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Human pluripotent stem cells (PSCs), which include both embryonic and induced pluripotent stem cells, are widely used in fundamental and applied biomedical research. They have been instrumental for better understanding development and cell differentiation processes, disease origin and progression and can aid in the discovery of new drugs. PSCs also hold great potential in regenerative medicine to treat or diminish the effects of certain debilitating diseases, such as degenerative disorders. However, some concerns have recently been raised over their safety for use in regenerative medicine. One of the major concerns is the fact that PSCs are prone to errors in passing the correct number of chromosomes to daughter cells, resulting in aneuploid cells. Aneuploidy, characterised by an imbalance in chromosome number, elicits the upregulation of different stress pathways that are deleterious to cell homeostasis, impair proper embryo development and potentiate cancer development. In this review, we will summarize known molecular mechanisms recently revealed to impair mitotic fidelity in human PSCs and the consequences of the decreased mitotic fidelity of these cells. We will finish with speculative views on how the physiological characteristics of PSCs can affect the mitotic machinery and how their suboptimal mitotic fidelity may be circumvented.

Researchers are exploring sex differences in experimental models of both development and disease-but are we doing enough? In this collection of Voices, we celebrate researchers who are asking this question and starting to offer mechanistic clues on sexually dimorphic differences seen in interorgan communication, metabolic disease, neurological disorders, and more., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Angelman Syndrome is a rare neurodevelopmental disorder caused by several (epi)genetic alterations. The patients present strong neurological impairment due to the absence of a functional maternal UBE3A gene in neurons. Here, we generated and characterized a new induced pluripotent stem cell (iPSC) line from a female child with Angelman syndrome harbouring a class II deletion. iPSCs were reprogrammed from fibroblasts using Sendai viruses. The new iPSCs express pluripotency markers, are capable of trilineage in vitro differentiation and have the expected imprinting status of Angelman syndrome. These iPSCs are a valuable tool to elucidate the pathophysiological mechanisms associated with this disease., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Ageing is the gradual decline in organismal fitness that occurs over time leading to tissue dysfunction and disease. At the cellular level, ageing is associated with reduced function, altered gene expression and a perturbed epigenome. Recent work has demonstrated that the epigenome is already rejuvenated by the maturation phase of somatic cell reprogramming, which suggests full reprogramming is not required to reverse ageing of somatic cells. Here we have developed the first "maturation phase transient reprogramming" (MPTR) method, where reprogramming factors are selectively expressed until this rejuvenation point then withdrawn. Applying MPTR to dermal fibroblasts from middle-aged donors, we found that cells temporarily lose and then reacquire their fibroblast identity, possibly as a result of epigenetic memory at enhancers and/or persistent expression of some fibroblast genes. Excitingly, our method substantially rejuvenated multiple cellular attributes including the transcriptome, which was rejuvenated by around 30 years as measured by a novel transcriptome clock. The epigenome was rejuvenated to a similar extent, including H3K9me3 levels and the DNA methylation ageing clock. The magnitude of rejuvenation instigated by MPTR appears substantially greater than that achieved in previous transient reprogramming protocols. In addition, MPTR fibroblasts produced youthful levels of collagen proteins, and showed partial functional rejuvenation of their migration speed. Finally, our work suggests that optimal time windows exist for rejuvenating the transcriptome and the epigenome. Overall, we demonstrate that it is possible to separate rejuvenation from complete pluripotency reprogramming, which should facilitate the discovery of novel anti-ageing genes and therapies., Competing Interests: DG, AP, FS, HO, CT, IH, IM No competing interests declared, TS is CEO and shareholder of Chronomics, WR is a consultant and shareholder of Cambridge Epigenetix. Is employed by Altos Labs, (© 2022, Gill et al.)

