NIH Public Access Author Manuscript Circ Res. Author manuscript; available in PMC 2012 June 10.

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1 NIH Public Access Author Manuscript Published in final edited form as: Circ Res June 10; 108(12): doi: /circresaha The IGF-1 Receptor Identifies a Pool of Human Cardiac Stem Cells with Superior Therapeutic Potential for Myocardial Regeneration Domenico D Amario 1, Mauricio Cabral-Da-Silva 1, Hanqiao Zheng 1, Claudia Fiorini 2, Polina Goichberg 1, Elisabeth Steadman 1, João Ferreira-Martins 1, Fumihiro Sanada 1, Marco Piccoli 1,2, Donato Cappetta 1, David A. D Alessandro 3, Robert E. Michler 3, Toru Hosoda 1, Luigi Anastasia 1,2, Marcello Rota 1, Annarosa Leri 1, Piero Anversa 1, and Jan Kajstura 1 1 Departments of Anesthesia and Medicine, and Division of Cardiovascular Medicine, Brigham and Women s Hospital, Harvard Medical School, Boston, MA Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, IRCCS Policlinico San Donato, San Donato Milanese, Milan, Italy 3 Department of Cardiothoracic Surgery, Montefiore Medical Center, Albert Einstein College of Medicine, New York, NY Abstract Rationale Age and coronary artery disease may negatively affect the function of human cardiac stem cells (hcscs) and their potential therapeutic efficacy for autologous cell transplantation in the failing heart. Objective Insulin-like growth factor 1 (IGF-1) and 2 (IGF-2), and angiotensin II (Ang II) and their receptors, IGF-1R, IGF-2R and AT1R, were characterized in c-kit-positive-hcscs to establish whether these systems would allow us to separate hcsc classes with different growth reserve in the aging and diseased myocardium. Methods and Results C-kit-positive-hCSCs were collected from myocardial samples obtained from 24 patients, 48 to 86 years of age, undergoing elective cardiac surgery for coronary artery disease. The expression of IGF-1R in hcscs recognized a young cell phenotype defined by long telomeres, high telomerase activity, enhanced cell proliferation and attenuated apoptosis. In addition to IGF-1, IGF-1R-positive-hCSCs secreted IGF-2 that promoted myocyte differentiation. Conversely, the presence of IGF-2R and AT1R, in the absence of IGF-1R, identified senescent hcscs with impaired growth reserve and increased susceptibility to apoptosis. The ability of IGF-1R-positive-hCSCs to regenerate infarcted myocardium was then compared with that of unselected c-kit-positive-hcscs. IGF-1R-positive-hCSCs improved cardiomyogenesis and Correspondence to: Piero Anversa, MD, or Jan Kajstura, PhD, Departments of Anesthesia and Medicine, 75 Francis Street, Brigham and Women s Hospital, Harvard Medical School, Boston, MA 02115; phone: ; panversa@partners.org or jkajstura@partners.org. Disclosures Dr. Anversa is a member of Autologous, LLC. In March 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.2 days. Subject Codes: [11], [130], [147], [154] This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 D Amario et al. Page 2 vasculogenesis. Pretreatment of IGF-1R-positive-hCSCs with IGF-2 resulted in the formation of more mature myocytes and superior recovery of ventricular structure. Conclusions hcscs expressing only IGF-1R synthesize both IGF-1 and IGF-2, which are potent modulators of stem cell replication, commitment to the myocyte lineage and myocyte differentiation, pointing to this hcsc subset as the ideal candidate cell for the management of human heart failure. Keywords IGFs; Angiotensin II; Stem cell growth and death; Stem cell senescence Methods Following the recognition that hematopoietic stem cells may improve the outcome of myocardial infarction in animal models, 1 bone marrow mononuclear cells, CD34-positive cells and mesenchymal stromal cells have been introduced clinically with rather consistent results. The intracoronary or intramyocardial injection of these cell classes has been shown to be safe and to produce a modest but significant enhancement in systolic function. 2 The identification of resident cardiac stem cells in the human heart, 3 together with the isolation of a complex pool of cardiac cells, namely the cardiospheres, 4 has created great expectation concerning the potential implementation of these categories of autologous cells for the management of the human disease. Preclinical studies have been completed and two phase 1 clinical trials are in progress in patients affected by acute (Identifier: NCT ) and chronic (Identifier: NCT ) ischemic cardiomyopathy. Although initial information is currently available on the efficacy of human cardiac stem cells (hcscs), 5 the age of the patient and the type and duration of the disease may affect the number and growth properties of these primitive cells. Telomere attrition, cellular senescence and apoptosis all contribute to decrease the compartment of functionally-competent hcscs in the old failing heart. 6,7 Because of the multiple variables that interfere with the growth behavior of stem cells in the aging, decompensated heart, the objective of the current study was to define whether specific surface receptors can be employed to distinguish hcscs with high and low replicative reserve and ability to form myocytes and coronary vessels within the damaged myocardium. Based on clinical and animal data on aging and ischemic heart failure, 8,9 the insulin-like growth factor (IGF) system and the renin-angiotensin system (RAS) were characterized in hcscs to establish whether they can be employed to distinguish pools of primitive cells with different therapeutic value for patients with ventricular dysfunction. Additionally, the effect of IGF-1, IGF-2 and angiotensin II (Ang II) on hcsc division, maturation and death was determined. A series of in vitro and in vivo assays were performed to evaluate the independent and combined function of IGF-1 receptor (IGF-1R), IGF-2 receptor (IGF-2R) and angiotensin type 1 receptor (AT1R) and their respective ligands in hcsc growth and repair capacity. Twenty-four human myocardial samples were used to isolate and expand hcscs and define their in vitro properties. Distinct classes of hcscs with low and high level of cell growth were then injected in the infarcted heart to establish their differences in cardiac repair. Protocols are described at

