Somatic Cell Lines Research Articles

At the Crossroads of Fate-Somatic Cell Lineage Specification in the Fetal Gonad.

The reproductive endocrine systems are vastly different between males and females. This sexual dimorphism of the endocrine milieu originates from sex-specific differentiation of the somatic cells in the gonads during fetal life. Most gonadal somatic cells arise from the adrenogonadal primordium. After separation of the adrenal and gonadal primordia, the gonadal somatic cells initiate sex-specific differentiation during gonadal sex determination with the specification of the supporting cell lineages: Sertoli cells in the testis vs granulosa cells in the ovary. The supporting cell lineages then facilitate the differentiation of the steroidogenic cell lineages, Leydig cells in the testis and theca cells in the ovary. Proper differentiation of these cell types defines the somatic cell environment that is essential for germ cell development, hormone production, and establishment of the reproductive tracts. Impairment of lineage specification and function of gonadal somatic cells can lead to disorders of sexual development (DSDs) in humans. Human DSDs and processes for gonadal development have been successfully modeled using genetically modified mouse models. In this review, we focus on the fate decision processes from the initial stage of formation of the adrenogonadal primordium in the embryo to the maintenance of the somatic cell identities in the gonads when they become fully differentiated in adulthood.

Transflammation: How Innate Immune Activation and Free Radicals Drive Nuclear Reprogramming.

Yamanaka and colleagues galvanized the field of stem cell biology and regenerative medicine by their generation of induced pluripotent stem cells. Evidence is emerging that activation of innate immune signaling is critical for efficient reprogramming to pluripotency and for the nuclear reprogramming occurring in transdifferentiation. Recent Advances: We have shown that innate immune signaling triggers a global change in the expression of epigenetic modifiers to enhance DNA accessibility. In this state of epigenetic plasticity, overexpression of lineage determination factors, and/or environmental cues and paracrine factors, can induce pluripotency, or can direct transdifferentiation to another somatic cell lineage. Accumulating evidence reveals that innate immune activation triggers the generation of reactive oxygen species and reactive nitrogen species, and that these free radicals are required for nuclear reprogramming to pluripotency or for transdifferentiation. We have discovered a limb of innate immune signaling that regulates DNA accessibility, in part, by the action of free radicals to induce post-translational modification of epigenetic modifiers. It is of scientific interest and clinical relevance to understand the mechanisms by which free radicals influence epigenetic plasticity, and how these mechanisms may be therapeutically modulated. Antioxid. Redox Signal. 00, 000-000.

Derivation of Haploid Neural Stem Cell Lines by Selection for a Pax6-GFP Reporter.

Haploid cells facilitate genetic screening of recessive mutations for a single set of chromosomes. Haploid embryonic stem cells (haESCs) have been achieved in several species and widely utilized in genetic screens. The fact that haESCs undergo substantial diploidization during differentiation has limited the screening to other haploid cell types. In this study, we report a method to establish haploid neural stem cells (haNSCs) by selection for a Pax6 reporter. We inserted a green fluorescence protein (GFP) marker gene by homologous recombination into the Pax6 locus of an haESC line. GFP-positive haploid cells could be sorted and further cultured in the NSC medium for more than 30 passages. The established haNSCs expressed neural lineage markers and could differentiate into neurons, oligodendroglia, and astrocytes. Our study shows the feasibility of deriving haploid proliferative somatic cell lines using a genetically encoded reporter that suggest a system for genetic screening of neural and retinal development.

Identification of molecular processes that differ among Scots pine somatic embryogenic cell lines leading to the development of normal or abnormal cotyledonary embryos

