Aneuploidy detection in pooled polar bodies using rapid nanopore sequencing.

PURPOSE: Various screening techniques have been developed for preimplantation genetic testing for aneuploidy (PGT-A) to reduce implantation failure and miscarriages in women undergoing in vitro fertilisation (IVF) treatment. Among these methods, the Oxford nanopore technology (ONT) has already been tested in several tissues. However, no studies have applied ONT to polar bodies, a cellular material that is less restrictively regulated for PGT-A in some countries. METHODS: We performed rapid short nanopore sequencing on pooled first and second polar bodies of 102 oocytes from women undergoing IVF treatment to screen for aneuploidy. An automated analysis pipeline was developed with the expectation of three chromatids per chromosome. The results were compared to those obtained by array-based comparative genomic hybridisation (aCGH). RESULTS: ONT and aCGH were consistent for 96% (98/102) of sample ploidy classification. Of those samples, 36 were classified as euploid, while 62 were classified as aneuploid. The four discordant samples were assessed as euploid using aCGH but classified as aneuploid using ONT. The concordance of the ploidy classification (euploid, gain, or loss) per chromosome was 92.5% (2169 of 2346 of analysed chromosomes) using aCGH and ONT and increased to 97.7% (2113/2162) without the eight samples assessed as highly complex aneuploid using ONT. CONCLUSION: The automated detection of the ploidy classification per chromosome and shorter duplications or deletions depending on the sequencing depth demonstrates an advantage of the ONT method over standard, commercial aCGH methods, which do not consider the presence of three chromatids in pooled polar bodies.

Oct4 controls basement membrane development during human embryogenesis.

Basement membranes (BMs) are sheet-like structures of extracellular matrix (ECM) that provide structural support for many tissues and play a central role in signaling. They are key regulators of cell behavior and tissue functions, and defects in their assembly or composition are involved in numerous human diseases. Due to the differences between human and animal embryogenesis, ethical concerns, legal constraints, the scarcity of human tissue material, and the inaccessibility of the in vivo condition, BM regulation during human embryo development has remained elusive. Using the post-implantation amniotic sac embryoid (PASE), we delineate BM assembly upon post-implantation development and BM disassembly during primitive streak (PS) cell dissemination. Further, we show that the transcription factor Oct4 regulates the expression of BM structural components and receptors and controls BM development by regulating Akt signaling and the small GTPase Rac1. These results represent a relevant step toward a more comprehensive understanding of early human development.

Aged-vascular niche hinders osteogenesis of mesenchymal stem cells through paracrine repression of Wnt-axis.

Age-induced decline in osteogenic potential of bone marrow mesenchymal stem cells (BMSCs) potentiates osteoporosis and increases the risk for bone fractures. Despite epidemiology studies reporting concurrent development of vascular and bone diseases in the elderly, the underlying mechanisms for the vascular-bone cross-talk in aging are largely unknown. In this study, we show that accelerated endothelial aging deteriorates bone tissue through paracrine repression of Wnt-driven-axis in BMSCs. Here, we utilize physiologically aged mice in conjunction with our transgenic endothelial progeria mouse model (Hutchinson-Gilford progeria syndrome; HGPS) that displays hallmarks of an aged bone marrow vascular niche. We find bone defects associated with diminished BMSC osteogenic differentiation that implicate the existence of angiocrine factors with long-term inhibitory effects. microRNA-transcriptomics of HGPS patient plasma combined with aged-vascular niche analyses in progeria mice reveal abundant secretion of Wnt-repressive microRNA-31-5p. Moreover, we show that inhibition of microRNA-31-5p as well as selective Wnt-activator CHIR99021 boosts the osteogenic potential of BMSCs through de-repression and activation of the Wnt-signaling, respectively. Our results demonstrate that the vascular niche significantly contributes to osteogenesis defects in aging and pave the ground for microRNA-based therapies of bone loss in elderly.

Evaluation of Nanopore Sequencing on Polar Bodies for Routine Pre-Implantation Genetic Testing for Aneuploidy.

