A latent cardiomyocyte regeneration potential in human heart disease.

Cardiomyocytes in the adult human heart show a regenerative capacity, with an annual renewal rate around 0.5%. Whether this regenerative capacity of human cardiomyocytes is employed in heart failure has been controversial. Using retrospective 14C birth dating we analyzed cardiomyocyte renewal in patients with end-stage heart failure. We show that cardiomyocyte generation is minimal in end-stage heart failure patients at rates 18-50 times lower compared to the healthy heart. However, patients receiving left ventricle support device therapy, who showed significant functional and structural cardiac improvement, had a >6-fold increase in cardiomyocyte renewal relative to the healthy heart. Our findings reveal a substantial cardiomyocyte regeneration potential in human heart disease, which could be exploited therapeutically.

Diploid hepatocytes drive physiological liver renewal in adult humans.

Physiological liver cell replacement is central to maintaining the organ's high metabolic activity, although its characteristics are difficult to study in humans. Using retrospective radiocarbon (14C) birth dating of cells, we report that human hepatocytes show continuous and lifelong turnover, allowing the liver to remain a young organ (average age <3 years). Hepatocyte renewal is highly dependent on the ploidy level. Diploid hepatocytes show more than 7-fold higher annual birth rates than polyploid hepatocytes. These observations support the view that physiological liver cell renewal in humans is mainly dependent on diploid hepatocytes, whereas polyploid cells are compromised in their ability to divide. Moreover, cellular transitions between diploid and polyploid hepatocytes are limited under homeostatic conditions. With these findings, we present an integrated model of homeostatic liver cell generation in humans that provides fundamental insights into liver cell turnover dynamics.

Evidence for postnatal neurogenesis in the human amygdala.

The human amygdala is involved in processing of memory, decision-making, and emotional responses. Previous studies suggested that the amygdala may represent a neurogenic niche in mammals. By combining two distinct methodological approaches, lipofuscin quantification and 14C-based retrospective birth dating of neurons, along with mathematical modelling, we here explored whether postnatal neurogenesis exists in the human amygdala. We investigated post-mortem samples of twelve neurologically healthy subjects. The average rate of lipofuscin-negative neurons was 3.4%, representing a substantial proportion of cells substantially younger than the individual. Mass spectrometry analysis of genomic 14C-concentrations in amygdala neurons compared with atmospheric 14C-levels provided evidence for postnatal neuronal exchange. Mathematical modelling identified a best-fitting scenario comprising of a quiescent and a renewing neuronal population with an overall renewal rate of >2.7% per year. In conclusion, we provide evidence for postnatal neurogenesis in the human amygdala with cell turnover rates comparable to the hippocampus.

Cell generation dynamics underlying naive T-cell homeostasis in adult humans.

Thymic involution and proliferation of naive T cells both contribute to shaping the naive T-cell repertoire as humans age, but a clear understanding of the roles of each throughout a human life span has been difficult to determine. By measuring nuclear bomb test-derived 14C in genomic DNA, we determined the turnover rates of CD4+ and CD8+ naive T-cell populations and defined their dynamics in healthy individuals ranging from 20 to 65 years of age. We demonstrate that naive T-cell generation decreases with age because of a combination of declining peripheral division and thymic production during adulthood. Concomitant decline in T-cell loss compensates for decreased generation rates. We investigated putative mechanisms underlying age-related changes in homeostatic regulation of CD4+ naive T-cell turnover, using mass cytometry to profile candidate signaling pathways involved in T-cell activation and proliferation relative to CD31 expression, a marker of thymic proximity for the CD4+ naive T-cell population. We show that basal nuclear factor κB (NF-κB) phosphorylation positively correlated with CD31 expression and thus is decreased in peripherally expanded naive T-cell clones. Functionally, we found that NF-κB signaling was essential for naive T-cell proliferation to the homeostatic growth factor interleukin (IL)-7, and reduced NF-κB phosphorylation in CD4+CD31- naive T cells is linked to reduced homeostatic proliferation potential. Our results reveal an age-related decline in naive T-cell turnover as a putative regulator of naive T-cell diversity and identify a molecular pathway that restricts proliferation of peripherally expanded naive T-cell clones that accumulate with age.

