Modelling the developmental origins of health and disease in the early embryo

Theriogenology 67 (2007) 43–53 www.theriojournal.com

Modelling the developmental origins of health and disease in the early embryo Kevin D. Sinclair *, Ravinder Singh School of Biosciences, University of Nottingham, Sutton Bonington Campus, Leicestershire, LE12 5RD, UK

Abstract The concept that certain adult diseases, such as hypertension, type 2 diabetes and dyslipidaemia can originate from events occurring in utero arose from epidemiological studies in humans but has since been supported by numerous animal-based studies. Referred to as the ‘‘developmental origins of health and disease’’ or ‘‘DOHaD’’ hypothesis, nutritional studies to date have largely focused on two experimental paradigms involving either calorie or protein restriction for varying intervals during pregnancy, where the favoured animal models have been the sheep and rat. In recent times, attention has been directed towards the earliest stages of gestation, where there is emerging evidence to indicate that the pre-implantation embryo may be particularly sensitive to environmentally induced perturbations leading to impaired health in adulthood. In this article, we make the case for hESCs as a model of the human pre-implantation embryo. Working with comparatively large populations of embryonic cells from the species of clinical interest, the scope exists to investigate the effects of specific genetic manipulations or combinations of metabolites against contrasting genetic backgrounds, where the consequences can be evaluated in downstream tissue specific progenitor and/or terminally differentiated cells. In order to fully realize these potentials, however, both derivation and culture conditions need to be harmonized and refined so as to preclude the requirement for feeder cells and serum. # 2006 Elsevier Inc. All rights reserved. Keywords: Fetal programming; Epigenetics; Human embryonic stem cells

1. Introduction The concept that late onset diseases can originate from events occurring in utero arose from the initial retrospective cohort studies of Barker et al. [1,2], who assessed relationships between size at birth, hypertension and ischemic heart disease in adult humans. Further studies were to establish inverse relationships between birth weight and the incidence of other diseases such as stroke, type 2 diabetes and dyslipidaemia and * Corresponding author. Tel.: +44 115 951 6053; fax: +44 115 951 6060. E-mail address: [email protected] (K.D. Sinclair). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.09.017

these observations ultimately led to the ‘‘developmental origins of adult health and disease’’ or ‘‘DOHaD’’ hypothesis [3]. In the decade or so that followed, numerous animal-based studies have sought to determine the effects of altered fetal nutrition on indices of long-term health (reviewed by ref. [4]). These studies have largely focused on two experimental paradigms involving either calorie or protein restriction for varying intervals during pregnancy and up to weaning, where the favoured animal models have been the sheep and rat. Whilst confirming the principle that nutrient restriction during in utero development can compromise physiological processes that determine long-term health, these studies have suffered from the following limitations. With notable exceptions to be identified later, they have:

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(i) provided little information on the effects of specific dietary nutrients, (ii) failed to establish a mechanistic base for these effects and (iii) generated little information that could reliably be extrapolated to early human development (reviewed by ref. [5]). There is also, currently, insufficient information on which to formulate a consensus regarding the effects of nutrient restriction during specific periods of in utero development, although there is emerging evidence that the earliest stages of pregnancy may be most sensitive to environmentally induced perturbations [6]. To that end, research efforts in the collaborating laboratories of Drs. K.D. Sinclair and L.E. Young at Nottingham are focussed on how specific dietary nutrients can alter epigenetic processes during gametogenesis and preimplantation development where, in addition to in vivo studies with rodents and large animal species, investigations have been initiated with human embryonic stem cells (hESC), which we believe can serve as a useful model for the human embryo [7,8]. In the present article, we provide a brief overview of some of the unresolved issues and recent concepts to emerge from studies addressing the DOHaD hypothesis, leading to an evaluation of models for developmental programming in the early embryo. A brief overview of hESC biology and culture is provided and, partially drawing from the authors’ own experiences, the potential of these cells to serve as a model for the human embryo assessed in relation to the use of contrasting animal models. 2. Developmental origins of adult health and disease Epidemiological evidence has led to the formation of a number of hypotheses that attempt to explain DOHaD (reviewed by ref. [5]). Whilst some hypotheses adhere closely to the empirical evidence (e.g. effects of catchup growth during infancy [9]), others attempt to explain this phenomenon in a broader evolutionary context in which DOHaD is viewed as an unfortunate consequence of a series of ill-defined mechanistic responses on the part of the developing organism as it attempts to adapt to changing ecological conditions [10–12]. Although an element of controversy surrounds these hypotheses, they have served to usefully focus the research efforts of a rather diverse group of biological scientists which include human epidemiologists, physiologists, reproductive biologists and, latterly, molecular geneticists. From the current debate emerge a number of poorly addressed issues that are central to the theme of the present review.