Women who inherit heterozygous mutations in the BRCA2 gene have an increased risk of developing cancer, mainly breast and ovarian tumors. A particular BRCA2 mutation (c.156_157insAlu) is exclusively found in families of Portuguese ancestry and is present in approximately 30% of all Portuguese families with hereditary breast and ovarian cancers. We report the generation and characterization of the first iPSC line from a female donor harboring the Portuguese BRCA2 founder mutation. Skin fibroblasts were reprogrammed using a non-integrative Sendai virus. These iPSCs are a valuable tool to study the origin of BRCA2-associated cancer in its earliest phases., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

How BRCA1 germline mutations predispose to cancer remains poorly understood. Induced pluripotent stem cells (iPSCs) represent an emerging model to investigate the molecular mechanisms underlying malignant transformation in primary cells from individuals who are carriers of deleterious mutations in the BRCA1 gene. Here we report the generation and characterization of iPSC lines from a female donor harboring a germline c.3612delA mutation in the BRCA1 gene and her daughter who does not carry the mutation. Skin fibroblasts were reprogrammed using non-integrative Sendai virus and characterized for their pluripotent properties. These iPSCs are a valuable cellular model for personalized pre-clinical research in the context of BRCA1 mutant hereditary cancers., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Pluripotent stem cells (PSCs) are central to development as they are the precursors of all cell types in the embryo. Therefore, maintaining a stable karyotype is essential, both for their physiological role as well as for their use in regenerative medicine. Karyotype abnormalities in PSCs in culture are common but the underlying causes remain unknown. To gain insight, we explore the composition of the centromere and kinetochore in human embryonic and induced PSCs. Centromere function depends on CENP-A nucleosome-defined chromatin. We show that while PSCs maintain abundant pools of CENP-A, CENP-C and CENP-T, these essential centromere components are strongly reduced at stem cell centromeres. Outer kinetochore recruitment is also impaired to a lesser extent, indicating an overall weaker kinetochore while the inner centromere protein Aurora B remains unaffected. We further show that, similar to differentiated human cells, CENP-A chromatin assembly in PSCs requires transition into G1 phase. Finally, reprogramming experiments indicate that reduction of centromeric CENP-A levels is an early event during dedifferentiation, coinciding with global chromatin remodelling. Our characterization of centromeres in human stem cells suggests a possible link between impaired centromere function and stem cell aneuploidies.

Global DNA demethylation is an integral part of reprogramming processes in vivo and in vitro, but whether it occurs in the derivation of induced pluripotent stem cells (iPSCs) is not known. Here, we show that iPSC reprogramming involves both global and targeted demethylation, which are separable mechanistically and by their biological outcomes. Cells at intermediate-late stages of reprogramming undergo transient genome-wide demethylation, which is more pronounced in female cells. Global demethylation requires activation-induced cytidine deaminase (AID)-mediated downregulation of UHRF1 protein, and abolishing demethylation leaves thousands of hypermethylated regions in the iPSC genome. Independently of AID and global demethylation, regulatory regions, particularly ESC enhancers and super-enhancers, are specifically targeted for hypomethylation in association with transcription of the pluripotency network. Our results show that global and targeted DNA demethylation are conserved and distinct reprogramming processes, presumably because of their respective roles in epigenetic memory erasure and in the establishment of cell identity., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mouse embryonic stem cells are dynamic and heterogeneous. For example, rare cells cycle through a state characterized by decondensed chromatin and expression of transcripts, including the Zscan4 cluster and MERVL endogenous retrovirus, which are usually restricted to preimplantation embryos. Here, we further characterize the dynamics and consequences of this transient cell state. Single-cell transcriptomics identified the earliest upregulated transcripts as cells enter the MERVL/Zscan4 state. The MERVL/Zscan4 transcriptional network was also upregulated during induced pluripotent stem cell reprogramming. Genome-wide DNA methylation and chromatin analyses revealed global DNA hypomethylation accompanying increased chromatin accessibility. This transient DNA demethylation was driven by a loss of DNA methyltransferase proteins in the cells and occurred genome-wide. While methylation levels were restored once cells exit this state, genomic imprints remained hypomethylated, demonstrating a potential global and enduring influence of endogenous retroviral activation on the epigenome., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Epigenetic aging clocks have been widely used to validate rejuvenation effects during cellular reprogramming. However, these predictions are unverifiable because the true biological age of reprogrammed cells remains unknown. We present an analytical framework to consider rejuvenation predictions from the uncertainty perspective. Our analysis reveals that the DNA methylation profiles across reprogramming are poorly represented in the aging data used to train clock models, thus introducing high epistemic uncertainty in age estimations. Moreover, predictions of different published clocks are inconsistent, with some even suggesting zero or negative rejuvenation. While not questioning the possibility of age reversal, we show that the high clock uncertainty challenges the reliability of rejuvenation effects observed during in vitro reprogramming before pluripotency and throughout embryogenesis. Conversely, our method reveals a significant age increase after in vivo reprogramming. We recommend including uncertainty estimation in future aging clock models to avoid the risk of misinterpreting the results of biological age prediction. [ABSTRACT FROM AUTHOR]