Several coniferous species can today be propagated through somatic embryogenesis, but for species belonging to the Pinus genus, there are still problems related to the small number of genotypes from which embryogenic cultures can be established and the low yield of high-quality cotyledonary embryos. In order to pinpoint molecular processes that might be disturbed during somatic embryogenesis in Scots pine, we have analyzed the expression pattern of selected transcripts during development of somatic embryos in a normal and an abnormal cell line of Scots pine. The selected transcripts have been previously shown to be differentially expressed during early zygotic embryogenesis in Scots pine (Merino et al. 2016). Based on the transcripts that accumulated differentially between the two cell lines, we conclude that the apical–basal polarization in early somatic embryos and the transition from the morphogenic phase to the maturation phase are disturbed in the abnormal cell line. A comparison between transcript accumulation during somatic and zygotic embryogenesis highlighted problems with the continuous embryo degeneration processes in embryogenic cultures of Scots pine. Transcripts that were highly abundant during the cleavage process in zygotic embryos showed different accumulation patterns during somatic embryogenesis in Scots pine compared to those in Norway spruce. Furthermore, altering the expression of these transcripts in embryogenic cultures of Norway spruce resulted in a changed proliferation pattern of the embryos so that they became more similar to proliferating somatic embryos of Scots pine. Taken together, our results provide a deeper understanding of the deviations in abnormal cell lines and indicate that embryogenic cultures of Scots pine are proliferating by a cleavage-like process.

Counterpoint: The Potential Harms of Human Gene Editing Using CRISPR-Cas9.

The anticipated benefits of human gene editing with the use of CRISPR (clustered interspaced short palindromic repeats)-Cas9 are both familiar and contested. First and foremost is the expectation of cures for blood disorders, lung diseases, cancers, and other maladies as clinician-scientists master various insertion, disruption, and deletion techniques. In addition to these potential therapeutic benefits, for some, there are the potential benefits of human enhancement as investigators learn to modify specific genetic traits in an effort to improve healthy individuals. Importantly, these proximate and distant potential benefits might be obtained through somatic cell or germ line gene modification. With germ line gene modification (which involves inserting, deleting, or replacing the DNA of human sperm, eggs, or embryos), there is the added potential benefit that changes made to the human genome (especially those aimed at correcting disease-causing and sometimes life-limiting genetic mutations) will be inherited by future generations. This would obviate the need for repeat somatic cell modifications from one generation to the next. The potential benefits of gene editing, however, are neither guaranteed nor risk-free. The potential harms include off-target changes (as might happen with the inactivation of essential genes), the inappropriate activation of cancer-causing genes, and the rearrangement of chromosomes. Additionally, there are the risks of on-target changes with unintended consequences, the creation of mosaics of altered and unaltered cells, and the introduction of changes that generate an immune response. In addition to these potential medical harms, there are also potential social harms. There is, for example, the risk that the introduction and eventual wide utilization of gene editing technology will exacerbate existing inequalities resulting in human rights abuses, a new wave of eugenics, increased discrimination and increased stigmatization. As such, the overarching risks with human gene editing by use of CRISPR-Cas9 are two-fold. First, there is the risk …

Mosaicism for GNAS methylation defects associated with pseudohypoparathyroidism type 1B arose in early post-zygotic phases

BackgroundPseudohypoparathyroidism type 1B (PHP1B; MIM#603233) is a rare imprinting disorder (ID), associated with the GNAS locus, characterized by parathyroid hormone (PTH) resistance in the absence of other endocrine or physical abnormalities. Sporadic PHP1B cases, with no known underlying primary genetic lesions, could represent true stochastic errors in early embryonic maintenance of methylation. Previous data confirmed the existence of different degrees of methylation defects associated with PHP1B and suggested the presence of mosaicism, a phenomenon already described in the context of other IDs.ResultsWith respect to mosaic conditions, the study of multiple tissues is a necessary approach; thus, we investigated somatic cell lines (peripheral blood and buccal epithelium and cells from the urine sediment) descending from different germ layers from 19 PHP patients (11 spor-PHP1B, 4 GNAS mutated PHP1A, and 4 PHP with no GNAS (epi)genetic defects) and 5 healthy controls. We identified 11 patients with epigenetic defects, further subdivided in groups with complete or partial methylation defects. The recurrence of specific patterns of partial methylation defects limited to specific CpGs was confirmed by checking methylation profiles of spor-PHP1B patients diagnosed in our lab (n = 56). Underlying primary genetic defects, such as uniparental disomy or deletion, potentially causative for the detected partial methylation were excluded in all samples.ConclusionsOur data showed no differences of methylation levels between organs and tissues from the same patient, so we concluded that the epimutation occurred in early post-zygotic phases and that the partial defects were mosaics. The number of patients with no detectable (epi)genetic GNAS defects was too small to exclude epimutations occurring in later post-zygotic phases, affecting only selected tissues different from blood, thus leading to underdiagnosis during routine molecular diagnosis. Finally, we found no correlation between methylation ratios, representing the proportion of epimutated cells, and the clinical presentation, further confirming the hypothesis of a threshold effect of the GNAS loss of imprinting leading to an “all-or-none” phenotype.

