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Purpose: Among the genome-editing methods for repairing disease-causing mutations resulting in autosomal dominant retinitis pigmentosa, homology-independent targeted integration (HITI)-mediated gene insertion of the normal form of the causative gene is useful because it allows the development of mutation-agnostic therapeutic products. In this study, we aimed for the rapid optimization and validation of HITI-treatment gene constructs of this approach in developing HITI-treatment constructs for various causative target genes in mouse models of retinal degeneration., Methods: We constructed the Cas9-driven HITI gene cassettes in plasmid vectors to treat the mouse Rho gene. A workflow utilizing in vivo electroporation was established to validate the efficacy of these constructs. Single-cell genotyping was conducted to evaluate allelic donor gene insertion. The therapeutic potency of HITI-treatment plasmid and adeno-associated virus (AAV) vectors was examined by section immunohistochemistry and optomotor response (OMR) in Rho+/P23H mutant mice. We also targeted mouse Prph2 to examine the workflow., Results: The optimized HITI-treatment constructs for mouse Rho genes achieved gene insertion in 80% to 90% of transduced mouse rod photoreceptor cells. This construct effectively suppressed degeneration and induced visual restoration in mutant mice. HITI-treatment constructs for the Rhodopsin gene demonstrated efficacy in AAV vectors and are adaptable for the mouse Prph2 gene locus., Conclusions: The study showcases a workflow for the rapid optimization and validation of highly effective HITI-treatment gene constructs against dominant-negative inheritance in inherited retinal dystrophy. These findings suggest the potential utility of this approach in developing HITI-treatment constructs for various target genes, advancing gene therapy products for diverse genetic disorders.

To systematically characterize the loss of tissue integrity and organ dysfunction resulting from aging, we produced an in-depth spatial transcriptomic profile of nine tissues in male mice during aging. We showed that senescence-sensitive spots (SSSs) colocalized with elevated entropy in organizational structure and that the aggregation of immunoglobulin-expressing cells is a characteristic feature of the microenvironment surrounding SSSs. Immunoglobulin G (IgG) accumulated across the aged tissues in both male and female mice, and a similar phenomenon was observed in human tissues, suggesting the potential of the abnormal elevation of immunoglobulins as an evolutionarily conserved feature in aging. Furthermore, we observed that IgG could induce a pro-senescent state in macrophages and microglia, thereby exacerbating tissue aging, and that targeted reduction of IgG mitigated aging across various tissues in male mice. This study provides a high-resolution spatial depiction of aging and indicates the pivotal role of immunoglobulin-associated senescence during the aging process., Competing Interests: Declaration of interests Y.G., C.N., Y.S., Z.L., H.D., and F.W. are employees of BGI. J.C.I.B. and C.R.E. are employees of Altos Labs., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In a rigorous 40-month study, we evaluated the geroprotective effects of metformin on adult male cynomolgus monkeys, addressing a gap in primate aging research. The study encompassed a comprehensive suite of physiological, imaging, histological, and molecular evaluations, substantiating metformin's influence on delaying age-related phenotypes at the organismal level. Specifically, we leveraged pan-tissue transcriptomics, DNA methylomics, plasma proteomics, and metabolomics to develop innovative monkey aging clocks and applied these to gauge metformin's effects on aging. The results highlighted a significant slowing of aging indicators, notably a roughly 6-year regression in brain aging. Metformin exerts a substantial neuroprotective effect, preserving brain structure and enhancing cognitive ability. The geroprotective effects on primate neurons were partially mediated by the activation of Nrf2, a transcription factor with anti-oxidative capabilities. Our research pioneers the systemic reduction of multi-dimensional biological age in primates through metformin, paving the way for advancing pharmaceutical strategies against human aging., Competing Interests: Declaration of interests J.C.I.B., S.H., C.R.E., P.R., and A.H. are employees of Altos Labs. S.H. is a founder of the non-profit Epigenetic Clock Development Foundation, which has licensed several of his patents from UC Regents and distributes the mammalian methylation array., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Local gene therapies, including in vivo genome editing, are highly anticipated for the treatment of genetic diseases in skin, especially the epidermis. While the adeno-associated virus (AAV) is a potent vector for in vivo gene delivery, the lack of efficient gene delivery methods has limited its clinical applications., Objective: To optimize the AAV gene delivery system with higher gene delivery efficiency and specificity for epidermis and keratinocytes (KCs), using AAV capsid and promoter engineering technologies., Methods: AAV variants with mutations in residues reported to be critical to determine the tropism of AAV2 for KCs were generated by site-directed mutagenesis of AAVDJ. The infection efficiency and specificity for KCs of these variants were compared with those of previously reported AAVs considered to be suitable for gene delivery to KCs in vitro and in vivo. Additionally, we generated an epidermis-specific promoter using the most recent short-core promoter and compared its specificity with existing promoters., Results: A novel AAVDJ variant capsid termed AAVDJK2 was superior to the existing AAVs in terms of gene transduction efficiency and specificity for epidermis and KCs in vitro and in vivo. A novel tissue-specific promoter, termed the K14 SCP3 promoter, was superior to the existing promoters in terms of gene transduction efficiency and specificity for KCs., Conclusion: The combination of the AAVDJK2 capsid and K14 SCP3 promoter improves gene delivery to epidermis in vivo and KCs in vitro. The novel AAV system may benefit experimental research and development of new epidermis-targeted gene therapies., Competing Interests: Conflict of Interest M.K. has a patent pending for AAV capsids and the tissue-specific promoter. The remaining authors have no conflicts of interest to declare., (Copyright © 2024. Published by Elsevier B.V.)

Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells., (© 2024. The Author(s).)

Defect in the SMN1 gene causes spinal muscular atrophy (SMA), which shows loss of motor neurons, muscle weakness and atrophy. While current treatment strategies, including small molecules or viral vectors, have shown promise in improving motor function and survival, achieving a definitive and long-term correction of SMA's endogenous mutations and phenotypes remains highly challenging. We have previously developed a CRISPR-Cas9 based homology-independent targeted integration (HITI) strategy, enabling unidirectional DNA knock-in in both dividing and non-dividing cells in vivo. In this study, we demonstrated its utility by correcting an SMA mutation in mice. When combined with Smn1 cDNA supplementation, it exhibited long-term therapeutic benefits in SMA mice. Our observations may provide new avenues for the long-term and efficient treatment of inherited diseases., (© 2024. The Author(s).)

Background: Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. Multiple identified mutations in nexilin (NEXN) have been suggested to be linked with severe DCM. However, the exact association between multiple mutations of Nexn and DCM remains unclear. Moreover, it is critical for the development of precise and effective therapeutics in treatments of DCM., Results: In our study, Nexn global knockout mice and mice carrying human equivalent G645del mutation are studied using functional gene rescue assays. AAV-mediated gene delivery is conducted through systemic intravenous injections at the neonatal stage. Heart tissues are analyzed by immunoblots, and functions are assessed by echocardiography. Here, we identify functional components of Nexilin and demonstrate that exogenous introduction could rescue the cardiac function and extend the lifespan of Nexn knockout mouse models. Similar therapeutic effects are also obtained in G645del mice, providing a promising intervention for future clinical therapeutics., Conclusions: In summary, we demonstrated that a single injection of AAV-Nexn was capable to restore the functions of cardiomyocytes and extended the lifespan of Nexn knockout and G645del mice. Our study represented a long-term gene replacement therapy for DCM that potentially covers all forms of loss-of-function mutations in NEXN., (© 2024. The Author(s).)

Background: CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks., Results: Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells., Conclusions: Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing., (© 2024. The Author(s).)

