By: Dr. Susana Chuva de Sousa Lopes, recipient of the De Snoo award 2017
One of the most exciting and controversial discoveries over the past decade is that conditions experienced early in life – even before birth – can have long-lasting impacts on health and ageing 1,2. Longitudinal studies on humans and experimental studies using animal models also suggest that these effects may span several generations 3. There is an increasing body of evidence that shows that maternal effects on health are caused by epigenetic changes in the embryo that remain stable throughout life and possibly could be passed on to subsequent generations 4,5. For example, a longitudinal study of individuals that were in utero during the Dutch Famine at the end of the Second World War has demonstrated local and genome-wide changes in DNA methylation that can be linked to increased risk of cardiovascular and metabolic disease 6,7. These effects appear to primarily affect embryos exposed during the first trimester, and recently studies in humans point to the importance of pre-implantation and early implantation stages, when drastic changes in DNA methylation occur 8,9.
Aims of the project
Recent technical developments regarding single cell transcriptomics and (DNA) methylomics 10,11 as well as the development of a protocol to mimic implantation in mice 12 opens exciting opportunities to study hypothesis driven questions related to the theory of developmental origins of health and disease.
We aim to achieve two key objectives:
- To develop a peri-implantation model using human pre-implantation embryos using a novel synthetic 3D-scaffold to allow the embryo to develop in vitro during the first 14 days. Using a novel synthetic hydrogel as 3D-scaffold will would allow us to develop a novel method to keep the embryos in a stiff-environment that can mimic the endometrium and promote physiological implantation. We plan to seed the hydrogel with human endometrium cells (containing endothelial cells and glandular cells) and characterise in detail the implantation during until day 14 of development.
- If successful, we will study in great detail how (metabolic) changes in the culture environment will influence the epigenetics of the human embryo. For this we will perform single cell transcriptomics and methylomics as well as other molecular techniques (immunostaining and QPCR) to compare standard culture conditions with those that are known to change DNA methylation (folic acid, vitamin A and vitamin C).
Use of a 3D-synthetic scaffold to allow embryo development in vitro during 14-days
The extended culture model has been developed under the supervision of one of the researchers that was part of the team that has developed and published the mouse implantation model 12. The human (frozen-thawed) embryos at day 3 post fertilization (dpf) have been first cultured to the blastocyst stage (day 5), graded them using the Gardner and Schoolcraft grading system and then cultured in a two-step protocol to mimic implantation (from day 5 to day 14) in 2D and in the 3D-scaffold. The results of the 2D and 3D-scaffold have been evaluated by immunofluorescence to assess morphology and lineage segregation (extraembryonic lineages, endoderm ectoderm, mesoderm and germline) using different markers. In the 2D- system, the embryo survives better than in the 3D-system. A manuscript with the results obtained from the 2D-system has been submitted and the results of the 3D system is expected to be submitted by the end of 2018. Interestingly, we noticed that when using our extended culture method, aneuploid blastocysts excluded the abnormal cells by day 14. These important finding, is being explored and a manuscript is being prepared.
The benefits of including endometrium cells (endothelial cells and or glandular cells) in the 3D-scaffold, as not been investigated yet. But those experiments will be performed in the continuation of the project, as one PhD has been recruited to continue with this project.
Influence of metabolic changes in the epigenetics of the human blastocycst
In collaboration with the Hubrecht Institute (Prof. van Oudenaarden), we are characterising the transcriptomics and methylomics at single cell level during the preimplantation period. Most importantly, we performed single cell isolation of cells from individual sibling and from those we have requested and obtained the genomic DNA (exome) from the parental bucal swabs. This has allowed us to identify the existing SNPs in the embryos. This work is in progress and will be the base line to then investigate the transcriptomics and methylomics using the extended protocol on sibeling embryos and to test 3 culture conditions known to change DNA methylation (folic acid, vitamin A and vitamin C).
1 Bateson, P. et al. Nature 430, 419-421 (2004); 2 Gluckman, P. D. et al. Lancet 373, 1654-1657 (2009); 3 Daxinger, L. & Whitelaw, E. Nature reviews. Genetics 13, 153-162 (2012); 4 Gluckman, P. D. et al. Nature reviews. Endocrinology 5, 401-408 (2009); 5 Petronis, A. Nature 465, 721-727 (2010); 6 Heijmans, B. T. et al. PNAS 105, 17046-17049 (2008); 7 Tobi, E. W. et al. PloS one 7, e37933 (2012); 8 Guo, H. et al. Nature 511 (2014); 9 Smith, Z. D. et al. Nature 511, 611-615 (2014); 10 Lefevre, A. & Blachere, T. Methods in molecular biology 1222, 209-226 (2015); 11 Yan, L. et al. Nature structural & molecular biology 20, 1131-1139 (2013); 12 Bedzhov, I. et al. Nature protocols 9, 2732-2739 (2014).