3 D Amario et al. Page 3 Results Growth Factor Receptor Systems in hcscs Three complementary methodologies were employed to document the presence of three growth factor-receptor systems in c-kit-positive hcscs. IGF-1-IGF-1R, IGF-2-IGF-2R and RAS were identified in these cells by qrt-pcr, FACS analysis and immunocytochemistry. These protocols were introduced to exclude that protein products were derived from uptake of ligands from the circulation, raising question on the origin of IGF-1, IGF-2 and Ang II and the ability of hcscs to synthesize these growth factors. Transcripts for IGF-1, IGF-1R, IGF-2, IGF-2R, angiotensinogen (Aogen), renin, angiotensin-converting enzyme (ACE) and AT1R were found in all cases, while AT2R mrna was undetectable (Figure 1A and Online Figure I). Importantly, IGF-1, IGF-1R, IGF-2, IGF-2R, AT1R and AT2R proteins were present in hcscs. These ligand and receptor epitopes were detected by FACS (Figure 1B), immunolabeling and confocal microscopy (Figure 1C), and Western blotting (Figure 1D). However, ACE was not detectable by Western blotting. ELISA was employed to document the synthesis and secretion of IGF-1, IGF-2, and Ang II in hcscs. For this purpose, these growth factors were measured in both hcscs and culture medium (Figure 1E). The localization of Aogen, renin, ACE and Ang II in the cytoplasm of hcscs was established by confocal microscopy (Figure 1F). The function of IGF-1 is largely mediated by binding to the receptor tyrosine kinase IGF-1R. 10 Alternative splicing occurs at the 5 and 3 ends of the Igf1 gene giving rise to several isoforms. All isoforms produce an identical mature form of IGF-1 that prior to secretion may undergo cleavage with release of the E peptide. 11 Although not all E peptides are cleaved prior to the secretion of IGF-1, it is generally believed that mature IGF-1 is the main mediator of the actions of IGF-1 through IGF-1R. 12 The Eb-peptide fragments, IBE1 and IBE2, are produced by IGF-1 class B isoforms and stimulate cell growth. 11,12 However, this E peptide extension is unique to the IGF-1 class B. For IGF-1 class A and C, splicing results in a frame shift and a premature termination of translation. Based on qrt-pcr, the predominant IGF-1 isoform in hcscs and human myocardium was IGF-1A (Figure 1G and 1H). Thus, hcscs possess three growth factor-receptor systems which may have implications in defining the biological properties of these cells. Age, Diabetes and hcscs A cohort of 24 patients affected by chronic coronary artery disease was studied. These patients underwent elective bypass surgery for multi-vessel coronary atherosclerosis and largely preserved cardiac function. Age varied from 48 to 86 years and both genders were represented: 10 women and 14 men. Type 2 diabetes was present in 7 women and 4 men and hypertension in 4 women and 10 men (Online Table I). At surgery, the right atrial appendage was sampled and c-kit-positive hcscs were isolated and propagated in vitro (Figure 2A). At P5-P6, expanded hcscs were characterized by FACS analysis. At these passages, 90±3% hcscs were c-kit-positive and lineage negative. hcscs did not express markers of hematopoietic stem cells and bone marrow derived cells. Epitopes of mesenchymal stromal cells were also absent (Figure 2B). Transcription factors and cytoplasmic proteins specific of myocytes, smooth muscle cells (SMCs) and endothelial cells (ECs) were rarely observed (Figure 2C). Aging was associated with changes in expression of IGF-1R, IGF-2R and AT1R in hcscs. The fraction of IGF-1R-positive hcscs decreased with age, while the proportion of IGF-2Rand AT1R-positive cells increased. A comparable impact of age was observed on the intracellular content of IGF-1, IGF-2 and Ang II (Figure 2D). Type 2 diabetes further

4 D Amario et al. Page 4 IGFs and hcsc Growth increases the number of hcscs positive for IGF-2R and AT1R. Gender differences did not result in changes in receptor and ligand expression (Online Figure II). Our IRB precluded the acquisition of clinical data regarding the actual degree of ventricular dysfunction and the duration of coronary artery disease. Thus, aging upregulates the RAS and the IGF-2-IGF-2R axis, and attenuates the IGF-1-IGF-1R pathway in hcscs; diabetes has an additive negative effect on these parameters. To define the role of IGF-1R and IGF-2R in hcscs, cells from 6-10 patients were randomly selected and studied further molecularly, biochemically and functionally. These analyses included the assessment of cell proliferation, differentiation, senescence, apoptosis, telomere-telomerase axis, and downstream effectors related to IGF-1R signaling. These in vitro assays were complemented with in vivo protocols. Auto-phosphorylation of IGF-1R leads to recruitment of the insulin receptor substrate protein (IRS) that upregulates PI3K and Akt. According to its cytoplasmic or nuclear localization phospho-akt favors cellular hypertrophy, differentiation or proliferation. 13,14 IGF-2 is the predominant form of IGF in humans; it binds to IGF-1R, IGF-2R, and to the insulin receptor A isoform. 15 Binding of IGF-2 to IGF-2R promotes IGF-2 degradation and prevents its interaction with IGF-1R. 16 To determine the role of IGF-1R and IGF-2R in hcsc growth, cells expressing only one of these two receptors, or both, were sorted by FACS (Figure 3A) and characterized (Figure 3B). Population doubling time (PDT) was shorter in IGF-1R-positive, longer in IGF-2Rpositive and intermediate in IGF-1R-IGF-2R-positive hcscs (Figure 3C). Since PDT defines the growth kinetics of cells but does not assess the fraction of cycling cells, this variable was obtained by BrdU labeling: IGF-1R-positive hcscs showed levels of BrdU labeling higher than IGF-1R-IGF-2R-positive and IGF-2R-positive cells (Figure 3D). Longterm culture of IGF-1R-positive hcscs up to 20 passages did not result in alterations in the cell karyotype (Figure 3E). Additionally, the fraction of IGF-1R-positive and IGF-2Rpositive hcscs was preserved during their expansion in culture (Figure 3F). Subsequently, the impact of each ligand, IGF-1 and IGF-2, on hcsc division was established. IGF-1 increased the proliferation of hcscs expressing IGF-1R alone or together with IGF-2R. IGF-2 had similar consequences but of smaller magnitude (Figure 3G). Conversely, IGF-1 or IGF-2 failed to enhance the degree of BrdU labeling of IGF-2Rpositive hcscs. To document that the growth promoting effects of IGF-1 and IGF-2 on hcscs were mediated by activation of IGF-1R, these protocols were repeated in the presence of IGF-1R blocking antibody. Under this condition, hcsc division was largely inhibited (Figure 3H). Thus, IGF-1 and IGF-2 promote hcsc division by activating IGF-1R, while IGF-2R has no influence on hcsc replication. The differential response of IGF-1R-positive IGF-2R-negative hcscs to IGF-1 and IGF-2 was characterized by the level and timing of phosphorylation of Akt at Ser473. This posttranslation modification was selected because its subcellular distribution in the cytoplasm and in the nucleus determines cellular division and hypertrophy, respectively. 13 IGF-1 and IGF-2 both stimulated IGF-1R but the active state of the receptor was significantly higher and more prolonged in the presence of IGF-1 (Figure 3I). Importantly, nearly 10% of hcscs exposed to IGF-1 and IGF-2 showed a nuclear localization of Akt at 10 minutes. However, at 1 hour, the fraction of Akt-labeled nuclei decreased markedly with IGF-2 and remained elevated with IGF-1 (Figure 3J).