A central question for regenerative neuroscience is whether synthetic neural circuits, such as those built from two species, can function in an intact brain. Here, we apply blastocyst complementation to selectively build and test interspecies neural circuits. Despite approximately 10-20 million years of evolution, and prominent species differences in brain size, rat pluripotent stem cells injected into mouse blastocysts develop and persist throughout the mouse brain. Unexpectedly, the mouse niche reprograms the birth dates of rat neurons in the cortex and hippocampus, supporting rat-mouse synaptic activity. When mouse olfactory neurons are genetically silenced or killed, rat neurons restore information flow to odor processing circuits. Moreover, they rescue the primal behavior of food seeking, although less well than mouse neurons. By revealing that a mouse can sense the world using neurons from another species, we establish neural blastocyst complementation as a powerful tool to identify conserved mechanisms of brain development, plasticity, and repair., Competing Interests: Declaration of interests K.K.B. is on the SAB of Gameto Therapeutics. B.T.T. is a current employee of Janssen Research and Development. P.O. is a founder of Theracast. J.C.I.B is a founding scientist and director of the San Diego Institute of Science, Altos Labs., (Copyright © 2024. Published by Elsevier Inc.)

Sirtuins are pro-longevity genes with chromatin modulation potential, but how these properties are connected is not well understood. Here, we generated a panel of isogeneic human stem cell lines with SIRT1-SIRT7 knockouts and found that any sirtuin deficiency leads to accelerated cellular senescence. Through large-scale epigenomic analyses, we show how sirtuin deficiency alters genome organization and that genomic regions sensitive to sirtuin deficiency are preferentially enriched in active enhancers, thereby promoting interactions within topologically associated domains and the formation of de novo enhancer-promoter loops. In all sirtuin-deficient human stem cell lines, we found that chromatin contacts are rewired to promote aberrant activation of the placenta-specific gene PAPPA, which controls the pro-senescence effects associated with sirtuin deficiency and serves as a potential aging biomarker. Based on our survey of the 3D chromatin architecture, we established connections between sirtuins and potential target genes, thereby informing the development of strategies for aging interventions., Competing Interests: Declaration of interests J.C.I.B. is an employee of Altos Labs., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tissue regeneration following an injury requires dynamic cell-state transitions that allow for establishing the cell identities required for the restoration of tissue homeostasis and function. Here, we present a biochemical intervention that induces an intermediate cell state mirroring a transition identified during normal differentiation of myoblasts and other multipotent and pluripotent cells to mature cells. When applied in somatic differentiated cells, the intervention, composed of one-carbon metabolites, reduces some dedifferentiation markers without losing the lineage identity, thus inducing limited reprogramming into a more flexible cell state. Moreover, the intervention enabled accelerated repair after muscle injury in young and aged mice. Overall, our study uncovers a conserved biochemical transitional phase that enhances cellular plasticity in vivo and hints at potential and scalable biochemical interventions of use in regenerative medicine and rejuvenation interventions that may be more tractable than genetic ones., Competing Interests: Declaration of interests Patent applications have been filed related to the subject matter of this publication. R.H.-B., C.W., C.Z., S.M., F.H., V.L., I.G., S.S., M.Y., Y.S., A.A.