Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair.

The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit(pos) cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kit(pos) eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage.

Sox2 controls neural stem cell self-renewal through a Fos-centered gene regulatory network.

The Sox2 transcription factor is necessary for the long-term self-renewal of neural stem cells (NSCs). Its mechanism of action is still poorly defined. To identify molecules regulated by Sox2, and acting in mouse NSC maintenance, we transduced, into Sox2-deleted NSC, genes whose expression is strongly downregulated following Sox2 loss (Fos, Jun, Egr2), individually or in combination. Fos alone rescued long-term proliferation, as shown by in vitro cell growth and clonal analysis. Furthermore, pharmacological inhibition by T-5224 of FOS/JUN AP1 complex binding to its targets decreased cell proliferation and expression of the putative target Suppressor of cytokine signaling 3 (Socs3). Additionally, Fos requirement for efficient long-term proliferation was demonstrated by the reduction of NSC clones capable of long-term expansion following CRISPR/Cas9-mediated Fos inactivation. Previous work showed that the Socs3 gene is strongly downregulated following Sox2 deletion, and its re-expression by lentiviral transduction rescues long-term NSC proliferation. Fos appears to be an upstream regulator of Socs3, possibly together with Jun and Egr2; indeed, Sox2 re-expression in Sox2-deleted NSC progressively activates both Fos and Socs3 expression; in turn, Fos transduction activates Socs3 expression. Based on available SOX2 ChIPseq and ChIA-PET data, we propose a model whereby Sox2 is a direct activator of both Socs3 and Fos, as well as possibly Jun and Egr2; furthermore, we provide direct evidence for FOS and JUN binding on Socs3 promoter, suggesting direct transcriptional regulation. These results provide the basis for developing a model of a network of interactions, regulating critical effectors of NSC proliferation and long-term maintenance.

Sox2-dependent maintenance of mouse oligodendroglioma involves the Sox2-mediated downregulation of Cdkn2b, Ebf1, Zfp423, and Hey2.

Cancer stem cells (CSC) are essential for tumorigenesis. The transcription factor Sox2 is overexpressed in brain gliomas, and is essential to maintain CSC. In mouse high-grade glioma pHGG cells in culture, Sox2 deletion causes cell proliferation arrest and inability to reform tumors after transplantation in vivo; in Sox2-deleted cells, 134 genes are derepressed. To identify genes mediating Sox2 deletion effects, we overexpressed into pHGG cells nine among the most derepressed genes, and identified four genes, Ebf1, Hey2, Zfp423, and Cdkn2b, that strongly reduced cell proliferation in vitro and brain tumorigenesis in vivo. CRISPR/Cas9 mutagenesis of each gene, individually or in combination (Ebf1 + Cdkn2b), significantly antagonized the proliferation arrest caused by Sox2 deletion. The same genes also repressed clonogenicity in primary human glioblastoma-derived CSC-like lines. These experiments identify a network of critical tumor suppressive Sox2-targets whose inhibition by Sox2 is involved in glioma CSC maintenance, defining new potential therapeutic targets.

The Coup-TFII orphan nuclear receptor is an activator of the γ-globin gene.

The human fetal γ-globin gene is repressed in the adult stage through complex regulatory mechanisms involving transcription factors and epigenetic modifiers. Reversing γ-globin repression, or maintaining its expression by manipulating regulatory mechanisms, has become a major clinical goal in the treatment of ß-hemoglobinopathies. Here, we identify the orphan nuclear receptor Coup-TFII (NR2F2/ARP-1) as an embryonic/fetal stage activator of γ-globin expression. We show that Coup-TFII is expressed in early erythropoiesis of yolk sac origin, together with embryonic/fetal globins. When overexpressed in adult cells (including peripheral blood cells from human healthy donors and ß039 thalassemic patients) Coup-TFII activates the embryonic/fetal globins genes, overcoming the repression imposed by the adult erythroid environment. Conversely, the knock-out of Coup-TFII increases the ß/γ+ß globin ratio. Molecular analysis indicates that Coup-TFII binds in vivo to the ß-locus and contributes to its conformation. Overall, our data identify Coup-TFII as a specific activator of the γ-globin gene.