BACKGROUND: Preimplantation genetic testing for aneuploidy (PGT-A) using polar body (PB) biopsy offers a clinical benefit by reducing the number of embryo transfers and miscarriage rates but is currently not cost-efficient. Nanopore sequencing technology opens possibilities by providing cost-efficient and fast sequencing results with uncomplicated sample preparation work flows. METHODS: In this comparative experimental study, 102 pooled PB samples (99 passing QC) from 20 patients were analyzed for aneuploidy using nanopore sequencing technology and compared with array comparative genomic hybridization (aCGH) results generated as part of the clinical routine. Samples were sequenced on a Nanopore MinION machine. Whole-chromosome copy-numbers were called by custom bioinformatic analysis software. Automatically called results were compared to aCGH results. RESULTS: Overall, 96/99 samples were consistently detected as euploid or aneuploid in both methods (concordance = 97.0%, sensitivity = 0.957, specificity = 1.0, positive predictive value = 1.0, negative predictive value = 0.906). On the chromosomal level, concordance reached 98.7%. Chromosomal aneuploidies analyzed in this trial covered all 23 chromosomes with 98 trisomies, and 97 monosomies in 70 aCGH samples.The whole nanopore work flow is feasible in under 5 h (for one sample) with a maximum time of 16 h (for 12 samples), enabling fresh PB-euploid embryo transfer. A material cost of US$ 165 (EUR 150)/sample possibly enables cost-efficient aneuploidy screening. CONCLUSIONS: This is the first study systematically comparing nanopore sequencing with standard methods for the detection of PB aneuploidy. High concordance rates confirmed the feasibility of nanopore technology for this application. Additionally, the fast and cost-efficient work flow reveals the clinical utility of this technology, making it clinically attractive for PB PGT-A.

2'-O-ribose methylation levels of ribosomal RNA distinguish different types of growth arrest in human dermal fibroblasts.

The 2'-O-methylation (2'-O-Me) of ribosomal RNA (rRNA) shows plasticity that is potentially associated with cell phenotypes. We used RiboMeth-seq profiling to reveal growth arrest-specific 2'-O-Me patterns in primary human dermal fibroblasts from three different donors. We exposed cells to hydrogen peroxide to induce cellular senescence and to high cell densities to promote quiescence by contact inhibition. We compared both modes of cell cycle arrest to proliferating cells and could indeed distinguish these conditions by their overall 2'-O-Me patterns. Methylation levels at a small fraction of sites showed plasticity and correlated with the expression of specific small nucleolar RNAs (snoRNAs) but not with expression of fibrillarin. Moreover, we observed subtle senescence-associated alterations in ribosome biogenesis. Knockdown of the snoRNA SNORD87, which acts as a guide for modification of a hypermethylated position in non-proliferating cells, was sufficient to boost cell proliferation. Conversely, depletion of SNORD88A, SNORD88B and SNORD88C, which act as guides for modification of a hypomethylated site, caused decreased proliferation without affecting global protein synthesis or apoptosis. Taken together, our findings provide evidence that rRNA modifications can be used to distinguish and potentially influence specific growth phenotypes of primary cells.

Aberrant Lipid Metabolism in Macrophages Is Associated with Granuloma Formation in Sarcoidosis.

Rationale: Chronic sarcoidosis is a complex granulomatous disease with limited treatment options that can progress over time. Understanding the molecular pathways contributing to disease would aid in new therapeutic development. Objectives: To understand whether macrophages from patients with nonresolving chronic sarcoidosis are predisposed to macrophage aggregation and granuloma formation and whether modulation of the underlying molecular pathways influence sarcoidosis granuloma formation. Methods: Macrophages were cultivated in vitro from isolated peripheral blood CD14+ monocytes and evaluated for spontaneous aggregation. Transcriptomics analyses and phenotypic and drug inhibitory experiments were performed on these monocyte-derived macrophages. Human skin biopsies from patients with sarcoidosis and a myeloid Tsc2-specific sarcoidosis mouse model were analyzed for validatory experiments. Measurements and Main Results: Monocyte-derived macrophages from patients with chronic sarcoidosis spontaneously formed extensive granulomas in vitro compared with healthy control participants. Transcriptomic analyses separated healthy and sarcoidosis macrophages and identified an enrichment in lipid metabolic processes. In vitro patient granulomas, sarcoidosis mouse model granulomas, and those directly analyzed from lesional patient skin expressed an aberrant lipid metabolism profile and contained increased neutral lipids. Conversely, a combination of statins and cholesterol-reducing agents reduced granuloma formation both in vitro and in vivo in a sarcoidosis mouse model. Conclusions: Together, our findings show that altered lipid metabolism in sarcoidosis macrophages is associated with its predisposition to granuloma formation and suggest cholesterol-reducing therapies as a treatment option in patients.

Form follows function: Nuclear morphology as a quantifiable predictor of cellular senescence.