2.1. Critical periods of development The aforementioned hypotheses draw support from the long-term follow-up studies of human populations subjected to famine during the Second World War, for example, in The Netherlands during the winter of 1944– 45 [13]. One study of that cohort assessed the effects of maternal undernutrition during early, mid and late gestation in 50 year-old subjects who were born as term singletons [14]. These authors found that the timing of nutritional insult determined the nature of adult disease, which they hypothesized reflected critical periods of development for specific organs. The incidence of impaired glucose tolerance, for example, was greatest in subjects exposed to famine during late gestation. In contrast, the incidence of coronary heart disease, dyslipidaemia and obesity were greatest after exposure to famine during early gestation. Where studies with animals have limited nutrient restriction to specific periods of gestation (Fig. 1) their findings broadly support the observations from the Dutch famine cohort. For example, calorie restriction during late but not early gestation in sheep led to increased obesity and insulin resistance as young adults [15]. In contrast, protein restriction in rats and calorie (i.e. global nutrient) restriction in sheep during early pregnancy have both been observed to increase systolic or mean arterial blood pressure [16–19] although other studies suggest that nutrient restriction during any stage of pregnancy can program adult hypertension [20]. Nutrient restriction during early pregnancy is interesting clinically for a number of reasons, but not least because up to 80% of women encounter symptoms of nausea and vomiting (‘morning sickness’) leading to modest weight loss between 4 and 12 weeks of gestation [21], a phenomenon largely overlooked by many working in the DOHaD field. Paradoxically, mild forms of morning sickness leading to modest weight loss are associated with positive pregnancy outcomes (e.g. reduced risks of miscarriage, perinatal death, low birth weight and congenital heart defects) [22], leading some investigators to question if the absence of ‘morning sickness’ is teratogenic [23]. Indeed, moderate nutrient restriction from 4 to 12 weeks of gestation in mature sheep can increase placental development in ewes of good but not poor body condition [24] and there is evidence of similar responses associated with increased fetal growth in humans [25]. The fact that women also develop cravings and aversions suggests that, during this very sensitive period of embryonic development, intake is modified in an attempt to avoid the consumption of foodstuffs that contain potential

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Fig. 1. Nutritionally sensitive periods during early mammalian development. Periods during which nutrient deprivation can lead to altered cardiovascular development and hypertension [16–20], obesity and diabetes [31], hypothalamic–pituitary–adrenal activity [88], growth hormone and insulin metabolism [89,90] and reproductive development [91] (LPD, low protein diet).

teratogens [22], which are more likely to arise in the omnivorous diet. Regrettably, the severity and/or timing of nutrient restriction in studies with both sheep and rats (Fig. 1) are such that they can shed no light on the longer-term health implications of morning sickness. If morning sickness truly functions as a protective mechanism during embryonic development then its timing is intriguing for two reasons. Firstly, it approximates the period of primordial germ cell migration, gonadal colonization and mitotic proliferation, which sequentially occurs between weeks 3 and 12 of gestation in humans [26]. Secondly, such a protective mechanism is not operative during pre-implantation development. Both periods, at least in the mouse, witness dramatic genome wide epigenetic modifications to DNA and associated histone proteins that, in the first instance, serve to erase epimutations and re-establish totipotency in the germ line and, in the second instance, facilitate syngamy and the activation of zygotic transcription [27]. It is the very nature and extent of these modifications that has led some commentators to postulate that this very early period of mammalian development is perhaps most vulnerable to environmentally induced epigenetic disregulation [5,28]. 2.2. Nature of environmental perturbations Both the protein restriction model in the rat and the global nutrient restriction model in the sheep have helped to establish the principle of DOHaD under