Sox2 conditional mutation in mouse causes ataxic symptoms, cerebellar vermis hypoplasia, and postnatal defects of Bergmann glia.

Sox2 is a transcription factor active in the nervous system, within different cell types, ranging from radial glia neural stem cells to a few specific types of differentiated glia and neurons. Mutations in the human SOX2 transcription factor gene cause various central nervous system (CNS) abnormalities, involving hippocampus and eye defects, as well as ataxia. Conditional Sox2 mutation in mouse, with different Cre transgenes, previously recapitulated different essential features of the disease, such as hippocampus and eye defects. In the cerebellum, Sox2 is active from early embryogenesis in the neural progenitors of the cerebellar primordium; Sox2 expression is maintained, postnatally, within Bergmann glia (BG), a differentiated cell type essential for Purkinje neurons functionality and correct motor control. By performing Sox2 Cre-mediated ablation in the developing and postnatal mouse cerebellum, we reproduced ataxia features. Embryonic Sox2 deletion (with Wnt1Cre) leads to reduction of the cerebellar vermis, known to be commonly related to ataxia, preceded by deregulation of Otx2 and Gbx2, critical regulators of vermis development. Postnatally, BG is progressively disorganized, mislocalized, and reduced in mutants. Sox2 postnatal deletion, specifically induced in glia (with GLAST-CreERT2), reproduces the BG defect, and causes (milder) ataxic features. Our results define a role for Sox2 in cerebellar function and development, and identify a functional requirement for Sox2 within postnatal BG, of potential relevance for ataxia in mouse mutants, and in human patients.

Sox2 is required for embryonic development of the ventral telencephalon through the activation of the ventral determinants Nkx2.1 and Shh.

The Sox2 transcription factor is active in stem/progenitor cells throughout the developing vertebrate central nervous system. However, its conditional deletion at E12.5 in mouse causes few brain developmental problems, with the exception of the postnatal loss of the hippocampal radial glia stem cells and the dentate gyrus. We deleted Sox2 at E9.5 in the telencephalon, using a Bf1-Cre transgene. We observed embryonic brain defects that were particularly severe in the ventral, as opposed to the dorsal, telencephalon. Important tissue loss, including the medial ganglionic eminence (MGE), was detected at E12.5, causing the subsequent impairment of MGE-derived neurons. The defect was preceded by loss of expression of the essential ventral determinants Nkx2.1 and Shh, and accompanied by ventral spread of dorsal markers. This phenotype is reminiscent of that of mice mutant for the transcription factor Nkx2.1 or for the Shh receptor Smo. Nkx2.1 is known to mediate the initial activation of ventral telencephalic Shh expression. A partial rescue of the normal phenotype at E14.5 was obtained by administration of a Shh agonist. Experiments in Medaka fish indicate that expression of Nkx2.1 is regulated by Sox2 in this species also. We propose that Sox2 contributes to Nkx2.1 expression in early mouse development, thus participating in the region-specific activation of Shh, thereby mediating ventral telencephalic patterning induction.

SOHLH1 and SOHLH2 control Kit expression during postnatal male germ cell development.