5 D Amario et al. Page 5 IGFs and hcsc Differentiation IGFs and hcsc Death In several cell systems, IGF-1 and IGF-2 promote cell differentiation rather than cell proliferation. 10 Although these opposite processes of cell growth may be cell context dependent, whether activation of IGF-1R by IGF-1 or IGF-2 regulates exclusively hcsc division or induces also the commitment of hcscs to the myocyte lineage is presently unknown. Additionally, the response of hcscs to IGFs may be time dependent; the early activation of IGF-1R by IGF-1 or IGF-2 and cell multiplication (see Figure 3G) may be followed by hcsc differentiation. The availability of hcscs positive only for IGF-1R allowed us to precisely define the role of IGF-1R in these primitive cells. Lineage negative hcscs were exposed to IGF-1 or IGF-2 for 12 and 36 hours and the expression of GATA4, Nkx2.5, and α-sarcomeric actin (α-sa) was determined by qrtpcr. After IGF-2 stimulation of IGF-1R-positive hcscs, the quantity of mrna for GATA4, Nkx2.5 and α-sa did not change at 12 hours, but increased at 36 hours. Similarly, transcripts of L-type Ca 2+ channels, α-subunit of voltage gated Na+ channels type V, and ryanodine receptor increased from 12 to 24 hours. IGF-1 had smaller effects on hcsc differentiation at 24 hours (Figure 4A). Changes at the protein level were evaluated at 12, 24 and 48 hours after exposure to IGF-1 or IGF-2. Following IGF-2 stimulation, the percentage of IGF-1R-positive hcscs expressing GATA4, Nkx2.5 and α-sa increased progressively from 12, to 24 and 48 hours. A comparable behavior was observed with respect to L-type Ca 2+ channels, α-subunit of voltage gated Na+ channels type V, and ryanodine receptor. Myofibrils with sarcomere striations were observed in the subsarcolemmal region (Figure 4B and 4C). FACS analysis at 48 hours confirmed the lineage specification of hcscs (Figure 4D). The impact of IGF-1 on IGF-1R-positive hcscs and myocyte commitment was less pronounced than that of IGF-2 and was mostly apparent at 48 hours. However, at this time point, there was a marked increase in the concentration of IGF-2 in the medium (Figure 4E), mediated by the synthesis and secretion of this growth factor by activation of IGF-1R in hcscs. Cardiomyocyte differentiation by IGF-1 or IGF-2 stimulation of hcscs positive for both IGF-1R and IGF-2R or positive only for IGF-2R was rather modest (Figure III in the online-only Data Supplement). The critical role of IGF-2 in the activation of IGF-1R and myocyte commitment of hcscs was confirmed by the use of neutralizing IGF-1R antibody, which interfered with myocyte differentiation and largely preserved the primitive cell phenotype (Figure 4F). Thus, the transition from the replicative state to lineage specification of hcscs appears to involve the autocrine/paracrine release of IGF-2. The impact of IGF-2 on the downstream effector of IGF-1R, mtor, were determined; by exposing hcscs to IGF-2 in the presence of the mtor inhibitor, rapamycin. Under this condition, the expression of genes conditioning myocyte differentiation were markedly attenuated; they included Nkx2.5, GATA4, L-type Ca 2+ channels, α-sarcomeric actin, and the α-subunit of voltage gated Na + channels type V (Figure 4G). Although the anti-apoptotic effects of IGF-1 on cardiac stem cells have repeatedly been documented, 14 the role of IGF-2 in hcsc survival or death has not been defined. This is a relevant question in view of the importance of IGF-2 in the maturation of myocytes and the fact that this growth factor is the predominant form of IGFs in humans. 15 Ang II was employed as trigger for apoptotic cell death. 8

6 D Amario et al. Page 6 At baseline, apoptosis, measured by TdT labeling, was modest and lower in IGF-1R-positive hcscs than in IGF-1R-IGF-2R- and IGF-2R-positive hcscs. Exposure to Ang II at 10 9 M for a period of 12 hours increased apoptosis in all three hcsc categories, but predominantly in IGF-2R- and IGF-1R-IGF-2R-positive hcscs. The addition of IGF-1 attenuated cell death in IGF-1R- and IGF-1R-IGF-2R-positive cells (Figure 5A through 5C). Conversely, IGF-2 increased apoptosis in hcscs expressing IGF-2R alone or together with IGF-1R, raising the possibility that IGF-2 and Ang II may have a synergistic effect on the activation of hcsc death. The differential response to Ang II of IGF-1R-, IGF-2R- and IGF-1R- IGF-2R-positive hcscs reflected comparable differences in the expression of AT1R in these three stem cell classes, as measured by FACS analysis (Figure 5D). Moreover, to establish the specificity of IGF-2 in the induction of cell apoptosis, IGF-2R-positive hcscs were challenged with IGF-2 alone in the presence of IGF-2R blocking antibody. Inhibition of IGF-2R function completely abrogated the apoptotic effect of IGF-2 (Figure 5E). Thus, activation of IGF-2R by IGF-2 initiates apoptosis opposing the survival effects promoted by IGF-1. IGFs, RAS and the Telomere-Telomerase Axis in hcscs The growth of stem cells is regulated by the length of their telomeres and the level of telomerase activity which restores in part the telomere DNA lost following each cell division. 17 These two variables of stem cell expansion were measured in a subset of 12 patients, from 48 to 86 years of age, to define the replication growth reserve hcscs expressing only IGF-1R, IGF-2R or AT1R; these receptors characterize the role of IGFs and Ang II in hcsc function. This information was critical for the recognition of the hcsc compartment which possessed the highest potential for myocardial regeneration in the diseased heart. In all 12 cases, IGF-1R-positive hcscs had longer telomeres than IGF-2R-positive cells; AT1R-positive hcscs had the shortest telomeres (Figure 6A and 6B). Surprisingly, a pool of hcscs with essentially normal telomere length, 9-10 kbp, was present in the human heart at all ages. Therefore, aging results in an increase in the pool of hcscs with short telomeres, i.e., AT1R-positive hcscs, and in a decrease in the pool of hcscs with long telomeres, i.e., IGF-1R-positive hcscs (see Figure 2D). However, age does not deplete the compartment of highly functional resident stem cells, suggesting that the old heart retains a considerable growth reserve for cell turnover and tissue repair. Measurements of telomerase activity in the three classes of hcscs in each of the 12 cases showed a comparable behavior (Figure 6C). IGF-1R-positive hcscs had the highest enzyme activity while IGF-2R-positive and AT1R-positive hcscs showed a reduced value, confirming that IGF-1R-positive hcscs constitute the most powerful cell subset. This notion was consistent with the expression of the senescence-associated protein p16 INK4a in hcscs; p16 INK4a expression was significantly higher in IGF-2R-positive and AT1Rpositive hcscs than in IGF-1R-positive hcscs (Figure 6D). Collectively, these findings suggest that a non-selected hcsc population should have a more limited regeneration potential than the restricted, highly enriched pool of IGF-1R-positive hcscs. Additionally, stimulation of IGF-1R-positive hcscs with IGF-2 may enhance their in vivo differentiation into mature cardiomyocytes, offering therapeutic options for the repair process of the infarcted heart. Myocardial Regeneration after Infarction To determine the therapeutic efficacy of unselected hcscs (Un-hCSCs), IGF-1R-positive hcscs (IGF-1R-hCSCs), and IGF-2 activated IGF-1R-positive hcscs (Ac-IGF-1R-hCSCs), cells were infected with a lentivirus carrying EGFP and delivered intramyocardially shortly