Enlarged or irregularly shaped nuclei are frequently observed in tissue cells undergoing senescence. However, it remained unclear whether this peculiar morphology is a cause or a consequence of senescence and how informative it is in distinguishing between proliferative and senescent cells. Recent research reveals that nuclear morphology can act as a predictive biomarker of senescence, suggesting an active role for the nucleus in driving senescence phenotypes. By employing deep learning algorithms to analyze nuclear morphology, accurate classification of cells as proliferative or senescent is achievable across various cell types and species both in vitro and in vivo. This quantitative imaging-based approach can be employed to establish links between senescence burden and clinical data, aiding in the understanding of age-related diseases, as well as assisting in disease prognosis and treatment response.

Metabolic support by macrophages sustains colonic epithelial homeostasis.

The intestinal epithelium has a high turnover rate and constantly renews itself through proliferation of intestinal crypt cells, which depends on insufficiently characterized signals from the microenvironment. Here, we showed that colonic macrophages were located directly adjacent to epithelial crypt cells in mice, where they metabolically supported epithelial cell proliferation in an mTORC1-dependent manner. Specifically, deletion of tuberous sclerosis complex 2 (Tsc2) in macrophages activated mTORC1 signaling that protected against colitis-induced intestinal damage and induced the synthesis of the polyamines spermidine and spermine. Epithelial cells ingested these polyamines and rewired their cellular metabolism to optimize proliferation and defense. Notably, spermine directly stimulated proliferation of colon epithelial cells and colon organoids. Genetic interference with polyamine production in macrophages altered global polyamine levels in the colon and modified epithelial cell proliferation. Our results suggest that macrophages act as "commensals" that provide metabolic support to promote efficient self-renewal of the colon epithelium.

An mTORC1-Dependent Mouse Model for Cardiac Sarcoidosis.

Background Sarcoidosis is an inflammatory, granulomatous disease of unknown cause affecting multiple organs, including the heart. Untreated, unresolved granulomatous inflammation can lead to cardiac fibrosis, arrhythmias, and eventually heart failure. Here we characterize the cardiac phenotype of mice with chronic activation of mammalian target of rapamycin (mTOR) complex 1 signaling in myeloid cells known to cause spontaneous pulmonary sarcoid-like granulomas. Methods and Results The cardiac phenotype of mice with conditional deletion of the tuberous sclerosis 2 (TSC2) gene in CD11c+ cells (TSC2fl/flCD11c-Cre; termed TSC2KO) and controls (TSC2fl/fl) was determined by histological and immunological stains. Transthoracic echocardiography and invasive hemodynamic measurements were performed to assess myocardial function. TSC2KO animals were treated with either everolimus, an mTOR inhibitor, or Bay11-7082, a nuclear factor-kB inhibitor. Activation of mTOR signaling was evaluated on myocardial samples from sudden cardiac death victims with a postmortem diagnosis of cardiac sarcoidosis. Chronic activation of mTORC1 signaling in CD11c+ cells was sufficient to initiate progressive accumulation of granulomatous infiltrates in the heart, which was associated with increased fibrosis, impaired cardiac function, decreased plakoglobin expression, and abnormal connexin 43 distribution, a substrate for life-threatening arrhythmias. Mice treated with the mTOR inhibitor everolimus resolved granulomatous infiltrates, prevented fibrosis, and improved cardiac dysfunction. In line, activation of mTOR signaling in CD68+ macrophages was detected in the hearts of sudden cardiac death victims who suffered from cardiac sarcoidosis. Conclusions To our best knowledge this is the first animal model of cardiac sarcoidosis that recapitulates major pathological hallmarks of human disease. mTOR inhibition may be a therapeutic option for patients with cardiac sarcoidosis.

SHP2 promotes sarcoidosis severity by inhibiting SKP2-targeted ubiquitination of TBET in CD8+ T cells.