How Kit expression is regulated in the germline remains unknown. SOHLH1 and SOHLH2, two bHLH transcription factors specifically expressed in germ cells, are involved in spermatogonia and oocyte differentiation. In the male, deletion of each factor causes loss of Kit-expressing spermatogonia in the prepuberal testis. In the female, SOHLH1 and SOHLH2 ablations cause oocyte loss in the neonatal ovary. To investigate whether Kit expression is regulated by these two factors in male germ cells, we examined SOHLH1 and SOHLH2 expression during fetal and postnatal mouse development. We found a strong positive correlation between Kit and the two transcription factors only in postnatal spermatogonia. SOHLH2 was enriched in undifferentiated spermatogonia, whereas SOHLH1 expression was maximal at Kit-dependent stages. Expression of SOHLH1, but not SOHLH2, was increased in postnatal mitotic germ cells by treatment with all-trans retinoic acid. We found that E-box sequences within the Kit promoter and its first intron can be transactivated in transfection experiments overexpressing Sohlh1 or Sohlh2. Co-transfection of both factors showed a cooperative effect. EMSA experiments showed that SOHLH1 and SOHLH2 can independently and cooperatively bind an E-box-containing probe. In vivo co-immunoprecipitations indicated that the two proteins interact and overexpression of both factors increases endogenous Kit expression in embryonic stem cells. SOHLH1 was found by ChIP analysis to occupy an E-box-containing region within the Kit promoter in spermatogonia chromatin. Our results suggest that SOHLH1 and SOHLH2 directly stimulate Kit transcription in postnatal spermatogonia, thus activating the signaling involved in spermatogonia differentiation and spermatogenetic progression.

Verification of the in vivo activity of three distinct cis-acting elements within the Gata1 gene promoter-proximal enhancer in mice.

The transcription factor GATA1 is essential for erythroid and megakaryocytic cell differentiation. Gata1 hematopoietic regulatory domain (G1HRD) has been shown to recapitulate endogenous Gata1 gene expression in transgenic mouse assays in vivo. G1HRD contains a promoter-proximal enhancer composed of a GATA-palindrome motif, four CP2-binding sites and two CACCC boxes. We prepared transgenic reporter mouse lines in which green fluorescent protein and ß-galactosidase expression are driven by wild-type G1HRD (as a positive control) and the G1HRD harboring mutations within these cis-acting elements (as the experimental conditions), respectively. Exploiting this transgenic dual reporter (TDR) assay, we show here that in definitive erythropoiesis, G1HRD activity was markedly affected by individual mutations in the GATA-palindrome motif and the CACCC boxes. Mutation of CP2-binding sites also moderately decreased G1HRD activity. The combined mutation of the CP2-binding sites and the GATA-palindrome motif resulted in complete loss of G1HRD activity. In contrast, in primitive erythroid cells, individual mutations of each element did not affect G1HRD activity; G1HRD activity was abolished only when these three mutations were combined. These results thus show that all three elements independently and cooperatively contribute to G1HRD activity in vivo in definitive erythropoiesis, although these are contributing redundantly to primitive erythropoiesis.

Cardiomyogenesis in the developing heart is regulated by c-kit-positive cardiac stem cells.

RATIONALE: Embryonic and fetal myocardial growth is characterized by a dramatic increase in myocyte number, but whether the expansion of the myocyte compartment is dictated by activation and commitment of resident cardiac stem cells (CSCs), division of immature myocytes or both is currently unknown. OBJECTIVE: In this study, we tested whether prenatal cardiac development is controlled by activation and differentiation of CSCs and whether division of c-kit-positive CSCs in the mouse heart is triggered by spontaneous Ca(2+) oscillations. METHODS AND RESULTS: We report that embryonic-fetal c-kit-positive CSCs are self-renewing, clonogenic and multipotent in vitro and in vivo. The growth and commitment of c-kit-positive CSCs is responsible for the generation of the myocyte progeny of the developing heart. The close correspondence between values computed by mathematical modeling and direct measurements of myocyte number at E9, E14, E19 and 1 day after birth strongly suggests that the organogenesis of the embryonic heart is dependent on a hierarchical model of cell differentiation regulated by resident CSCs. The growth promoting effects of c-kit-positive CSCs are triggered by spontaneous oscillations in intracellular Ca(2+), mediated by IP3 receptor activation, which condition asymmetrical stem cell division and myocyte lineage specification. CONCLUSIONS: Myocyte formation derived from CSC differentiation is the major determinant of cardiac growth during development. Division of c-kit-positive CSCs in the mouse is promoted by spontaneous Ca(2+) spikes, which dictate the pattern of stem cell replication and the generation of a myocyte progeny at all phases of prenatal life and up to one day after birth.