Publisher Correction: Dynamics of oligodendrocyte generation in multiple sclerosis.

In this Letter, the vertical error bars were missing from Fig. 3b and 3c. This figure has been corrected online.

Dynamics of oligodendrocyte generation in multiple sclerosis.

Oligodendrocytes wrap nerve fibres in the central nervous system with layers of specialized cell membrane to form myelin sheaths1. Myelin is destroyed by the immune system in multiple sclerosis, but myelin is thought to regenerate and neurological function can be recovered. In animal models of demyelinating disease, myelin is regenerated by newly generated oligodendrocytes, and remaining mature oligodendrocytes do not seem to contribute to this process2-4. Given the major differences in the dynamics of oligodendrocyte generation and adaptive myelination between rodents and humans5-9, it is not clear how well experimental animal models reflect the situation in multiple sclerosis. Here, by measuring the integration of 14C derived from nuclear testing in genomic DNA10, we assess the dynamics of oligodendrocyte generation in patients with multiple sclerosis. The generation of new oligodendrocytes was increased several-fold in normal-appearing white matter in a subset of individuals with very aggressive multiple sclerosis, but not in most subjects with the disease, demonstrating an inherent potential to substantially increase oligodendrocyte generation that fails in most patients. Oligodendrocytes in shadow plaques-thinly myelinated lesions that are thought to represent remyelinated areas-were old in patients with multiple sclerosis. The absence of new oligodendrocytes in shadow plaques suggests that remyelination of lesions occurs transiently or not at all, or that myelin is regenerated by pre-existing, and not new, oligodendrocytes in multiple sclerosis. We report unexpected oligodendrocyte generation dynamics in multiple sclerosis, and this should guide the use of current, and the development of new, therapies.

Meningioma growth dynamics assessed by radiocarbon retrospective birth dating.

It is not known how long it takes from the initial neoplastic transformation of a cell to the detection of a tumor, which would be valuable for understanding tumor growth dynamics. Meningiomas show a broad histological, genetic and clinical spectrum, are usually benign and considered slowly growing. There is an intense debate regarding their age and growth pattern and when meningiomas should be resected. We have assessed the age and growth dynamics of 14 patients with meningiomas (WHO grade I: n=6 with meningothelial and n=6 with fibrous subtype, as well as n=2 atypical WHO grade II meningiomas) by combining retrospective birth-dating of cells by analyzing incorporation of nuclear-bomb-test-derived 14C, analysis of cell proliferation, cell density, MRI imaging and mathematical modeling. We provide an integrated model of the growth dynamics of benign meningiomas. The mean age of WHO grade I meningiomas was 22.1±6.5years, whereas atypical WHO grade II meningiomas originated 1.5±0.1years prior to surgery (p<0.01). We conclude that WHO grade I meningiomas are very slowly growing brain tumors, which are resected in average two decades after time of origination.

The Lifespan and Turnover of Microglia in the Human Brain.

The hematopoietic system seeds the CNS with microglial progenitor cells during the fetal period, but the subsequent cell generation dynamics and maintenance of this population have been poorly understood. We report that microglia, unlike most other hematopoietic lineages, renew slowly at a median rate of 28% per year, and some microglia last for more than two decades. Furthermore, we find no evidence for the existence of a substantial population of quiescent long-lived cells, meaning that the microglia population in the human brain is sustained by continuous slow turnover throughout adult life.

Impact of fat mass and distribution on lipid turnover in human adipose tissue.