Sarcoidosis is an interstitial lung disease (ILD) characterized by interferon-γ (IFN-γ) and T-box expressed in T cells (TBET) dysregulation. Although one-third of patients progress from granulomatous inflammation to severe lung damage, the molecular mechanisms underlying this process remain unclear. Here, we found that pharmacological inhibition of phosphorylated SH2-containing protein tyrosine phosphatase-2 (pSHP2), a facilitator of aberrant IFN-γ abundance, decreased large granuloma formation and macrophage infiltration in the lungs of mice with sarcoidosis-like disease. Positive treatment outcomes were dependent on the effective enhancement of TBET ubiquitination within CD8+ T cells. Mechanistically, we identified a posttranslational modification pathway in which the E3 F-box protein S-phase kinase-associated protein 2 (SKP2) targets TBET for ubiquitination in T cells under normal conditions. However, this pathway was disrupted by aberrant pSHP2 signaling in CD8+ T cells from patients with progressive pulmonary sarcoidosis and end-stage disease. Ex vivo inhibition of pSHP2 in CD8+ T cells from patients with end-stage sarcoidosis enhanced TBET ubiquitination and suppressed IFN-γ and collagen synthesis. Therefore, these studies provided new mechanistic insights into the SHP2-dependent posttranslational regulation of TBET and identified SHP2 inhibition as a potential therapeutic intervention against severe sarcoidosis. Furthermore, these studies also suggest that the small-molecule SHP2 inhibitor SHP099 might be used as a therapeutic measure against human diseases linked to TBET or ubiquitination.

Pluripotent Stem Cell-Derived In Vitro Gametogenesis and Synthetic Embryos-It Is Never Too Early for an Ethical Debate.

Recently, 2 branches of the wide area of synthetic biology-in vitro gametogenesis and synthetic embryo development-have gained considerable attention. Rodent induced pluripotent stem cells derived via reprogramming of somatic cells can in vitro be differentiated into gametes to produce fertile offspring. And even synthetic embryos with organ progenitors were generated ex utero entirely from murine pluripotent stem cells. The use of these approaches in basic research, which is rightfully accompanied by an ethical discussion, will allow hitherto unattainable insights into the processes of the beginning of life. There is a broad international consensus that currently the application of these technologies in human-assisted reproduction must be considered to be unsafe and unethical. However, newspaper headlines also addressed the putatively resulting paradigm shift in human reproduction and thereby raised expectations in patients. Due to unsolved biological and technological obstacles, most scientists do not anticipate translation of any of these approaches into human reproductive medicine, if ever, for the next 10 years. Still, whereas the usage of synthetic embryos for reproductive purposes should be banned, in the context of in vitro-derived human gametes it is not too early to initiate the evaluation of the ethical implications, which could still remain assuming all technological hurdles can ever be cleared.

Multipotent fetal stem cells in reproductive biology research.

Due to the limited accessibility of the in vivo situation, the scarcity of the human tissue, legal constraints, and ethical considerations, the underlying molecular mechanisms of disorders, such as preeclampsia, the pathological consequences of fetomaternal microchimerism, or infertility, are still not fully understood. And although substantial progress has already been made, the therapeutic strategies for reproductive system diseases are still facing limitations. In the recent years, it became more and more evident that stem cells are powerful tools for basic research in human reproduction and stem cell-based approaches moved into the center of endeavors to establish new clinical concepts. Multipotent fetal stem cells derived from the amniotic fluid, amniotic membrane, chorion leave, Wharton´s jelly, or placenta came to the fore because they are easy to acquire, are not associated with ethical concerns or covered by strict legal restrictions, and can be banked for autologous utilization later in life. Compared to adult stem cells, they exhibit a significantly higher differentiation potential and are much easier to propagate in vitro. Compared to pluripotent stem cells, they harbor less mutations, are not tumorigenic, and exhibit low immunogenicity. Studies on multipotent fetal stem cells can be invaluable to gain knowledge on the development of dysfunctional fetal cell types, to characterize the fetal stem cells migrating into the body of a pregnant woman in the context of fetomaternal microchimerism, and to obtain a more comprehensive picture of germ cell development in the course of in vitro differentiation experiments. The in vivo transplantation of fetal stem cells or their paracrine factors can mediate therapeutic effects in preeclampsia and can restore reproductive organ functions. Together with the use of fetal stem cell-derived gametes, such strategies could once help individuals, who do not develop functional gametes, to conceive genetically related children. Although there is still a long way to go, these developments regarding the usage of multipotent fetal stem cells in the clinic should continuously be accompanied by a wide and detailed ethical discussion.

BDNF and KISS-1 Levels in Maternal Serum, Umbilical Cord, and Placenta: The Potential Role of Maternal Levels as Effect Biomarker.