Copper(i) complexes with phosphine derived from sparfloxacin. Part III: multifaceted cell death and preliminary study of liposomal formulation of selected copper(i) complexes.

The cytotoxic effect of iodide or thiocyanate copper(i) complexes (1-PSf, 2-PSf, 3-PSf, 4-PSf) with phosphine derived from sparfloxacin (HSf) and 2,9-dimethyl-1,10-phenanthroline (dmp) or 2,2'-biquinoline (bq) as diimine auxiliary ligands was proved in vitro on somatic (MRC-5) and neoplastic (MCF7) human cell lines. Differences in mode of action were investigated in-depth for the selected dmp and bq complexes (1-PSf, 3-PSf, respectively) by elucidation of the following: (i) the efficiency to produce reactive oxygen species (ROS) in biological systems (cyclic voltammetry); (ii) their impact on mitochondrial membrane potential; (iii) potency for the activation of caspases 3 and 9; (iv) influence on the degree of DNA degradation (comet assay). It was concluded that the apoptosis of cancer cells is directly connected to the caspase-dependent mitochondrial pathway and supported by ROS production along with irreversible DNA fragmentation. Finally, it was demonstrated that the selected copper(i) complex encapsulated inside liposomes (1-PSf-L) exhibited enhanced accumulation inside cancer cells. This resulted in its higher cytotoxicity against cancer cells with therapeutic index of ca. 60. Increased selective accumulation in active neoplasm with simultaneous enhanced bioavailability and reduced systemic toxicity of liposomal formulation of copper(i) complexes can result in the development of new copper-based therapeutics and their successful implementation in anticancer chemotherapy.

Nickel(ii) inhibits the oxidation of DNA 5-methylcytosine in mammalian somatic cells and embryonic stem cells.

Nickel is found widely in the environment. It is an essential microelement but also toxic. However, nickel displays only weak genotoxicity and mutagenicity. Exploration of the epigenetic toxicity of nickel is extremely interesting. Iron(ii)- and 2-oxoglutarate-dependent Tet dioxygenases are a class of epigenetic enzymes that catalyze the oxidation of DNA 5-methylcytosine (5mC). Thus, they are critical for DNA demethylation and, importantly, are involved with nuclear reprogramming, embryonic development, and regulation of gene expression. Here, we demonstrated that nickel(ii) dramatically inhibits Tet proteins-mediated oxidation of DNA 5mC in cells ranging from somatic cell lines to embryonic stem cells, as manifested by the consistent observation of a significant decrease in 5-hydroxymethylcytosine, a critical intermediate resulting from the oxidation of 5mC. The inhibitory effects of nickel(ii) were concentration- and time-dependent. Using HEK293T cells overexpressing Tet proteins and ascorbic acid-stimulated Tet-proficient ES cells, we observed that nickel(ii) significantly reduced DNA demethylation at the global level. Interestingly, we also showed that nickel(ii) might affect the naïve or ground state of pluripotent embryonic stem cells. Here we show, for the first time, that nickel(ii) represses the oxidation of DNA 5mC and potentially alters the Tet proteins-regulated DNA methylation landscape in human cells. These findings provide new insights into the epigenetic toxicology of nickel.

Genetic screening and multipotency in rhesus monkey haploid neural progenitor cells.

Haploid embryonic stem cells (haESCs) have been extensively applied in forward and reverse genetic screening. However, a mammalian haploid somatic cell line is difficult to achieve because of spontaneous diploidization in differentiation. As a non-human primate species, monkeys are widely used in basic and pre-clinical research in which haploid cells are restricted to ESCs. Here, we report that rhesus monkey haESCs in an optimized culture medium show naïve-state pluripotency and stable haploidy. This model facilitated the derivation of haploid neural progenitor cells (haNPCs), which maintained haploidy and differentiation potential into neurons and glia for a long period in vitro High-throughput trapping mutations can be efficiently introduced into haNPCs via piggyBac transposons. This system proves useful when identifying gene targets of neural toxicants via a proof-of-concept experiment. Using CRISPR/Cas9 editing, we confirmed that B4GALT6, from the candidate gene list, is a resistance gene of A803467 (a tetrodotoxin-like toxicant). This model is the first non-human primate haploid somatic cell line with proliferative ability, multipotency and an intact genome, thus providing a cellular resource for recessive genetic and potential drug screening.

