Lorraine E. Young

Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture

Manipulation or non-physiological embryo culture environments can lead to defective fetal programming in livestock. Our demonstration of reduced fetal methylation and expression of ovine IGF2R suggests pre-implantation embryo procedures may be vulnerable to epigenetic alterations in imprinted genes. This highlights the potential benefits of epigenetic diagnostic screening in developing embryo procedures.

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.

Somatic cell nuclear transfer

Cloning by nuclear transfer from adult somatic cells is a remarkable demonstration of developmental plasticity. When a nucleus is placed in oocyte cytoplasm, the changes in chromatin structure that govern differentiation can be reversed, and the nucleus can be made to control development to term.

Drug evaluation in cardiomyocytes derived from human induced pluripotent stem cells carrying a long QT syndrome type 2 mutation

Aims Congenital long QT syndromes (LQTSs) are associated with prolonged ventricular repolarization and sudden cardiac death. Limitations to existing clinical therapeutic management strategies prompted us to develop a novel human in vitro drug-evaluation system for LQTS type 2 (LQT2) that will complement the existing in vitro and in vivo models. Methods and results Skin fibroblasts from a patient with a KCNH2 G1681A mutation (encodes IKr potassium ion channel) were reprogrammed to human induced pluripotent stem cells (hiPSCs), which were subsequently differentiated to functional cardiomyocytes. Relative to controls (including the patients mother), multi-electrode array and patch-clamp electrophysiology of LQT2–hiPSC cardiomyocytes showed prolonged field/action potential duration. When LQT2–hiPSC cardiomyocytes were exposed to E4031 (an IKr blocker), arrhythmias developed and these presented as early after depolarizations (EADs) in the action potentials. In contrast to control cardiomyocytes, LQT2–hiPSC cardiomyocytes also developed EADs when challenged with the clinically used stressor, isoprenaline. This effect was reversed by β-blockers, propranolol, and nadolol, the latter being used for the patients therapy. Treatment of cardiomyocytes with experimental potassium channel enhancers, nicorandil and PD118057, caused action potential shortening and in some cases could abolish EADs. Notably, combined treatment with isoprenaline (enhancers/isoprenaline) caused EADs, but this effect was reversed by nadolol. Conclusions Findings from this paper demonstrate that patient LQT2–hiPSC cardiomyocytes respond appropriately to clinically relevant pharmacology and will be a valuable human in vitro model for testing experimental drug combinations.

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.

Evaluation of Gestational Deficiencies in Cloned Sheep Fetuses and Placentae

Abstract Sheep fetal development at 35 days of gestation was examined following natural mating, in vitro production (IVP) of fertilized embryos, or somatic cell nuclear transfer (NT). Five crossbred (Blackface × Black Welsh) and four purebred (Black Welsh) fetuses and their associated placentae produced by natural mating were morphologically normal and consistent with each other. From 10 ewes receiving 21 IVP embryos, 17 fetuses (81%) were recovered, and 15 of these (88%) were normal. The NT fetuses were derived from two Black Welsh fetal fibroblast cell lines (BLW1 and 6). Transfer of 21 BLW1 and 22 BLW6 NT embryos into 12 and 11 ewes, respectively, yielded 7 (33%) and 8 (36%) fetuses, respectively. Only three (43%) BLW1 and two (25%) BLW6 NT fetuses were normal, with the rest being developmentally retarded. The NT fetal and placental deficiencies included liver enlargement, dermal hemorrhaging, and lack of placental vascular development reflected by reduced or absent cotyledonary structures. Fibroblasts isolated from normal and abnormal cloned fetuses did not differ in their karyotype from sexually conceived fetuses or nuclear donor cell lines. Our results demonstrate that within the first quarter of gestation, cloned fetuses are characterized by a high incidence of developmental retardation and placental insufficiency. These deficiencies are not linked to gross defects in chromosome number.

Non-conservation of mammalian preimplantation methylation dynamics

In mammals, active demethylation of sperm pronuclear DNA shortly after fertilisation is thought to be important for reprogramming subsequent embryonic development [1–3]. A further passive loss of methylation has been observed as DNA replicates between 2-cell and morula stages, with somatic cell levels being re-established at or after the blastocyst stage when differentiated lineages are first formed [1,3,4]. We now demonstrate non-conservation in the DNA methylation dynamics of the sheep embryo and suggest this challenges the perceived role of DNA methylation in mammalian preimplantation development. A dramatic loss of cytosine methylation from the male pronucleus has previously been observed in mouse, pig and cow with a 5-methylcytosine antibody [1]. By contrast, we did not observe loss of methylation from either pronucleus in the in vivo-derived ovine zygote (Figure 1A). With the same immunostaining technique, we observed asymmetry in pronuclear methylation in mouse embryos as reported previously, ruling out procedural differences (Figure 1B). Examination of pronuclear stages revealed demethylation in the human zygote (Figure 1C) and no demethylation in the rabbit zygote (Figure 1D). In contrast with a previous study [1], we observed only partial asymmetric demethylation in the bovine zygote (Figure 1E). Collectively, this suggests that demethylation of the paternal genome is not an obligate requirement for early mammalian development. Also in contrast to the mouse and cow [1], we observed no passive demethylation throughout sheep preimplantation development upon purely visual inspection, but rather an apparent increase between the 8-cell and morula stages (Figure 2A–E). Only at the blastocyst stage is demethylation visible in the sheep trophectoderm, whereas the cells of the inner cell mass (ICM) remain methylated (Figure 2F). This might be important as trophectoderm cells are the first differentiated cell type to form during development and trophoblast-specific gene expression is essential for embryonic nutrition and implantation. Quantification of confocal images (Figure 2G), demonstrates a significant decrease in methylation intensity from the 2-cell to the 8-cell stage (43%, p < 0.05). However, nuclear size also decreases between these stages (41%, p < 0.01), thus the ratio of mean methylation intensity to nuclear size was not different. Moreover, quantification revealed that the apparent increase observed between the 8-cell and morula stages is also a visual artefact. A simple explanation is that methylation levels do not increase but nuclear intensities appear higher due to the increased nuclear compaction (55% size reduction from 8-cell to morula (p < 0.001, Figure 2G); we …