Reactivation of retroelements in the human genome has been linked to aging. However, whether the epigenetic state of specific retroelements can predict chronological age remains unknown. We provide evidence that locus‐specific retroelement DNA methylation can be used to create retroelement‐based epigenetic clocks that accurately measure chronological age in the immune system, across human tissues, and pan‐mammalian species. We also developed a highly accurate retroelement epigenetic clock compatible with EPICv.2.0 data that was constructed from CpGs that did not overlap with existing first‐ and second‐generation epigenetic clocks, suggesting a unique signal for epigenetic clocks not previously captured. We found retroelement‐based epigenetic clocks were reversed during transient epigenetic reprogramming, accelerated in people living with HIV‐1, and responsive to antiretroviral therapy. Our findings highlight the utility of retroelement‐based biomarkers of aging and support a renewed emphasis on the role of retroelements in geroscience. [ABSTRACT FROM AUTHOR]

Objective: By assessing the genetic diversity and associated selective traits of Kirghiz sheep (KIR), we aim to uncover the mechanisms that contribute to sheep's adaptability to the Pamir Plateau environment. Methods: This study utilized Illumina Ovine SNP50 BeadChip data from KIR residing in the Pamir Plateau, Qira Black sheep (QBS) inhabiting the Taklamakan Desert, and commonly introduced breeds including Dorper sheep (DOR), Suffolk sheep (SUF), and Hu sheep (HU). The data was analyzed using principal component analysis, phylogenetic analysis, population admixture analysis, kinship matrix analysis, linkage disequilibrium analysis, and selective signature analysis. We employed four methods for selective signature analysis: fixation index (Fst), cross-population extended homozygosity (XP-EHH), integrated haplotype score (iHS), and nucleotide diversity (Pi). These methods aim to uncover the genetic mechanisms underlying the germplasm resources of Kirghiz sheep, enhance their production traits, and explore their adaptation to challenging environmental conditions. Results: The test results unveiled potential selective signals associated with adaptive traits and growth characteristics in sheep under harsh environmental conditions, and annotated the corresponding genes accordingly. These genes encompass various functionalities such as adaptations associated with plateau, cold, and arid environment (ETAA1, UBE3D, TLE4, NXPH1, MAT2B, PPARGC1A, VEGFA, TBX15 and PLXNA4), wool traits (LMO3, TRPS1, EPHA5), body size traits (PLXNA2, EFNA5), reproductive traits (PPP3CA, PDHA2, NTRK2), and immunity (GATA3). Conclusion: Our study identified candidate genes associated with the production traits and adaptation to the harsh environment of the Pamir Plateau in Kirghiz sheep. These findings provide valuable resources for local sheep breeding programs. The objective of this study is to offer valuable insights for the sustainable development of the Kirghiz sheep industry. [ABSTRACT FROM AUTHOR]

The CYP46A1 gene codes for the cholesterol 24-hydroxylase, a cytochrome P450 specifically expressed in neurons and responsible for the majority of cholesterol turnover in the central nervous system. Previously, we have demonstrated the critical participation of Sp transcription factors in the CYP46A1 response to histone deacetylase (HDAC) inhibitors, and in this study we investigated the involvement of intracellular signaling pathways in the trichostatin A (TSA) effect. Our results show that pretreatment of neuroblastoma cells with chemical inhibitors of mitogen-activated kinase kinase (MEK)1 significantly potentiates the TSA-dependent induction of cholesterol 24-hydroxylase, whereas inhibition of protein phosphatases by okadaic acid (OA) or overexpression of MEK1 partially impairs the TSA effect without affecting histone hyperacetylation at the promoter. Immunoblotting revealed that TSA treatment decreases ERK1/2 phosphorylation concomitantly with a decrease in Sp3 binding activity, which are both reversed by pretreatment with OA. Chromatin immunoprecipitation analysis demonstrated that TSA induces the release of p-ERK1/2 from the CYP46A1 proximal promoter, whereas pretreatment with OA restores the co-occupancy of Sp3-ERK1/2 in the same promoter fragments. We demonstrate for the first time the participation of MEK-ERK1/2 signaling pathway in HDAC inhibitor-dependent induction of cytochrome P450 gene expression, underlying the importance of this regulatory signaling mechanism in the control of brain cholesterol elimination.