Sox6 enhances erythroid differentiation in human erythroid progenitors.

Sox6 belongs to the Sry (sex-determining region Y)-related high-mobility-group-box family of transcription factors, which control cell-fate specification of many cell types. Here, we explored the role of Sox6 in human erythropoiesis by its overexpression both in the erythroleukemic K562 cell line and in primary erythroid cultures from human cord blood CD34+ cells. Sox6 induced significant erythroid differentiation in both models. K562 cells underwent hemoglobinization and, despite their leukemic origin, died within 9 days after transduction; primary erythroid cultures accelerated their kinetics of erythroid maturation and increased the number of cells that reached the final enucleation step. Searching for direct Sox6 targets, we found SOCS3 (suppressor of cytokine signaling-3), a known mediator of cytokine response. Sox6 was bound in vitro and in vivo to an evolutionarily conserved regulatory SOCS3 element, which induced transcriptional activation. SOCS3 overexpression in K562 cells and in primary erythroid cells recapitulated the growth inhibition induced by Sox6, which demonstrates that SOCS3 is a relevant Sox6 effector.

The ephrin A1-EphA2 system promotes cardiac stem cell migration after infarction.

RATIONALE: Understanding the mechanisms that regulate trafficking of human cardiac stem cells (hCSCs) may lead to development of new therapeutic approaches for the failing heart. OBJECTIVE: We tested whether the motility of hCSCs in immunosuppressed infarcted animals is controlled by the guidance system that involves the interaction of Eph receptors with ephrin ligands. METHODS AND RESULTS: Within the cardiac niches, cardiomyocytes expressed preferentially the ephrin A1 ligand, whereas hCSCs possessed the EphA2 receptor. Treatment of hCSCs with ephrin A1 resulted in the rapid internalization of the ephrin A1-EphA2 complex, posttranslational modifications of Src kinases, and morphological changes consistent with the acquisition of a motile cell phenotype. Ephrin A1 enhanced the motility of hCSCs in vitro, and their migration in vivo following acute myocardial infarction. At 2 weeks after infarction, the volume of the regenerated myocardium was 2-fold larger in animals injected with ephrin A1-activated hCSCs than in animals receiving control hCSCs; this difference was dictated by a greater number of newly formed cardiomyocytes and coronary vessels. The increased recovery in myocardial mass with ephrin A1-treated hCSCs was characterized by further restoration of cardiac function and by a reduction in arrhythmic events. CONCLUSIONS: Ephrin A1 promotes the motility of EphA2-positive hCSCs, facilitates their migration to the area of damage, and enhances cardiac repair. Thus, in situ stimulation of resident hCSCs with ephrin A1 or their ex vivo activation before myocardial delivery improves cell targeting to sites of injury, possibly providing a novel strategy for the management of the diseased heart.

A highly conserved SOX6 double binding site mediates SOX6 gene downregulation in erythroid cells.

The Sox6 transcription factor plays critical roles in various cell types, including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells, we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar, evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo, and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied, in erythroid cells, by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation, mediated by the double Sox6 binding site within its own promoter, may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation.

Defective erythroid maturation in gelsolin mutant mice.