Brain-derived neurotrophic factor (BDNF) and kisspeptin-1 (KISS-1) regulate placental development and fetal growth. The predictive value of maternal serum BDNF and KISS-1 concentrations for placental and umbilical cord levels has not yet been explored. The influence of prenatal lead (Pb) and cadmium (Cd) exposure and maternal iron status on BDNF and KISS-1 levels is also unclarified and of concern. In a pilot cross-sectional study with 65 mother-newborn pairs, we analyzed maternal and cord serum levels of pro-BDNF, mature BDNF, and KISS-1, BDNF, and KISS-1 gene expression in placenta, Pb and Cd in maternal and umbilical cord blood (erythrocytes), and placenta. We conducted a series of in vitro experiments using human primary trophoblast cells (hTCs) and BeWo cells to verify main findings of the epidemiological analysis. Strong and consistent correlations were observed between maternal serum levels of pro-BDNF, mature BDNF, and KISS-1 and corresponding levels in umbilical serum and placental tissue. Maternal red blood cell Pb levels were inversely correlated with serum and placental KISS-1 levels. Lower expression and release of KISS-1 was also observed in Pb-exposed BeWo cells. In vitro Pb exposure also reduced cellular BDNF levels. Cd-treated BeWo cells showed increased pro-BDNF levels. Low maternal iron status was positively associated with low BDNF levels. Iron-deficient hTCs and BeWo cells showed a consistent decrease in the release of mature BDNF. The correlations between maternal BDNF and KISS-1 levels, placental gene expression, and umbilical cord serum levels, respectively, indicate the strong potential of maternal serum as predictive matrix for BDNF and KISS-1 levels in placentas and fetal sera. Pb exposure and iron status modulate BDNF and KISS-1 levels, but a clear direction of modulations was not evident. The associations need to be confirmed in a larger sample and validated in terms of placental and neurodevelopmental function. Supplementary Information: The online version contains supplementary material available at 10.1007/s12403-023-00565-w.

Duodenal macrophages control dietary iron absorption via local degradation of transferrin.

Iron is an essential cellular metal that is important for many physiological functions including erythropoiesis and host defense. It is absorbed from the diet in the duodenum and loaded onto transferrin (Tf), the main iron transport protein. Inefficient dietary iron uptake promotes many diseases, but mechanisms regulating iron absorption remain poorly understood. By assessing mice that harbor a macrophage-specific deletion of the tuberous sclerosis complex 2 (Tsc2), a negative regulator of mechanistic target of rapamycin complex 1 (mTORC1), we found that these mice possessed various defects in iron metabolism, including defective steady-state erythropoiesis and a reduced saturation of Tf with iron. This iron deficiency phenotype was associated with an iron import block from the duodenal epithelial cells into the circulation. Activation of mTORC1 in villous duodenal CD68+ macrophages induced serine protease expression and promoted local degradation of Tf, whereas the depletion of macrophages in mice increased Tf levels. Inhibition of mTORC1 with everolimus or serine protease activity with nafamostat restored Tf levels and Tf saturation in the Tsc2-deficient mice. Physiologically, Tf levels were regulated in the duodenum during the prandial process and Citrobacter rodentium infection. These data suggest that duodenal macrophages determine iron transfer to the circulation by controlling Tf availability in the lamina propria villi.

Mimicking Tumor Cell Heterogeneity of Colorectal Cancer in a Patient-derived Organoid-Fibroblast Model.

BACKGROUND & AIMS: Patient-derived organoid cancer models are generated from epithelial tumor cells and reflect tumor characteristics. However, they lack the complexity of the tumor microenvironment, which is a key driver of tumorigenesis and therapy response. Here, we developed a colorectal cancer organoid model that incorporates matched epithelial cells and stromal fibroblasts. METHODS: Primary fibroblasts and tumor cells were isolated from colorectal cancer specimens. Fibroblasts were characterized for their proteome, secretome, and gene expression signatures. Fibroblast/organoid co-cultures were analyzed by immunohistochemistry and compared with their tissue of origin, as well as on gene expression levels compared with standard organoid models. Bioinformatics deconvolution was used to calculate cellular proportions of cell subsets in organoids based on single-cell RNA sequencing data. RESULTS: Normal primary fibroblasts, isolated from tumor adjacent tissue, and cancer associated fibroblasts retained their molecular characteristics in vitro, including higher motility of cancer associated compared with normal fibroblasts. Importantly, both cancer-associated fibroblasts and normal fibroblasts supported cancer cell proliferation in 3D co-cultures, without the addition of classical niche factors. Organoids grown together with fibroblasts displayed a larger cellular heterogeneity of tumor cells compared with mono-cultures and closely resembled the in vivo tumor morphology. Additionally, we observed a mutual crosstalk between tumor cells and fibroblasts in the co-cultures. This was manifested by considerably deregulated pathways such as cell-cell communication and extracellular matrix remodeling in the organoids. Thrombospondin-1 was identified as a critical factor for fibroblast invasiveness. CONCLUSION: We developed a physiological tumor/stroma model, which will be vital as a personalized tumor model to study disease mechanisms and therapy response in colorectal cancer.