Derivation of Human Induced Pluripotent Stem Cell (iPSC) Lines and Mechanism of Pluripotency: Historical Perspective and Recent Advances.

Derivation of human embryonic stem cell (hES) lines in 1998 was not only a major technological breakthrough in the field of regenerative medicine; it also triggered a passionate debate about the ethical issues associated with the utilization of human embryos for derivation of hESC lines. Successful derivation of induced pluripotent stem cell (iPS) lines from human somatic cells with defined reprogramming factors by Shinya Yamanaka`s group in 2007 was another breakthrough that generated enormous excitement and hope for the development of donor-specific personalized cell replacement therapies (CRT) without the ethical dilemma associated with it. As we approach twentieth anniversary of derivation of hESC lines and the tenth anniversary of isolation of donor-specific iPSC lines, this manuscript summarizes the key advances in pluripotent stem cell (PSC) research field that led to derivation of human iPSC lines, different methodologies for derivation iPSC lines and characterization of the mechanism of reprogramming. We will also review progress towards generating donor-specific somatic cell lineages from iPSC lines, especially the functional immune cell lineages, and progress towards advancing these findings to the clinic. Finally, we will discuss the challenges, such as genome instability and inherent immunogenicity of hPSC lines that need to be addressed to develop safe and effective iPSC-based CRT.

Inherent Immunogenicity or Lack Thereof of Pluripotent Stem Cells: Implications for Cell Replacement Therapy.

Donor-specific induced pluripotent stem cells (iPSCs) offer opportunities for personalized cell replacement therapeutic approaches due to their unlimited self-renewal potential and ability to differentiate into different somatic cells. A significant progress has been made toward generating iPSC lines that are free of integrating viral vectors, development of xeno-free culture conditions, and differentiation of pluripotent stem cells (PSCs) into functional somatic cell lineages. Since donor-specific iPSC lines are genetically identical to the individual, they are expected to be immunologically matched and these iPSC lines and their cellular derivatives are not expected to be immunologically rejected. However, studies in mouse models, utilizing rejection of teratomas as a model, have claimed that syngenic iPSC lines, especially the iPSC lines derived with integrating viral vectors, could be inherently immunogenic. This manuscript reviews current understanding of inherent immunogenicity of PSC lines, especially that of the human iPSC lines and their cellular derivatives, and strategies to overcome it.

Developmental Restriction of Retrotransposition Activated in Arabidopsis by Environmental Stress

Retrotransposons (RTs) can rapidly increase in copy number due to periodic bursts of transposition. Such bursts are mutagenic and thus potentially deleterious. However, certain transposition-induced gain-of-function or regulatory mutations may be of selective advantage. How an optimal balance between these opposing effects arises is not well characterized. Here, we studied transposition bursts of a heat-activated retrotransposon family in Arabidopsis. We recorded a high inter and intraplant variation in the number and chromosomal position of new insertions, which usually did not affect plant fertility and were equally well transmitted through male and female gametes, even though 90% of them were within active genes. We found that a highly heterogeneous distribution of these new retroelement copies result from a combination of two mechanisms, of which the first prevents multiple transposition bursts in a given somatic cell lineage that later contributes to differentiation of gametes, and the second restricts the regulatory influence of new insertions toward neighboring chromosomal DNA. As a whole, such regulatory characteristics of this family of RTs ensure its rapid but stepwise accumulation in plant populations experiencing transposition bursts accompanied by high diversity of chromosomal sites harboring new RT insertions.

Cell cycle arrest in plants: what distinguishes quiescence, dormancy and differentiated G1?

