Extensive elimination of acinar cells during normal postnatal pancreas growth.

While programmed cell death plays important roles during morphogenetic stages of development, post-differentiation organ growth is considered an efficient process whereby cell proliferation increases cell number. Here we demonstrate that early postnatal growth of the pancreas unexpectedly involves massive acinar cell elimination. Measurements of cell proliferation and death in the human pancreas in comparison to the actual increase in cell number predict daily elimination of 0.7% of cells, offsetting 88% of cell formation over the first year of life. Using mouse models, we show that death is associated with mitosis, through a failure of dividing cells to generate two viable daughters. In p53-deficient mice, acinar cell death and proliferation are reduced, while organ size is normal, suggesting that p53-dependent developmental apoptosis triggers compensatory proliferation. We propose that excess cell turnover during growth of the pancreas, and presumably other organs, facilitates robustness to perturbations and supports maintenance of tissue architecture.

The DNA methylome of human vascular endothelium and its use in liquid biopsies.

BACKGROUND: Vascular endothelial cells (VECs) are an essential component of each tissue, contribute to multiple pathologies, and are targeted by important drugs. Yet, there is a shortage of biomarkers to assess VEC turnover. METHODS: To develop DNA methylation-based liquid biopsies for VECs, we determined the methylome of VECs isolated from freshly dissociated human tissues. FINDINGS: A comparison with a human cell-type methylome atlas yielded thousands of loci that are uniquely unmethylated in VECs. These sites are typically gene enhancers, often residing adjacent to VEC-specific genes. We also identified hundreds of genomic loci that are differentially methylated in organotypic VECs, indicating that VECs feeding specific organs are distinct cell types with a stable epigenetic identity. We established universal and lung-specific VEC markers and evaluated their presence in circulating cell-free DNA (cfDNA). Nearly 2.5% of cfDNA in the plasma of healthy individuals originates from VECs. Sepsis, graft versus host disease, and cardiac catheterization are associated with elevated levels of VEC-derived cfDNA, indicative of vascular damage. Lung-specific VEC cfDNA is selectively elevated in patients with chronic obstructive pulmonary disease (COPD) or lung cancer, revealing tissue-specific vascular turnover. CONCLUSIONS: VEC cfDNA biomarkers inform vascular dynamics in health and disease, potentially contributing to early diagnosis and monitoring of pathologies, and assessment of drug activity. FUNDING: This work was supported by the Beutler Research Program, Helmsley Charitable Trust, JDRF, Grail and the DON Foundation (to Y.D.). Y.D holds the Walter & Greta Stiel Chair in heart studies. B.G., R.S., J.M., D.N., T.K., and Y.D. filed patents on cfDNA analysis.

Novel cfDNA Methylation Biomarkers Reveal Delayed Cardiac Cell Death after Open-heart Surgery.

The use of cardiopulmonary bypass (CPB) is thought to cause delayed cardiac damage. DNA methylation-based liquid biopsies are novel biomarkers for monitoring acute cardiac cell death. We assessed cell-free DNA molecules as markers for cardiac damage after open-heart surgery. Novel cardiomyocyte-specific DNA methylation markers were applied to measure cardiac cfDNA in the plasma of 42 infants who underwent open-heart surgery. Cardiac cfDNA was elevated following surgery, reflecting direct surgery-related tissue damage, and declined thereafter in most patients. The concentration of cardiac cfDNA post-surgery correlated with the duration of CPB and aortic cross clamping. Strikingly, cardiac cfDNA at 6 h predicted duration of mechanical ventilation and maximal vasoactive-inotropic score better than did maximal troponin levels. Cardiac cfDNA reveals heart damage associated with CPB, and can be used to monitor cardiac cell death, to predict clinical outcome of surgery and to assess performance of cardioprotective interventions.

B cell-derived cfDNA after primary BNT162b2 mRNA vaccination anticipates memory B cells and SARS-CoV-2 neutralizing antibodies.

BACKGROUND: Much remains unknown regarding the response of the immune system to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination. METHODS: We employed circulating cell-free DNA (cfDNA) to assess the turnover of specific immune cell types following administration of the Pfizer/BioNTech vaccine. FINDINGS: The levels of B cell cfDNA after the primary dose correlated with development of neutralizing antibodies and memory B cells after the booster, revealing a link between early B cell turnover-potentially reflecting affinity maturation-and later development of effective humoral response. We also observed co-elevation of B cell, T cell, and monocyte cfDNA after the booster, underscoring the involvement of innate immune cell turnover in the development of humoral and cellular adaptive immunity. Actual cell counts remained largely stable following vaccination, other than a previously demonstrated temporary reduction in neutrophil and lymphocyte counts. CONCLUSIONS: Immune cfDNA dynamics reveal the crucial role of the primary SARS-CoV-2 vaccine in shaping responses of the immune system following the booster vaccine. FUNDING: This work was supported by a generous gift from Shlomo Kramer. Supported by grants from Human Islet Research Network (HIRN UC4DK116274 and UC4DK104216 to R.S. and Y.D.), Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, The Alex U Soyka Pancreatic Cancer Fund, The Israel Science Foundation, the Waldholtz/Pakula family, the Robert M. and Marilyn Sternberg Family Charitable Foundation, the Helmsley Charitable Trust, Grail, and the DON Foundation (to Y.D.). Y.D. holds the Walter and Greta Stiel Chair and Research Grant in Heart Studies. I.F.-F. received a fellowship from the Glassman Hebrew University Diabetes Center.

