Cinzia Allegrucci

DNA methylation, insulin resistance, and blood pressure in offspring determined by maternal periconceptional B vitamin and methionine status

A complex combination of adult health-related disorders can originate from developmental events that occur in utero. The periconceptional period may also be programmable. We report on the effects of restricting the supply of specific B vitamins (i.e., B12 and folate) and methionine, within normal physiological ranges, from the periconceptional diet of mature female sheep. We hypothesized this would lead to epigenetic modifications to DNA methylation in the preovulatory oocyte and/or preimplantation embryo, with long-term health implications for offspring. DNA methylation is a key epigenetic contributor to maintenance of gene silencing that relies on a dietary supply of methyl groups. We observed no effects on pregnancy establishment or birth weight, but this modest early dietary intervention led to adult offspring that were both heavier and fatter, elicited altered immune responses to antigenic challenge, were insulin-resistant, and had elevated blood pressure–effects that were most obvious in males. The altered methylation status of 4% of 1,400 CpG islands examined by restriction landmark genome scanning in the fetal liver revealed compelling evidence of a widespread epigenetic mechanism associated with this nutritionally programmed effect. Intriguingly, more than half of the affected loci were specific to males. The data provide the first evidence that clinically relevant reductions in specific dietary inputs to the methionine/folate cycles during the periconceptional period can lead to widespread epigenetic alterations to DNA methylation in offspring, and modify adult health-related phenotypes.

Improved Human Embryonic Stem Cell Embryoid Body Homogeneity and Cardiomyocyte Differentiation from a Novel V‐96 Plate Aggregation System Highlights Interline Variability

Although all human ESC (hESC) lines have similar morphology, express key pluripotency markers, and can differentiate toward primitive germ layers in vitro, the lineage‐specific developmental potential may vary between individual lines. In the current study, four hESC lines were cultured in the same feeder‐free conditions to provide a standardized platform for interline analysis. A high‐throughput, forced‐aggregation system involving centrifugation of defined numbers of hESCs in V‐96 plates (V‐96FA) was developed to examine formation, growth, and subsequent cardiomyocyte differentiation from >22,000 EBs. Homogeneity of EBs formed by V‐96FA in mouse embryo fibroblast‐conditioned medium was significantly improved compared with formation in mass culture (p < .02; Levenes test). V‐96FA EB formation was successful in all four lines, although significant differences in EB growth were observed during the first 6 days of differentiation (p = .044 to .001; one‐way analysis of variance [ANOVA]). Cardiomyocyte differentiation potential also varied; 9.5% ± 0.9%, 6.6% ± 2.4%, 5.2% ± 3.1%, and 1.6% ± 1.0% beating EBs were identified for HUES‐7, NOTT2, NOTT1, and BG01, respectively (p = .008; one‐way ANOVA). Formation of HUES‐7 V‐96FA EBs in defined medium containing activin A and basic fibroblast growth factor resulted in 23.6% ± 3.6% beating EBs, representing a 13.1‐fold increase relative to mass culture (1.8% ± 0.7%), consistent with an observed 14.8‐fold increase in MYH6 (αMHC) expression by real‐time polymerase chain reaction. In contrast, no beating areas were derived from NOTT1‐EBs and BG01‐EBs formed in defined medium. Thus, the V‐96FA system highlighted interline variability in EB growth and cardiomyocyte differentiation but, under the test conditions described, identified HUES‐7 as a line that can respond to cardiomyogenic stimulation.

Pig Epiblast Stem Cells Depend on Activin/Nodal Signaling for Pluripotency and Self-Renewal

Activin/Nodal signaling is required for maintaining pluripotency and self-renewal of mouse epiblast stem cells and human embryonic stem cells (hESC). In this study, we investigated whether this signaling mechanism is also operative in cultured epiblasts derived from Days 10.5-12 pig embryos. Pig epiblast stem cell lines (pEpiSC) were established on mouse feeder layers and medium supplemented with basic fibroblast growth factor (bFGF). pEpiSC express the core pluripotency factors OCT4 (or POU5F1), NANOG, SOX2, and NODAL, but they do not express REX1 or alkaline phosphatase activity. Blocking leukemia inhibitory factor (LIF)/JAK/STAT3 pathway by adding the specific JAK I inhibitor 420099 and an anti-LIF antibody over 3 passages did not affect pluripotency of pEpiSC. In contrast, cells grown with the Alk-5 inhibitor SB431542, which blocks Activin/Nodal pathway, differentiated readily toward the neural lineage. pEpiSC are pluripotent, as established by their differentiation potential to ectoderm, mesoderm, and endoderm. These cells can be induced to differentiate toward trophectoderm and to germ cell precursors in response to bone morphogenetic protein 4 (BMP-4). In conclusion, our study demonstrates that pig epiblasts express the core pluripotency genes and that the capacity for maintaining self-renewal in pEpiSC depends on Activin/Nodal signaling. This study provides further evidence that maintenance of pluripotency via Activin/Nodal signal is conserved in mammals.