Cholesterol metabolism in the brain is distinct from that in other tissues due to the fact that cholesterol itself is unable to pass across the blood-brain barrier. Elimination of brain cholesterol is mainly dependent on a neuronal-specific cytochrome P450, CYP46A1, catalyzing the conversion of cholesterol into 24(S)-hydroxycholesterol (24OHC), which is able to pass the blood-brain barrier. A suitable model for studying this elimination from human neuronal cells has not been described previously. It is shown here that differentiated Ntera2/clone D1 (NT2) cells express the key genes involved in brain cholesterol homeostasis including CYP46A1, and that the expression profiles of the genes observed during neuronal differentiation are those expected to occur in vivo. Thus there was a decrease in the mRNA levels corresponding to cholesterol synthesis enzymes and a marked increase in the mRNA level of CYP46A1. The latter increase was associated with increased levels of CYP46A1 protein and increased production of 24OHC. The magnitude of the secretion of 24OHC from the differentiated NT2 cells into the medium was similar to that expected to occur under in vivo conditions. An alternative to elimination of cholesterol by the CYP46A1 mechanism is elimination by CYP27A1, and the product of this enzyme, 27-hydroxycholesterol (27OHC), is also known to pass the blood-brain barrier. The CYP27A1 protein level decreased during the differentiation of the NT2 cells in parallel with decreased production of 27OHC. The ratio between 24OHC and 27OHC in the medium from the cultured cells increased, by a factor of 13, during the differentiation process. The results suggest that progenitor cells eliminate cholesterol in the form of 27OHC while neurogenesis induces a change to the CYP46A1 dependent pathway. Furthermore this study demonstrates that differentiated NT2 cells are suitable for studies of cholesterol homeostasis in human neurons., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

CYP46A1 is a neuron-specific cytochrome P450 that plays a pivotal role in maintaining cholesterol homeostasis in the CNS. However, the molecular mechanisms underlying human CYP46A1 expression are still poorly understood, partly because of the lack of a cellular model that expresses high levels of CYP46A1. Our previous studies demonstrated that specificity protein (Sp) transcription factors control CYP46A1 expression, and are probably responsible for cell-type specificity. Herein, we have differentiated Ntera2/cloneD1 cells into post-mitotic neurons and identified for the first time a human cell model that expresses high levels of CYP46A1 mRNA. Our results show a decrease in Sp1 protein levels, concomitant with the increase in CYP46A1 mRNA levels. This decrease was correlated with changes in the ratio of Sp proteins associated to the CYP46A1 proximal promoter. To examine if the increase in (Sp3+Sp4)/Sp1 ratio was observed in other Sp-regulated promoters, we have selected four genes--reelin, glutamate receptor subunit zeta-1, glutamate receptor subunit epsilon-1 and μ-opioid receptor--known to be expressed in the human brain and analyzed the Sp proteins binding pattern to the promoter of these genes, in undifferentiated and differentiated Ntera2/cloneD1. Our data indicate that the dissociation of Sp1 from promoter regions is a common feature amongst Sp-regulated genes that are up-regulated after neuronal differentiation., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Human PDHA2 is a testis-specific gene that codes for the E(1)α subunit of Pyruvate Dehydrogenase Complex (PDC), a crucial enzyme system in cell energy metabolism. Since activation of the PDHA2 gene in somatic cells could be a new therapeutic approach for PDC deficiency, we aimed to identify the regulatory mechanisms underlying the human PDHA2 gene expression. Functional deletion studies revealed that the -122 to -6 promoter region is indispensable for basal expression of this TATA-less promoter, and suggested a role of an epigenetic program in the control of PDHA2 gene expression. Indeed, treatment of SH-SY5Y cells with the hypomethylating agent 5-Aza-2'-deoxycytidine (DAC) promoted the reactivation of the PDHA2 gene, by inducing the recruitment of the RNA polymerase II to the proximal promoter region and the consequent increase in PDHA2 mRNA levels. Bisulfite sequencing analysis revealed that DAC treatment induced a significant demethylation of the CpG island II (nucleotides +197 to +460) in PDHA2 coding region, while the promoter region remained highly methylated. Taken together with our previous results that show an in vivo correlation between PDHA2 expression and the demethylation of the CpG island II in testis germ cells, the present results show that internal methylation of the PDHA2 gene plays a part in its repression in somatic cells. In conclusion, our data support the novel finding that methylation of the PDHA2 coding region can inhibit gene transcription. This represents a key mechanism for absence of PDHA2 expression in somatic cells and a target for PDC therapy.

