Fumihiro Sanada

Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial.

BACKGROUND c-kit-positive, lineage-negative cardiac stem cells (CSCs) improve post-infarction left ventricular (LV) dysfunction when administered to animals. We undertook a phase 1 trial (Stem Cell Infusion in Patients with Ischemic cardiOmyopathy [SCIPIO]) of autologous CSCs for the treatment of heart failure resulting from ischaemic heart disease. METHODS In stage A of the SCIPIO trial, patients with post-infarction LV dysfunction (ejection fraction [EF] ≤40%) before coronary artery bypass grafting were consecutively enrolled in the treatment and control groups. In stage B, patients were randomly assigned to the treatment or control group in a 2:3 ratio by use of a computer-generated block randomisation scheme. 1 million autologous CSCs were administered by intracoronary infusion at a mean of 113 days (SE 4) after surgery; controls were not given any treatment. Although the study was open label, the echocardiographic analyses were masked to group assignment. The primary endpoint was short-term safety of CSCs and the secondary endpoint was efficacy. A per-protocol analysis was used. This study is registered with ClinicalTrials.gov, number NCT00474461. FINDINGS This study is still in progress. 16 patients were assigned to the treatment group and seven to the control group; no CSC-related adverse effects were reported. In 14 CSC-treated patients who were analysed, LVEF increased from 30·3% (SE 1·9) before CSC infusion to 38·5% (2·8) at 4 months after infusion (p=0·001). By contrast, in seven control patients, during the corresponding time interval, LVEF did not change (30·1% [2·4] at 4 months after CABG vs 30·2% [2·5] at 8 months after CABG). Importantly, the salubrious effects of CSCs were even more pronounced at 1 year in eight patients (eg, LVEF increased by 12·3 ejection fraction units [2·1] vs baseline, p=0·0007). In the seven treated patients in whom cardiac MRI could be done, infarct size decreased from 32·6 g (6·3) by 7·8 g (1·7; 24%) at 4 months (p=0·004) and 9·8 g (3·5; 30%) at 1 year (p=0·04). INTERPRETATION These initial results in patients are very encouraging. They suggest that intracoronary infusion of autologous CSCs is effective in improving LV systolic function and reducing infarct size in patients with heart failure after myocardial infarction, and warrant further, larger, phase 2 studies. FUNDING University of Louisville Research Foundation and National Institutes of Health.

Evidence for Human Lung Stem Cells

BACKGROUND Although progenitor cells have been described in distinct anatomical regions of the lung, description of resident stem cells has remained elusive. METHODS Surgical lung-tissue specimens were studied in situ to identify and characterize human lung stem cells. We defined their phenotype and functional properties in vitro and in vivo. RESULTS Human lungs contain undifferentiated human lung stem cells nested in niches in the distal airways. These cells are self-renewing, clonogenic, and multipotent in vitro. After injection into damaged mouse lung in vivo, human lung stem cells form human bronchioles, alveoli, and pulmonary vessels integrated structurally and functionally with the damaged organ. The formation of a chimeric lung was confirmed by detection of human transcripts for epithelial and vascular genes. In addition, the self-renewal and long-term proliferation of human lung stem cells was shown in serial-transplantation assays. CONCLUSIONS Human lungs contain identifiable stem cells. In animal models, these cells participate in tissue homeostasis and regeneration. They have the undemonstrated potential to promote tissue restoration in patients with lung disease. (Funded by the National Institutes of Health.).

Cardiomyogenesis in the Adult Human Heart

Rationale: The ability of the human heart to regenerate large quantities of myocytes remains controversial, and the extent of myocyte renewal claimed by different laboratories varies from none to nearly 20% per year. Objective: To address this issue, we examined the percentage of myocytes, endothelial cells, and fibroblasts labeled by iododeoxyuridine in postmortem samples obtained from cancer patients who received the thymidine analog for therapeutic purposes. Additionally, the potential contribution of DNA repair, polyploidy, and cell fusion to the measurement of myocyte regeneration was determined. Methods and Results: The fraction of myocytes labeled by iododeoxyuridine ranged from 2.5% to 46%, and similar values were found in fibroblasts and endothelial cells. An average 22%, 20%, and 13% new myocytes, fibroblasts, and endothelial cells were generated per year, suggesting that the lifespan of these cells was approximately 4.5, 5, and 8 years, respectively. The newly formed cardiac cells showed a fully differentiated adult phenotype and did not express the senescence-associated protein p16INK4a. Moreover, measurements by confocal microscopy and flow cytometry documented that the human heart is composed predominantly of myocytes with 2n diploid DNA content and that tetraploid and octaploid nuclei constitute only a small fraction of the parenchymal cell pool. Importantly, DNA repair, ploidy formation, and cell fusion were not implicated in the assessment of myocyte regeneration. Conclusions: Our findings indicate that the human heart has a significant growth reserve and replaces its myocyte and nonmyocyte compartment several times during the course of life.

