line with the findings in cancer cells, where Abcg2 identified mainly fast cycling tumor progenitor cells.13 Our data provide convincing evidence demonstrating that Abcg2 may play a functional role in regulating the proliferation capacity of cSP cells, although the precise mechanisms are unknown. Further investigation, therefore, is warranted to dissect the molecular mechanisms by which Abcg2 facilitates the cell cycle progression in cardiac progenitor cells.
In addition to enhancing cell proliferation, we show that Abcg2 expression is necessary for protecting cSP cells from undergoing apoptosis and necrosis, specifically under conditions of increased oxidative stress. A similar prosurvival effect of Abcg2 was identified in trophoblast and hematopoietic stem cells.14,23 Moreover, Martin et al recently demonstrated that expression of Abcg2 induced low levels of oxidative stress in C2C12 myoblasts, which resulted in upregulation of cytoprotective and oxidative stress pathways.15 The cytoprotective effect of such Abcg2-mediated ROS preconditioning was also confirmed in mouse embryonic fibroblasts that displayed reduced oxidative stress–induced cell death, when transfected with Abcg2.15 Our data are in complete agreement with this concept by demonstrating increased tolerance of oxidative stress in Abcg2-competent WT cSP cells as compared to cSP cells lacking Abcg2.
Consistent with the notion that Abcg2 maintains progenitor cells in a proliferative stage and is downregulated during lineage-specific differentiation, overexpression of Abcg2 prevented cSP cells from undergoing cardiomyogenic differentiation. Our data are supported by the recently published work in hematopoietic and retinal stem cells demonstrating highly regulated Abcg2 expression during stem cell differentiation with a sharp decline during lineage commitment.4,5 In contrast, overexpression of Abcg2 blocks the differentiation of hematopoietic and retinal stem cells, indicating a functional role of Abcg2 in the maintenance of the stem cell pool.4,5 In both cell types, overexpression of Abcg2 leads to increased cell expansion and adversely affects their lineage commitment.
We have shown that overexpression of Abcg2 not only promotes proliferation and survival of cSP cells but, at the same time, also inhibits cellular differentiation. Taken together, our data suggest that Abcg2 is essential to the fate and function of cSP cells. As such, the tight regulation of Abcg2 expression may be critical for maintaining progenitor cells in either a proproliferative or prodifferentiation state. Moreover, such regulation may be especially essential following tissue injury, during which a rapid increase in cSP cell proliferation is observed to replenish tissue SP cell pools.20 Dysregulation of Abcg2 expression may also result in uncontrolled cell growth or cell death. To date, the exact mechanism by which Abcg2 prevents stem cells from lineage commitment remains to be elucidated. Considering the primary function of Abcg2 as a detoxifying transmembrane pump, however, it is tempting to speculate that active extrusion of key molecules of the differentiation-promoting pathway might be involved in this process.
In summary, our study highlights the importance of Abcg2 in regulating the function and homeostasis of cSP cells that goes beyond its traditional role as dye efflux transporter. Manipulation of Abcg2 expression and function may be of particular importance in promoting cardiac regeneration following injury by both endogenous and exogenously delivered cSP cells. Given the role of Abcg2 in the proliferation of cancer cells, as well as cSP cells, there is great potential for cardiac toxicity with emerging chemotherapeutic agents specifically targeting Abcg2. Further investigation into the role of Abcg2 in cSP cells is of clinical importance to limit cancer drug–induced cardiac toxicity and to promote cardiac regeneration.
We thank Drs Richard C. Mulligan and Alejandro B. Balazs for help in the purification of SP cells and for useful discussions. Grigoriy Losyev at the Brigham and Women’s Hospital Cardiovascular and Laura B. Prickett at the Massachusetts General Hospital FACS Cores are acknowledged for assistance with cell sorting.
Sources of Funding
This work was supported by NIH grants HL71775, HL86967, HL73756, and HL 88533 (to R.L.). A.O. and G.C.F. were supported by an American Heart Association Northeast Affiliate Predoctoral Fellowship and a Sarnoff Cardiovascular Research Foundation Fellowship, respectively.
↵*These authors contributed equally to this work.
Original received February 25, 2008; revision received August 27, 2008; accepted August 28, 2008.
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