Somatic Cell Lines Research Articles

Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness.

Pluripotent stem cells are defined by their self-renewal capacity, which is the ability of the stem cells to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into any somatic cell lineage. However, understanding the mechanisms that control stem cell fitness versus the pluripotent cell identity is challenging. To investigate the interplay between these two aspects of pluripotency, we performed four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSC self-renewal conditions, and the dissolution of the primed pluripotency identity during early differentiation. Comparative analyses led to the discovery of genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further discovered a core set of factors that control both stem cell fitness and pluripotent identity, including a network of chromatin factors that safeguard pluripotency. Our unbiased and systematic screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide rich datasets for exploring pluripotent cell identity versus cell fitness, and offer a valuable model for categorizing gene function in broad biological contexts.

The Evolutionary Interplay of Somatic and Germline Mutation Rates.

Novel sequencing technologies are making it increasingly possible to measure the mutation rates of somatic cell lineages. Accurate germline mutation rate measurement technologies have also been available for a decade, making it possible to assess how this fundamental evolutionary parameter varies across the tree of life. Here, we review some classical theories about germline and somatic mutation rate evolution that were formulated using principles of population genetics and the biology of aging and cancer. We find that somatic mutation rate measurements, while still limited in phylogenetic diversity, seem consistent with the theory that selection to preserve the soma is proportional to life span. However, germline and somatic theories make conflicting predictions regarding which species should have the most accurate DNA repair. Resolving this conflict will require carefully measuring how mutation rates scale with time and cell division and achieving a better understanding of mutation rate pleiotropy among cell types.

The expression profiles of piRNAs and their interacting Piwi proteins in cellular model of renal development: Focus on Piwil1 in mitosis

Piwi proteins and Piwi interacting RNAs, piRNAs, presented in germline cells play a role in transposon silencing during germline development. In contrast, the role of somatic Piwi proteins and piRNAs still remains obscure. Here, we characterize the expression pattern and distribution of piRNAs in human renal cells in terms of their potential role in kidney development. Further, we show that all PIWI genes are expressed at the RNA level, however, only PIWIL1 gene is detected at the protein level by western blotting in healthy and cancerous renal cells. So far, the expression of human Piwil1 protein has only been shown in testes and cancer cells, but not in healthy somatic cell lines. Since we observe only Piwil1 protein, the regulation of other PIWI genes is probably more intricated, and depends on environmental conditions. Next, we demonstrate that downregulation of Piwil1 protein results in a decrease in the rate of cell proliferation, while no change in the level of apoptotic cells is observed. Confocal microscopy analysis reveals that Piwil1 protein is located in both cellular compartments, cytoplasm and nucleus in renal cells. Interestingly, in nucleus region Piwil1 is observed close to the spindle during all phases of mitosis in all tested cell lines. It strongly indicates that Piwil1 protein plays an essential role in proliferation of somatic cells. Moreover, involvement of Piwil1 in cell division could, at least partly, explain invasion and metastasis of many types of cancer cells with upregulation of PIWIL1 gene expression. It also makes Piwil1 protein as a potential target in the anticancer therapy.

Mapping the Development of Human Spermatogenesis Using Transcriptomics-Based Data: A Scoping Review.