Effect of Limited DNA Methylation Reprogramming in the Normal Sheep Embryo on Somatic Cell Nuclear Transfer

Abstract Active demethylation of cytosine residues in the sperm genome before forming a functional zygotic nucleus is thought to be an important function of the oocyte cytoplasm for subsequent embryonic development in the mouse. Conversely, this event does not occur in the sheep or rabbit zygote and occurs only partially in the cow. The aim of this study was to investigate the effect of limited methylation reprogramming in the normal sheep embryo on reprogramming somatic nuclei. Sheep fibroblast somatic nuclei were partially demethylated after electrofusion with recipient sheep oocytes and undergo a stepwise passive loss of DNA methylation during early development, as determined by 5-methylcytosine immunostaining on interphase embryonic nuclei. A similar decrease takes place with in vivo-derived sheep embryos up to the eight-cell stage, although nuclear transfer embryos exhibit a consistently higher level of methylation at each stage. Between the eight-cell and blastocyst stages, DNA methylation levels in nuclear transfer embryos are comparable with those derived in vivo, but the distribution of methylated DNA is abnormal in a high proportion. By correlating DNA methylation with developmental potential at individual stages, our results suggest that somatic nuclei that do not undergo rapid reorganization of their DNA before the first mitosis fail to develop within two to three cell cycles and that the observed methylation defects in early cleavage stages more likely occur as a direct consequence of failed nuclear reorganization than in failed demethylation capacity. However, because only embryos with reorganized chromatin appear to survive the 16-cell and morula stages, failure to demethylate the trophectoderm cells of the blastocyst is likely to directly impact on developmental potential by altering programmed patterns of gene expression in extra–embryonic tissues. Thus, both remodeling of DNA and epigenetic reprogramming appear critical for development of both fertilized and nuclear transfer embryos.

Automated, scalable culture of human embryonic stem cells in feeder-free conditions

Large‐scale manufacture of human embryonic stem cells (hESCs) is prerequisite to their widespread use in biomedical applications. However, current hESC culture strategies are labor‐intensive and employ highly variable processes, presenting challenges for scaled production and commercial development. Here we demonstrate that passaging of the hESC lines, HUES7, and NOTT1, with trypsin in feeder‐free conditions, is compatible with complete automation on the CompacT SelecT, a commercially available and industrially relevant robotic platform. Pluripotency was successfully retained, as evidenced by consistent proliferation during serial passage, expression of stem cell markers (OCT4, NANOG, TRA1‐81, and SSEA‐4), stable karyotype, and multi‐germlayer differentiation in vitro, including to pharmacologically responsive cardiomyocytes. Automation of hESC culture will expedite cell‐use in clinical, scientific, and industrial applications. Biotechnol. Bioeng. 2009;102: 1636–1644.

Conservation of IGF2-H19 and IGF2R imprinting in sheep: effects of somatic cell nuclear transfer

In different mammalian species, in vitro culture and manipulation can lead to aberrant fetal and peri-natal development. It has been postulated that these diverse abnormalities are caused by epigenetic alterations and that these could affect genes that are regulated by genomic imprinting. To explore this hypothesis relative to somatic cell nuclear transfer in sheep, we investigated whether the ovine H19-IGF2 and IGF2R loci are imprinted and analysed their DNA methylation status in cloned lambs. A comparison between parthenogenetic and control concepti established that imprinting at these two growth-related loci is evolutionarily conserved in sheep. As in humans and mice, IGF2R and H19 comprise differentially methylated regions (DMRs) that are methylated on one of the two parental alleles predominantly. In tongue tissue from 12 out of 13 cloned lambs analysed, the DMR in the second intron of IGF2R had strongly reduced levels of DNA methylation. The DMR located upstream of the ovine H19 gene was found to be similarly organised as in humans and mice, with multiple CTCF binding sites. At this DMR, however, aberrant methylation was observed in only one of the cloned lambs. Although the underlying mechanisms remain to be determined, our data indicate that somatic cell nuclear transfer procedures can lead to epigenetic deregulation at imprinted loci.