BACKGROUND: During late differentiation, erythroid cells undergo profound changes involving actin filament remodeling. One of the proteins controlling actin dynamics is gelsolin, a calcium-activated actin filament severing and capping protein. Gelsolin-null (Gsn(-/-)) mice generated in a C57BL/6 background are viable and fertile.1 DESIGN AND METHODS: We analyzed the functional roles of gelsolin in erythropoiesis by: (i) evaluating gelsolin expression in murine fetal liver cells at different stages of erythroid differentiation (using reverse transcription polymerase chain reaction analysis and immunohistochemistry), and (ii) characterizing embryonic and adult erythropoiesis in Gsn(-/-) BALB/c mice (morphology and erythroid cultures). RESULTS: In the context of a BALB/c background, the Gsn(-/-) mutation causes embryonic death. Gsn(-/-) embryos show defective erythroid maturation with persistence of circulating nucleated cells. The few Gsn(-/-) mice reaching adulthood fail to recover from phenylhydrazine-induced acute anemia, revealing an impaired response to stress erythropoiesis. In in vitro differentiation assays, E13.5 fetal liver Gsn(-/-) cells failed to undergo terminal maturation, a defect partially rescued by Cytochalasin D, and mimicked by administration of Jasplakinolide to the wild-type control samples. CONCLUSIONS: In BALB/c mice, gelsolin deficiency alters the equilibrium between erythrocyte actin polymerization and depolymerization, causing impaired terminal maturation. We suggest a non-redundant role for gelsolin in terminal erythroid differentiation, possibly contributing to the Gsn(-/-) mice lethality observed in mid-gestation.

Bone marrow-derived cells can acquire cardiac stem cells properties in damaged heart.

Experimental data suggest that cell-based therapies may be useful for cardiac regeneration following ischaemic heart disease. Bone marrow (BM) cells have been reported to contribute to tissue repair after myocardial infarction (MI) by a variety of humoural and cellular mechanisms. However, there is no direct evidence, so far, that BM cells can generate cardiac stem cells (CSCs). To investigate whether BM cells contribute to repopulate the Kit(+) CSCs pool, we transplanted BM cells from transgenic mice, expressing green fluorescent protein under the control of Kit regulatory elements, into wild-type irradiated recipients. Following haematological reconstitution and MI, CSCs were cultured from cardiac explants to generate 'cardiospheres', a microtissue normally originating in vitro from CSCs. These were all green fluorescent (i.e. BM derived) and contained cells capable of initiating differentiation into cells expressing the cardiac marker Nkx2.5. These findings indicate that, at least in conditions of local acute cardiac damage, BM cells can home into the heart and give rise to cells that share properties of resident Kit(+) CSCs.

Spontaneous calcium oscillations regulate human cardiac progenitor cell growth.

RATIONALE: The adult heart possesses a pool of progenitor cells stored in myocardial niches, but the mechanisms involved in the activation of this cell compartment are currently unknown. OBJECTIVE: Ca2+ promotes cell growth raising the possibility that changes in intracellular Ca2+ initiate division of c-kit-positive human cardiac progenitor cells (hCPCs) and determine their fate. METHODS AND RESULTS: Ca2+ oscillations were identified in hCPCs and these events occurred independently from coupling with cardiomyocytes or the presence of extracellular Ca2+. These findings were confirmed in the heart of transgenic mice in which enhanced green fluorescent protein was under the control of the c-kit promoter. Ca2+ oscillations in hCPCs were regulated by the release of Ca2+ from the endoplasmic reticulum through activation of inositol 1,4,5-triphosphate receptors (IP3Rs) and the reuptake of Ca2+ by the sarco-/endoplasmic reticulum Ca2+ pump (SERCA). IP3Rs and SERCA were highly expressed in hCPCs, whereas ryanodine receptors were not detected. Although Na+-Ca2+ exchanger, store-operated Ca2+ channels and plasma membrane Ca2+ pump were present and functional in hCPCs, they had no direct effects on Ca2+ oscillations. Conversely, Ca2+ oscillations and their frequency markedly increased with ATP and histamine which activated purinoceptors and histamine-1 receptors highly expressed in hCPCs. Importantly, Ca2+ oscillations in hCPCs were coupled with the entry of cells into the cell cycle and 5-bromodeoxyuridine incorporation. Induction of Ca2+ oscillations in hCPCs before their intramyocardial delivery to infarcted hearts was associated with enhanced engraftment and expansion of these cells promoting the generation of a large myocyte progeny. CONCLUSION: IP3R-mediated Ca2+ mobilization control hCPC growth and their regenerative potential.

