Christian Arranto

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.).

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.

Tracking Chromatid Segregation to Identify Human Cardiac Stem Cells that Regenerate Extensively the Infarcted Myocardium

Rationale: According to the immortal DNA strand hypothesis, dividing stem cells selectively segregate chromosomes carrying the old template DNA, opposing accumulation of mutations resulting from nonrepaired replication errors and attenuating telomere shortening. Objective: Based on the premise of the immortal DNA strand hypothesis, we propose that stem cells retaining the old DNA would represent the most powerful cells for myocardial regeneration. Methods and Results: Division of human cardiac stem cells (hCSCs) by nonrandom and random segregation of chromatids was documented by clonal assay of bromodeoxyuridine-tagged hCSCs. Additionally, their growth properties were determined by a series of in vitro and in vivo studies. We report that a small class of hCSCs retain during replication the mother DNA and generate 2 daughter cells, which carry the old and new DNA, respectively. hCSCs with immortal DNA form a pool of nonsenescent cells with longer telomeres and higher proliferative capacity. The self-renewal and long-term repopulating ability of these cells was shown in serial-transplantation assays in the infarcted heart; these cells created a chimeric organ, composed of spared rat and regenerated human cardiomyocytes and coronary vessels, leading to a remarkable restoration of cardiac structure and function. The documentation that hCSCs divide by asymmetrical and symmetrical chromatid segregation supports the view that the human heart is a self-renewing organ regulated by a compartment of resident hCSCs. Conclusions: The impressive recovery in ventricular hemodynamics and anatomy mediated by clonal hCSCs carrying the “mother” DNA underscores the clinical relevance of this stem cell class for the management of heart failure in humans.

Inositol 1, 4, 5-Trisphosphate Receptors and Human Left Ventricular Myocytes

Background— Little is known about the function of inositol 1,4,5-trisphosphate receptors (IP3Rs) in the adult heart experimentally. Moreover, whether these Ca2+ release channels are present and play a critical role in human cardiomyocytes remains to be defined. IP3Rs may be activated after G&agr;q-protein–coupled receptor stimulation, affecting Ca2+ cycling, enhancing myocyte performance, and potentially favoring an increase in the incidence of arrhythmias. Methods and Results— IP3R function was determined in human left ventricular myocytes, and this analysis was integrated with assays in mouse myocytes to identify the mechanisms by which IP3Rs influence the electric and mechanical properties of the myocardium. We report that IP3Rs are expressed and operative in human left ventricular myocytes. After G&agr;q-protein–coupled receptor activation, Ca2+ mobilized from the sarcoplasmic reticulum via IP3Rs contributes to the decrease in resting membrane potential, prolongation of the action potential, and occurrence of early afterdepolarizations. Ca2+ transient amplitude and cell shortening are enhanced, and extrasystolic and dysregulated Ca2+ elevations and contractions become apparent. These alterations in the electromechanical behavior of human cardiomyocytes are coupled with increased isometric twitch of the myocardium and arrhythmic events, suggesting that G&agr;q-protein–coupled receptor activation provides inotropic reserve, which is hampered by electric instability and contractile abnormalities. Additionally, our findings support the notion that increases in Ca2+ load by IP3Rs promote Ca2+ extrusion by forward-mode Na+/Ca2+ exchange, an important mechanism of arrhythmic events. Conclusions— The G&agr;q-protein/coupled receptor/IP3R axis modulates the electromechanical properties of the human myocardium and its propensity to develop arrhythmias.

Late Na + current and protracted electrical recovery are critical determinants of the aging myopathy

The aging myopathy manifests itself with diastolic dysfunction and preserved ejection fraction. We raised the possibility that, in a mouse model of physiological aging, defects in electromechanical properties of cardiomyocytes are important determinants of the diastolic characteristics of the myocardium, independently from changes in structural composition of the muscle and collagen framework. Here we show that an increase in the late Na+ current (INaL) in aging cardiomyocytes prolongs the action potential (AP) and influences temporal kinetics of Ca2+ cycling and contractility. These alterations increase force development and passive tension. Inhibition of INaL shortens the AP and corrects dynamics of Ca2+ transient, cell contraction and relaxation. Similarly, repolarization and diastolic tension of the senescent myocardium are partly restored. Thus, INaL offers inotropic support, but negatively interferes with cellular and ventricular compliance, providing a new perspective of the biology of myocardial aging and the aetiology of the defective cardiac performance in the elderly.

Response to Letter Regarding Article “Inositol 1,4,5-Trisphosphate Receptors and Human Left Ventricular Myocytes”

We thank Drs Heidrich and colleagues for their comments on our study discussing the role of inositol 1,4,5-trisphosphate receptors (IP3Rs) in ventricular myocytes.1 We documented that IP3Rs are present and operative in the rodent and human ventricular myocardium and that stimulation of Gαq-protein-coupled receptors promotes IP3R-mediated Ca2+ release. This adaptation provides inotropic support but favors electric instability. As pointed out by Dr Heidrich and colleagues, our report did not address the contribution of endogenous IP3R-regulatory proteins2 to the electrophysiological and contractile properties of ventricular myocytes. Specifically, they raised the possibility that chromogranin B, a sarcoplasmic reticulum Ca2+-binding protein, interacts with IP3Rs3 and modulates myocyte behavior after Gαq-protein-coupled receptor stimulation. The relationship between chromogranin B and IP3Rs …

Response to Letter Regarding Article, “Human Cardiac Stem Cell Differentiation Is Regulated by a Mircrine Mechanism”

We appreciate the interest of Dr Sluijter in our study that defined the role of miR-499 in the activation of the myogenic molecular program in human cardiac stem cells.1 We agree that the description of multiple classes of stem/progenitor cells in the adult heart has created controversy in the field. However, the human cardiomyocyte progenitor cells described by Sluijter and collaborators2 are not stem cells, but immature myocytes. Our conclusion is not based on the pattern of miR expression, but on the premise that human cardiomyocyte progenitor cells …