A comprehensive review: synergizing stem cell and embryonic development knowledge in mouse and human integrated stem cell-based embryo models.

Mammalian stem cell-based embryo models have emerged as innovative tools for investigating early embryogenesis in both mice and primates. They not only reduce the need for sacrificing mice but also overcome ethical limitations associated with human embryo research. Furthermore, they provide a platform to address scientific questions that are otherwise challenging to explore in vivo. The usefulness of a stem cell-based embryo model depends on its fidelity in replicating development, efficiency and reproducibility; all essential for addressing biological queries in a quantitative manner, enabling statistical analysis. Achieving such fidelity and efficiency requires robust systems that demand extensive optimization efforts. A profound understanding of pre- and post-implantation development, cellular plasticity, lineage specification, and existing models is imperative for making informed decisions in constructing these models. This review aims to highlight essential differences in embryo development and stem cell biology between mice and humans, assess how these variances influence the formation of partially and fully integrated stem cell models, and identify critical challenges in the field.

Efficient generation of ETX embryoids that recapitulate the entire window of murine egg cylinder development.

The murine embryonic-trophoblast-extra-embryonic endoderm (ETX) model is an integrated stem cell-based model to study early postimplantation development. It is based on the self-assembly potential of embryonic, trophoblast, and hypoblast/primitive/visceral endoderm-type stem cell lines (ESC, TSC, and XEN, respectively) to arrange into postimplantation egg cylinder-like embryoids. Here, we provide an optimized method for reliable and efficient generation of ETX embryoids that develop into late gastrulation in static culture conditions. It is based on transgenic Gata6-overproducing ESCs and modified assembly and culture conditions. Using this method, up to 43% of assembled ETX embryoids exhibited a correct spatial distribution of the three stem cell derivatives at day 4 of culture. Of those, 40% progressed into ETX embryoids that both transcriptionally and morphologically faithfully mimicked in vivo postimplantation mouse development between E5.5 and E7.5. The ETX model system offers the opportunity to study the murine postimplantation egg cylinder stages and could serve as a source of various cell lineage precursors.

Fungi attacking historic wood of Fort Conger and the Peary Huts in the High Arctic.

Historic wooden structures in Polar Regions are being adversely affected by decay fungi and a warming climate will likely accelerate degradation. Fort Conger and the Peary Huts at Lady Franklin Bay in northern Ellesmere Island are important international heritage sites associated with early exploration in the High Arctic. Fort Conger, built by Adolphus Greely and expedition members during the First International Polar Year in 1881, was dismantled and used by Robert Peary and his expedition crew in the early 1900's to build several smaller shelters. These historic structures remain at the site but are deteriorating. This investigation examines the fungi associated with wood decay in the historic woods. Soft rot was observed in all 125 wood samples obtained from the site. The major taxa found associated with the decayed wood were Coniochaeta (18%), Phoma (13%) Cadophora (12%), Graphium (9%), and Penicillium (9%) as well as many other Ascomycota that are known to cause soft rot in wood. Micromorphological observations using scanning electron microscopy of historic wooden timbers that were in ground contact revealed advanced stages of type I soft rot. No wood destroying Basidiomycota were found. Identification of the fungi associated with decay in these historic woods is a first step to better understand the unusual decomposition processes underway in this extreme environment and will aid future research to help control decay and preserve this important cultural heritage.

REX1 is the critical target of RNF12 in imprinted X chromosome inactivation in mice.

In mice, imprinted X chromosome inactivation (iXCI) of the paternal X in the pre-implantation embryo and extraembryonic tissues is followed by X reactivation in the inner cell mass (ICM) of the blastocyst to facilitate initiation of random XCI (rXCI) in all embryonic tissues. RNF12 is an E3 ubiquitin ligase that plays a key role in XCI. RNF12 targets pluripotency protein REX1 for degradation to initiate rXCI in embryonic stem cells (ESCs) and loss of the maternal copy of Rnf12 leads to embryonic lethality due to iXCI failure. Here, we show that loss of Rex1 rescues the rXCI phenotype observed in Rnf12-/- ESCs, and that REX1 is the prime target of RNF12 in ESCs. Genetic ablation of Rex1 in Rnf12-/- mice rescues the Rnf12-/- iXCI phenotype, and results in viable and fertile Rnf12-/-:Rex1-/- female mice displaying normal iXCI and rXCI. Our results show that REX1 is the critical target of RNF12 in XCI.