7 D Amario et al. Page 7 after coronary artery ligation in immunosuppressed rats. Infarcted immunosuppressed rats injected with PBS were used as controls. IGF-2 stimulated IGF-2R-positive cells were not included in this analysis in view of their limited growth capacity extensively characterized in vitro. Additionally, animals injected with PBS constituted the most appropriate negative control. At 10 days after surgery and cell delivery, large areas of tissue regeneration composed of newly-formed EGFP-positive human myocytes and coronary vessels replaced the infarcted myocardium of the left ventricle (LV). Cardiac repair was not observed in untreated infarcts (Figure 7A through 7D), suggesting that myocyte and vessel formation in this group of animals was restricted to the surviving myocardium without invasion of the scarred region of the wall. 3,18,19 Infarct size, measured by the number of rat myocytes lost as a result of permanent coronary occlusion, involved nearly 70% of the cells of the free wall of the LV (Figure 7E). In all cases, the band of newly-formed human tissue was distributed in the mid-portion of the infarct and only occasionally reached the epi- or endomyocardium (Figure 7D). The human origin of the regenerated myocytes was confirmed by the detection of human DNA sequences with an Alu probe, together with the identification of human X-chromosomes by Q-FISH and the expression of human troponin I (Figure 7F through 7H). Similarly, the regenerated human coronary arterioles and capillaries were Alu-positive and carried human X-chromosomes (Figure 7I and 7J). In the three cell treated infarcts, the newly-formed packed myocytes occupied ~84% of the regenerated tissue, while arterioles and capillaries accounted for ~8% (Figure 7K). The specificity of the recorded signal for EGFP, α-sa, human troponin I, and Alu was validated by spectral analysis (Online Figure IV). The human myocardium comprised 7, 12 and 15 mm 3 of tissue following treatment with Un-hCSCs, IGF-1R-hCSCs and Ac-IGF-1R-hCSCs, respectively (Figure 8A), indicating that the unselected pool of hcscs was associated with a smaller degree of tissue reconstitution. The increased recovery in myocardial mass in the other two cases resulted in attenuation of ventricular dilation and thinning of the wall in the spared and infarcted region of the LV. Additionally, wall thickness-to-chamber radius ratio and left ventricular mass-tochamber volume ratio were largely restored (Figure 8B). Differences were found in the magnitude and characteristics of the human myocardium formed by IGF-1R-hCSCs and Ac-IGF-1R-hCSCs. The latter led to a 30% higher degree of tissue regeneration which was the product of a 2.7-fold larger myocyte volume and a 40% lower myocyte number (Figure 8C). The prevailing hypertrophic response observed with Ac-IGF-1R-hCSCs was consistent with the in vitro findings and the lower level of myocyte replication measured by Ki67 in the regenerated myocardium (Figure 8D). Moreover, capillary length density per mm 3 of myocardium was higher in these hearts, reflecting a commensurate magnitude of vascularization dictated by the larger myocyte cross-sectional area (Figure 8E). Treatment with IGF-1R-hCSCs and Ac-IGF-1R-hCSCs was coupled with reduced hypertrophy of spared rat myocytes (Figure 8F) and less negative LV remodeling (see Figure 8B). Functionally, myocardial regeneration was characterized by the reappearance of contraction in the infarcted region of the LV and superior improvement in LV systolic pressure and positive and negative dp/dt (Figure 8G). Additionally, calculated diastolic wall stress was significantly decreased in these animals. Thus, IGF-1R-positive hcscs provide a remarkable recovery of the structure and function of the infarcted heart which is further enhanced by IGF-2 activation. Although similar changes in ventricular function were observed with IGF-1R-hCSCs and Ac-IGF-1R-hCSCs, the phenotypical properties of the regenerated cardiomyocytes differed with these protocols.

8 D Amario et al. Page 8 Discussion In summary, as illustrated schematically (Figure 8H), binding of IGF-1 and IGF-2 to IGF-1R activates the PI3K-Akt pathway leading to Akt phosphorylation at Ser473, which is more prolonged with IGF-1 than with IGF-2. Additionally, the presence of IGF-1R is coupled with enhanced telomerase activity and preservation of telomere integrity, which, in turn, favor hcsc division and survival. Commitment of hcscs to the myocyte lineage involves the rapamycin-dependent and the rapamycin-independent mtor signaling mechanisms which condition myocyte maturation. Conversely, binding of Ang II to AT1R and IGF-2 to IGF-2R leads to hcsc apoptosis, possibly mediated by PKC phosphorylation of p The results of the current study indicate that isolation and expansion of c-kit-positive hcscs from small samples of human myocardium yields a heterogeneous cell population composed of stem cell subsets with considerably different growth reserve in vitro and in vivo. The stem cell antigen c-kit is expressed in a population of hematopoietic stem cells that are capable of differentiating into cardiomyocytes and coronary vessels, replacing in part large myocardial infarcts with restoration of ventricular performance. 1 Similarly, the c-kit receptor tyrosine kinase identifies a pool of cardiac cells which reside in niches, 21 possess the properties of stem cells and regenerate in vivo infarcted tissue with contracting myocardium. 3,22 However, our findings show that the association of c-kit with distinct proteins on the membrane of hcscs conditions functional differences within an apparently uniform cell compartment. The behavior of hcscs is dictated by a specific surface phenotype which permits the selective isolation of young highly dividing hcscs from the pool of c-kit-positive cells. Different membrane receptors affect the phenotypic plasticity of hcscs and their ability to compensate myocyte loss by forming new efficiently contracting parenchymal cells. Aging progressively decreases the compartment of hcscs with high regenerative potential and progressively increases the pool of stem cells with minimal or no ability to divide and acquire cardiac cell lineages. The loss of hcsc function with aging is mediated partly by an imbalance between factors promoting growth and survival, and factors enhancing oxidative stress, telomere attrition and apoptosis. Three growth-factor receptor systems seem to have a critical role in hcsc replication, differentiation, senescence and death: IGF-1-IGF-1R, IGF-2-IGF-2R and RAS. The IGF-1-IGF-1R induces hcsc division, upregulates telomerase activity, maintains telomere length, hinders replicative senescence and preserves the population of functionally-competent CSCs in animals 23 and, as demonstrated here, in humans. The expression of IGF-1R and the synthesis of IGF-1 are attenuated in aging hcscs possibly diminishing the ability of IGF-1 to activate cell proliferation and interfere with oxidative damage and telomeric shortening. 24 In progenitor cells, IGF-1 is generally linked to the protection of the primitive phenotype 25 while IGF-2 induces the acquisition of the committed state, a phenomenon that has been identified in the present study in hcscs. The presence of IGF-2 conditions the osteogenic differentiation of mesenchymal stromal cells 26 and the formation of skeletal myoblasts by satellite cells. 27 The function of IGF-1R-positive hcscs is regulated by both IGF-1 and IGF-2 which exert opposite effects on the fate of these cells. Importantly, cardiomyocytes and fibroblasts surrounding hcscs have the ability to secrete the IGF-1 and IGF-2 ligand, 28,29 suggesting that a cross-talk occurs within the cardiac niches where myocytes and fibroblasts function as supporting cells. 21,30 These cells may dictate the developmental decision of hcscs according to the needs of the organ. Based on the current data in humans and previous findings in animals, 8,18,19 IGF-1 may activate IGF-1R-positive hcscs which possess high telomerase activity and rather intact telomeres, favoring their migration out of