Quiescence is a fundamental feature of plant life, which enables plasticity, renewal and fidelity of the somatic cell line. Cellular quiescence is defined by arrest in a particular phase of the cell cycle, typically G1 or G2; however, the regulation of quiescence and proliferation can also be considered across wider scales in space and time. As such, quiescence is a defining feature of plant development and phenology, from meristematic stem cell progenitors to terminally differentiated cells, as well as dormant or suppressed seeds and buds. While the physiology of each of these states differs considerably, each is referred to as 'cell cycle arrest' or 'G1 arrest'. Here the physiology and molecular regulation of (1) meristematic quiescence, (2) dormancy and (3) terminal differentiation (cell cycle exit) are considered in order to determine whether and how the molecular decisions guiding these nuclear states are distinct. A brief overview of the canonical cell cycle regulators is provided, and the genetic and genomic, as well as physiological, evidence is considered regarding two primary questions: (1) Are the canonical cell cycle regulators superior or subordinate in the regulation of quiescence? (2) Are these three modes of quiescence governed by distinct molecular controls? Meristematic quiescence, dormancy and terminal differentiation are each predominantly characterized by G1 arrest but regulated distinctly, at a level largely superior to the canonical cell cycle. Meristematic quiescence is intrinsically linked to non-cell-autonomous regulation of meristem cell identity, and particularly through the influence of ubiquitin-dependent proteolysis, in partnership with reactive oxygen species, abscisic acid and auxin. The regulation of terminal differentiation shares analogous features with meristematic quiescence, albeit with specific activators and a greater role for cytokinin signalling. Dormancy meanwhile appears to be regulated at the level of chromatin accessibility, by Polycomb group-type histone modifications of particular dormancy genes.

Carbohydrate-mediated responses during zygotic and early somatic embryogenesis in the endangered conifer, Araucaria angustifolia.

Three zygotic developmental stages and two somatic Araucaria angustifolia cell lines with contrasting embryogenic potential were analyzed to identify the carbohydrate-mediated responses associated with embryo formation. Using a comparison between zygotic and somatic embryogenesis systems, the non-structural carbohydrate content, cell wall sugar composition and expression of genes involved in sugar sensing were analyzed, and a network analysis was used to identify coordinated features during embryogenesis. We observed that carbohydrate-mediated responses occur mainly during the early stages of zygotic embryo formation, and that during seed development there are coordinated changes that affect the development of the different structures (embryo and megagametophyte). Furthermore, sucrose and starch accumulation were associated with the responsiveness of the cell lines. This study sheds light on how carbohydrate metabolism is influenced during zygotic and somatic embryogenesis in the endangered conifer species, A. angustifolia.

Gene editing tools: state-of-the-art and the road ahead for the model and non-model fishes.

Advancements in the DNA sequencing technologies and computational biology have revolutionized genome/transcriptome sequencing of non-model fishes at an affordable cost. This has led to a paradigm shift with regard to our heightened understandings of structure-functional relationships of genes at a global level, from model animals/fishes to non-model large animals/fishes. Whole genome/transcriptome sequencing technologies were supplemented with the series of discoveries in gene editing tools, which are being used to modify genes at pre-determined positions using programmable nucleases to explore their respective in vivo functions. For a long time, targeted gene disruption experiments were mostly restricted to embryonic stem cells, advances in gene editing technologies such as zinc finger nuclease, transcriptional activator-like effector nucleases and CRISPR (clustered regulatory interspaced short palindromic repeats)/CRISPR-associated nucleases have facilitated targeted genetic modifications beyond stem cells to a wide range of somatic cell lines across species from laboratory animals to farmed animals/fishes. In this review, we discuss use of different gene editing tools and the strategic implications in fish species for basic and applied biology research.

Global gene expression profiling and senescence biomarker analysis of hESC exposed to H2O2 induced non-cytotoxic oxidative stress