In vitro maturation (IVM) is a promising fertility restoration strategy for patients with nonobstructive azoospermia or for prepubertal boys to obtain fertilizing-competent spermatozoa. However, in vitro spermatogenesis is still not achieved with human immature testicular tissue. Knowledge of various human testicular transcriptional profiles from different developmental periods helps us to better understand the testis development. This scoping review aims to describe the testis development and maturation from the fetal period towards adulthood and to find information to optimize IVM. Research papers related to native and in vitro cultured human testicular cells and single-cell RNA-sequencing (scRNA-seq) were identified and critically reviewed. Special focus was given to gene ontology terms to facilitate the interpretation of the biological function of related genes. The different consecutive maturation states of both the germ and somatic cell lineages were described. ScRNA-seq regularly showed major modifications around 11 years of age to eventually reach the adult state. Different spermatogonial stem cell (SSC) substates were described and scRNA-seq analyses are in favor of a paradigm shift, as the Adark and Apale spermatogonia populations could not distinctly be identified among the different SSC states. Data on the somatic cell lineage are limited, especially for Sertoli cells due technical issues related to cell size. During cell culture, scRNA-seq data showed that undifferentiated SSCs were favored in the presence of an AKT-signaling pathway inhibitor. The involvement of the oxidative phosphorylation pathway depended on the maturational state of the cells. Commonly identified cell signaling pathways during the testis development and maturation highlight factors that can be essential during specific maturation stages in IVM.

The hagfish genome and the evolution of vertebrates

As the only surviving lineages of jawless fishes, hagfishes and lampreys provide a crucial window into early vertebrate evolution1–3. Here we investigate the complex history, timing and functional role of genome-wide duplications4–7 and programmed DNA elimination8,9 in vertebrates in the light of a chromosome-scale genome sequence for the brown hagfish Eptatretus atami. Combining evidence from syntenic and phylogenetic analyses, we establish a comprehensive picture of vertebrate genome evolution, including an auto-tetraploidization (1RV) that predates the early Cambrian cyclostome–gnathostome split, followed by a mid–late Cambrian allo-tetraploidization (2RJV) in gnathostomes and a prolonged Cambrian–Ordovician hexaploidization (2RCY) in cyclostomes. Subsequently, hagfishes underwent extensive genomic changes, with chromosomal fusions accompanied by the loss of genes that are essential for organ systems (for example, genes involved in the development of eyes and in the proliferation of osteoclasts); these changes account, in part, for the simplification of the hagfish body plan1,2. Finally, we characterize programmed DNA elimination in hagfish, identifying protein-coding genes and repetitive elements that are deleted from somatic cell lineages during early development. The elimination of these germline-specific genes provides a mechanism for resolving genetic conflict between soma and germline by repressing germline and pluripotency functions, paralleling findings in lampreys10,11. Reconstruction of the early genomic history of vertebrates provides a framework for further investigations of the evolution of cyclostomes and jawed vertebrates.

Strategies for single base gene editing in an immortalized human cell line by CRISPR/Cas9 technology

The use of CRISPR/Cas9 system has rapidly grown in the last years. Here, the optimization of gene editing of a single-nucleotide polymorphism in a human non-malignant somatic cell line of thyrocytes (Nthy-Ori) was described highlighting strategies for overcoming the problems concerning the delivery and off-targets. We employed both lentivirus and chemical lipids as delivery agents and two strategies for creating the double-strand breaks (DSB). The former induced a DSB by a classical Cas9 nuclease (standard strategy), while the second one employed a modified Cas9 creating a single-strand break (SSB). The knock-in was carried out using a single-stranded donor oligonucleotide or the HR410-PA donor vector (HR). The desired cells could be obtained by combining the double nickase system with the HR vector transfected chemically. This result could be due to the type of DSB, likely processed mainly by non-homologous end joining when blunt (standard strategy) and by HR when overhanging (double nickase). Our results showed that the double nickase is suitable for knocking-in the immortalized Nthy-Ori cell line, while the standard CRISPR/Cas9 system is suitable for gene knock-out creating in/del mutations.

Conditional Depletion of STN1 in Mouse Embryonic Fibroblasts.

