Mouse embryonic stem cells as a model system for periconceptional developmental programming

Abstract

The Developmental Origins of Health and Disease (DOHaD) hypothesis proposes that maternal environment during pregnancy induces changes in early development, and influences adult offspring health and chronic disease risk. Such developmental programming may occur within the preimplantation embryo. In rodents, adverse maternal environments such as advanced reproductive age and undernutrition have been shown to induce permanent and irreversible changes in the preimplantation embryo which, following implantation and term delivery, subsequently predisposes offspring to late onset non-communicable diseases such as cardiometabolic dysfunction. Disease probability in adult offspring has been correlated with perturbations in lineage specification and metabolism in preimplantation embryos. However, limited cell numbers and the inaccessibility of post-implantation embryos have restricted our ability to explore the underlying mechanisms that predispose to adult disease. To overcome these issues, the current study utilised mouse embryonic stem cells (mESCs) derived from inbred C57BL/6 mice as a model system. This presented the opportunity to overcome the aforementioned limitations and to characterise the pluripotent population of cells within the embryo (at E3.5) in order to investigate the effects of advanced maternal age (AMA) and maternal low protein diet (LPD, 9% casein). Furthermore, with this model it was possible to consider the effects of LPD on the efficiency of neural induction (NI).

Embryos retrieved from Old (7-8 months) dams exhibited developmental delay (i.e. an increased proportion of morulae and a reduced proportion of blastocysts) compared to those recovered from Young (7-8 weeks) dams. Mouse ESCs derived from blastocysts of Old and Young dams (i) showed no differences in mESC derivation efficiency, (ii) displayed dominance towards male sex and (iii) generally displayed a similar pattern of gene expression for pluripotency (except Sox2), differentiation, early apoptosis and Dnmt (except Dnmt3b) markers. However, aneuploidy was greater in Old male mESCs, which were less proliferative and viable compared to Young male mESC lines. Old mESC lines were sexually dimorphic with respect to viability. Aneuploidy and apoptosis was greater in Old Female than Old Male mESC lines.

Global metabolomics of derived male LPD (9% casein) and normal protein diet, NPD (18% casein) mESC lines (undertaken in collaboration with Metabolon Inc.) showed evidence of altered glucose and fatty acid (FA) metabolism, while amino acid levels generally remained similar between the two dietary groups. Within the glycolytic pathway, LPD mESC lines exhibited increased levels of glucose 6-phosphate (G-6-P) and fructose 6-phosphate (F-6-P), and reduced downstream metabolites including fructose-1,6-bisphosphate (F-1,6-bP). Further analysis suggested reduced activity of phosphofructokinase (PFK), a key glycolytic enzyme that is allosterically regulated of LPD mESC lines. Interestingly, unlike previously reported in vivo data, LPD mESC lines displayed increased levels of n-3 and n-6 polyunsaturated FAs (PUFAs).

In separate experiments, derived male LPD and NPD mESC lines were expanded in serum-free (KO/SR) mESC medium and differentiated towards neural lineages (with Biotalentum Ltd.) using an optimised 14-day neural induction (NI) protocol, under standard NI medium (S-NIM) conditions. Preliminary results suggest increased NI efficiency of LPD mESC lines compared to NPD mESC lines. This was illustrated by precocious morphological appearance of rosettes and neurites, increased and protracted gene, and qualitative protein, expression for neural stem cell (NSC) and mature neuronal markers of differentiating LPD lines; all during the 14-day NI protocol.

Collectively, these data indicate that advanced maternal age and maternal diet can determine pluripotent mESC phenotype. Despite protracted periods of culture, the effects of adverse maternal environment on the developmental programming of blastocysts could be observed in derived mESC lines and cell populations derived following NI. These findings encourage further investigation of AMA and LPD on mechanistic adaptations in neural and other cell lineages to provide greater mechanistic insights into the induction of later disease using mESC as an in vitro model system, thereby reducing animal use.

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