BackgroundHuman embryonic stem cells (hESCs) potentially offer new routes to study, on the basis of the Developmental Origins of Health and Disease (DOHaD) concept, how the maternal environment during pregnancy influences the offspring’s health and can predispose to chronic disease in later life. Reactive oxygen species (ROS), antioxidant defences and cellular redox status play a key function in gene expression regulation and are involved in diabetes and metabolic syndromes as in ageing.MethodsWe have, therefore, designed an in vitro cell model of oxidative stress by exposing hESCs to hydrogen peroxide (H2O2) during 72 h, in order to resemble the period of preimplantation embryonic development.ResultsWe have analysed the global gene expression profiles of hESCs (HUES3) exposed to non-cytotoxic H2O2 concentrations, using Illumina microarray HT-12 v4, and we found the differential expression of 569 upregulated and 485 downregulated genes. The most affected gene ontology categories were those related with RNA processing and splicing, oxidation reduction and sterol metabolic processes. We compared our findings with a published RNA-seq profiling dataset of human embryos developed in vitro, thereupon exposed to oxidative stress, and we observed that one of the common downregulated genes between this publication and our data, NEDD1, is involved in centrosome structure and function.ConclusionsTherefore, we assessed the presence of supernumerary centrosomes and showed that the percentage of cells with more than two centrosomes increased acutely with H2O2 treatment in hESCs (HUES3 and 7) and in a control somatic cell line (Hs27), inducing a premature entry into senescence.

Gene editing in birds takes flight

The application of gene editing (GE) technology to create precise changes to the genome of bird species will provide new and exciting opportunities for the biomedical, agricultural and biotechnology industries, as well as providing new approaches for producing research models. Recent advances in modifying both the somatic and germ cell lineages in chicken indicate that this species, and conceivably soon other avian species, has joined a growing number of model organisms in the gene editing revolution.

Environmental influences on ovarian dysgenesis - developmental windows sensitive to chemical exposures.

A woman's reproductive health and ability to have children directly affect numerous aspects of her life, from personal well-being and socioeconomic standing, to morbidity and lifespan. In turn, reproductive health depends on the development of correctly functioning ovaries, a process that starts early during fetal life. Early disruption to ovarian programming can have long-lasting consequences, potentially manifesting as disease much later in adulthood. A growing body of evidence suggests that exposure to chemicals early in life, including endocrine-disrupting chemicals, can cause a range of disorders later in life, such as those described in the ovarian dysgenesis syndrome hypothesis. In this Review, we discuss four specific time windows during which the ovary is particularly sensitive to disruption by exogenous insults: gonadal sex determination, meiotic division, follicle assembly and the first wave of follicle recruitment. To date, most evidence points towards the germ cell lineage being the most vulnerable to chemical exposure, particularly meiotic division and follicle assembly. Environmental chemicals and pharmaceuticals, such as bisphenols or mild analgesics (including paracetamol), can also affect the somatic cell lineages. This Review summarizes our current knowledge pertaining to environmental chemicals and pharmaceuticals, and their potential contributions to the development of ovarian dysgenesis syndrome. We also highlight knowledge gaps that need addressing to safeguard female reproductive health.

Hedgehog signaling establishes precursors for germline stem cell niches by regulating cell adhesion.

Stem cells require different types of supporting cells, or niches, to control stem cell maintenance and differentiation. However, little is known about how those niches are formed. We report that in the development of the Drosophila melanogaster ovary, the Hedgehog (Hh) gradient sets differential cell affinity for somatic gonadal precursors to specify stromal intermingled cells, which contributes to both germline stem cell maintenance and differentiation niches in the adult. We also report that Traffic Jam (an orthologue of a large Maf transcription factor in mammals) is a novel transcriptional target of Hh signaling to control cell-cell adhesion by negative regulation of E-cadherin expression. Our results demonstrate the role of Hh signaling in niche establishment by segregating somatic cell lineages for differentiation.

Discovery of novel determinants of endothelial lineage using chimeric heterokaryons.

We wish to identify determinants of endothelial lineage. Murine embryonic stem cells (mESC) were fused with human endothelial cells in stable, non-dividing, heterokaryons. Using RNA-seq, it is possible to discriminate between human and mouse transcripts in these chimeric heterokaryons. We observed a temporal pattern of gene expression in the ESCs of the heterokaryons that recapitulated ontogeny, with early mesodermal factors being expressed before mature endothelial genes. A set of transcriptional factors not known to be involved in endothelial development was upregulated, one of which was POU class 3 homeobox 2 (Pou3f2). We confirmed its importance in differentiation to endothelial lineage via loss- and gain-of-function (LOF and GOF). Its role in vascular development was validated in zebrafish embryos using morpholino oligonucleotides. These studies provide a systematic and mechanistic approach for identifying key regulators in directed differentiation of pluripotent stem cells to somatic cell lineages.