The CTC1-STN1-TEN1 (CST) complex is a single-strand DNA-binding protein complex that plays an important role in genome maintenance in various model eukaryotes. Dysfunction of CST is the underlying cause of the rare genetic disorder known as Coats plus disease. In addition, down regulation of STN1 promotes colorectal cancer development in mice. While prior studies have utilized RNAi to knock down CST components in mammalian cells, this approach is associated with off-target effects. Attempts to employ CRISPR/Cas9-based knockout of CST components in somatic cell lines have been unsuccessful due to CST's indispensable role in DNA replication and cell proliferation. To address these challenges, we outline a novel approach utilizing a Cre-loxP-based conditional knockout in mouse embryonic fibroblasts (MEFs). This method offers an alternative means to investigate the function and characteristics of the CST complex in mammalian systems, potentially shedding new light on its roles in genome maintenance. Key features • Conditional depletion of mammalian STN1 using mouse embryonic fibroblast (MEFs). • Analysis of oxidative damage sensitivity using STN1-depleted MEFs. • This protocol requires Stn1flox/flox mice.

Generating Retinas through Guided Pluripotent Stem Cell Differentiation and Direct Somatic Cell Reprogramming.

Retinal degeneration diseases affect millions of people worldwide but are among the most difficult eye diseases to cure. Studying the mechanisms and developing new therapies for these blinding diseases requires researchers to have access to many retinal cells. In recent years there has been substantial advances in the field of biotechnology in generating retinal cells and even tissues in vitro, either through programmed sequential stem cell differentiation or direct somatic cell lineage reprogramming. The resemblance of these in vitro-generated retinal cells to native cells has been increasingly utilized by researchers. With the help of these in vitro retinal models, we now have a better understanding of human retinas and retinal diseases. Furthermore, these in vitro-generated retinal cells can be used as donor cells which solves a major hurdle in the development of cell replacement therapy for retinal degeneration diseases, while providing a promising option for patients suffering from these diseases. In this review, we summarize the development of pluripotent stem cell-to-retinal cell differentiation methods, the recent advances in generating retinal cells through direct somatic cell reprogramming, and the translational applications of retinal cells generated in vitro. Finally, we discuss the limitations of the current protocols and possible future directions for improvement.

Unveiling the roles of Sertoli cells lineage differentiation in reproductive development and disorders: a review.

In mammals, gonadal somatic cell lineage differentiation determines the development of the bipotential gonad into either the ovary or testis. Sertoli cells, the only somatic cells in the spermatogenic tubules, support spermatogenesis during gonadal development. During embryonic Sertoli cell lineage differentiation, relevant genes, including WT1, GATA4, SRY, SOX9, AMH, PTGDS, SF1, and DMRT1, are expressed at specific times and in specific locations to ensure the correct differentiation of the embryo toward the male phenotype. The dysregulated development of Sertoli cells leads to gonadal malformations and male fertility disorders. Nevertheless, the molecular pathways underlying the embryonic origin of Sertoli cells remain elusive. By reviewing recent advances in research on embryonic Sertoli cell genesis and its key regulators, this review provides novel insights into sex determination in male mammals as well as the molecular mechanisms underlying the genealogical differentiation of Sertoli cells in the male reproductive ridge.

Ethylene dimethanesulfonate effects on gene promoter activities related to the endocrine function of immortalized Leydig cell lines R2C and MA-10

Ethylene dimethanesulfonate (EDS) is a molecule with known selective cytotoxicity on adult Leydig cells. A single intraperitoneal injection in rats but not mice, leads to male androgen deprivation and infertility. In vitro studies using rat and mouse immortalized Leydig cell lines, showed similar effects of cell death promoted by EDS in rat cells as seen in vivo, and suggest that EDS affects gene transcription, which could firstly compromise steroidogenesis before the apoptosis process. Using gene reporter assay, this study aimed to investigate EDS effects on the promoter activity of genes important for endocrine function (Star, Insl3) and response to toxic agents (Gsta3) in immortalized Leydig cell lines (rat R2C and mouse MA-10 cells), as well as identify possible EDS-responsive elements in the Star gene promoter. EDS exposure of R2C and MA-10 Leydig cells increased Gsta3 promoter activity after 4 h of treatment and decreased Insl3 promoter activity only in R2C cells after 24 h of treatment. EDS also decreased Star promoter activity in both Leydig cell lines. Using R2C cells, the EDS-responsive region in the Star promoter was located between −400 and −195 bp. This suggests that this region and the associated transcription factors, which include MEF2, might be targeted by EDS. Additional somatic gonadal cell lines expressing Star were used and EDS did not affect Star promoter activity in DC3 granulosa cells while Star promoter activity was increased in MSC-1 Sertoli cells after 24 h of treatment. This study contributes to the knowledge regarding the mechanism of EDS action in Leydig cells, and in other gonadal cell lineages, and brings new light regarding the rats and mice differential susceptibility to EDS effects, in addition to providing new avenues for experimental approaches to better understand Leydig cell function and dynamics in different rodent species.