-V., C.R.E., P.R., S.H., and J.C.I.B. are employees of Altos Labs., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Adrenal glands, vital for steroid secretion and the regulation of metabolism, stress responses and immune activation, experience age-related decline, impacting systemic health. However, the regulatory mechanisms underlying adrenal aging remain largely uninvestigated. Here we established a single-nucleus transcriptomic atlas of both young and aged primate suprarenal glands, identifying lipid metabolism and steroidogenic pathways as core processes impacted by aging. We found dysregulation in centripetal adrenocortical differentiation in aged adrenal tissues and cells in the zona reticularis region, responsible for producing dehydroepiandrosterone sulfate (DHEA-S), were highly susceptible to aging, reflected by senescence, exhaustion and disturbed hormone production. Remarkably, LDLR was downregulated in all cell types of the outer cortex, and its targeted inactivation in human adrenal cells compromised cholesterol uptake and secretion of dehydroepiandrosterone sulfate, as observed in aged primate adrenal glands. Our study provides crucial insights into endocrine physiology, holding therapeutic promise for addressing aging-related adrenal insufficiency and delaying systemic aging., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Human pluripotent stem cell-derived kidney organoids offer unprecedented opportunities for studying polycystic kidney disease (PKD), which still has no effective cure. Here, we developed both in vitro and in vivo organoid models of PKD that manifested tubular injury and aberrant upregulation of renin-angiotensin aldosterone system. Single-cell analysis revealed that a myriad of metabolic changes occurred during cystogenesis, including defective autophagy. Experimental activation of autophagy via ATG5 overexpression or primary cilia ablation significantly inhibited cystogenesis in PKD kidney organoids. Employing the organoid xenograft model of PKD, which spontaneously developed tubular cysts, we demonstrate that minoxidil, a potent autophagy activator and an FDA-approved drug, effectively attenuated cyst formation in vivo. This in vivo organoid model of PKD will enhance our capability to discover novel disease mechanisms and validate candidate drugs for clinical translation., Competing Interests: Declaration of interests A Singapore patent application no.10202303337R has been filed on November 24, 2023 for this work. C.R.E. and J.C.I.B. are employees of Altos Labs., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Regeneration across tissues and organs exhibits significant variation throughout the body and undergoes a progressive decline with age. To decode the relationships between aging and regenerative capacity, we conducted a comprehensive single-cell transcriptome analysis of regeneration in eight tissues from young and aged mice. We employed diverse analytical models to study tissue regeneration and unveiled the intricate cellular and molecular mechanisms underlying the attenuated regenerative processes observed in aged tissues. Specifically, we identified compromised stem cell mobility and inadequate angiogenesis as prominent contributors to this age-associated decline in regenerative capacity. Moreover, we discovered a unique subset of Arg1 + macrophages that were activated in young tissues but suppressed in aged regenerating tissues, suggesting their important role in age-related immune response disparities during regeneration. This study provides a comprehensive single-cell resource for identifying potential targets for interventions aimed at enhancing regenerative outcomes in the aging population., Competing Interests: Declaration of interests J.C.I.B. is an employee of Altos Labs., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Aging, often considered a result of random cellular damage, can be accurately estimated using DNA methylation profiles, the foundation of pan-tissue epigenetic clocks. Here, we demonstrate the development of universal pan-mammalian clocks, using 11,754 methylation arrays from our Mammalian Methylation Consortium, which encompass 59 tissue types across 185 mammalian species. These predictive models estimate mammalian tissue age with high accuracy (r > 0.96). Age deviations correlate with human mortality risk, mouse somatotropic axis mutations and caloric restriction. We identified specific cytosines with methylation levels that change with age across numerous species. These sites, highly enriched in polycomb repressive complex 2-binding locations, are near genes implicated in mammalian development, cancer, obesity and longevity. Our findings offer new evidence suggesting that aging is evolutionarily conserved and intertwined with developmental processes across all mammals., (© 2023. The Author(s).)