experimental conditions and both have gone some way towards identifying nutritional sensitive periods during development (Fig. 1). The very nature of these models, however, has rendered it difficult to gain a meaningful insight into the specific nutrients involved. Although it has been shown that both glycine [29] and folic acid [30] supplementation throughout gestation ameliorates the effects of protein restriction in the rat, these responses are known to arise primarily as a consequence of excessive levels of methionine which are a feature of these diets [20]. Likewise, although the study of Waterland and Jirtle [31] confirms a role for specific ‘B’ Vitamins (i.e. Folate, B12 and choline) and methyl donating metabolites (e.g. betaine) during pregnancy and lactation on post-weaning phenotypic outcomes in the viable yellow agouti (Avy) mouse, levels of incorporation of these nutrients were well above the physiological norm and so have little clinical relevance. A significant observation from this latter study was the observation that, although supplementation with the aforementioned methyl donors was extended throughout pregnancy and lactation, DNA methylation in tissues from all three germ layers was affected, indicating that the effects are likely to have been induced during early embryo development. We have previously commented on the supra-physiological concentrations of a number of methyl cycle relevant nutrients found in a range of commercially available animal and human embryo culture media [8]. With concentrations of folate up to 400-fold, Vitamin B12 up

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to 3000-fold and methionine up to 12-fold that found in normal human serum or follicular fluid, serious concerns about the normality of embryos or cells cultured under such conditions needs to be addressed, a point reinforced by Kane [32] who observed that the removal of Vitamin B12 from Hams F10 significantly improved blastocyst yields in the rabbit. 2.3. Effects of specific micronutrients Most animal studies to date have tended to focus on the effects of global nutrient or protein restriction during pregnancy. Whilst the teratogenic effects of various micronutrient imbalances are well documented (e.g. Vitamins A and K, essential and nonessential metals including Mn, Zn, Pb and Cd [33,34]), long-term programming effects have generally not been investigated. The exception is iron deficiency during pregnancy, which is known to alter blood pressure in adult rats [35]. The effects of dietary fatty acids during pregnancy on long-term programming effects are also poorly chronicled but are worthy of investigation. Current research efforts at Nottingham are directed towards understanding the effects of specific nutrients known to influence single carbon metabolism, as we believe that altered input to these pathways can lead to epigenetic modifications to DNA. Details of these metabolic pathways have been provided elsewhere [36] and so only a brief overview is provided here. S-Adenosyl methionine (SAM) is a universal donor of methyl groups providing one-carbon moieties for a number of biochemical reactions including DNA methylation. In a reaction that involves the transfer of an adenosyl group from ATP, SAM is synthesized from the essential amino acid methionine, which in turn is generated by the remethylation of homocysteine (hcy) by one of two enzymes, namely methionine synthase (MTR) or betaine-homocysteine methyl transferase (BHMT), the methyl donor for the former reaction being 5-methyl tetrahydofolate (Fig. 2). The non-protein amino acid hcy is a central intermediate in the metabolism of sulfur in all animals. It is derived from the hydrolysis of S-adenosyl homocysteine (SAH) and, in addition to its remethylation, can also be catabolized to cystathionine via the transsulfuration pathway. In mammals Vitamin B12 acts as a cofactor in the remethylation of hcy catalysed by cytoplasmic MTR. The cobalamin form of the cofactor is methylated by 5-methyltetrahydrofolate, producing enzyme bound methylacobalamin and tetrahydrofolate. Key dietary derived inputs to these cycles, therefore,

Fig. 2. Combined methionine and folate cycles. Metabolites: methionine (Met), S-adenosyl methionine (SAM), S-adenosyl homocysteine (SAH), homocysteine (hcy), dihydrofolate (DHF), tetrahydrofolate (THF), serine (Ser), glycine (Gly), methylcobalamin (B12), pyridoxal phosphate (B6). Enzymes: methionine synthase [1], methionine adenosyl trasnferase [2], SAM-dependent methyltransferases [3], SAHhydrolase [4], betaine-homocysteine methyltransferase [5], dihydrofolate reductase [6], serine hydroxymethyl transferase [7], 5,10methylene THF reductase [8], cystathionine b-synthase [9], cystathionine g-lyase [10].