Differences in white adipose tissue (WAT) lipid turnover between the visceral (vWAT) and subcutaneous (sWAT) depots may cause metabolic complications in obesity. Here we compare triglyceride age and, thereby, triglyceride turnover in vWAT and sWAT biopsies from 346 individuals and find that subcutaneous triglyceride age and storage capacity are increased in overweight or obese individuals. Visceral triglyceride age is only increased in excessively obese individuals and associated with a lower lipid removal capacity. Thus, although triglyceride storage capacity in sWAT is higher than in vWAT, the former plateaus at substantially lower levels of excess WAT mass than vWAT. In individuals with central or visceral obesity, lipid turnover is selectively increased in vWAT. Obese individuals classified as 'metabolically unhealthy' (according to ATPIII criteria) who have small subcutaneous adipocytes exhibit reduced triglyceride turnover. We conclude that excess WAT results in depot-specific differences in lipid turnover and increased turnover in vWAT and/or decreased turnover in sWAT may result in metabolic complications of overweight or obesity.

Antibody-secreting plasma cells persist for decades in human intestine.

Plasma cells (PCs) produce antibodies that mediate immunity after infection or vaccination. In contrast to PCs in the bone marrow, PCs in the gut have been considered short lived. In this study, we studied PC dynamics in the human small intestine by cell-turnover analysis in organ transplants and by retrospective cell birth dating measuring carbon-14 in genomic DNA. We identified three distinct PC subsets: a CD19+ PC subset was dynamically exchanged, whereas of two CD19- PC subsets, CD45+ PCs exhibited little and CD45- PCs no replacement and had a median age of 11 and 22 yr, respectively. Accumulation of CD45- PCs during ageing and the presence of rotavirus-specific clones entirely within the CD19- PC subsets support selection and maintenance of protective PCs for life in human intestine.

Dynamics of Cell Generation and Turnover in the Human Heart.

The contribution of cell generation to physiological heart growth and maintenance in humans has been difficult to establish and has remained controversial. We report that the full complement of cardiomyocytes is established perinataly and remains stable over the human lifespan, whereas the numbers of both endothelial and mesenchymal cells increase substantially from birth to early adulthood. Analysis of the integration of nuclear bomb test-derived (14)C revealed a high turnover rate of endothelial cells throughout life (>15% per year) and more limited renewal of mesenchymal cells (<4% per year in adulthood). Cardiomyocyte exchange is highest in early childhood and decreases gradually throughout life to <1% per year in adulthood, with similar turnover rates in the major subdivisions of the myocardium. We provide an integrated model of cell generation and turnover in the human heart.

Dynamics of oligodendrocyte generation and myelination in the human brain.

The myelination of axons by oligodendrocytes has been suggested to be modulated by experience, which could mediate neural plasticity by optimizing the performance of the circuitry. We have assessed the dynamics of oligodendrocyte generation and myelination in the human brain. The number of oligodendrocytes in the corpus callosum is established in childhood and remains stable after that. Analysis of the integration of nuclear bomb test-derived (14)C revealed that myelin is exchanged at a high rate, whereas the oligodendrocyte population in white matter is remarkably stable in humans, with an annual exchange of 1/300 oligodendrocytes. We conclude that oligodendrocyte turnover contributes minimally to myelin modulation in human white matter and that this instead may be carried out by mature oligodendrocytes, which may facilitate rapid neural plasticity.

The age and genomic integrity of neurons after cortical stroke in humans.

It has been unclear whether ischemic stroke induces neurogenesis or neuronal DNA rearrangements in the human neocortex. Using immunohistochemistry; transcriptome, genome and ploidy analyses; and determination of nuclear bomb test-derived (14)C concentration in neuronal DNA, we found neither to be the case. A large proportion of cortical neurons displayed DNA fragmentation and DNA repair a short time after stroke, whereas neurons at chronic stages after stroke showed DNA integrity, demonstrating the relevance of an intact genome for survival.

Neurogenesis in the striatum of the adult human brain.