Using DNA methylation profiles ( n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species.

Charcot-Marie-Tooth disease is a chronic hereditary motor and sensory polyneuropathy targeting Schwann cells and/or motor neurons. Its multifactorial and polygenic origin portrays a complex clinical phenotype of the disease with a wide range of genetic inheritance patterns. The disease-associated gene GDAP1 encodes for a mitochondrial outer membrane protein. Mouse and insect models with mutations in Gdap1 have reproduced several traits of the human disease. However, the precise function in the cell types affected by the disease remains unknown. Here, we use induced-pluripotent stem cells derived from a Gdap1 knockout mouse model to better understand the molecular and cellular phenotypes of the disease caused by the loss-of-function of this gene. Gdap1-null motor neurons display a fragile cell phenotype prone to early degeneration showing (1) altered mitochondrial morphology, with an increase in the fragmentation of these organelles, (2) activation of autophagy and mitophagy, (3) abnormal metabolism, characterized by a downregulation of Hexokinase 2 and ATP5b proteins, (4) increased reactive oxygen species and elevated mitochondrial membrane potential, and (5) increased innate immune response and p38 MAP kinase activation. Our data reveals the existence of an underlying Redox-inflammatory axis fueled by altered mitochondrial metabolism in the absence of Gdap1. As this biochemical axis encompasses a wide variety of druggable targets, our results may have implications for developing therapies using combinatorial pharmacological approaches and improving therefore human welfare. A Redox-immune axis underlying motor neuron degeneration caused by the absence of Gdap1. Our results show that Gdap1 -/- motor neurons have a fragile cellular phenotype that is prone to degeneration. Gdap1 -/- iPSCs differentiated into motor neurons showed an altered metabolic state: decreased glycolysis and increased OXPHOS. These alterations may lead to hyperpolarization of mitochondria and increased ROS levels. Excessive amounts of ROS might be the cause of increased mitophagy, p38 activation and inflammation as a cellular response to oxidative stress. The p38 MAPK pathway and the immune response may, in turn, have feedback mechanisms, leading to the induction of apoptosis and senescence, respectively. CAC, citric acid cycle; ETC, electronic transport chain; Glc, glucose; Lac, lactate; Pyr, pyruvate., (© 2023. The Author(s).)