9 D Amario et al. Page 9 the niche area to regions of myocyte replacement. Defects in telomerase activity and telomere length oppose lodging and motility of progenitor cells in the skin 31 and in the heart. 8 Additionally, IGF-2, formed via an autocrine/paracrine mechanism, 32 may promote the transition of amplifying cells to a class of myocytes with a more mature phenotype, structurally and mechanically. Consistent with these observations, skeletal muscle differentiation is strictly dependent on an autocrine loop initiated by the muscle secretion of IGF-2 that binds to IGF-1R in myoblasts. 33 This effector pathway targets transcriptional regulators of MyoD and the myogenin promoter. 34,35 A similar mechanism appears to mediate cardiomyocyte differentiation, although the signaling cascade located downstream IGF-1R in hcscs remains to be determined. Thus, the recognition that hcscs expressing only IGF-1R synthesize both IGF-1 and IGF-2, which are potent modulators of stem cell replication, commitment to the myocyte lineage and myocyte differentiation, points to this hcsc subset as the ideal candidate cell for the management of human heart failure. The main function of IGF-2R is related to the clearance of IGF-2. Binding of IGF-2 to IGF-2R limits ligand bioavailability, inducing IGF-2 degradation and, thereby, preventing its interaction with IGF-1R. 16 In the current study, we have identified a novel function of IGF-2; ligand binding to IGF-2R promotes apoptosis of hcscs and enhances the effects of Ang II on cell death. The high level of expression of AT1R in IGF-2R-positive hcscs defines the surface phenotype of senescent cells, greatly prone to apoptotic death. Under conditions in which hcsc survival is essential for the well-being of the organ and organism, the upregulation of death genes appears to be a paradoxical response. However, preservation of the steady state may conform to an intrinsic mechanism aiming at the maintenance of a constant number of functionally-competent hcscs within the myocardial niches. Apoptosis of hcscs may be regarded as a biological adaptation that removes unwanted old cells and concurrently triggers replication of young hcscs and their commitment to the myocyte lineage. IGFs are bound to IGF-binding proteins (IGFBPs), which modulate IGF ligand-receptor interactions and, thereby, their function. IGFPBs prolong the half-life of IGFs, but this process can result in the inhibition or stimulation of IGF1R and IGF-2R pathway. 36 Although the importance of IGFBPs is largely unknown, these proteins add a level of complexity to the study of IGFs. Recently, IGFBP-4 has been shown to enhance cardiomyocyte differentiation in vitro, while its deletion attenuates cardiomyogenesis in vitro and in vivo. 37 This role of IGFBP-4 is independent from its IGF-binding activity and is mediated by its direct interaction with Wnt receptors. The documentation that a local RAS is present in hcscs and the formation of Ang II, together with the expression of AT1R, increases with age in hcscs provides evidence in favor of the role of this octapeptide in hcsc senescence and death. Ang II may contribute to the age-dependent accumulation of oxidative damage in the heart. 38 Inhibition of Ang II positively interferes with heart failure and prolongs life in humans. 39 Ang II generates reactive oxygen species (ROS) and the most prominent form of DNA damage induced by free radicals is 8-OH-dG. In the presence of Ang II, 8-OH-dG increases more in old than in young progenitor cells. 8 8-OH-dG accumulates at the GGG triplets of telomeres resulting in telomeric shortening and uncapping. 40 Loss of telomere integrity is the major determinant of cellular senescence and death. Conversely, IGF-1 interferes with ROS generation, 38 decreases oxidative stress with age, 23 and can repair DNA damage by homologous recombination. 41 Cardiac restricted overexpression of IGF-1 delays the aging myopathy and the manifestations of heart failure in transgenic mice. 23 Thus, changes in the proportion of these growth factor receptor systems in hcscs condition their growth reserve and potential

10 D Amario et al. Page 10 therapeutic efficacy, a phenomenon documented here after infarction and the delivery of hcsc subsets. In summary, our results suggest that the clinical implementation of autologous hcscs in patients with acute and chronic ischemic cardiomyopathy necessitates a rather sophisticated approach which involves the characterization of the molecular signature of the cells to be delivered. Although extremely large numbers of different classes of bone marrow progenitor cells are currently being administered to patients, and hcscs are now entering the scene of cell therapy for the decompensated heart, a careful analysis of the phenotypic properties of the cells to be used has to be considered. The quality of the cells may be the critical factor for successful clinical outcome rather than cell number. Supplementary Material Acknowledgments Refer to Web version on PubMed Central for supplementary material. We are grateful to the Cytogenetics Core Laboratory of Dana Farber Harvard Cancer Center, Boston, for the evaluation of the karyotype of hcscs (P30 CA006516). Sources of Funding This work was supported by NIH grants. Non-standard Abbreviations and Acronyms hcscs IGF-1 IGF-1R IGF-2 IGF-2R RAS Ang II ACE Angiotensinogen AT1R AT2R TERT mtor References human cardiac stem cells insulin-like growth factor-1 insulin-like growth factor-1 receptors insulin-like growth factor-2 insulin-like growth factor-2 receptors renin-angiotensin system angiotensin II angiotensin converting enzyme Aogen angiotensin II type 1 receptors angiotensin II type 2 receptors telomerase protein mammalian target of rapamycin 1. Anversa P, Leri A, Kajstura J. Cardiac regeneration. J Am Coll Cardiol. 2006; 47: [PubMed: ] 2. Abdel-Latif A, Bolli R, Tleyjeh IM, Montori VM, Perin EC, Hornung CA, Zuba-Surma EK, Al- Mallah M, Dawn B. Adult bone marrow-derived cells for cardiac repair: a systematic review and meta-analysis. Arch Intern Med. 2007; 167: [PubMed: ]