Allele-specific control of replication timing and genome organization during development.

DNA replication occurs in a defined temporal order known as the replication-timing (RT) program. RT is regulated during development in discrete chromosomal units, coordinated with transcriptional activity and 3D genome organization. Here, we derived distinct cell types from F1 hybrid musculus × castaneus mouse crosses and exploited the high single-nucleotide polymorphism (SNP) density to characterize allelic differences in RT (Repli-seq), genome organization (Hi-C and promoter-capture Hi-C), gene expression (total nuclear RNA-seq), and chromatin accessibility (ATAC-seq). We also present HARP, a new computational tool for sorting SNPs in phased genomes to efficiently measure allele-specific genome-wide data. Analysis of six different hybrid mESC clones with different genomes (C57BL/6, 129/sv, and CAST/Ei), parental configurations, and gender revealed significant RT asynchrony between alleles across ∼12% of the autosomal genome linked to subspecies genomes but not to parental origin, growth conditions, or gender. RT asynchrony in mESCs strongly correlated with changes in Hi-C compartments between alleles but not as strongly with SNP density, gene expression, imprinting, or chromatin accessibility. We then tracked mESC RT asynchronous regions during development by analyzing differentiated cell types, including extraembryonic endoderm stem (XEN) cells, four male and female primary mouse embryonic fibroblasts (MEFs), and neural precursor cells (NPCs) differentiated in vitro from mESCs with opposite parental configurations. We found that RT asynchrony and allelic discordance in Hi-C compartments seen in mESCs were largely lost in all differentiated cell types, accompanied by novel sites of allelic asynchrony at a considerably smaller proportion of the genome, suggesting that genome organization of homologs converges to similar folding patterns during cell fate commitment.

An expandable embryonic stem cell-derived Purkinje neuron progenitor population that exhibits in vivo maturation in the adult mouse cerebellum.

The directed differentiation of patient-derived induced pluripotent stem cells into cell-type specific neurons has inspired the development of therapeutic discovery for neurodegenerative diseases. Many forms of ataxia result from degeneration of cerebellar Purkinje cells, but thus far it has not been possible to efficiently generate Purkinje neuron (PN) progenitors from human or mouse pluripotent stem cells, let alone to develop a methodology for in vivo transplantation in the adult cerebellum. Here, we present a protocol to obtain an expandable population of cerebellar neuron progenitors from mouse embryonic stem cells. Our protocol is characterized by applying factors that promote proliferation of cerebellar progenitors. Cerebellar progenitors isolated in culture from cell aggregates contained a stable subpopulation of PN progenitors that could be expanded for up to 6 passages. When transplanted into the adult cerebellum of either wild-type mice or a strain lacking Purkinje cells (L7cre-ERCC1 knockout), GFP-labeled progenitors differentiated in vivo to establish a population of calbindin-positive cells in the molecular layer with dendritic trees typical of mature PNs. We conclude that this protocol may be useful for the generation and maturation of PNs, highlighting the potential for development of a regenerative medicine approach to the treatment of cerebellar neurodegenerative diseases.

Ancient DNA analysis identifies marine mollusc shells as new metagenomic archives of the past.

Marine mollusc shells enclose a wealth of information on coastal organisms and their environment. Their life history traits as well as (palaeo-) environmental conditions, including temperature, food availability, salinity and pollution, can be traced through the analysis of their shell (micro-) structure and biogeochemical composition. Adding to this list, the DNA entrapped in shell carbonate biominerals potentially offers a novel and complementary proxy both for reconstructing palaeoenvironments and tracking mollusc evolutionary trajectories. Here, we assess this potential by applying DNA extraction, high-throughput shotgun DNA sequencing and metagenomic analyses to marine mollusc shells spanning the last ~7,000 years. We report successful DNA extraction from shells, including a variety of ancient specimens, and find that DNA recovery is highly dependent on their biomineral structure, carbonate layer preservation and disease state. We demonstrate positive taxonomic identification of mollusc species using a combination of mitochondrial DNA genomes, barcodes, genome-scale data and metagenomic approaches. We also find shell biominerals to contain a diversity of microbial DNA from the marine environment. Finally, we reconstruct genomic sequences of organisms closely related to the Vibrio tapetis bacteria from Manila clam shells previously diagnosed with Brown Ring Disease. Our results reveal marine mollusc shells as novel genetic archives of the past, which opens new perspectives in ancient DNA research, with the potential to reconstruct the evolutionary history of molluscs, microbial communities and pathogens in the face of environmental changes. Other future applications include conservation of endangered mollusc species and aquaculture management.