come in the form of choline, folate, Vitamins B6, B12 and methionine. 2.4. Mechanistic basis of responses Few studies into the aetiology of the DOHaD have attempted to establish the mechanistic basis of this phenomenon. Instead, most have and continue to direct their efforts to understanding how various disease states are ultimately manifest (reviewed by ref. [4]). In contrast, we have formulated the hypothesis that maternal nutrition prior to or during pregnancy can program fetal development and adult health via heritable epigenetic changes in DNA methylation at specific gene loci [37]. This hypothesis does not exclude the possibility of epigenetic mechanisms involving additional interactions between non-transcribed RNAs and covalent modifications to associated histone proteins (discussed by ref. [5]). Support for such a mechanistic base has come from studies with the protein restriction model in rats, where reductions in DNA methylation at the promoter regions of both peroxisomal proliferator-activated receptor alpha (PPARa) and glucocorticoid receptor (GR) genes are associated with significant increases in gene expression [30]. Other studies with rodents have reported both epigenetic and phenotypic effects in offspring of dams transiently exposed to endocrine disrupting compounds during pregnancy [38,39].

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Evidence of a direct link between early embryo culture and epigenetic modifications to DNA, leading to altered imprinted gene expression, has now been established in both the sheep [40] and mouse [41]. Recently, we have shown that maternal nitrogen metabolism prior to mating in sheep can influence developmental outcomes and expression of the type II insulin-like growth factor gene following the transfer of in vitro cultured embryos [42]. This phenomenon in ruminants, often referred to as the ‘large offspring syndrome’ (LOS), has features reminiscent of a number of naturally occurring overgrowth syndromes in humans, the incidence of which is reported to increase following the use of assisted reproduction [43–45]. Whilst such phenomena would not normally be considered in the context of DOHaD, they do serve to illustrate the environmental lability of acquired epigenetic modifications during the earliest stages of mammalian development. 3. Human ES cells: characteristics and culture Human embryonic stem cells were first derived from the inner cell mass of human blastocysts by Thomson et al. almost a decade ago [46]. Since then more than 200 hESC lines have been derived worldwide [47]. Importantly, these lines have invariably been established from embryos supernumerary to IVF treatment [48]. Human ES cells exhibit two unique features: (i) a karyotypically and epigenetically stable continued selfrenewal capacity and (ii) an ability to differentiate via precursor cells into terminally differentiated cells of the three germ layers both in vitro and in vivo [49]. Morphologically, hESCs are characterized by a high nucleo-cytoplasmic ratio. Other properties of these cells include the expression of cell surface antigens such as cell stage specific embryonic antigens (e.g. SSEA-3 and -4), tumour rejection antigen (e.g. TRA-1-60 and -81), germ cell tumour marker (e.g. GCTM-2) and alkaline phosphatase. The expression of specific pluripotency transcription factors (e.g. Oct-4, Nanog and Sox-2) are required to maintain an undifferentiated state in these cells, whereas high levels of telomerase, a ribonucleoprotein, are responsible for maintaining telomere length which is correlated with immortality in mammalian tissue culture cells. Other pluripotent factors expressed by these cells include FGF-4 (a member of fibroblast growth factor family of signalling peptides) and Rex-1 (a zinc finder transcription factor) [49]. Unlike mouse ES cells which require leukaemia inhibitory factor, hESCs have a specific requirement for basic fibroblast growth factor for self-renewal [49].

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3.1. Differentiation Since their discovery numerous reports have emerged describing the ability of hESCs to differentiate into a wide array of cell types including cardiomyocytes [50], neurons [51] endothelial cells [52], islets [53], hematopoietic cells [54], trophoblasts [55] and germ cells [56]. For example, hESCs can be directed to differentiate into cardiomyocytes by cultures supplemented with hepatocyte growth factor, epidermal growth factor, basic FGF and retinoic acid [57]. Identified by the appearance of spontaneous contractions and electrophysiological measurements, these cardiomyocytes were found to be similar to those derived in vivo. These cells exhibit chronotropic responses following administration of adrenergic and muscarinic agonists and express a number of cardiacrestricted proteins and transcription factors, suggesting close physical and functional similarity to early-stage cardiac tissue. It has been shown that decreased expression of pluripotency factors such as Nanog, Oct-4 and Sox-2 and increased activity of lineage specific transcription factors such as BRY and MESP accompanies the differentiation into mesodermal progenitors, which subsequently differentiate into several types of cardiac cells such as conduction cells, cardiac muscle cells and endothelial cells under the influence of lineage-decision transcription factors such as GAT4, HF-1b and HOXB5 [58]. The differentiation potential of hESCs has raised hopes concerning their therapeutic value for the treatment of severe degenerative diseases such as Parkinson’s disease, diabetes, heart disease and spinal cord injury, but their capacity to differentiate into many cell lineages also enhances their value as a model for the human pre-implantation embryo in studies addressing the DOHaD hypothesis. 3.2. Culture To realize the full potential of hESCs, either for therapeutic or research purposes, reproducible and reliable production under well-defined, harmonized and controlled conditions is crucial. To date, hESCs have been cultured predominately in mitotically inactived, irradiated mice embryonic feeders (MEFs), thereby enhancing the risk of xenogenic pathogen/viral transfer. Several recent reports describe the development of feeder-free culture systems which utilize MatrigelTM or laminin-coated surfaces and MEF conditioned medium [59,60]. However, these approaches do not overcome the risk of pathogen transfer and also make it difficult to control the quality and reproducibility of the medium.