11 D Amario et al. Page Bearzi C, Rota M, Hosoda T, Tillmanns J, Nascimbene A, De Angelis A, Yasuzawa-Amano S, Trofimova I, Siggins RW, Lecapitaine N, Cascapera S, Beltrami AP, D Alessandro DA, Zias E, Quaini F, Urbanek K, Michler RE, Bolli R, Kajstura J, Leri A, Anversa P. Human cardiac stem cells. Proc Natl Acad Sci USA. 2007; 104: [PubMed: ] 4. Smith RR, Barile L, Cho HC, Leppo MK, Hare JM, Messina E, Giacomello A, Abraham MR, Marbán E. Regenerative potential of cardiosphere-derived cells expanded from percutaneous endomyocardial biopsy specimens. Circulation. 2007; 115: [PubMed: ] 5. Bolli R, Chugh AR, D Amario D, Stoddard MF, Ikram S, Wagner SG, Beache GM, Leri A, Hosoda T, Loughran JH, Goihberg P, Fiorini C, Solankhi NK, Fahsah I, Chatterjee A, Elmore JB, Rokosh DG, Slaughter MS, Kajstura J, Anversa P. Use of cardiac stem cells for the treatment of heart failure: translation from bench to the clinical setting. Circ Res. 2010:e33. Abstract. 6. Chimenti C, Kajstura J, Torella D, Urbanek K, Heleniak H, Colussi C, Di Meglio F, Nadal-Ginard B, Frustaci A, Leri A, Maseri A, Anversa P. Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure. Circ Res. 2003; 93: [PubMed: ] 7. Urbanek K, Torella D, Sheikh F, De Angelis A, Nurzynska D, Silvestri F, Beltrami CA, Bussani R, Beltrami AP, Quaini F, Bolli R, Leri A, Kajstura J, Anversa P. Myocardial regeneration by activation of multipotent cardiac stem cells in ischemic heart failure. Proc Natl Acad Sci U S A. 2005; 102: [PubMed: ] 8. Gonzalez A, Rota M, Nurzynska D, Misao Y, Tillmanns J, Ojaimi C, Padin-Iruegas ME, Müller P, Esposito G, Bearzi C, Vitale S, Dawn B, Sanganalmath SK, Baker M, Hintze TH, Bolli R, Urbanek K, Hosoda T, Anversa P, Kajstura J, Leri A. Activation of cardiac progenitor cells reverses the failing heart senescent phenotype and prolongs lifespan. Circ Res. 2008; 102: [PubMed: ] 9. Opie LH, Commerford PJ, Gersh BJ, Pfeffer MA. Controversies in ventricular remodeling. Lancet. 2006; 367: [PubMed: ] 10. Baserga R. The contradictions of the insulin-like growth factor 1 receptor. Oncogene. 2000; 19: [PubMed: ] 11. Favelyukis S, Till JH, Hubbard SR, Miller WT. Structure and autoregulation of the insulin-line growth factor 1 receptor kinase. Nat Struct Biol. 2001; 8: [PubMed: ] 12. Li W, Miller WT. Role of the activation loop tyrosines in regulation of the insulin-like growth factor 1 receptor-tyrosine kinase. J Biol Chem. 2006; 281: [PubMed: ] 13. Walsh K. Akt signaling and growth of the heart. Circulation. 2006; 113: [PubMed: ] 14. Heller Brown J, Del Re DP, Sussman MA. The Rac and Rho hall of fame. A decade of hypertrophic signaling hits. Circ Res. 2006; 98: [PubMed: ] 15. Denley A, Bonython ER, Booker GW, Cosgrove LJ, Forbes BE, Ward CW, Wallace JC. Structural determinants for high-affinity binding of insulin-like growth factor II to insulin receptor (IR)-A, the exon 11 minus isoform of the IR. Mol Endocrinol. 2004; 18: [PubMed: ] 16. Ghosh P, Dahms NM, Kornfeld S. Mannose 6-phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol. 2003; 4: [PubMed: ] 17. Chan SW, Blackburn EH. New ways not to make ends meet: telomerase, DNA damage proteins and heterochromatin. Oncogene. 2002; 21: [PubMed: ] 18. Rota M, Padin-Iruegas ME, Misao Y, De Angelis A, Maestroni S, Ferreira-Martins J, Fiumana E, Rastaldo R, Arcarese ML, Mitchell TS, Boni A, Bolli R, Urbanek K, Hosoda T, Anversa P, Leri A, Kajstura J. Local activation or implantation of cardiac progenitor cells rescues scarred infarcted myocardium improving cardiac function. Circ Res. 2008; 103: [PubMed: ] 19. Padin-Iruegas ME, Misao Y, Davis ME, Segers VF, Esposito G, Tokunou T, Urbanek K, Hosoda T, Rota M, Anversa P, Leri A, Lee RT, Kajstura J. Cardiac progenitor cells and biotinylated insulin-like growth factor-1 nanofibers improve endogenous and exogenous myocardial regeneration after infarction. Circulation. 2009; 120: [PubMed: ] 20. Leri A, Claudio PP, Li Q, Wang X, Reiss K, Wang S, Malhotra A, Kajstura J, Anversa P. Stretchmediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the