Universal lung epithelium DNA methylation markers for detection of lung damage in liquid biopsies.

BACKGROUND: Circulating biomarkers for lung damage are lacking. Lung epithelium-specific DNA methylation patterns can potentially report the presence of lung-derived cell-free DNA (cfDNA) in blood, as an indication of lung cell death. METHODS: We sorted human lung alveolar and bronchial epithelial cells from surgical specimens, and obtained their methylomes using whole-genome bisulfite sequencing. We developed a PCR sequencing assay determining the methylation status of 17 loci with lung-specific methylation patterns, and used it to assess lung-derived cfDNA in the plasma of healthy volunteers and patients with lung disease. RESULTS: Loci that are uniquely unmethylated in alveolar or bronchial epithelial cells are enriched for enhancers controlling lung-specific genes. Methylation markers extracted from these methylomes revealed that normal lung cell turnover probably releases cfDNA into the air spaces, rather than to blood. People with advanced lung cancer show a massive elevation of lung cfDNA concentration in blood. Among individuals undergoing bronchoscopy, lung-derived cfDNA is observed in the plasma of those later diagnosed with lung cancer, and to a lesser extent in those diagnosed with other lung diseases. Lung cfDNA is also elevated in patients with acute exacerbation of COPD compared with patients with stable disease, and is associated with future exacerbation and mortality in these patients. CONCLUSIONS: Universal cfDNA methylation markers of normal lung epithelium allow for mutation-independent, sensitive and specific detection of lung-derived cfDNA, reporting on ongoing lung injury. Such markers can find broad utility in the study of normal and pathologic human lung dynamics.

Liquid biopsy reveals collateral tissue damage in cancer.

Cancer inflicts damage to surrounding normal tissues, which can culminate in fatal organ failure. Here, we demonstrate that cell death in organs affected by cancer can be detected by tissue-specific methylation patterns of circulating cell-free DNA (cfDNA). We detected elevated levels of hepatocyte-derived cfDNA in the plasma of patients with liver metastases originating from different primary tumors, compared with cancer patients without liver metastases. In addition, patients with localized pancreatic or colon cancer showed elevated hepatocyte cfDNA, suggesting liver damage inflicted by micrometastatic disease, by primary pancreatic tumor pressing the bile duct, or by a systemic response to the primary tumor. We also identified elevated neuron-, oligodendrocyte-, and astrocyte-derived cfDNA in a subpopulation of patients with brain metastases compared with cancer patients without brain metastasis. Cell type-specific cfDNA methylation markers enabled the identification of collateral tissue damage in cancer, revealing the presence of metastases in specific locations and potentially assisting in early cancer detection.

Remote immune processes revealed by immune-derived circulating cell-free DNA.

Blood cell counts often fail to report on immune processes occurring in remote tissues. Here, we use immune cell type-specific methylation patterns in circulating cell-free DNA (cfDNA) for studying human immune cell dynamics. We characterized cfDNA released from specific immune cell types in healthy individuals (N = 242), cross sectionally and longitudinally. Immune cfDNA levels had no individual steady state as opposed to blood cell counts, suggesting that cfDNA concentration reflects adjustment of cell survival to maintain homeostatic cell numbers. We also observed selective elevation of immune-derived cfDNA upon perturbations of immune homeostasis. Following influenza vaccination (N = 92), B-cell-derived cfDNA levels increased prior to elevated B-cell counts and predicted efficacy of antibody production. Patients with eosinophilic esophagitis (N = 21) and B-cell lymphoma (N = 27) showed selective elevation of eosinophil and B-cell cfDNA, respectively, which were undetectable by cell counts in blood. Immune-derived cfDNA provides a novel biomarker for monitoring immune responses to physiological and pathological processes that are not accessible using conventional methods.

Digital Droplet PCR for Monitoring Tissue-Specific Cell Death Using DNA Methylation Patterns of Circulating Cell-Free DNA.