Human embryonic stem cell methyl cycle enzyme expression: modelling epigenetic programming in assisted reproduction?

To investigate a possible mechanism for inducing epigenetic defects in the preimplantation embryo, a human embryonic stem cell model was developed, and gene expression of the key methyl cycle enzymes, MAT2A, MAT2B, GNMT, SAHH, CBS, CGL, MTR, MTRR, BHMT, BHMT2, mSHMT, cSHMT and MTHFR was demonstrated, while MAT1 was barely detectable. Several potential acceptors of cycle-generated methyl groups, the DNA methyltransferases (DNMT1, DNMT3A, DNMT3B and DNMT3L), glycine methyltransferase and the polyamine biosynthetic enzymes, SAM decarboxylase and ornithine decarboxylase, were also expressed. Expression of folate receptor alpha suggests a propensity for folate metabolism. Methotrexate-induced depletion of folate resulted in elevated intracellular homocysteine concentration after 7 days in culture and a concomitant increase in cysteine and glutathione, indicating clearance of homocysteine through the transulphuration pathway. These studies indicate that altered methyl group metabolism provides a potential mechanism for inducing epigenetic changes in the preimplantation embryo.

Axolotl Nanog activity in mouse embryonic stem cells demonstrates that ground state pluripotency is conserved from urodele amphibians to mammals

Cells in the pluripotent ground state can give rise to somatic cells and germ cells, and the acquisition of pluripotency is dependent on the expression of Nanog. Pluripotency is conserved in the primitive ectoderm of embryos from mammals and urodele amphibians, and here we report the isolation of a Nanog ortholog from axolotls (axNanog). axNanog does not contain a tryptophan repeat domain and is expressed as a monomer in the axolotl animal cap. The monomeric form is sufficient to regulate pluripotency in mouse embryonic stem cells, but axNanog dimers are required to rescue LIF-independent self-renewal. Our results show that protein interactions mediated by Nanog dimerization promote proliferation. More importantly, they demonstrate that the mechanisms governing pluripotency are conserved from urodele amphibians to mammals.

Overcoming Drug Resistance and Treating Advanced Prostate Cancer.

Most of the prostate cancers (PCa) in advanced stage will progress to castration-resistant prostate cancer (CRPC). Within CRPC group, 50-70% of the patients will develop bone metastasis in axial and other regions of the skeleton. Once PCa cells spread to the bone, currently, no treatment regimens are available to eradicate the metastasis, and cancer-related death becomes inevitable. In 2012, it is estimated that there will be 28,170 PCa deaths in the United States. Thus, PCa bone metastasis-associated clinical complications and treatment resistance pose major clinical challenges. In this review, we will present recent findings on the molecular and cellular pathways that are responsible for bone metastasis of PCa. We will address several novel mechanisms with a focus on the role of bone and bone marrow microenvironment in promoting PCa metastasis, and will further discuss why prostate cancer cells preferentially metastasize to the bone. Additionally, we will discuss novel roles of several key pathways, including angiogenesis and extracellular matrix remodeling in bone marrow and stem cell niches with their relationship to PCa bone metastasis and poor treatment response. We will evaluate how various chemotherapeutic drugs and radiation therapies may allow aggressive PCa cells to gain advantageous mutations leading to increased survival and rendering the cancer cells to become resistant to treatment. The novel concept relating several key survival and invasion signaling pathways to stem cell niches and treatment resistance will be reviewed. Lastly, we will provide an update of several recently developed novel drug candidates that target metastatic cancer microenvironments or niches, and discuss the advantages and significance provided by such therapeutic approaches in pursuit of overcoming drug resistance and treating advanced PCa.

Modulation of Pluripotency in the Porcine Embryo and iPS Cells

The establishment of the pluripotent ICM during early mammalian development is characterized by the differential expression of the transcription factors NANOG and GATA4/6, indicative of the epiblast and hypoblast, respectively. Differences in the mechanisms regulating the segregation of these lineages have been reported in many species, however little is known about this process in the porcine embryo. The aim of this study was to investigate the signalling pathways participating in the formation of the porcine ICM, and to establish whether their modulation can be used to increase the developmental potential of pluripotent cells. We show that blocking MEK signalling enhances the proportion of NANOG expressing cells in the ICM, but does not prevent the segregation of GATA-4 cells. Interestingly, inhibition of FGF signalling does not alter the segregation of NANOG and GATA-4 cells, but affects the number of ICM cells. This indicates that FGF signalling participates in the formation of the founders of the ICM. Inhibition of MEK signalling combined with GSK3β inhibition and LIF supplementation was used to modulate pluripotency in porcine iPS (piPS) cells. We demonstrate that under these stringent culture conditions piPS cells acquire features of naive pluripotency, characterized by the expression of STELLA and REX1, and increased in vitro germline differentiation capacity. We propose that small molecule inhibitors can be used to increase the homogeneity of induced pluripotent stem cell cultures. These improved culture conditions will pave the way for the generation of germline competent stem cells in this species.