Selective Eradication of Colon Cancer Cells Harboring PI3K and/or MAPK Pathway Mutations in 3D Culture by Combined PI3K/AKT/mTOR Pathway and MEK Inhibition.

Colorectal cancer (CRC) is the second deadliest cancer in the world. Besides APC and p53 alterations, the PI3K/AKT/MTOR and MAPK pathway are most commonly mutated in CRC. So far, no treatment options targeting these pathways are available in routine clinics for CRC patients. We systematically analyzed the response of CRC cells to the combination of small molecular inhibitors targeting the PI3K and MAPK pathways. We used CRC cells in 2D, 3D spheroid, collagen gel cultures and freshly isolated organoids for drug response studies. Readout for drug response was spheroid or organoid growth, spheroid outgrowth, metabolic activity, Western blotting and immunofluorescence. We found profound tumor cell destruction under treatment with a combination of Torin 1 (inhibiting mTOR), MK2206 (targeting AKT) and selumetinib (inhibiting MEK) in 3D but not in 2D. Induction of cell death was due to apoptosis. Western blot analysis revealed efficient drug action. Gedatolisib, a dual PI3K/mTOR inhibitor, could replace Torin1/MK2206 with similar efficiency. The presence of PI3K and/or RAS-RAF-MAPK pathway mutations accounted for treatment responsiveness. Here, we identified a novel, efficient therapy, which induced proliferation stop and tumor cell destruction in vitro based on the genetic background. These preclinical findings show promise to further test this combi-treatment in vivo in mice and to potentially develop a mutation specific targeted therapy for CRC patients.

Amniotic Fluid Stem Cells: What They Are and What They Can Become.

In the last two decades, fetal amniotic fluid stem cells progressively attracted attention in the context of both basic research and the development of innovative therapeutic concepts. They exhibit broadly multipotent plasticity with the ability to differentiate into cells of all three embryonic germ layers and low immunogenicity. They are convenient to maintain, highly proliferative, genomically stable, non-tumorigenic, perfectly amenable to genetic modifications, and do not raise ethical concerns. However, it is important to note that among the various fetal amniotic fluid cells, only c-Kit+ amniotic fluid stem cells represent a distinct entity showing the full spectrum of these features. Since amniotic fluid additionally contains numerous terminally differentiated cells and progenitor cells with more limited differentiation potentials, it is of highest relevance to always precisely describe the isolation procedure and characteristics of the used amniotic fluid-derived cell type. It is of obvious interest for scientists, clinicians, and patients alike to be able to rely on up-todate and concisely separated pictures of the utilities as well as the limitations of terminally differentiated amniotic fluid cells, amniotic fluid-derived progenitor cells, and c-Kit+ amniotic fluid stem cells, to drive these distinct cellular models towards as many individual clinical applications as possible.

Premature aging in genetic diseases: what conclusions can be drawn for physiological aging.

According to current views the major hallmarks of physiological aging may be subdivided into three categories, primary causes of cellular damage (genomic instability, telomere attrition, loss of proteostasis, epigenetic alterations and compromised macroautophagy), antagonistic hallmarks that represent response to damage (deregulated nutrient sensing, cellular senescence, mitochondrial dysfunction) and integrative hallmarks that represent culprits of the phenotype (stem cell exhaustion, altered intercellular communication, chronic inflammation, dysbiosis). In contrast to physiological aging, premature aging diseases are driven by one or two distinct primary causes of aging, such as genomic instability in the case of Werner syndrome (WS), each displaying other hallmarks of aging to a variable extent. In this review we will focus on primary causes of well-investigated premature aging diseases Hutchinson-Gilford progeria syndrome (HGPS), WS, and Cockayne syndrome (CS) and for each provide an overview of reported aging hallmarks to elucidate resemblance to physiological aging on the mechanistic level and in the context of characteristic age-related diseases. Ubiquitous and tissue specific animal models of premature aging diseases will be discussed as useful tools to decipher fundamental aging-related mechanisms and develop intervention strategies to combat premature aging and age-related diseases.