STAT3-dependent long non-coding RNA Lncenc1 contributes to mouse ES cells pluripotency via stabilizing Klf4 mRNA.

Embryonic stem cells (ESCs) preserve the unique ability to differentiate into any somatic cell lineage while maintaining their self-renewal potential, relying on a complex interplay of extracellular signals regulating the expression/activity of pluripotency transcription factors and their targets. Leukemia inhibitory factor (LIF)-activated STAT3 drives ESCs' stemness by a number of mechanisms, including the transcriptional induction of pluripotency factors such as Klf4 and the maintenance of a stem-like epigenetic landscape. However, it is unknown if STAT3 directly controls stem-cell specific non-coding RNAs, crucial to balance pluripotency and differentiation. Applying a bioinformatic pipeline, here we identify Lncenc1 in mouse ESCs as an STAT3-dependent long non-coding RNA that supports pluripotency. Lncenc1 acts in the cytoplasm as a positive feedback regulator of the LIF-STAT3 axis by competing for the binding of microRNA-128 to the 3'UTR of the Klf4 core pluripotency factor mRNA, enhancing its expression. Our results unveil a novel non-coding RNA-based mechanism for LIF-STAT3-mediated pluripotency.

Epigenetic inheritance is unfaithful at intermediately methylated CpG sites

DNA methylation at the CpG dinucleotide is considered a stable epigenetic mark due to its presumed long-term inheritance through clonal expansion. Here, we perform high-throughput bisulfite sequencing on clonally derived somatic cell lines to quantitatively measure methylation inheritance at the nucleotide level. We find that although DNA methylation is generally faithfully maintained at hypo- and hypermethylated sites, this is not the case at intermediately methylated CpGs. Low fidelity intermediate methylation is interspersed throughout the genome and within genes with no or low transcriptional activity, and is not coordinately maintained between neighbouring sites. We determine that the probabilistic changes that occur at intermediately methylated sites are likely due to DNMT1 rather than DNMT3A/3B activity. The observed lack of clonal inheritance at intermediately methylated sites challenges the current epigenetic inheritance model and has direct implications for both the functional relevance and general interpretability of DNA methylation as a stable epigenetic mark.

Generation of a human haploid neural stem cell line for genome-wide genetic screening.

Haploid embryonic stem cells (haESCs) have been established in many species. Differentiated haploid cell line types in mammals are lacking due to spontaneous diploidization during differentiation that compromises lineage-specific screens. To derive human haploid neural stem cells (haNSCs) to carry out lineage-specific screens. Human haNSCs were differentiated from human extended haESCs with the help of Y27632 (ROCK signaling pathway inhibitor) and a series of cytokines to reduce diploidization. Neuronal differentiation of haNSCs was performed to examine their neural differentiation potency. Global gene expression analysis was con-ducted to compare haNSCs with diploid NSCs and haESCs. Fluorescence activated cell sorting was performed to assess the diploidization rate of extended haESCs and haNSCs. Genetic manipulation and screening were utilized to evaluate the significance of human haNSCs as genetic screening tools. Human haESCs in extended pluripotent culture medium showed more compact and smaller colonies, a higher efficiency in neural differentiation, a higher cell survival ratio and higher stability in haploidy maintenance. These characteristics effectively facilitated the derivation of human haNSCs. These human haNSCs can be generated by differentiation and maintain haploidy and multipotency to neurons and glia in the long term in vitro. After PiggyBac transfection, there were multiple insertion sites in the human haNSCs' genome, and the insertion sites were evenly spread across all chromosomes. In addition, after the cells were treated with manganese, we were able to generate a list of manganese-induced toxicity genes, demonstrating their utility as genetic screening tools. This is the first report of a generated human haploid somatic cell line with a complete genome, proliferative ability and neural differentiation potential that provides cell resources for recessive inheritance and drug targeted screening.