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An alternative approach is to use human feeder cells derived from embryonic fibroblasts [61], human adult marrow cells [62], foreskin fibroblasts [63] or ES-derived autogenic feeder cells [64]. The use of an additional cell line, irrespective of origin, nevertheless introduces variability during culture. The presence of a second cell population during culture also renders it difficult to assess the direct effects of nutrient interventions in hESCs. Recently, feeder-free and serum-free culture systems based on medium containing serum replacement and a combination of growth factors has been reported [65]. Human ES cells grown under these conditions maintain all ES features. However, serum replacement contains bovine proteins, thereby reducing the value of such systems for the production of clinical-grade stem cells or for research purposes. More recently, a well-defined serum-free system that uses human laminin-coated surfaces and contains only human and recombinant proteins has been reported [66]. A better understanding of the mechanisms underlying self-renewal will be vital for isolating specific factors required for maintaining the pluripotent population of cells and for establishing defined media universally suitable for the culture of genetically and epigenetically normal hESCs. In addition, an improved understanding of self-renewal mechanisms will facilitate tighter control over early spontaneous differentiation thereby enhancing controlled and directed differentiation. The use of common hESC culture conditions for maintenance of multiple lines will enhance the validity of hESCs as a model for developmental research and may also facilitate the transfer of specific differentiation protocols between the lines. 3.3. Genetic and epigenetic stability An important concern regarding the use of these cells for transplantation and as a model for developmental studies, has been the probable risk of chromosomal aberrations and epimutations following prolonged cultivation [67]. For example, mouse ES cells (mESCs) are known to develop chromosomal errors during culture [68,69] and a recent study using comparative genomic hybridization and karyotyping of three hESC lines (HS181, HS235 and HS237) revealed the occurrence of an aberrant X chromosome at passage 61 in HS237, an abnormality not detected up to passage 35 in this cell line [70]. The other cell lines analysed in this study (i.e. HS181 and HS235) were stable through to at least passage 39 and 59 respectively. Chromosomal aberrations could arise as a result of passaging techniques (e.g. enzymatic vs. mechanical isolation),

through use of feeder layers, matrices of animal origin and/or serum. They may also be present in the preimplantation embryo at the time of ESC derivation [71]. Imprinting anomalies have been reported following the culture of mESCs [72]. However, early indications suggest a high degree of epigenetic stability among imprinted genes in high-passage number hESCs [73]. The epigenetic status of non-imprinted loci in hESCs during culture is poorly defined at present, but this needs to be established in order to fully validate the use of these cells as a model of the human preimplantation embryo. 4. Modelling the pre-implantation embryo: what hESCs have to offer As with isogenic strains of mice, the clonal characteristics of ESCs offer several advantages over populations of embryos from out-bred strains, the use of which in humans is not possible in any case. The first of these pertains to statistical power in analysis, which is enhanced in a large population of embryonic cells that are genetically identical. The structured use of more than one strain (in the case of mice) or line (in the case of hESCs) can dramatically reduce the amount of replication required to achieve a given level of statistical precision, whilst ensuring that genetic variation in the population is represented. Metabolic responses to nutrient inputs vary between individuals as a consequence of polymorphic variation in relevant enzymes and this can be precisely modelled across several hESC lines (discussed later). However, as with inbred strains of mice, the simultaneous culture of several hESC lines, particularly given the disparity of culture conditions (discussed earlier), is beyond the capabilities of many laboratories at present. Directed differentiation of hESCs along selected pathways would facilitate investigations into nutritional programming during specific early human developmental events along with the regulatory signals for the identification of target genes for nutrient interventions. The differentiation of specific hESC lines into cells of all the three germ layers has been reported and validated by the expression of differentiation marker genes, such as a-fetoprotein and albumin (endoderm), a-cardiac and muscle actin (mesoderm) and tubulin-III and neurofilament (ectoderm), by several authors [65,66]. However, certain culture conditions may limit the ability of these cells to differentiate into particular cell types. For instance, late-passage mouse ES cells, maintained on MatrigelTM to avoid the use of feeder layers, showed increased aneuploidy and impaired