Human Cardiac Stem Cell Differentiation Is Regulated by a Mircrine Mechanism

Background— Cardiac stem cells (CSCs) delivered to the infarcted heart generate a large number of small fetal-neonatal cardiomyocytes that fail to acquire the differentiated phenotype. However, the interaction of CSCs with postmitotic myocytes results in the formation of cells with adult characteristics. Methods and Results— On the basis of results of in vitro and in vivo assays, we report that the commitment of human CSCs (hCSCs) to the myocyte lineage and the generation of mature working cardiomyocytes are influenced by microRNA-499 (miR-499), which is barely detectable in hCSCs but is highly expressed in postmitotic human cardiomyocytes. miR-499 traverses gap junction channels and translocates to structurally coupled hCSCs favoring their differentiation into functionally competent cells. Expression of miR-499 in hCSCs represses the miR-499 target genes Sox6 and Rod1, enhancing cardiomyogenesis in vitro and after infarction in vivo. Although cardiac repair was detected in all cell-treated infarcted hearts, the aggregate volume of the regenerated myocyte mass and myocyte cell volume were greater in animals injected with hCSCs overexpressing miR-499. Treatment with hCSCs resulted in an improvement in ventricular function, consisting of a better preservation of developed pressure and positive and negative dP/dt after infarction. An additional positive effect on cardiac performance occurred with miR-499, pointing to enhanced myocyte differentiation/hypertrophy as the mechanism by which miR-499 potentiated the restoration of myocardial mass and function in the infarcted heart. Conclusions— The recognition that miR-499 promotes the differentiation of hCSCs into mechanically integrated cardiomyocytes has important clinical implications for the treatment of human heart failure.

Cardiomyogenesis in the Aging and Failing Human Heart

Background— Two opposite views of cardiac growth are currently held; one views the heart as a static organ characterized by a large number of cardiomyocytes that are present at birth and live as long as the organism, and the other views the heart a highly plastic organ in which the myocyte compartment is restored several times during the course of life. Methods and Results— The average age of cardiomyocytes, vascular endothelial cells (ECs), and fibroblasts and their turnover rates were measured by retrospective 14C birth dating of cells in 19 normal hearts 2 to 78 years of age and in 17 explanted failing hearts 22 to 70 years of age. We report that the human heart is characterized by a significant turnover of ventricular myocytes, ECs, and fibroblasts, physiologically and pathologically. Myocyte, EC, and fibroblast renewal is very high shortly after birth, decreases during postnatal maturation, remains relatively constant in the adult organ, and increases dramatically with age. From 20 to 78 years of age, the adult human heart entirely replaces its myocyte, EC, and fibroblast compartment ≈8, ≈6, and ≈8 times, respectively. Myocyte, EC, and fibroblast regeneration is further enhanced with chronic heart failure. Conclusions— The human heart is a highly dynamic organ that retains a remarkable degree of plasticity throughout life and in the presence of chronic heart failure. However, the ability to regenerate cardiomyocytes, vascular ECs, and fibroblasts cannot prevent the manifestations of myocardial aging or oppose the negative effects of ischemic and idiopathic dilated cardiomyopathy.

Functionally Competent Cardiac Stem Cells Can Be Isolated From Endomyocardial Biopsies of Patients With Advanced Cardiomyopathies

Rationale: Two categories of cardiac stem cells (CSCs) with predominantly myogenic (mCSC) and vasculogenic (vCSC) properties have been characterized in the human heart. However, it is unknown whether functionally competent CSCs of both classes are present in the myocardium of patients affected by end-stage cardiac failure, and whether these cells can be harvested from relatively small myocardial samples. Objective: To establish whether a clinically relevant number of mCSCs and vCSCs can be isolated and expanded from endomyocardial biopsies of patients undergoing cardiac transplantation or left ventricular assist device implantation. Methods and Results: Endomyocardial biopsies were collected with a bioptome from the right side of the septum of explanted hearts or the apical LV core at the time of left ventricular assist device implantation. Two to 5 biopsies from each patient were enzymatically dissociated, and, after expansion, cells were sorted for c-kit (mCSCs) or c-kit and KDR (vCSCs) and characterized. mCSCs and vCSCs constituted 97% and 3% of the c-kit population, respectively. Population doubling time averaged 27 hours in mCSCs and vCSCs; 5×106 mCSCs and vCSCs were obtained in 28 and 41 days, respectively. Both CSC classes possessed significant growth reserve as documented by high telomerase activity and relatively long telomeres. mCSCs formed mostly cardiomyocytes, and vCSCs endothelial and smooth muscle cells. Conclusions: The growth properties of mCSCs and vCSCs isolated from endomyocardial biopsies from patients with advanced heart failure were comparable to those obtained previously from larger myocardial samples of patients undergoing elective cardiac surgery.