Creating Ex Situ Protected Bioreservoirs as a Powerful Strategy for the Reproductive Biotechnology-Mediated Rescue of Threatened Polish Livestock Breeds

The current article presents the state of the art of the creation of bioreservoirs of cryopreserved somatic and stem cell lines and cryopreserved or lyophilized germplasm-based resources of selected farm animal species. It also presents the agricultural, biological and biotechnological determinants, and showcases the importance of the National Research Institute of Animal Production (NRIAP) in Poland in this process. The aforementioned bioreservoirs serve as an innovative research tool used for cryogenically or lyophilizogenically assisted and species-specific ex situ conservation. The latter ensures, in the long term, not only restitution, but also perpetuation of sustainable biodiversity that underlies genotypic and phenotypic, intra- and inter-population variability within pure-breeding herds of the national conserved livestock breeds.

H3K9 methylation patterns during somatic embryogenic competence expression in tamarillo (Solanum betaceum Cav.)

The capacity to regenerate is intrinsic to plants and is the basis of natural asexual propagation and artificial cloning. Despite there are different ways of plant regeneration, they all require a change in cell fate and pluripotency reacquisition, in particular somatic embryogenesis. The mechanisms underlying somatic cell reprogramming for embryogenic competence acquisition, expression and maintenance remain not fully understood. These complex processes have been often associated with epigenetic markers, mainly DNA methylation, while little is known about the possible role of histone modifications. In the present study, the dynamics of global levels and distribution patterns of histone H3 methylation at lysine 9 (H3K9), a major repressive histone modification, were analyzed in somatic embryogenesis-induced cell lines with different embryogenic capacities and during somatic embryo initiation, in the woody species Solanum betaceum. Quantification of global H3K9 methylation showed similar levels in the three types of proliferating calli (embryogenic, long-term and non-embryogenic), kept in high sucrose and auxin-containing medium. Microscopic analyzes revealed heterogeneous cell organization and different cell types, particularly evident in embryogenic callus. The H3K9 dimethylation (H3K9me2) immunofluorescence signal was lower in nuclei of cells showing embryogenic-like and proliferating features, while labeling was higher in vacuolated, non-embryogenic cells with higher proliferation rates. By auxin removal, somatic embryo development was promoted in the embryogenic cell line. During the initiation of this process, increasing levels of global H3K9 methylation were found, together with increasing H3K9me2 immunofluorescence signals, especially in cells of the developing embryo. These results suggest that H3K9 methylation is involved in somatic embryo development, a developmental pathway in which this epigenetic mark could play a role in the gene transcription variation that is associated with embryogenic competence expression in S. betaceum. Altogether, these data provide new insights into the role of this epigenetic mark in somatic embryogenesis in trees, where scarce information is available.

Cohesin SMC1β promotes closed chromatin and controls TERRA expression at spermatocyte telomeres.

Previous data showed that meiotic cohesin SMC1β protects spermatocyte telomeres from damage. The underlying reason, however, remained unknown as the expressions of telomerase and shelterin components were normal in Smc1β -/- spermatocytes. Here. we report that SMC1β restricts expression of the long noncoding RNA TERRA (telomeric repeat containing RNA) in spermatocytes. In somatic cell lines increased TERRA was reported to cause telomere damage through altering telomere chromatin structure. In Smc1β -/- spermatocytes, we observed strongly increased levels of TERRA which accumulate on damaged chromosomal ends, where enhanced R-loop formation was found. This suggested a more open chromatin configuration near telomeres in Smc1β -/- spermatocytes, which was confirmed by ATAC-seq. Telomere-distal regions were not affected by the absence of SMC1β but RNA-seq revealed increased transcriptional activity in telomere-proximal regions. Thus, SMC1β promotes closed chromatin specifically near telomeres and limits TERRA expression in spermatocytes.