Cell death involves the release of short DNA fragments into blood, termed circulating cell-free DNA (cfDNA). Sequencing of cfDNA in the plasma has recently emerged as a liquid biopsy for detecting fetal chromosomal aberrations, tumor DNA, and graft rejection. However, in cases where cfDNA is derived from tissues with a normal genome, its primary sequence is not informative regarding the tissue of origin. We developed a method of determining the tissue origins of cfDNA, allowing inference of tissue-specific cell death, based on tissue-specific methylation patterns. We have previously described a version of the method that uses next generation sequencing (NGS) to determine methylation patterns in specific marker loci. Here we describe a rapid and simple procedure for cfDNA methylation analysis using droplet digital PCR (ddPCR) on bisulfite treated cfDNA to accurately count the number of molecules carrying a specific methylation signature. Specificity and sensitivity of the assay increases by simultaneously interrogating four to six cytosines in the same molecule using two fluorescent probes. cfDNA methylation analysis using ddPCR can find multiple applications in the non-invasive study of human tissue dynamics in health and disease. © 2019 by John Wiley & Sons, Inc.

Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease.

Methylation patterns of circulating cell-free DNA (cfDNA) contain rich information about recent cell death events in the body. Here, we present an approach for unbiased determination of the tissue origins of cfDNA, using a reference methylation atlas of 25 human tissues and cell types. The method is validated using in silico simulations as well as in vitro mixes of DNA from different tissue sources at known proportions. We show that plasma cfDNA of healthy donors originates from white blood cells (55%), erythrocyte progenitors (30%), vascular endothelial cells (10%) and hepatocytes (1%). Deconvolution of cfDNA from patients reveals tissue contributions that agree with clinical findings in sepsis, islet transplantation, cancer of the colon, lung, breast and prostate, and cancer of unknown primary. We propose a procedure which can be easily adapted to study the cellular contributors to cfDNA in many settings, opening a broad window into healthy and pathologic human tissue dynamics.

Vascular development in the vertebrate pancreas.

The vertebrate pancreas is comprised of a highly branched tubular epithelium, which is intimately associated with an extensive and specialized vasculature. While we know a great deal about basic vascular anatomy of the adult pancreas, as well as islet capillaries, surprisingly little is known about the ontogeny of its blood vessels. Here, we analyze development of the pancreatic vasculature in the mouse embryo. We show that pancreatic epithelial branches intercalate with the fine capillary plexus of the surrounding pancreatic mesenchyme. Endothelial cells (ECs) within this mesenchyme are heterogeneous from the onset of organogenesis. Pancreatic arteries take shape before veins, in a manner analogous to early embryonic vessels. The main central artery forms during mid-gestation, as a result of vessel coalescence and remodeling of a vascular plexus. In addition, we show that vessels in the forming pancreas display a predictable architecture that is dependent on VEGF signaling. Over-expression of VEGF disrupts vascular patterning and arteriovenous differentiation within the developing pancreas. This study constitutes a first-time in-depth cellular and molecular characterization of pancreatic blood vessels, as they coordinately grow along with the pancreatic epithelium.

Identification of tissue-specific cell death using methylation patterns of circulating DNA.

Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic ß-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

Short-term overexpression of VEGF-A in mouse beta cells indirectly stimulates their proliferation and protects against diabetes.

AIMS/HYPOTHESIS: Vascular endothelial growth factor (VEGF) has been recognised by loss-of-function experiments as a pleiotropic factor with importance in embryonic pancreas development and postnatal beta cell function. Chronic, nonconditional overexpression of VEGF-A has a deleterious effect on beta cell development and function. We report, for the first time, a conditional gain-of-function study to evaluate the effect of transient VEGF-A overexpression by adult pancreatic beta cells on islet vasculature and beta cell proliferation and survival, under both normal physiological and injury conditions. METHODS: In a transgenicmouse strain, overexpressing VEGF-A in a doxycycline-inducible and beta cell-specific manner, we evaluated the ability of VEGF-A to affect islet vessel density, beta cell proliferation and protection of the adult beta cell mass from toxin-induced injury. RESULTS: Short-term VEGF-A overexpression resulted in islet hypervascularisation, increased beta cell proliferation and protection from toxin-mediated beta cell death, and thereby prevented the development of hyperglycaemia. Extended overexpression of VEGF-A led to impaired glucose tolerance, elevated fasting glycaemia and a decreased beta cell mass. CONCLUSIONS/INTERPRETATION: Overexpression of VEGF-A in beta cells time-dependently affects glycometabolic control and beta cell protection and proliferation. These data nourish further studies to examine the role of controlled VEGF delivery in (pre)clinical applications aimed at protecting and/or restoring the injured beta cell mass.

Gastrin: a distinct fate of neurogenin3 positive progenitor cells in the embryonic pancreas.