Distinct metabolic conditions rewire circadian-clock-controlled signaling pathways leading to the de novo construction of signal transduction networks. However, it remains unclear whether metabolic hallmarks unique to pluripotent stem cells (PSCs) are connected to clock functions. Reprogramming somatic cells to a pluripotent state, here we highlighted non-canonical functions of the circadian repressor CRY1 specific to PSCs. Metabolic reprogramming, including AMPK inactivation and SREBP1 activation, was coupled with the accumulation of CRY1 in PSCs. Functional assays verified that CRY1 is required for the maintenance of self-renewal capacity, colony organization, and metabolic signatures. Genome-wide occupancy of CRY1 identified CRY1-regulatory genes enriched in development and differentiation in PSCs, albeit not somatic cells. Last, cells lacking CRY1 exhibit differential gene expression profiles during induced PSC (iPSC) reprogramming, resulting in impaired iPSC reprogramming efficiency. Collectively, these results suggest the functional implication of CRY1 in pluripotent reprogramming and ontogenesis, thereby dictating PSC identity., Competing Interests: Declaration of interests These authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The ontogeny and dynamics of mtDNA heteroplasmy remain unclear due to limitations of current mtDNA sequencing methods. We developed individual Mitochondrial Genome sequencing (iMiGseq) of full-length mtDNA for ultra-sensitive variant detection, complete haplotyping, and unbiased evaluation of heteroplasmy levels, all at the individual mtDNA molecule level. iMiGseq uncovered unappreciated levels of heteroplasmic variants in single cells well below the conventional NGS detection limit and provided accurate quantitation of heteroplasmy level. iMiGseq resolved the complete haplotype of individual mtDNA in single oocytes and revealed genetic linkage of de novo mutations. iMiGseq detected sequential acquisition of detrimental mutations, including large deletions, in defective mtDNA in NARP/Leigh syndrome patient-derived induced pluripotent stem cells. iMiGseq identified unintended heteroplasmy shifts in mitoTALEN editing, while showing no appreciable level of unintended mutations in DdCBE-mediated mtDNA base editing. Therefore, iMiGseq could not only help elucidate the mitochondrial etiology of diseases, but also evaluate the safety of various mtDNA editing strategies., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Maternal mitochondria are the sole source of mtDNA for every cell of the offspring. Heteroplasmic mtDNA mutations inherited from the oocyte are a common cause of metabolic diseases and associated with late-onset diseases. However, the origin and dynamics of mtDNA heteroplasmy remain unclear. We used our individual Mitochondrial Genome sequencing (iMiGseq) technology to study mtDNA heterogeneity, quantitate single nucleotide variants (SNVs) and large structural variants (SVs), track heteroplasmy dynamics, and analyze genetic linkage between variants at the individual mtDNA molecule level in single oocytes and human blastoids. Our study presented the first single-mtDNA analysis of the comprehensive heteroplasmy landscape in single human oocytes. Unappreciated levels of rare heteroplasmic variants well below the detection limit of conventional methods were identified in healthy human oocytes, of which many are reported to be deleterious and associated with mitochondrial disease and cancer. Quantitative genetic linkage analysis revealed dramatic shifts of variant frequency and clonal expansions of large SVs during oogenesis in single-donor oocytes. iMiGseq of a single human blastoid suggested stable heteroplasmy levels during early lineage differentiation of naïve pluripotent stem cells. Therefore, our data provided new insights of mtDNA genetics and laid a foundation for understanding mtDNA heteroplasmy at early stages of life., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Transgenerational epigenetic inheritance in mammals remains a debated subject. Here, we demonstrate that DNA methylation of promoter-associated CpG islands (CGIs) can be transmitted from parents to their offspring in mice. We generated DNA methylation-edited mouse embryonic stem cells (ESCs), in which CGIs of two metabolism-related genes, the Ankyrin repeat domain 26 and the low-density lipoprotein receptor, were specifically methylated and silenced. DNA methylation-edited mice generated by microinjection of the methylated ESCs exhibited abnormal metabolic phenotypes. Acquired methylation of the targeted CGI and the phenotypic traits were maintained and transmitted across multiple generations. The heritable CGI methylation was subjected to reprogramming in parental PGCs and subsequently reestablished in the next generation at post-implantation stages. These observations provide a concrete step toward demonstrating transgenerational epigenetic inheritance in mammals, which may have implications in our understanding of evolutionary biology as well as the etiology, diagnosis, and prevention of non-genetically inherited human diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Exercise benefits the whole organism, yet, how tissues across the body orchestrally respond to exercise remains enigmatic. Here, in young and old mice, with or without exercise, and exposed to infectious injury, we characterized the phenotypic and molecular adaptations to a 12-month exercise across 14 tissues/organs at single-cell resolution. Overall, exercise protects tissues from infectious injury, although more effectively in young animals, and benefits aged individuals in terms of inflammaging suppression and tissue rejuvenation, with structural improvement in the central nervous system and systemic vasculature being the most prominent. In vascular endothelial cells, we found that readjusting the rhythmic machinery via the core circadian clock protein BMAL1 delayed senescence and facilitated recovery from infectious damage, recapitulating the beneficial effects of exercise. Our study underscores the effect of exercise in reconstituting the youthful circadian clock network and provides a foundation for further investigating the interplay between exercise, aging, and immune challenges across the whole organism., Competing Interests: The authors declare no competing interests., (© 2023.)