We investigated whether the CYP46A1 gene, a neuronal-specific cytochrome P450, responsible for the majority of brain cholesterol turnover, is subject to transcriptional modulation through modifications in histone acetylation. We demonstrated that inhibition of histone deacetylase activity by trichostatin A (TSA), valproic acid and sodium butyrate caused a potent induction of both CYP46A1 promoter activity and endogenous expression. Silencing of Sp transcription factors through specific small interfering RNAs, or impairing Sp binding to the proximal promoter, by site-directed mutagenesis, led to a significant decrease in TSA-mediated induction of CYP46A1 expression/promoter activity. Electrophoretic mobility shift assay, DNA affinity precipitation assays and chromatin immunoprecipitation assays were used to determine the multiprotein complex recruited to the CYP46A1 promoter, upon TSA treatment. Our data showed that a decrease in Sp3 binding at particular responsive elements, can shift the Sp1/Sp3/Sp4 ratio, and favor the detachment of histone deacetylase (HDAC) 1 and HDAC2 and the recruitment of p300/CBP. Moreover, we observed a dynamic change in the chromatin structure upon TSA treatment, characterized by an increase in the local recruitment of euchromatic markers and RNA polymerase II. Our results show the critical participation of an epigenetic program in the control of CYP46A1 gene transcription, and suggest that brain cholesterol catabolism may be affected upon treatment with HDAC inhibitors.

The major mechanism of brain cholesterol elimination is the conversion of cholesterol into 24S-hydroxycholesterol by CYP46A1, a neuron-specific cytochrome P450. Since increasing evidence suggests that upregulation of CYP46A1 may be relevant for the treatment of Alzheimer's disease, we aim to identify the molecular mechanisms involved in CYP46A1 transcription. Our previous studies demonstrated the role of Sp transcription factors in basal expression and histone deacetylase (HDAC) inhibitor-dependent derepression of CYP46A1. Here, we show that the demethylating agent 5'-Aza-2'-deoxycytidine (DAC) is a CYP46A1 inducer and that pre-treatment with DAC causes a marked synergistic activation of CYP46A1 transcription by trichostatin A. Surprisingly, bisulfite sequencing analysis revealed that the CYP46A1 core promoter is completely unmethylated in both human brain and non-neuronal human tissues where CYP46A1 is not expressed. Therefore, we have investigated Sp expression levels by western blot and real-time PCR, and their binding patterns to the CYP46A1 promoter, by electrophoretic mobility shift assay and chromatin immunoprecipitation assays, after DAC treatment. Our results showed that DAC decreases not only Sp1 and Sp3 protein levels, but also the binding activity of Sp3 to the +1 region of the CYP46A1 locus. Concomitantly, HDAC1 and HDAC2 were also significantly dissociated from the promoter. In conclusion, DAC induces CYP46A1 gene expression, in a DNA methylation-independent mechanism, decreasing Sp3/HDAC binding to the proximal promoter. Furthermore, by affecting the expression of the Sp3 transcription factor in neuronal cells, DAC might affect not only brain cholesterol metabolism, but also the expression of many other neuronal genes.