FGF treatment of host embryos injected with ES cells increases rates of chimaerism.

In spite of the emergence of genome editing tools, ES cell mediated transgenesis remains the most controllable way of creating genetically modified animals. Although tetraploid (4N) complementation of 4N host embryos and ES cells, is the only method guaranteeing that offspring are entirely ES cell derived, this technique is challenging, not always successful and difficult to implement in some laboratory settings. The current study shows that pretreatment of host blastocysts with FGF4 prior to ES cell injection can provide an alternative method for the generation of animals displaying high rates of chimaerism. Chimaerism assessment in E11 fetuses and born pups shows that a large percentage of resulting conceptuses show a high ES cell contribution from implantation onwards and that developing pups do not necessitate c-section for delivery.

Characterization of Histone Modifications Associated with Inactive X-Chromosome in Trophoblast Stem Cells, eXtra-Embryonic Endoderm Cells and in In Vitro Derived Undifferentiated and Differentiated Epiblast Like Stem Cells.

In mouse, X-chromosome inactivation (XCI) can either be imprinted or random. Imprinted XCI (iXCI) is considered unstable and depending on continuous Xist expression, whereas random XCI (rXCI) is stably maintained even in the absence of Xist. Here we have systematically examined epigenetic modifications associated with the inactive X-chromosome (Xi) in Trophoblast Stem cells, eXtra-Embryonic Endoderm Cells, undifferentiated and differentiated Epiblast Like Stem Cells in order to understand intrinsic differences in epigenetic mechanisms involved in silencing of the inactive X-chromosome in lineages presenting iXCI and rXCI. Whereas euchromatic histone modifications are predominantly lost from the Xi territory in all cell types, the accumulation of heterochromatic modifications diverges in between the analysed cell lineages. Particularly, only the Xi of multipotent Trophoblast (iXCI) and Epiblast stem cells (rXCI) display a visible accumulation of Polycomb Repressive Complexes (PRCs), in contrast to the Xi in differentiated Epiblast Like Stem Cells and eXtra-embryonic Endoderm cells. Despite this, the histone modifications catalysed by PRCs, ubH2AK119 and H3K27me3, remain the best heterochromatic markers for the Xi in all assessed lineages. Heterochromatic chromatin modifications associated with the Xi are a reflection of the epigenetic landscape of the entire genome of the assessed cell regardless whether XCI is imprinted or random.

Different flavors of X-chromosome inactivation in mammals.

Dosage compensation of X-linked gene products between the sexes in therians has culminated in the inactivation of one of the two X chromosomes in female cells. Over the years, the mouse has been the preferred animal model to study this X-chromosome inactivation (XCI) process in placental mammals (eutherians). Similar to the imprinted inactivation of the paternally inherited X chromosome (Xp) in marsupials (methatherians), the Xp is inactivated during early mouse development. In this eutherian model, cell derivatives of the primitive endoderm (PE) and trophectoderm (TE) will continue to display this imprinted form of XCI. Cells developing from the mouse epiblast will reactivate the Xp, and subsequently initiate XCI of either the Xp or the maternally inherited Xm, in a random manner. Examination of XCI in other eutherians and in metatherians, however, indicates clear differences in the form and timing of XCI. This review highlights and discusses imprinted and random XCI from such a comparative viewpoint.

Nurse-led educative consultation setting personalized tertiary prevention goals after cardiovascular rehabilitation: evaluation of patient satisfaction and long-term effects.