Stem cells play central roles in tissue development, homeostasis, and regeneration. Decades of scientific research have uncovered processes of stem cell decline in tissue and organismal aging, and more recently, pioneering technologies permit the dissection of its underlying mechanisms and inform therapeutic development for aging and aging-associated disorders. In this review, we elucidate aging-related features across different somatic stem cell types, with a specific focus on epigenetic changes, loss of protein homeostasis, and systemic influencing factors, including chronic inflammation, circadian rhythm dysregulation, and metabolic disorder. Our survey of organismal stem cell aging summarizes its underlying biological implications, points to potential biomarkers of stem cell aging, and discusses stem cell-based therapeutic strategies with the potential for promoting healthy aging and combating aging and age-related diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

Apolipoprotein E4 (APOEε4) is the major allelic risk factor for late-onset sporadic Alzheimer's disease (sAD). Inflammation is increasingly considered as critical in sAD initiation and progression. Identifying brain molecular mechanisms that could bridge these two risk factors remain unelucidated. Leveraging induced pluripotent stem cell (iPSC)-based strategies, we demonstrate that APOE controls inflammation in human astrocytes by regulating Transgelin 3 (TAGLN3) expression and, ultimately, nuclear factor κB (NF-κB) activation. We uncover that APOE4 specifically downregulates TAGLN3, involving histone deacetylases activity, which results in low-grade chronic inflammation and hyperactivated inflammatory responses. We show that APOE4 exerts a dominant negative effect to prime astrocytes toward a pro-inflammatory state that is pharmacologically reversible by TAGLN3 supplementation. We further confirm that TAGLN3 is downregulated in the brain of patients with sAD. Our findings highlight the APOE-TAGLN3-NF-κB axis regulating neuroinflammation in human astrocytes and reveal TAGLN3 as a molecular target to modulate neuroinflammation, as well as a potential biomarker for AD., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Constitutive heterochromatin is responsible for genome repression of DNA enriched in repetitive sequences, telomeres, and centromeres. During physiological and pathological premature aging, heterochromatin homeostasis is profoundly compromised. Here, we showed that LINE-1 ( Long Interspersed Nuclear Element-1; L1 ) RNA accumulation was an early event in both typical and atypical human progeroid syndromes. L1 RNA negatively regulated the enzymatic activity of the histone-lysine N -methyltransferase SUV39H1 (suppression of variegation 3-9 homolog 1), resulting in heterochromatin loss and onset of senescent phenotypes in vitro. Depletion of L1 RNA in dermal fibroblast cells from patients with different progeroid syndromes using specific antisense oligonucleotides (ASOs) restored heterochromatin histone 3 lysine 9 and histone 3 lysine 27 trimethylation marks, reversed DNA methylation age, and counteracted the expression of senescence-associated secretory phenotype genes such as p16 , p21 , activating transcription factor 3 ( ATF3 ), matrix metallopeptidase 13 ( MMP13 ), interleukin 1a ( IL1a ), BTG anti-proliferation factor 2 ( BTG2 ), and growth arrest and DNA damage inducible beta ( GADD45b ). Moreover, systemic delivery of ASOs rescued the histophysiology of tissues and increased the life span of a Hutchinson-Gilford progeria syndrome mouse model. Transcriptional profiling of human and mouse samples after L1 RNA depletion demonstrated that pathways associated with nuclear chromatin organization, cell proliferation, and transcription regulation were enriched. Similarly, pathways associated with aging, inflammatory response, innate immune response, and DNA damage were down-regulated. Our results highlight the role of L1 RNA in heterochromatin homeostasis in progeroid syndromes and identify a possible therapeutic approach to treat premature aging and related syndromes.

The diverse functions of WASP, the deficiency of which causes Wiskott-Aldrich syndrome (WAS), remain poorly defined. We generated three isogenic WAS models using patient induced pluripotent stem cells and genome editing. These models recapitulated WAS phenotypes and revealed that WASP deficiency causes an upregulation of numerous RNA splicing factors and widespread altered splicing. Loss of WASP binding to splicing factor gene promoters frequently leads to aberrant epigenetic activation. WASP interacts with dozens of nuclear speckle constituents and constrains SRSF2 mobility. Using an optogenetic system, we showed that WASP forms phase-separated condensates that encompasses SRSF2, nascent RNA and active Pol II. The role of WASP in gene body condensates is corroborated by ChIPseq and RIPseq. Together our data reveal that WASP is a nexus regulator of RNA splicing that controls the transcription of splicing factors epigenetically and the dynamics of the splicing machinery through liquid-liquid phase separation., (© 2022. The Author(s).)

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient in vivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression in vivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

It is widely believed that cellular senescence plays a critical role in both aging and cancer, and that senescence is a fundamental, permanent growth arrest that somatic cells cannot avoid. Here we show that Myc plays an important role in self-renewal of esophageal epithelial cells, contributing to their resistance to cellular senescence. Myc is homogeneously expressed in basal cells of the esophageal epithelium and Myc positively regulates their self-renewal by maintaining their undifferentiated state. Indeed, Myc knockout induced a loss of the undifferentiated state of esophageal epithelial cells resulting in cellular senescence while forced MYC expression promoted oncogenic cell proliferation. A superoxide scavenger counteracted Myc knockout-induced senescence, therefore suggesting that a mitochondrial superoxide takes part in inducing senescence. Taken together, these analyses reveal extremely low levels of cellular senescence and senescence-associated phenotypes in the esophageal epithelium, as well as a critical role for Myc in self-renewal of basal cells in this organ. This provides new avenues for studying and understanding the links between stemness and resistance to cellular senescence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hishida, Vazquez-Ferrer, Hishida-Nozaki, Takemoto, Hatanaka, Yoshida, Prieto, Sahu, Takahashi, Reddy, O’Keefe, Rodriguez Esteban, Knoepfler, Nuñez Delicado, Castells, Campistol, Kato, Nakagawa and Izpisua Belmonte.)