In most mammals, neurons are added throughout life in the hippocampus and olfactory bulb. One area where neuroblasts that give rise to adult-born neurons are generated is the lateral ventricle wall of the brain. We show, using histological and carbon-14 dating approaches, that in adult humans new neurons integrate in the striatum, which is adjacent to this neurogenic niche. The neuronal turnover in the striatum appears restricted to interneurons, and postnatally generated striatal neurons are preferentially depleted in patients with Huntington's disease. Our findings demonstrate a unique pattern of neurogenesis in the adult human brain.

Evaluation of intracavity optogalvanic spectroscopy for radiocarbon measurements.

Ever since the first publication of intracavity optogalvanic spectroscopy (ICOGS) in 2008, this novel technique for measuring the (14)C/(12)C ratio in carbon dioxide has rendered considerable attention. As a result, there are currently at least five different research groups pursuing research on ICOGS. With a claimed limit of detection of 10(-15) ((14)C/(12)C), i.e., in the same order as accelerator mass spectroscopy, achieved with a relatively inexpensive and uncomplicated table-top system, ICOGS has major scientific and commercial implications. However, during the past 5 years, no research group has been able to reproduce these results or present additional proof for ICOGS's capability of unambiguous (14)C detection, including the authors of the original publication. Starting in 2010, our group has set up a state-of-the-art ICOGS laboratory and has investigated the basic methodology of ICOGS in general and tried to reproduce the reported experiments in particular. We have not been able to reproduce the reported results concerning the optogalvanic signals dependence on (14)C concentration and wavelength and, ultimately, not seen any evidence of the capability of ICOGS to unambiguously detect (14)C at all. Instead, we have found indications that the reported results can be products of measurement uncertainties and mistakes. Furthermore, our results strongly indicate that the reported limit of detection is likely to be overestimated by at least 2 orders of magnitude, based on the results presented in the original publication. Hence, we conclude that the original reports on ICOGS cannot be confirmed and therefore must be in error.

Dynamics of hippocampal neurogenesis in adult humans.

Adult-born hippocampal neurons are important for cognitive plasticity in rodents. There is evidence for hippocampal neurogenesis in adult humans, although whether its extent is sufficient to have functional significance has been questioned. We have assessed the generation of hippocampal cells in humans by measuring the concentration of nuclear-bomb-test-derived ¹4C in genomic DNA, and we present an integrated model of the cell turnover dynamics. We found that a large subpopulation of hippocampal neurons constituting one-third of the neurons is subject to exchange. In adult humans, 700 new neurons are added in each hippocampus per day, corresponding to an annual turnover of 1.75% of the neurons within the renewing fraction, with a modest decline during aging. We conclude that neurons are generated throughout adulthood and that the rates are comparable in middle-aged humans and mice, suggesting that adult hippocampal neurogenesis may contribute to human brain function.

Dynamics of human adipose lipid turnover in health and metabolic disease.

Adipose tissue mass is determined by the storage and removal of triglycerides in adipocytes. Little is known, however, about adipose lipid turnover in humans in health and pathology. To study this in vivo, here we determined lipid age by measuring (14)C derived from above ground nuclear bomb tests in adipocyte lipids. We report that during the average ten-year lifespan of human adipocytes, triglycerides are renewed six times. Lipid age is independent of adipocyte size, is very stable across a wide range of adult ages and does not differ between genders. Adipocyte lipid turnover, however, is strongly related to conditions with disturbed lipid metabolism. In obesity, triglyceride removal rate (lipolysis followed by oxidation) is decreased and the amount of triglycerides stored each year is increased. In contrast, both lipid removal and storage rates are decreased in non-obese patients diagnosed with the most common hereditary form of dyslipidaemia, familial combined hyperlipidaemia. Lipid removal rate is positively correlated with the capacity of adipocytes to break down triglycerides, as assessed through lipolysis, and is inversely related to insulin resistance. Our data support a mechanism in which adipocyte lipid storage and removal have different roles in health and pathology. High storage but low triglyceride removal promotes fat tissue accumulation and obesity. Reduction of both triglyceride storage and removal decreases lipid shunting through adipose tissue and thus promotes dyslipidaemia. We identify adipocyte lipid turnover as a novel target for prevention and treatment of metabolic disease.