This study aimed to evaluate the perception and long-term effects of an educative consultation performed before cardiac rehabilitation discharge. The patient and the referring nurse summed up the educative interventions, and filled a personalized form summarizing tertiary prevention goals. Fifty patients were contacted by mail at 11 ± 1 months, and called at 4.2 ± 0.2 years after discharge, to evaluate their satisfaction and assess cardiovascular risk factors (CVRF) control. Mail response rate was 82%, and 90% of patients believed that it had encouraged them to adopt a healthier lifestyle. Almost half the number of patients declared that they considered the nurse intervention as positive. Telephone response rate was 54%. Most long-term effects were better than usually reported in the field of multidisciplinary secondary prevention of CVRF. Patients felt that this educational action was positive, even though highlighting this role of nurses seems necessary. Additional controlled trials are needed to provide rigorous validation of this strategy.

Expression profiles of cohesins, shugoshins and spindle assembly checkpoint genes in rhesus macaque oocytes predict their susceptibility for aneuploidy during embryonic development.

High frequencies of chromosomal anomalies are reported in human and non-human primate in vitro-produced preimplantation embryos. It is unclear why certain embryos develop aneuploidies while others remain euploid. A differential susceptibility to aneuploidy is most likely a consequence of events that occur before oocyte collection. One hypothesis is that the relative transcript levels of cohesins, shugoshins and spindle assembly checkpoint genes are correlated with the occurrence of chromosomal anomalies. Transcript levels of these genes were quantified in individual oocytes that were either mature (group 1, low aneuploidy rate) or immature (group 2, high aneuploidy rate) at retrieval, utilizing TaqMan-based real-time PCR. The transcript level in each oocyte was categorized as absent, below the median or above the median in order to conduct comparisons. Statistically significant differences were observed between group 1 and group 2 for SGOL1 and BUB1. There were more oocytes with SGOL1 expression levels above the median in group 1, while oocytes lacking BUB1 were only observed in group 1. These findings suggest that higher SGOL1 levels in group 1 oocytes could better protect against a premature separation of sister chromatids than in embryos derived from group 2 oocytes. The absence of BUB1 transcripts in group 1 was frequently associated with reduced expression of either mitotic cohesins or shugoshins. We hypothesize that ablation of BUB1 could induce mitotic arrest in oocytes that fail to express a complete complement of cohesins and shugoshins, thereby reducing the number of developing aneuploid preimplantation embryos.

Fibromyalgia and related conditions: electromyogram profile during isometric muscle contraction.

OBJECTIVES: To evaluate electromyogram (EMG) profiles in patients with three related conditions: fibromyalgia, chronic fatigue syndrome, and depression. METHODS: We studied 44 healthy volunteers, 22 patients with fibromyalgia, 11 patients with chronic fatigue syndrome, and 10 patients admitted for depression. The trapezius electromyogram was recorded during maximally sustained, bilateral, 90 degrees abduction of the shoulders. EMG signal frequency and amplitude were measured throughout the test. RESULTS: In the fibromyalgia group, isometric contraction duration was significantly shorter than in the other two patient groups (P<0.001) and the EMG frequency and amplitude pattern indicated premature discontinuation of the muscle contraction. Findings in the chronic fatigue patients were similar to those in the healthy controls. The patients with depression had a distinctive EMG profile characterized by excessive initial motor-unit recruitment with a shift in the frequency spectrum. CONCLUSIONS: Fibromyalgia was associated with a specific EMG pattern indicating premature discontinuation of the muscle contraction. Therefore, maximal voluntary muscle contraction tests may be of limited value for assessing function in fibromyalgia patients. Chronic fatigue syndrome patients had similar EMG findings to those in the healthy controls. The EMG alterations in the patients with depression were consistent with manifestations of psychomotor retardation.

Incidence of chromosomal mosaicism in morphologically normal nonhuman primate preimplantation embryos.

OBJECTIVE: To establish the exact rates of chromosomal mosaicism in morphologically normal rhesus macaque embryos by determining the chromosomal complement of all blastomeres. DESIGN: Retrospective rhesus monkey IVF study. SETTING: Academic laboratory and primate research center. PATIENT(S): Young fertile rhesus macaque females. INTERVENTION(S): Morphologically normal in vitro-produced rhesus macaque embryos were dissociated and cytogenetically assessed using a five-color fluorescent in situ hybridization assay developed for rhesus macaque chromosomes homologous to human chromosomes 13, 16, 18, X, and Y. MAIN OUTCOME MEASURE(S): The incidence and extent of chromosomal mosaicism in rhesus macaque preimplantation embryos. RESULT(S): Seventy-seven preimplantation embryos, displaying normal morphology and development, from 17 young rhesus macaque females were analyzed. Overall, 39 embryos (50.6%) were normal, 14 embryos (18.2%) were completely abnormal, and 24 embryos (31.2%) were mosaic. Of the 226 blastomeres analyzed in the mosaic group, 110 blastomeres (48.7%) were normal. CONCLUSION(S): The observed rate of mosaicism in good-quality rhesus embryos resembles previously documented frequencies in poor-quality human preimplantation embryos. A high incidence of mosaicism may limit the diagnostic accuracy of preimplantation genetic diagnosis.