Large-Scale Polymorphism Analysis of Dog Leukocyte Antigen Class I and Class II Genes (DLA-88, DLA-12/88L and DLA-DRB1) and Comparison of the Haplotype Diversity between Breeds in Japan.

Polymorphisms of canine leukocyte antigen (DLA) class I (DLA-88 and DLA-12/88L) and class II (DLA-DRB1) genes are important for disease susceptibility studies, but information on the genetic diversity among dog breeds is still lacking. To better elucidate the polymorphism and genetic diversity between breeds, we genotyped DLA-88, DLA-12/88L, and DLA-DRB1 loci using 829 dogs of 59 breeds in Japan. Genotyping by Sanger sequencing identified 89, 43, and 61 alleles in DLA-88, DLA-12/88L, and DLA-DRB1 loci, respectively, and a total of 131 DLA-88-DLA-12/88L-DLA-DRB1 haplotypes (88-12/88L-DRB1) were detected more than once. Of the 829 dogs, 198 were homozygotes for one of the 52 different 88-12/88L-DRB1 haplotypes (homozygosity rate: 23.8%). Statistical modeling suggests that 90% of the DLA homozygotes or heterozygotes with one or other of the 52 different 88-12/88L-DRB1 haplotypes within somatic stem cell lines would benefit graft outcome after 88-12/88L-DRB1-matched transplantation. As previously reported for DLA class II haplotypes, the diversity of 88-12/88L-DRB1 haplotypes varied remarkably between breeds but was relatively conserved within most breeds. Therefore, the genetic characteristics of high DLA homozygosity rate and poor DLA diversity within a breed are useful for transplantation therapy, but they may affect biological fitness as homozygosity progresses.

Comparative analysis of the cardiomyocyte differentiation potential of induced pluripotent stem cells reprogrammed from human atrial or ventricular fibroblasts.

Background: We had shown that cardiomyocytes (CMs) were more efficiently differentiated from human induced pluripotent stem cells (hiPSCs) when the hiPSCs were reprogrammed from cardiac fibroblasts rather than dermal fibroblasts or blood mononuclear cells. Here, we continued to investigate the relationship between somatic-cell lineage and hiPSC-CM production by comparing the yield and functional properties of CMs differentiated from iPSCs reprogrammed from human atrial or ventricular cardiac fibroblasts (AiPSC or ViPSC, respectively). Methods: Atrial and ventricular heart tissues were obtained from the same patient, reprogrammed into AiPSCs or ViPSCs, and then differentiated into CMs (AiPSC-CMs or ViPSC-CMs, respectively) via established protocols. Results: The time-course of expression for pluripotency genes (OCT4, NANOG, and SOX2), the early mesodermal marker Brachyury, the cardiac mesodermal markers MESP1 and Gata4, and the cardiovascular progenitor-cell transcription factor NKX2.5 were broadly similar in AiPSC-CMs and ViPSC-CMs during the differentiation protocol. Flow-cytometry analyses of cardiac troponin T expression also indicated that purity of the two differentiated hiPSC-CM populations (AiPSC-CMs: 88.23% ± 4.69%, ViPSC-CMs: 90.25% ± 4.99%) was equivalent. While the field-potential durations were significantly longer in ViPSC-CMs than in AiPSC-CMs, measurements of action potential duration, beat period, spike amplitude, conduction velocity, and peak calcium-transient amplitude did not differ significantly between the two hiPSC-CM populations. Yet, our cardiac-origin iPSC-CM showed higher ADP and conduction velocity than previously reported iPSC-CM derived from non-cardiac tissues. Transcriptomic data comparing iPSC and iPSC-CMs showed similar gene expression profiles between AiPSC-CMs and ViPSC-CMs with significant differences when compared to iPSC-CM derived from other tissues. This analysis also pointed to several genes involved in electrophysiology processes responsible for the physiological differences observed between cardiac and non-cardiac-derived cardiomyocytes. Conclusion: AiPSC and ViPSC were differentiated into CMs with equal efficiency. Detected differences in electrophysiological properties, calcium handling activity, and transcription profiles between cardiac and non-cardiac derived cardiomyocytes demonstrated that 1) tissue of origin matters to generate a better-featured iPSC-CMs, 2) the sublocation within the cardiac tissue has marginal effects on the differentiation process.