Epigenetic marks in somatic chromatin are remodelled to resemble pluripotent nuclei by amphibian oocyte extracts.

Reprogramming pluripotency after nuclear transplantation shows that molecules in oocytes can remodel somatic chromatin to a stem cell state. Here we report on an ex-ovo system using axolotl oocyte extracts to remodel epigenetic marks of somatic chromatin. Molecules present in axolotl oocyte extracts induce the reduction of the overall levels of H3K9me3, HP1α, and DNA methylation of somatic cells, and they increase the levels of H3K9ac. The levels of signal intensity detected in treated differentiated cells resemble those detected in embryonic stem cells, which are, in contrast, unaffected by these extracts. Analysis of specific genome sequences shows that somatic cells exposed to oocyte extracts undergo demethylation of LINE-1 repeats but Major Satellite repeats and the imprinted gene H19 remain unchanged. In addition, they induce demethylation of the Oct-4 promoter. Finally, the kinetics of activation of Oct-4 and Nanog expression from MEF nuclei treated in extracts suggests that these genes are subject to different levels of epigenetic control. The results demonstrate that axolotl oocyte extracts are a useful tool for studying epigenetic remodelling of somatic cells to a stem cell configuration, and for elucidating oocyte specific mechanisms of nuclear reprogramming.

Stem-cell consequences of embryo epigenetic defects

CONTEXT The genetic code in the DNA of virtually every somatic cell can produce the entire complement of encoded proteins. Acetylation of histones and methylation of histones and DNA cytosine residues are part of the complex epigenetic regulatory process determining lineage-specific gene expression by altering the local structure of chromatin. After fertilisation, sperm DNA exchanges protamines for histones recruited from oocyte cytoplasm, reconfiguring both parental genomes into an epigenetic state conducive to activating the embryonic developmental programme. The identification of epigenetic reprogramming mechanisms is a major interest, rekindled by the ability of at least some somatic cells to acquire totipotency after somatic-cell nuclear transfer. STARTING POINT Recently, Woo SukHwang and colleagues (Science 2004; 303: 1669-74) derived a human embryonic stem-cell line from embryo therapeutic cloning. Chad Cowan and colleagues (N Engl JMed 2004; 350: 1353-56) produced 17 new lines from embryos supernumerary to infertility treatments. However, increasing evidence from a range of mammals shows a propensity for epigenetic errors with embryo technologies. If paralleled in human embryos, the effect on tumorigenic and differentiation properties of embryonic stem cells needs to be established. WHERE NEXT? Identifying the mechanisms in the oocyte that reprogramme a somatic cell to the embryonic state might allow somatic cells to be reprogrammed ex ovo by in-vitro manipulation of the epigenome. Because the oocyte is designed to reprogramme the sperm genome, which is in a different chromatin state from a somatic cell, perhaps many of the epigenetic errors induced by somatic-cell nuclear transfer could be avoided by a more targeted approach.

Epigenetic reprogramming of breast cancer cells with oocyte extracts

BackgroundBreast cancer is a disease characterised by both genetic and epigenetic alterations. Epigenetic silencing of tumour suppressor genes is an early event in breast carcinogenesis and reversion of gene silencing by epigenetic reprogramming can provide clues to the mechanisms responsible for tumour initiation and progression. In this study we apply the reprogramming capacity of oocytes to cancer cells in order to study breast oncogenesis.ResultsWe show that breast cancer cells can be directly reprogrammed by amphibian oocyte extracts. The reprogramming effect, after six hours of treatment, in the absence of DNA replication, includes DNA demethylation and removal of repressive histone marks at the promoters of tumour suppressor genes; also, expression of the silenced genes is re-activated in response to treatment. This activity is specific to oocytes as it is not elicited by extracts from ovulated eggs, and is present at very limited levels in extracts from mouse embryonic stem cells. Epigenetic reprogramming in oocyte extracts results in reduction of cancer cell growth under anchorage independent conditions and a reduction in tumour growth in mouse xenografts.ConclusionsThis study presents a new method to investigate tumour reversion by epigenetic reprogramming. After testing extracts from different sources, we found that axolotl oocyte extracts possess superior reprogramming ability, which reverses epigenetic silencing of tumour suppressor genes and tumorigenicity of breast cancer cells in a mouse xenograft model. Therefore this system can be extremely valuable for dissecting the mechanisms involved in tumour suppressor gene silencing and identifying molecular activities capable of arresting tumour growth. These applications can ultimately shed light on the contribution of epigenetic alterations in breast cancer and advance the development of epigenetic therapies.