Epigenetics: definition, mechanisms and clinical perspective.

A vast array of successive epigenetic modifications ensures the creation of a healthy individual. Crucial epigenetic reprogramming events occur during germ cell development and early embryogenesis in mammals. As highlighted by the large offspring syndrome with in vitro conceived ovine and bovine animals, any disturbance during germ cell development or early embryogenesis has the potential to alter epigenetic reprogramming. Therefore the complete array of human assisted reproductive technology (ART), starting from ovarian hormonal stimulation to embryo uterine transfer, could have a profound impact on the epigenetic state of human in vitro produced individuals. Although some investigators have suggested an increased incidence of epigenetic abnormalities in in vitro conceived children, other researchers have refuted these allegations. To date, multiple reasons can be hypothesized why irrefutable epigenetic alterations as a result of ART have not been demonstrated yet.

Rhesus macaque embryos derived from MI oocytes maturing after retrieval display high rates of chromosomal anomalies.

BACKGROUND: Rhesus macaque and human preimplantation embryos display similar rates of chromosomal abnormalities. The aim of this study was to determine whether embryos developing from MI oocytes that mature post-retrieval display more chromosomal anomalies than those embryos that are generated from oocytes that are at MII at the time of retrieval. METHODS: Rhesus macaque oocytes were obtained after hormonal ovarian stimulation. Immediately after retrieval, the oocytes were classified according to their maturational status. Following in vitro fertilization, Day 3 embryos with good morphology and development derived from oocytes maturing post-retrieval and those from oocytes that were mature at the time of retrieval were cytogenetically assessed using a five-color fluorescent in situ fluorescent hybridization assay developed for rhesus macaque chromosomes homologous to human chromosomes 13, 16, 18, X and Y. RESULTS: Blastomeres from 53 embryos were analyzed. Of the 27 embryos that developed from oocytes that were mature at collection, 18 embryos were chromosomally normal (66.7%), while from the 26 embryos that developed from oocytes that matured post-retrieval, only 9 embryos were chromosomally normal (34.6%). CONCLUSIONS: These results indicate that embryos developing from oocytes maturing post-retrieval display high rates of chromosomal abnormalities and have therefore a reduced developmental competence. As a result, the clinical relevance of using immature oocytes that are retrieved after stimulated cycles in human IVF warrants further investigation.

Chromosomal instability in rhesus macaque preimplantation embryos.

OBJECTIVE: To establish a relevant animal model to systematically investigate chromosomal instability in human oocytes and preimplantation embryos. DESIGN: Prospective rhesus monkey IVF study. SETTING: Academic laboratory, Oregon National Primate Research Center and Caribbean Primate Research Center. ANIMAL(S): Young rhesus macaque females. INTERVENTION(S): In vitro produced entire rhesus macaque preimplantation embryos were cytogenetically assessed using a five-color fluorescent in situ hybridization assay developed for rhesus macaque chromosomes homologous to human chromosomes 13, 16, 18, X, and Y, using human bacterial artificial chromosome probes. MAIN OUTCOME MEASURE(S): Chromosomal abnormality rates in preimplantation embryos from young rhesus macaque females were established. RESULT(S): Fifty preimplantation embryos, displaying good morphology and normal development, were analyzed from 11 young rhesus macaque females. Overall, 27 embryos (54%) were normal, 11 embryos (22%) mosaic, 3 embryos (6%) chaotic, 2 embryos (4%) aneuploid, 3 embryos (6%) haploid, and 4 embryos (8%) triploid. CONCLUSION(S): These data indicate that in vitro produced rhesus macaque and human preimplantation embryos exhibit similar numerical chromosomal aberrations. Rhesus macaques appear to be a suitable animal model for investigating the origin of chromosomal instability observed in human preimplantation embryos.