Partial reprogramming by expression of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) for short periods of time restores a youthful epigenetic signature to aging cells and extends the life span of a premature aging mouse model. However, the effects of longer-term partial reprogramming in physiologically aging wild-type mice are unknown. Here, we performed various long-term partial reprogramming regimens, including different onset timings, during physiological aging. Long-term partial reprogramming lead to rejuvenating effects in different tissues, such as the kidney and skin, and at the organismal level; duration of the treatment determined the extent of the beneficial effects. The rejuvenating effects were associated with a reversion of the epigenetic clock and metabolic and transcriptomic changes, including reduced expression of genes involved in the inflammation, senescence and stress response pathways. Overall, our observations indicate that partial reprogramming protocols can be designed to be safe and effective in preventing age-related physiological changes. We further conclude that longer-term partial reprogramming regimens are more effective in delaying aging phenotypes than short-term reprogramming., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

In a recent issue of Nature, Kagawa et al. reported a highly efficient and robust protocol for generating human blastoids from naive human pluripotent stem cells. The blastoids resemble human blastocysts, follow the sequential lineage specification of blastocyst development, and can attach to endometrial cells with the polar trophectoderm to model implantation., Competing Interests: Declaration of interests Juan Carlos Izpisua Belmonte is a member of the Cell Advisory Board., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Extended/expanded pluripotent stem (EPS) cells can efficiently contribute to both embryonic and extraembryonic lineages in vitro and in vivo . Starting from these cells, we established a 3D differentiation system that enabled the generation of blastocyst-like structures (EPS-blastoids) through lineage segregation and self-organization. We also provide proof of concept that EPS-blastoids can be generated from adult cells via cellular reprogramming. EPS-blastoids provide a unique platform for studying early embryogenesis. For complete details on the use and execution of this protocol, please refer to Li et al. (2019)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Background: Strategies for monitoring the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are crucial for combating the pandemic. Detection and mutation surveillance of SARS-CoV-2 and other respiratory viruses require separate and complex workflows that rely on highly specialized facilities, personnel, and reagents. To date, no method can rapidly diagnose multiple viral infections and determine variants in a high-throughput manner., Methods: We describe a method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes, termed nanopore sequencing of isothermal rapid viral amplification for near real-time analysis (NIRVANA). It can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus and monitor mutations for up to 96 samples in real time., Findings: NIRVANA showed high sensitivity and specificity for SARS-CoV-2 in 70 clinical samples with a detection limit of 20 viral RNA copies per μL of extracted nucleic acid. It also detected the influenza A co-infection in two samples. The variant analysis results of SARS-CoV-2-positive samples mirror the epidemiology of coronavirus disease 2019 (COVID-19). Additionally, NIRVANA could simultaneously detect SARS-CoV-2 and pepper mild mottle virus (PMMoV) (an omnipresent virus and water-quality indicator) in municipal wastewater samples., Conclusions: NIRVANA provides high-confidence detection of both SARS-CoV-2 and other respiratory viruses and mutation surveillance of SARS-CoV-2 on the fly. We expect it to offer a promising solution for rapid field-deployable detection and mutational surveillance of pandemic viruses., Funding: M.L. is supported by KAUST Office of Sponsored Research (BAS/1/1080-01). This work is supported by KAUST Competitive Research Grant (URF/1/3412-01-01; M.L. and J.C.I.B.) and Universidad Catolica San Antonio de Murcia (J.C.I.B.). A.M.H. is supported by Saudi Ministry of Education (project 436)., Competing Interests: A patent application based on methods described in this paper has been filed by King Abdullah University of Science and Technology, in which C.B. and M.L. are listed as inventors. The authors declare no other competing interests., (© 2021 Elsevier Inc.)