Neurogenin3(+) (Ngn3(+)) progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach, where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors, but the lineage and regulators of pancreatic gastrin(+) cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin(+) cells in the developing pancreas co-express glucagon, ghrelin or pancreatic polypeptide, but many gastrin(+) cells do not express any other islet hormone. Pancreatic gastrin(+) cells express the transcription factors Nkx6.1, Nkx2.2 and low levels of Pdx1, and derive from Ngn3(+) endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3, Nkx2.2, NeuroD1 and Arx, but not Pax4 or Pax6. Finally, gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus, gastrin(+) cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells.

Conditional hypovascularization and hypoxia in islets do not overtly influence adult ß-cell mass or function.

It is generally accepted that vascularization and oxygenation of pancreatic islets are essential for the maintenance of an optimal ß-cell mass and function and that signaling by vascular endothelial growth factor (VEGF) is crucial for pancreas development, insulin gene expression/secretion, and (compensatory) ß-cell proliferation. A novel mouse model was designed to allow conditional production of human sFlt1 by ß-cells in order to trap VEGF and study the effect of time-dependent inhibition of VEGF signaling on adult ß-cell fate and metabolism. Secretion of sFlt1 by adult ß-cells resulted in a rapid regression of blood vessels and hypoxia within the islets. Besides blunted insulin release, ß-cells displayed a remarkable capacity for coping with these presumed unfavorable conditions: even after prolonged periods of blood vessel ablation, basal and stimulated blood glucose levels were only slightly increased, while ß-cell proliferation and mass remained unaffected. Moreover, ablation of blood vessels did not prevent ß-cell generation after severe pancreas injury by partial pancreatic duct ligation or partial pancreatectomy. Our data thus argue against a major role of blood vessels to preserve adult ß-cell generation and function, restricting their importance to facilitating rapid and adequate insulin delivery.

Blood vessels restrain pancreas branching, differentiation and growth.

How organ size and form are controlled during development is a major question in biology. Blood vessels have been shown to be essential for early development of the liver and pancreas, and are fundamental to normal and pathological tissue growth. Here, we report that, surprisingly, non-nutritional signals from blood vessels act to restrain pancreas growth. Elimination of endothelial cells increases the size of embryonic pancreatic buds. Conversely, VEGF-induced hypervascularization decreases pancreas size. The growth phenotype results from vascular restriction of pancreatic tip cell formation, lateral branching and differentiation of the pancreatic epithelium into endocrine and acinar cells. The effects are seen both in vivo and ex vivo, indicating a perfusion-independent mechanism. Thus, the vasculature controls pancreas morphogenesis and growth by reducing branching and differentiation of primitive epithelial cells.

Ngn3(+) endocrine progenitor cells control the fate and morphogenesis of pancreatic ductal epithelium.

During pancreas development, endocrine and exocrine cells arise from a common multipotent progenitor pool. How these cell fate decisions are coordinated with tissue morphogenesis is poorly understood. Here we have examined ductal morphology, endocrine progenitor cell fate and Notch signaling in Ngn3(-/-) mice, which do not produce islet cells. Ngn3 deficiency results in reduced branching and enlarged pancreatic duct-like structures, concomitant with Ngn3 promoter activation throughout the ductal epithelium and reduced Notch signaling. Conversely, forced generation of surplus endocrine progenitor cells causes reduced duct caliber and an excessive number of tip cells. Thus, endocrine progenitor cells normally provide a feedback signal to adjacent multipotent ductal progenitor cells that activates Notch signaling, inhibits further endocrine differentiation and promotes proper morphogenesis. These results uncover a novel layer of regulation coordinating pancreas morphogenesis and endocrine/exocrine differentiation, and suggest ways to enhance the yield of beta cells from stem cells.

Recognition and killing of human and murine pancreatic beta cells by the NK receptor NKp46.

Type 1 diabetes is an incurable disease that is currently treated by insulin injections or in rare cases by islet transplantation. We have recently shown that NKp46, a major killer receptor expressed by NK cells, recognizes an unknown ligand expressed by ß cells and that in the absence of NKp46, or when its activity is blocked, diabetes development is inhibited. In this study, we investigate whether NKp46 is involved in the killing of human ß cells that are intended to be used for transplantation, and we also thoroughly characterize the interaction between NKp46 and its human and mouse ß cell ligands. We show that human ß cells express an unknown ligand for NKp46 and are killed in an NKp46-dependent manner. We further demonstrate that the expression of the NKp46 ligand is detected on human ß cells already at the embryonic stage and that it appears on murine ß cells only following birth. Because the NKp46 ligand is detected on healthy ß cells, we wondered why type 1 diabetes does not develop in all individuals and show that NK cells are absent from the vicinity of islets of healthy mice and are detected in situ in proximity with ß cells in NOD mice. We also investigate the molecular mechanisms controlling NKp46 interactions with its ß cell ligand and demonstrate that the recognition is confined to the membrane proximal domain and stalk region of NKp46 and that two glycosylated residues of NKp46, Thr(125) and Asn(216), are critical for this recognition.