Characterization of Oogonial Stem Cells in Adult Mouse Ovaries with Age and Comparison to In Silico Data on Human Ovarian Aging.

Many adult somatic stem cell lineages are comprised of subpopulations that differ in gene expression, mitotic activity, and differentiation status. In this study, we explored if cellular heterogeneity also exists within oogonial stem cells (OSCs), and how chronological aging impacts OSCs. In OSCs isolated from mouse ovaries by flow cytometry and established in culture, we identified subpopulations of OSCs that could be separated based on differential expression of stage-specific embryonic antigen 1 (SSEA1) and cluster of differentiation 61 (CD61). Levels of aldehyde dehydrogenase (ALDH) activity were inversely related to OSC differentiation, whereas commitment of OSCs to differentiation through transcriptional activation of stimulated by retinoic acid gene 8 was marked by a decline in ALDH activity and in SSEA1 expression. Analysis of OSCs freshly isolated from ovaries of mice between 3 and 20 months of age revealed that these subpopulations were present and persisted throughout adult life. However, expression of developmental pluripotency associated 3 (Dppa3), an epigenetic modifier that promotes OSC differentiation into oocytes, was lost as the mice transitioned from a time of reproductive compromise (10 months) to reproductive failure (15 months). Further analysis showed that OSCs from aged females could be established in culture, and that once established the cultured cells reactivated Dppa3 expression and the capacity for oogenesis. Analysis of single-nucleus RNA sequence data sets generated from ovaries of women in their 20s versus those in their late 40s to early 50s showed that the frequency of DPPA3-expressing cells decreased with advancing age, and this was paralleled by reduced expression of several key meiotic differentiation genes. These data support the existence of OSC subpopulations that differ in gene expression profiles and differentiation status. In addition, an age-related decrease in Dppa3/DPPA3 expression, which is conserved between mice and humans, may play a role in loss of the ability of OSCs to maintain oogenesis with age.

The cohesin modifier ESCO2 is stable during DNA replication

Cohesion between sister chromatids by the cohesin protein complex ensures accurate chromosome segregation and enables recombinational DNA repair. Sister chromatid cohesion is promoted by acetylation of the SMC3 subunit of cohesin by the ESCO2 acetyltransferase, inhibiting cohesin release from chromatin. The interaction of ESCO2 with the DNA replication machinery, in part through PCNA-interacting protein (PIP) motifs in ESCO2, is required for full cohesion establishment. Recent reports have suggested that Cul4-dependent degradation regulates the level of ESCO2 protein following replication. To follow up on these observations, we have characterized ESCO2 stability in Xenopus egg extracts, a cell-free system that recapitulates cohesion establishment in vitro. We found that ESCO2 was stable during DNA replication in this system. Indeed, further challenging the system by inducing DNA damage signaling or increasing the number of nuclei undergoing DNA replication had no significant impact on the stability of ESCO2. In transgenic somatic cell lines, we also did not see evidence of GFP-ESCO2 degradation during S phase of the cell cycle using both flow cytometry and live-cell imaging. We conclude that ESCO2 is stable during DNA replication in both embryonic and somatic cells.