Activation of c-Jun N-terminal kinase (JNK) signaling pathway is a key event in apoptosis. The cellular mechanisms underlying the control of JNK catalytic activity before and immediately after stress in neuronal cells are still not completely understood. Under resting conditions the basal activity of JNK is low, since JNK is kept inactive by the presence of one or more endogenous repressors, including glutathione S-transferase pi (GSTpi). The aim of this study was to investigate the control of JNK signaling by GSTpi. We examined the modifications of GSTpi protein expression and oligomerization after UV irradiation-induced stress in human SH-SY5Y neuroblastoma cells. In parallel, we investigated the effect of UV irradiation on JNK activation and c-Jun phosphorylation, and whether apoptosis represents a functional consequence triggered by this signaling pathway. We show that in SH-SY5Y cells JNK phosphorylation and activation precedes c-Jun phosphorylation and caspase-3 cleavage. Importantly, the increase of JNK enzymatic activity correlates with the dissociation of GSTpi-JNK complexes and the increased concentration of GSTpi multimer forms. Results presented herein show for the first time direct interaction between JNK and GSTpi in SH-SY5Y neuroblastoma cells, and suggest that in these cells GSTpi may serve as a regulator of JNK catalytic activity. This work contributes to further elucidate the mechanisms underlying the regulation of JNK activity under stress conditions.

Brain defective cholesterol homeostasis has been associated with neurologic diseases, such as Alzheimer's and Huntington's disease. The elimination of cholesterol from the brain involves its conversion into 24(S)-hydroxycholesterol by CYP46A1, and the efflux of this oxysterol across the blood-brain barrier. Herein, we identified the regulatory elements and factors involved the human CYP46A1 expression. Functional 5'deletion analysis mapped a region spanning from nucleotides -236/-64 that is indispensable for basal expression of this TATA-less gene. Treatment of SH-SY5Y cells with mithramycin A resulted in a significant reduction of promoter activity, suggesting a role of Sp family of transcription factors in CYP46A1 regulation. Combination of Sp1, Sp3, and Sp4 over-expression studies in Drosophila SL-2 cells, and systematic promoter mutagenesis identified Sp3 and Sp4 binding to four GC-boxes as required and sufficient for high levels of promoter activity. Moreover, Sp3 and Sp4 were demonstrated to be the major components of the protein-DNA complexes observed in primary rat cortical extracts. Our results suggest that the cell-type specific expression of Sp transcription factors - substitution of Sp1 by Sp4 in neurons - is responsible for the basal expression of the CYP46A1 gene. This study delineates for the first time the mechanisms underlying the human CYP46A1 transcription and thereby elucidates potential pathways underlying cholesterol homeostasis in the brain.

Human pluripotent stem cells (hPSCs) are pivotal in regenerative medicine, yet their in vitro expansion often leads to genetic abnormalities, raising concerns about their safety in clinical applications. This study analyzed ten human embryonic stem cell lines across multiple passages to elucidate the dynamics of chromosomal abnormalities and single-nucleotide variants (SNVs) in 380 cancer-related genes. Prolonged in vitro culture resulted in 80% of the lines acquiring gains of chromosome 20q or 1q, both known for conferring an in vitro growth advantage. 70% of lines also acquired other copy number variants (CNVs) outside the recurrent set. Additionally, we detected 122 SNVs in 88 genes, with all lines acquiring at least one de novo SNV during culture. Our findings showed higher loads of both CNVs and SNVs at later passages, which were due to the cumulative acquisition of mutations over a longer time in culture, and not to an increased rate of mutagenesis over time. Importantly, we observed that SNVs and rare CNVs followed the acquisition of chromosomal gains in 1q and 20q, while most of the low-passage and genetically balanced samples were devoid of cancer-associated mutations. This suggests that recurrent chromosomal abnormalities are potential drivers for the acquisition of other mutations. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSCs) are a powerful tool for biomedical research, but their production presents challenges and safety concerns. Yamanaka and Takahashi revolutionised the field by demonstrating that somatic cells could be reprogrammed into pluripotent cells by overexpressing four key factors for a sufficient time. iPSCs are typically generated using viruses or virus-based methods, which have drawbacks such as vector persistence, risk of insertional mutagenesis, and oncogenesis. The application of less harmful nonviral vectors is limited as conventional plasmids cannot deliver the levels or duration of the factors necessary from a single transfection. Hence, plasmids that are most often used for reprogramming employ the potentially oncogenic Epstein–Barr nuclear antigen 1 (EBNA-1) system to ensure adequate levels and persistence of expression. In this study, we explored the use of nonviral SMAR DNA vectors to reprogram human fibroblasts into iPSCs. We show for the first time that iPSCs can be generated using nonviral plasmids without the use of EBNA-1 and that these DNA vectors can provide sufficient expression to induce pluripotency. We describe an optimised reprogramming protocol using these vectors that can produce high-quality iPSCs with comparable pluripotency and cellular function to those generated with viruses or EBNA-1 vectors. [ABSTRACT FROM AUTHOR]