12 D Amario et al. Page 12 local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell. J Clin Invest. 1998; 101: [PubMed: ] 21. Urbanek K, Cesselli D, Rota M, Nascimbene A, De Angelis A, Hosoda T, Bearzi C, Boni A, Bolli R, Kajstura J, Anversa P, Leri A. Stem cell niches in the adult mouse heart. Proc Natl Acad Sci U S A. 2006; 103: [PubMed: ] 22. Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K, Leri A, Kajstura J, Nadal-Ginard B, Anversa P. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003; 114: [PubMed: ] 23. Torella D, Rota M, Nurzynska D, Musso E, Monsen A, Shiraishi I, Zias E, Walsh K, Rosenzweig A, Sussman MA, Urbanek K, Nadal-Ginard B, Kajstura J, Anversa P, Leri A. Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression. Circ Res. 2004; 94: [PubMed: ] 24. Kenyon C. The plasticity of aging: insights from long-lived mutants. Cell. 2005; 120: [PubMed: ] 25. Ye P, D Ercole AJ. Insulin-like growth factor actions during development of neural stem cells and progenitors in the central nervous system. J Neurosci Res. 2006; 83:1 6. [PubMed: ] 26. Chen L, Jiang W, Huang J, He BC, Zuo GW, Zhang W, Luo Q, Shi Q, Zhang BQ, Wagner ER, Luo J, Tang M, Wietholt C, Luo X, Bi Y, Su Y, Liu B, Kim SH, He CJ, Hu Y, Shen J, Rastegar F, Huang E, Gao Y, Gao JL, Zhou JZ, Reid RR, Luu HH, Haydon RC, He TC, Deng ZL. Insulin-like growth factor 2 (IGF2) potentiates BMP9-induced osteogenic differentiation and bone formation. J Bone Miner Res In press. 27. Roel W, Broek T, Grefte S, Johannes W, Von den Hoff JW. Regulatory factors and cell populations involved in skeletal muscle regeneration. Journal of Cellular Physiology. 2010; 224:7 16. [PubMed: ] 28. Hu BS, Landeen LK, Aroonsakool N, Giles WR. An analysis of the effects of stretch on IGF-I secretion from rat ventricular fibroblasts. Am J Physiol Heart Circ Physiol. 2007; 293:H677 H683. [PubMed: ] 29. Blaauw E, van Nieuwenhoven FA, Willemsen P, Delhaas T, Prinzen FW, Snoeckx LH, van Bilsen M, van der Vusse GJ. Stretch-induced hypertrophy of isolated adult rabbit cardiomyocytes. Am J Physiol Heart Circ Physiol. 2010; 299:H780 H787. [PubMed: ] 30. Bearzi C, Leri A, Lo Monaco F, Rota M, Gonzalez A, Hosoda T, Pepe M, Qanud K, Ojaimi C, Bardelli S, D Amario D, D Alessandro DA, Michler RE, Dimmeler S, Zeiher AM, Urbanek K, Hintze TH, Kajstura J, Anversa P. Identification of a coronary vascular progenitor cell in the human heart. Proc Natl Acad Sci USA. 2009; 106: [PubMed: ] 31. Flores I, Cayuela ML, Blasco MA. Effects of telomerase and telomere length on epidermal stem cell behavior. Science. 2005; 309: [PubMed: ] 32. Rosenthal SM, Brunetti A, Brown EJ, Mamula PW, Goldfine ID. Regulation of insulin-like growth factor (IGF) I receptor expression during muscle cell differentiation. Potential autocrine role of IGF-II. J Clin Invest. 1991; 87: [PubMed: ] 33. Philippou A, Halapas A, Maridaki M, Koutsilieris M. Type I insulin-like growth factor receptor signaling in skeletal muscle regeneration and hypertrophy. J Musculoskelet Neuronal Interact. 2007; 7: [PubMed: ] 34. Wilson EM, Rotwein P. Control of MyoD function during initiation of muscle differentiation by an autocrine signaling pathway activated by insulin-like growth factor-ii. J Biol Chem. 2006; 281: [PubMed: ] 35. Erbay E, Park IH, Nuzzi PD, Schoenherr CJ, Chen J. IGF-II transcription in skeletal myogenesis is controlled by mtor and nutrients. J Cell Biol. 2003; 163: [PubMed: ] 36. Le Roith D. Regulation of proliferation and apoptosis by the insulin-like growth factor I receptor. Growth Horm IGF Res. 2000; 10(Suppl A):S12 S13. [PubMed: ] 37. Zhu W, Shiojima I, Ito Y, Li Z, Ikeda H, Yoshida M, Naito AT, Nishi J, Ueno H, Umezawa A, Minamino T, Nagai T, Kikuchi A, Asashima M, Komuro I. IGFBP-4 is an inhibitor of canonical Wnt signalling required for cardiogenesis. Nature. 2008; 454: [PubMed: ]

13 D Amario et al. Page Kajstura J, Fiordaliso F, Andreoli AM, Li B, Chimenti S, Medow MS, Limana F, Nadal-Ginard B, Leri A, Anversa P. IGF-1 overexpression inhibits the development of diabetic cardiomyopathy and angiotensin II-mediated oxidative stress. Diabetes. 2001; 50: [PubMed: ] 39. O Meara E, Clayton T, McEntegart MB, McMurray JJV, Pina IL, Granger CB, Ostergren J, Michelson EL, Solomon SD, Pocock S, Yusuf S, Swedberg K, Pfeffer MA, CHARM Investigators. Sex differences in clinical characteristics and prognosis in a broad spectrum of patients with heart failure. Circulation. 2007; 115: [PubMed: ] 40. Kawanishi S, Oikawa S. Mechanism of telomere shortening by oxidative stress. Ann NY Acad Sci. 2004; 1019: [PubMed: ] 41. Yang S, Chintapalli J, Sodagum L, Baskin S, Malhotra A, Reiss K, Meggs LG. Activated IGF-1R inhibits hyperglycemia-induced DNA damage and promotes DNA repair by homologous recombination. Am J Physiol Renal Physiol. 2005; 289:F1144 F1152. [PubMed: ]

14 D Amario et al. Page 14 What Is Known? Novelty and Significance Pathological changes in the heart and aging negatively affect the function of human cardiac stem cells (hcscs). The decrease of the proliferative activity and differentiation capacity of hcscs may be related to the changes in the growth factor receptor systems.. Human myocardial aging may be in part due to impairment in the behavior of hcscs. What New Information Does This Article Contribute? Young hcscs express IGF-1 receptors and this characteristic is associated with high telomerase activity and intact telomere length. IGF-2 modulates the differentiation of hcscs into functionally-competent cardiomyocytes by binding to IGF-1 receptors. Old hcscs have short telomeres, low telomerase activity and they synthesize Ang II, which by activating AT1 receptors, triggers apoptotic cell death. IGF-1 receptor positive hcscs have high regenerative ability and they restore large quantity of infarcted myocardium, representing a potent cell population for cardiac repair. The isolation and expansion of c-kit-positive hcscs from small samples of human myocardium yields a heterogeneous cell population composed of stem cell subsets with highly variable growth reserve in vitro and in vivo. The association of c-kit with distinct proteins on the membrane of hcscs leads to functional differences within an apparently uniform cell population The behavior of hcscs is dictated by a specific surface phenotype which permits selective isolation of young highly dividing hcscs from the pool of c-kit-positive cells. Different membrane receptors affect the phenotypic plasticity of hcscs and their ability to compensate myocyte loss by forming new efficiently contracting parenchymal cells.

15 D Amario et al. Page 15 Figure 1. Growth factor receptor systems in hcscs A, Transcripts for IGF-1, IGF-1R, IGF-2, IGF-2R, Aogen, renin, ACE and AT1R in c-kitpositive hcscs. Electrophoresis of PCR products is shown (for sequences see Online Figure I). B, Scatter plots of hcscs; the stem cell antigen c-kit is expressed together with IGF-1, IGF-1R, IGF-2, IGF-2R, AT1R and AT2R. Negative isotype control is also shown. C, Selected fields illustrating by immunolabeling and confocal microscopy that c-kit-positive hcscs (green) express IGF-1, IGF-1R, IGF-2, IGF-2R, AT1R and AT2R. D, Expression of IGF-1R, IGF-2R, angiotensinogen (Aogen), renin, AT1R and AT2R in hcscs by Western blotting. GAPDH, loading conditions. E, Concentration of IGF-1, IGF-2, and Ang II by ELISA in culture medium (Medium) and hcscs. F, Selected fields illustrating by immunolabeling and confocal microscopy that c-kit-positive hcscs express Aogen, renin, ACE, and Ang II. G, For the detection of IGF-1 isoforms, primers were designed between exons to avoid genomic DNA amplification. DNA sequences in the adjacent introns were examined to exclude binding of the primers to these regions. Primers 1F and 2F recognize class 1 and 2 isoforms, respectively. Primers AR, BR and CR identify transcripts of class A,

16 D Amario et al. Page 16 B and C. Primer 1=2R is common for class 1 and 2 isoforms whereas primers A=B=CR are shared by class A, B and C. H, PCR products and expression level of IGF-1 splice isoforms in hcscs and human myocardium (hheart). For sequences: see Online Figure I.