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differentiation into beating cardiomyocytes [74]. Similarly, MatrigelTM cultures were detrimental to embryoid body formation and subsequent cardiomyocyte differentiation in BG01 and HUES-7 hESC lines [75]. Optimal conditions that obviate the need for complex and non-specific culture components for directed differentiation into all cell types remain to be established. 4.1. The difficulty of endogenous reserves Studies into the effects of nutrient restriction are complicated by the fact that short-term deficiencies are buffered to varying degrees by the intra-cellular reserves of the nutrient(s) in question. This complication applies to both animal and cell culture studies but is confounded further in animal studies by the fact that nutrients can be stored by several tissue types. Consequently, both the rate and extent of nutrient release is dependent on both the tissue type and metabolites in question. The sequence of pathophysiological changes that occurs during micro-nutrient deprivation in animals was described by Underwood and Suttle [76] and is schematically represented in Fig. 3. A reciprocal relationship exists for nutrient excesses. By way of illustration, from our own unpublished studies and those of others [77] with sheep, a reduction in plasma Vitamin B12 concentrations to below 250 pmol/L (i.e. ‘deficiency’, Fig. 3.) can take between 4 and 6 weeks. Dysfunction, associated with elevated concentrations of plasma homocysteine and methylmalonic acid and altered tissue SAM:SAH ratios, can take a further two to four weeks to manifest depending on dietary levels of cobalt and sulphur. In

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contrast, it is possible in rodents to explant whole embryos (up to around 11–12 days post-coitum [78]), an approach commonly adopted in developmental toxicology [79]. Whole embryo culture affords more control on nutrient provision over periods of up to 48 h but suffers from the same basic limitations of tissue reserves described above. For example, in a study of the effects of iron deficiency during pregnancy on fetal cardiovascular development, dams had to be fed an iron deficient diet for 4 weeks prior to mating to ensure metabolic dysfunction in E10.5 whole rat embryos [80]. As for cell culture, whole embryo culture has the added complication at present of subjecting embryos to nonphysiological in vitro environments in the presence of high concentrations of serum which, at least during preimplantation development, is known to epigenetically modify the expression of a number of developmentally important genes [81]. Endogenous reserves of specific micronutrients can also be an issue with regard to ‘timing experiments’ in the pre-implantation embryo. For example, the culture of mouse zygotes in human tubal fluid in the presence or absence of folic acid had no effect on pre-implantation development [82]. In contrast, the inclusion of the antifolate drug, methotrexate, significantly impaired embryo development, an effect brought about primarily through the intracellular depletion of pyrimidines. These results indicate that, under normal circumstances, reserves of reduced folate already present in the oocyte at the time of IVF are sufficient for pre-implantation development, but that the embryo requires the capacity to re-cycle intracellular folates during the normal course of development. In principle, hESCs can overcome many of these limitations of the pre-implantation embryo because cells can be cultured for extended (>5 days) periods in micronutrient deficient media until the intracellular pools of the nutrients concerned are depleted sufficient to induce dysfunction. They also offer the practical advantage of yielding many more cells for metabolic and molecular analyses. The immediate challenge with these cells, however, is the development of chemically defined systems of culture that obviate the need for serum and feeder layers and that will allow the effects of specific nutrients to be determined in several lines cultured under comparable conditions [75]. 4.2. Nutrient–gene interactions

Fig. 3. Schematic sequence of patho-physiological changes in animals offered a nutrient deficient diet, illustrating temporal relationships between the processes of nutrient storage, transport and function with the onset of depletion leading to deficiency, dysfunction and ultimately disease (adapted from ref. [76]).

