Barbara Ogorek

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.

Myocyte Turnover in the Aging Human Heart

Rationale: The turnover of cardiomyocytes in the aging female and male heart is currently unknown, emphasizing the need to define human myocardial biology. Objective: The effects of age and gender on the magnitude of myocyte regeneration and the origin of newly formed cardiomyocytes were determined. Methods and Results: The interaction of myocyte replacement, cellular senescence, growth inhibition, and apoptosis was measured in normal female (n=32) and male (n=42) human hearts collected from patients 19 to 104 years of age who died from causes other than cardiovascular diseases. A progressive loss of telomeric DNA in human cardiac stem cells (hCSCs) occurs with aging and the newly formed cardiomyocytes inherit short telomeres and rapidly reach the senescent phenotype. Our data provide novel information on the superior ability of the female heart to sustain the multiple variables associated with the development of the senescent myopathy. At all ages, the female heart is equipped with a larger pool of functionally competent hCSCs and younger myocytes than the male myocardium. The replicative potential is higher and telomeres are longer in female hCSCs than in male hCSCs. In the female heart, myocyte turnover occurs at a rate of 10%, 14%, and 40% per year at 20, 60, and 100 years of age, respectively. Corresponding values in the male heart are 7%, 12%, and 32% per year, documenting that cardiomyogenesis involves a large and progressively increasing number of parenchymal cells with aging. From 20 to 100 years of age, the myocyte compartment is replaced 15 times in women and 11 times in men. Conclusions: The human heart is a highly dynamic organ regulated by a pool of resident hCSCs that modulate cardiac homeostasis and condition organ aging.

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.

Clonality of mouse and human cardiomyogenesis in vivo

An analysis of the clonality of cardiac progenitor cells (CPCs) and myocyte turnover in vivo requires genetic tagging of the undifferentiated cells so that the clonal marker of individual mother cells is traced in the specialized progeny. CPC niches in the atria and apex of the mouse heart were infected with a lentivirus carrying EGFP, and the destiny of the tagged cells was determined 1–5 months later. A common integration site was identified in isolated CPCs, cardiomyocytes, endothelial cells (ECs), and fibroblasts, documenting CPC self-renewal and multipotentiality and the clonal origin of the differentiated cell populations. Subsequently, the degree of EGFP-lentiviral infection of CPCs was evaluated 2–4 days after injection, and the number of myocytes expressing the reporter gene was measured 6 months later. A BrdU pulse-chasing protocol was also introduced as an additional assay for the analysis of myocyte turnover. Over a period of 6 months, each EGFP-positive CPC divided approximately eight times generating 230 cardiomyocytes; this value was consistent with the number of newly formed cells labeled by BrdU. To determine whether, human CPCs (hCPCs) are self-renewing and multipotent, these cells were transduced with the EGFP-lentivirus and injected after acute myocardial infarction in immunosuppressed rats. hCPCs, myocytes, ECs, and fibroblasts collected from the regenerated myocardium showed common viral integration sites in the human genome. Thus, our results indicate that the adult heart contains a pool of resident stem cells that regulate cardiac homeostasis and repair.

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.

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.

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.

Repair of the Injured Adult Heart Involves New Myocytes Potentially Derived From Resident Cardiac Stem Cells

Rationale: The ability of the adult heart to generate new myocytes after injury is not established. Objective: Our purpose was to determine whether the adult heart has the capacity to generate new myocytes after injury, and to gain insight into their source. Methods and Results: Cardiac injury was induced in the adult feline heart by infusing isoproterenol (ISO) for 10 days via minipumps, and then animals were allowed to recover for 7 or 28 days. Cardiac function was measured with echocardiography, and proliferative cells were identified by nuclear incorporation of 5-bromodeoxyuridine (BrdU; 7-day minipump infusion). BrdU was infused for 7 days before euthanasia at days 10, 17, and 38 or during injury and animals euthanized at day 38. ISO caused reduction in cardiac function with evidence of myocyte loss from necrosis. During this injury phase there was a significant increase in the number of proliferative cells in the atria and ventricle, but there was no increase in BrdU+ myocytes. cKit+ cardiac progenitor cells were BrdU labeled during injury. During the first 7 days of recovery there was a significant reduction in cellular proliferation (BrdU incorporation) but a significant increase in BrdU+ myocytes. There was modest improvement in cardiac structure and function during recovery. At day 38, overall cell proliferation was not different than control, but increased numbers of BrdU+ myocytes were found when BrdU was infused during injury. Conclusions: These studies suggest that ISO injury activates cardiac progenitor cells that can differentiate into new myocytes during cardiac repair.

Inhibition of Notch1-Dependent Cardiomyogenesis Leads to a Dilated Myopathy in the Neonatal Heart

Rationale Physiological hypertrophy in the developing heart has been considered the product of an increase in volume of preexisting fetal cardiomyocytes in the absence of myocyte formation. Objective In this study, we tested whether the mouse heart at birth has a pool of cardiac stem cells (CSCs) that differentiate into myocytes contributing to the postnatal expansion of the parenchymal cell compartment. Methods and Results We have found that the newborn heart contains a population of c-kit–positive CSCs that are lineage negative, self-renewing, and multipotent. CSCs express the Notch1 receptor and show the nuclear localization of its active fragment, N1ICD. In 60% of cases, N1ICD was coupled with the presence of Nkx2.5, indicating that the commitment of CSCs to the myocyte lineage is regulated by Notch1. Importantly, overexpression of N1ICD in neonatal CSCs significantly expanded the proportion of transit-amplifying myocytes. To establish whether these in vitro findings had a functional counterpart in vivo, the Notch pathway was blocked in newborn mice by administration of a &ggr;-secretase inhibitor. This intervention resulted in the development of a dilated myopathy and high mortality rates. Ventricular decompensation was characterized by a 62% reduction in amplifying myocytes, which resulted in a 54% decrease in myocyte number. After cessation of Notch blockade and recovery of myocyte regeneration, cardiac anatomy and function were largely restored. Conclusions Notch1 signaling is a critical determinant of CSC growth and differentiation; when this cascade of events is altered, cardiomyogenesis is impaired, physiological cardiac hypertrophy is prevented, and a life-threatening myopathy supervenes.