Cancers harness embryonic programs to evade aging and promote survival. Normally, sequences at chromosome ends called telomeres shorten with cell division, serving as a countdown clock to limit cell replication. Therefore, a crucial aspect of cancerous transformation is avoiding replicative aging by activation of telomere repair programs. Mouse embryonic stem cells (mESCs) activate a transient expression of the gene Zscan4, which correlates with chromatin de-condensation and telomere extension. Head and neck squamous cell carcinoma (HNSCC) cancers reactivate ZSCAN4, which in turn regulates the phenotype of cancer stem cells (CSCs). Our study reveals a new role for human ZSCAN4 in facilitating functional histone H3 acetylation at telomere chromatin. Next-generation sequencing indicates ZSCAN4 enrichment at telomere chromatin. These changes correlate with ZSCAN4-induced histone H3 acetylation and telomere elongation, while CRISPR/Cas9 knockout of ZSCAN4 leads to reduced H3 acetylation and telomere shortening. Our study elucidates the intricate involvement of ZSCAN4 and its significant contribution to telomere chromatin remodeling. These findings suggest that ZSCAN4 induction serves as a novel link between 'stemness' and telomere maintenance. Targeting ZSCAN4 may offer new therapeutic approaches to effectively limit or enhance the replicative lifespan of stem cells and cancer cells. [ABSTRACT FROM AUTHOR]

Aging and associated diseases are acute problems of modern biology and medicine. Although aging cannot currently be prevented, its impact on the lifespan and health of older adults can potentially be minimized through interventions aimed at returning cells to normal function. The constant search for ways to rejuvenate and improve the regenerative capacity of cells led to the discovery in 2016 of a partial reprogramming method based on short-term expression of reprogramming factors (Oct4, Sox2, Klf4, and c-Myc). As a result, the youthful epigenetic signature of aging cells is restored. The effectiveness of the method is shown as in the system in vitro and in vivo. The presented review discusses the main successes of partial reprogramming, as well as the problems and unresolved questions that researchers have encountered. Data on molecular changes during the process of partial reprogramming are discussed separately. The partial reprogramming method provides a wide range of opportunities for fundamental research on aging and rejuvenation. Further work in this direction may lead to the development of therapeutic strategies to alleviate age-related diseases and, thus, improve health and longevity. [ABSTRACT FROM AUTHOR]

Evaluation of the influence of primary and secondary aging on the manifestation of molecular and cellular hallmarks of aging is a challenging and currently unresolved issue. Our study represents the first demonstration of the distinct role of primary aging and chronic inflammation/physical inactivity – the most important drivers of secondary aging, in the regulation of transcriptomic and proteomic profiles in human skeletal muscle. To achieve this purpose, young healthy people (n = 15), young (n = 8) and older (n = 37) patients with knee/hip osteoarthritis, a model to study the effect of long‐term inactivity and chronic inflammation on the vastus lateralis muscle, were included in the study. It was revealed that widespread and substantial age‐related changes in gene expression in older patients relative to young healthy people (~4000 genes regulating mitochondrial function, proteostasis, cell membrane, secretory and immune response) were related to the long‐term physical inactivity and chronic inflammation rather than primary aging. Primary aging contributed mainly to the regulation of genes (~200) encoding nuclear proteins (regulators of DNA repair, RNA processing, and transcription), mitochondrial proteins (genes encoding respiratory enzymes, mitochondrial complex assembly factors, regulators of cristae formation and mitochondrial reactive oxygen species production), as well as regulators of proteostasis. It was found that proteins associated with aging were regulated mainly at the post‐transcriptional level. The set of putative primary aging genes and their potential transcriptional regulators can be used as a resource for further targeted studies investigating the role of individual genes and related transcription factors in the emergence of a senescent cell phenotype. [ABSTRACT FROM AUTHOR]