As alluded to earlier, studies at Nottingham are investigating the effects of specific nutrients known to influence single carbon metabolism on epigenetic

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mechanisms regulating early development. Our studies with hESCs began by confirming the expression of transcripts for all the methyl cycle enzymes listed in Fig. 2, together with transcripts for several other related enzymes [8]. The observation that methotrexate, a selective inhibitor of dihydrofolate reductase, significantly altered intracellular concentrations of a number of methyl cycle metabolites, including methionine, serine, glycine and sarcosine, confirms that at least some components of these cycles are functional in embryonic cells. At the time of writing, studies into the effects of nutrient interventions on SAM-mediated transmethylation reactions (including DNA methylation) in these cells are underway. In humans, metabolic responses to methyl nutrients are also known to differ between individuals as a result of polymorphic variation in at least four enzymes involved in these cycles. For example, the 677C to T polymorphism in methylene tetrahydrofolate reductase (MTHFR) reduces enzyme activity and is highly prevalent among Caucasians. Such individuals require high intakes of folic acid in order to reduce plasma hcy concentrations [83]. Similarly, the A to G polymorphism at position 2756 of the MTR gene and the A66G polymorphism in the associated methionine synthase reductase gene (needed to convert the inactive form of MTR to its active state) also occurs at a relatively high frequency in humans, where enzyme activities can be reduced to around 30% of that in wildtype [84,85]. The screening of 17 Harvard University hESC lines for polymorphisms in methyl cycle enzymes is currently underway at Nottingham. Preliminary evidence indicates that the genotypic frequencies for the 677C to T transition in MTHFR among these lines of 6, 47 and 47% [7] agree well with published frequencies of 11, 45 and 44% for TT, CT and CC, respectively among Caucasians [86]. These lines will therefore permit investigations into varying metabolite (e.g. folate and/or Vitamin B12) concentrations on epigenetic modifications to DNA and histone methylation in human embryonic cells and thus provide a unique insight into nutrient induced epimutations in human embryos from contrasting genetic backgrounds. The clinical significance of such studies was recently highlighted with the report that MTHFR genotype is linked to a woman’s likelihood of becoming pregnant following IVF, women homozygous for the mutation were less fertile [87]. Although the authors postulated that this was linked to embryo quality, in reality the effects of uterine receptivity cannot be ruled out. Studies with hESCs would assist in our understanding of such effects.

5. Conclusions There is accumulating evidence to suggest that the pre-implantation embryo may be particularly sensitive to environmentally induced perturbations leading to impaired health during adulthood. The effects discussed in this article should be viewed differently from the better established consequences of embryo manipulations and IVP, which are associated with gross alterations in utero development leading to phenomena such as the LOS in ruminants. The DOHaD hypothesis is concerned with more subtle, long-term programming effects, although some of the underlying epigenetic mechanisms may be similar to those encountered in LOS. This is the topic of ongoing investigations at the authors’ laboratory and elsewhere. In the present article we present the case for hESCs as a model for the human pre-implantation embryo. Working with comparatively large populations of embryonic cells from the species of clinical interest, the scope exists to investigate the effects of combinations of media constituents (i.e. nutrients) against contrasting genetic backgrounds. The consequences of these interventions can be further evaluated in downstream tissue specific progenitors and terminally differentiated cells. However, in order to fully utilize the potential of hESCs, derivation and culture methods need to be harmonized and refined so as to obviate the requirement for feeder cells, serum and animal proteins. Acknowledgements Original research is supported by a cooperative agreement from the National Institutes of Health, NICHD (U01-HD044638) and the Biotechnology and Biological Sciences Research Council (BBS/B/06164). References [1] Barker DJ, Osmond C. Low birth weight and hypertension. Br Med J 1988;297:134–5. [2] Barker DJ, Osmond C, Golding J, Kuh D, Wadsworth ME. Growth in utero, blood pressure in childhood and adult life, and mortality from cardiovascular disease. Br Med J 1989;298:564–7. [3] Barker DJ. The fetal and infant origins of disease. Eur J Clin Invest 1995;25:457–63. [4] McMillen IC, Robinson JS. Developmental origins of the metabolic syndrome: prediction, plasticity, and programming. Physiol Rev 2005;85:571–633. [5] Sinclair KD, Lea RG, Rees WD, Young LE. The developmental origins of health and disease: current theories and epigenetic mechanisms. Reprod Suppl 2007. [6] Fleming TP, Kwong WY, Porter R, Ursell E, Fesenko I, Wilkins A, et al. The embryo and its future. Biol Reprod 2004;71: 1046–54.

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