FOS Rescues Neuronal Differentiation of Sox2-Deleted Neural Stem Cells by Genome-Wide Regulation of Common SOX2 and AP1(FOS-JUN) Target Genes.

The transcription factor SOX2 is important for brain development and for neural stem cells (NSC) maintenance. Sox2-deleted (Sox2-del) NSC from neonatal mouse brain are lost after few passages in culture. Two highly expressed genes, Fos and Socs3, are strongly downregulated in Sox2-del NSC; we previously showed that Fos or Socs3 overexpression by lentiviral transduction fully rescues NSC's long-term maintenance in culture. Sox2-del NSC are severely defective in neuronal production when induced to differentiate. NSC rescued by Sox2 reintroduction correctly differentiate into neurons. Similarly, Fos transduction rescues normal or even increased numbers of immature neurons expressing beta-tubulinIII, but not more differentiated markers (MAP2). Additionally, many cells with both beta-tubulinIII and GFAP expression appear, indicating that FOS stimulates the initial differentiation of a "mixed" neuronal/glial progenitor. The unexpected rescue by FOS suggested that FOS, a SOX2 transcriptional target, might act on neuronal genes, together with SOX2. CUT&RUN analysis to detect genome-wide binding of SOX2, FOS, and JUN (the AP1 complex) revealed that a high proportion of genes expressed in NSC are bound by both SOX2 and AP1. Downregulated genes in Sox2-del NSC are highly enriched in genes that are also expressed in neurons, and a high proportion of the "neuronal" genes are bound by both SOX2 and AP1.

Repression of kit expression by Plzf in germ cells.

Male mice lacking expression of Plzf, a DNA sequence-specific transcriptional repressor, show progressive germ cell depletion due to exhaustion of the spermatogonial stem cell population. This is likely due to the deregulated expression of genes controlling the switch between spermatogonial self-renewal and differentiation. Here we show that Plzf directly represses the transcription of kit, a hallmark of spermatogonial differentiation. Plzf represses both endogenous kit expression and expression of a reporter gene under the control of the kit promoter region. A discrete sequence of the kit promoter, required for Plzf-mediated kit transcriptional repression, is bound by Plzf both in vivo and in vitro. A 3-bp mutation in this Plzf binding site abolishes the responsiveness of the kit promoter to Plzf repression. A significant increase in kit expression is also found in the undifferentiated spermatogonia isolated from Plzf(-/-) mice. Thus, we suggest that one mechanism by which Plzf maintains the pool of spermatogonial stem cells is through a direct repression of kit expression.

Green fluorescent protein transgene driven by Kit regulatory sequences is expressed in hematopoietic stem cells.

BACKGROUND: The transcriptional regulation of stem cell genes is still poorly understood. Kit, encoding the stem cell factor receptor, is a pivotal molecule for multiple types of stem/progenitor cells. We previously generated mouse lines expressing transgenic green fluorescent protein under the control of Kit promoter/first intron regulatory elements, and we demonstrated expression in hematopoietic progenitors. DESIGN AND METHODS: In the present work we investigated whether the transgene is also expressed in hematopoietic stem cells of adult bone marrow and fetal liver. To this purpose, we tested, in long-term repopulating assays, cell fractions expressing different levels of green fluorescent protein within Kit-positive or SLAM-selected populations. RESULTS: The experiments demonstrated transgene expression in both fetal and adult hematopoietic stem cells and indicated that the transgene is transcribed at distinctly lower levels in hematopoietic stem cells than in pluripotent and committed progenitors. CONCLUSIONS: These results, together with previous data, show that a limited subset of DNA sequences drives gene expression in number of stem cell types (hematopoietic stem cells, primordial germ cells, cardiac stem cells). Additionally, our results might help to further improve high level purification of hematopoietic stem cells for experimental purposes. Finally, as the Kit/green fluorescent protein transgene is expressed in multiple stem cell types, our transgenic model provides powerful in vivo system to track these cells during development and tissue regeneration.

Evolution of the c-kit-positive cell response to pathological challenge in the myocardium.

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.