Application of accelerator mass spectrometry to macromolecules: preclinical pharmacokinetic studies on a polybisphosphonate.

Data on the use of accelerator mass spectrometry (AMS) in conjunction with in vivo studies of macromolecular drugs are scarce. The present study shows the versatility of this technique when investigating the pharmacokinetics (PK) of a macromolecular drug candidate, a polybisphosphonate conjugate (ODX). The aforementioned is a polymer (molecular weight ~30 kDa) constituting a carbohydrate backbone with covalently linked ligands (aldendronate and aminoguanidine) and is intended for treatment of osteoporosis and the therapy of bone metastasis from prostate cancer. The conjugate is prepared through partial oxidation of the carbohydrate and sequential coupling of the ligands by reductive amination. (14)C was incorporated in the conjugate by means of coupling a commercially available (14)C-lysine in the conjugation sequence. Fifteen rats were injected intravenously with (14)C-labelled ODX (150 µg, 14 Bq/rat) and blood samples were collected at 1, 2, 4, 6, and 24 h post-injection (3 rats/time point). Liver, spleen and kidney samples were collected at 4 and 24 h post-injection. Blood from each time point (triplicate) were collected for AMS measurement determining the isotopic ratio ((14)C/(12)C) and consequently the drug concentration in blood. ODX showed a transient presence in blood circulation; 93% of the total dose was cleared from the circulation within 1 h. The half-life after 1 h was estimated to be about 3 h; 0.7% of the administered (14)C dose of ODX remained in circulation after 24 h. The major (14)C accumulation was in the liver, the spleen and the kidneys indicating the probable route of metabolism and excretion. This study demonstrates the versatility of AMS for pharmacological in vivo studies of macromolecules. Labelling with (14)C is relatively simple, inexpensive and the method requires minimal radioactivity, eliminating the need for radioprotection precautions in contrast to methods using scintillation counting.

Carotid plaque age is a feature of plaque stability inversely related to levels of plasma insulin.

BACKGROUND: The stability of atherosclerotic plaques determines the risk for rupture, which may lead to thrombus formation and potentially severe clinical complications such as myocardial infarction and stroke. Although the rate of plaque formation may be important for plaque stability, this process is not well understood. We took advantage of the atmospheric (14)C-declination curve (a result of the atomic bomb tests in the 1950s and 1960s) to determine the average biological age of carotid plaques. METHODOLOGY/PRINCIPAL FINDING: The cores of carotid plaques were dissected from 29 well-characterized, symptomatic patients with carotid stenosis and analyzed for (14)C content by accelerator mass spectrometry. The average plaque age (i.e. formation time) was 9.6±3.3 years. All but two plaques had formed within 5-15 years before surgery. Plaque age was not associated with the chronological ages of the patients but was inversely related to plasma insulin levels (p = 0.0014). Most plaques were echo-lucent rather than echo-rich (2.24±0.97, range 1-5). However, plaques in the lowest tercile of plaque age (most recently formed) were characterized by further instability with a higher content of lipids and macrophages (67.8±12.4 vs. 50.4±6.2, p = 0.00005; 57.6±26.1 vs. 39.8±25.7, p<0.0005, respectively), less collagen (45.3±6.1 vs. 51.1±9.8, p<0.05), and fewer smooth muscle cells (130±31 vs. 141±21, p<0.05) than plaques in the highest tercile. Microarray analysis of plaques in the lowest tercile also showed increased activity of genes involved in immune responses and oxidative phosphorylation. CONCLUSIONS/SIGNIFICANCE: Our results show, for the first time, that plaque age, as judge by relative incorporation of (14)C, can improve our understanding of carotid plaque stability and therefore risk for clinical complications. Our results also suggest that levels of plasma insulin might be involved in determining carotid plaque age.