Insulin-like growth factor-1 receptor identifies a pool of human cardiac stem cells with superior therapeutic potential for myocardial regeneration.

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 (IGF)-1, IGF-2, and angiotensin II (Ang II), as well as their receptors, IGF-1R, IGF-2R, and AT1R, were characterized in c-kit+ 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+ 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+ 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+ hCSCs to regenerate infarcted myocardium was then compared with that of unselected c-kit+ hCSCs. IGF-1R+ hCSCs improved cardiomyogenesis and vasculogenesis. Pretreatment of IGF-1R+ 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, which points to this hCSC subset as the ideal candidate cell for the management of human heart failure.

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 Ca2+ 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 Ca2+, 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 Ca2+ 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.

Dissecting the Molecular Relationship Among Various Cardiogenic Progenitor Cells

Rationale: Multiple progenitors derived from the heart and bone marrow (BM) have been used for cardiac repair. Despite this, not much is known about the molecular identity and relationship among these progenitors. To develop a robust stem cell therapy for the heart, it is critical to understand the molecular identity of the multiple cardiogenic progenitor cells. Objective: This study is the first report of high-throughput transcriptional profiling of cardiogenic progenitor cells carried out on an identical platform. Method and Results: Microarray-based transcriptional profiling was carried out for 3 cardiac (ckit+, Sca1+, and side population) and 2 BM (ckit+ and mesenchymal stem cell) progenitors, obtained from age- and sex-matched wild-type C57BL/6 mice. Analysis indicated that cardiac-derived ckit+ population was very distinct from Sca1+ and side population cells in the downregulation of genes encoding for cell–cell and cell–matrix adhesion proteins, and in the upregulation of developmental genes. Significant enrichment of transcripts involved in DNA replication and repair was observed in BM-derived progenitors. The BM ckit+ cells seemed to have the least correlation with the other progenitors, with enrichment of immature neutrophil–specific molecules. Conclusions: Our study indicates that cardiac ckit+ cells represent the most primitive population in the rodent heart. Primitive cells of cardiac versus BM origin differ significantly with respect to stemness and cardiac lineage–specific genes, and molecules involved in DNA replication and repair. The detailed molecular profile of progenitors reported here will serve as a useful reference to determine the molecular identity of progenitors used in future preclinical and clinical studies.

c-kit-Positive Cardiac Stem Cells Nested in Hypoxic Niches are Activated by Stem Cell Factor Reversing the Aging Myopathy

Rationale: Hypoxia favors stem cell quiescence, whereas normoxia is required for stem cell activation, but whether cardiac stem cell (CSC) function is regulated by the hypoxic/normoxic state of the cell is currently unknown. Objective: A balance between hypoxic and normoxic CSCs may be present in the young heart, although this homeostatic control may be disrupted with aging. Defects in tissue oxygenation occur in the old myocardium, and this phenomenon may expand the pool of hypoxic CSCs, which are no longer involved in myocyte renewal. Methods and Results: Here, we show that the senescent heart is characterized by an increased number of quiescent CSCs with intact telomeres that cannot re-enter the cell cycle and form a differentiated progeny. Conversely, myocyte replacement is controlled only by frequently dividing CSCs with shortened telomeres; these CSCs generate a myocyte population that is chronologically young but phenotypically old. Telomere dysfunction dictates their actual age and mechanical behavior. However, the residual subset of quiescent young CSCs can be stimulated in situ by stem cell factor reversing the aging myopathy. Conclusions: Our findings support the notion that strategies targeting CSC activation and growth interfere with the manifestations of myocardial aging in an animal model. Although caution has to be exercised in the translation of animal studies to human beings, our data strongly suggest that a pool of functionally competent CSCs persists in the senescent heart and that this stem cell compartment can promote myocyte regeneration effectively, partly correcting the aging myopathy.