17 D Amario et al. Page 17 Figure 2. Effects of age and diabetes on hcscs A, c-kit-positive (green) hcscs at P5. B and C, Scatter plots of c-kit-positive hcscs at P5- P6, showing the lack of hematopoietic and mesenchymal cell markers (B), and minimal levels of expression of cardiac cell lineages (C). D, Relationships of aging and receptor and ligand expression in hcscs. Diabetes affected further IGF-2R and AT1R expression. *P<0.05 vs. non-diabetic patients.

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19 D Amario et al. Page 19 Figure 3. IGFs and hcscs A, Bivariate distribution of c-kit, IGF-1R and IGF-2R in hcscs. B, Immunolabeling and confocal microscopy documenting the co-expression of IGF-1R (upper left) and IGF2R (upper central) in freshly sorted hcscs. Upper right, merge. Sorted hcscs continue to express IGF-1R (lower left) and IGF-2R (lower right) in culture. C, Phase contrast micrographs of hcsc classes and corresponding PDT values. *,**P<0.05 vs. IGF-1Rpositive and IGF-2R-positive hcscs, respectively. D, BrdU immunolabeling and confocal microscopy of hcscs and fraction of BrdU-labeled cells. *,**P<0.05 vs. IGF-1R-positive and IGF-2R-positive hcscs, respectively. E, Euploid set of chromosomes in a metaphase spread of hcscs at P20. F, Fraction of hcscs positive for IGF-1R and IGF2-R from P2 to P7. G, BrdU-labeling of hcsc classes in the presence (+) or absence (-) of IGF-1, or IGF-2. *P<0.05 vs. hcscs in the absence of IGF-1; P<0.05 vs. IGF-1R-positive hcscs. H, BrdUlabeling of IGF-1R-positive hcscs in the presence (+) or absence (-) of IGF-1, or IGF-2, and in the presence or absence of IGF-1R neutralizing antibody. *,**,***, P<0.05 vs. unsorted hcscs, absence of IGF-1, absence of IGF-1R antibody, absence of IGF-2, respectively. I, Expression of phospho-akt Ser473 in IGF-1R-positive IGF-2R-negative hcscs stimulated with IGF-1 or IGF-2 for 10 and 60 minutes. Control, untreated hcscs. GAPDH, loading conditions. OD, optical density. J, Nuclear localization of phospho- Akt Ser473 in IGF-1R-positive IGF-2R-negative hcscs stimulated with IGF-1 or IGF-2 for 10 and 60 minutes.

20 D Amario et al. Page 20 Figure 4. IGFs and hcsc differentiation A and B, Changes in GATA4, Nkx2.5, α-sa, L-type Ca 2+ channels (CACNA1c), α-subunit of voltage gated Na+ channels type V (Scn5a), and ryanodine receptor (RyR2) mrna (A) and protein (B) following IGF-1 and IGF-2 stimulation of hcscs. C, Developing myocytes show sarcomere striation in the subsarcolemmal region. D, FACS analysis of hcsc commitment to the myocyte lineage. E, Cytokine array of the medium collected after

21 D Amario et al. Page 21 stimulation of hcscs with IGF-1. F, Neutralizing antibody against IGF-1R (+) and expression of markers of myocyte commitment in hcscs following or IGF-2 (+) stimulation. G, Transcripts for myocyte specific genes in hcscs exposed to IGF-2 or IGF-2 together with rapamycin (RAPA).

22 D Amario et al. Page 22 Figure 5. IGFs and hcsc death A, TdT labeling (white) of IGF-2R-positive hcscs exposed to Ang II (c-kit, green). B, Apoptosis at baseline. *P<0.05 vs. IGF-1R-positive hcscs. C, Effects of Ang II together with IGF-1 or IGF-2 on apoptosis. *,**P<0.05 vs. Ang II alone, and Ang II in combination with IGF-1, respectively. D, Scatter plots documenting the fraction of AT1R-positive hcscs in each class of primitive cells. Quantitative results are also shown. *,**P<0.05 vs. IGF-1Rpositive hcscs, and IGF-2R-positive hcscs, respectively. E, IGF-2 stimulation and apoptosis in IGF-2R-positive hcscs. Neutralizing antibody against IGF-2R prevents IGF-2- mediated apoptosis. *,**P<0.05 vs. baseline, and IGF-2 only, respectively.

23 D Amario et al. Page 23 Figure 6. Telomere-telomerase system in hcscs A, hcscs expressing IGF-1R (left) have longer telomeres (magenta dots) than hcscs positive for IGF-2R (right). B, Telomere length in IGF-1R- (green), IGF-2R (blue) and AT1R- (red) positive hcscs measured by flow-fish. Example of flow-fish data (upper left), average values in the 12 patients (upper right), and individual values in each of the 12 patients (lower). C, Telomerase activity: average values (upper), and individual values in each of the 12 patients in each of the three classes of hcscs (lower). D, Localization of p16 INK4a (yellow) in c-kit-positive (green) IGF-2R-positive hcscs. Fraction of p16 INK4a - positive cells in the three classes of hcscs. *P<0.05 vs. IGF-1R-positive hcscs.

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25 D Amario et al. Page 25 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

26 D Amario et al. Page 26 Figure 7. Myocardial regeneration after infarction A-D, Myocardial scarring (A: collagen, white) is partially replaced by EGFP-positive (green) cardiomyocytes and coronary vessels (arrowheads) following delivery of Un-hCSCs (B), IGF-1R-hCSCs (C), and Ac-IGF-1R-hCSCs (D). Inserts show higher magnification of the region included in rectangles: myocytes (α-sa, red). E, Fraction of LV myocytes lost following coronary occlusion. F-H, Localization of Alu DNA sequences (F: white dots in nuclei), human X-chromosome (G: bright blue dot in nuclei), and human troponin I (H, red) in regenerated myocytes following delivery of IGF-1R-hCSCs (F and H) or Ac-IGF-1RhCSCs (G). I and J, Human coronary arterioles (I and J) and capillaries (J) show Alu DNA sequences (I) or are positive for EGFP and carry human X-chromosome (J). α-sma, red; vwf, magenta. K, Volume composition of the regenerated human myocardium. O.I., other interstitium.

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28 D Amario et al. Page 28 Figure 8. Remodeling of the infarcted heart A, Extent of myocardial recovery 10 days after the delivery of Un-hCSCs, IGF-1R-hCSCs, and Ac-IGF-1R-hCSCs. B, Cardiac anatomy. C, Characteristics of newly-formed myocytes. D, Localization of Ki67 (yellow) in regenerated cardiomyocytes (left). Quantitative data (right). E, Capillary length density in the regenerated myocardium. F, Volume of spared myocytes. G, M-mode echocardiography and hemodynamic parameters. *,**,***P<0.05 vs. sham-operated (SO), infarcted untreated (MI), Un-hCSCs, respectively. H, Scheme, see text.