Deciphering and overcoming epigenetic erosion at imprinted loci in mouse and human naive pluripotent stem cells

During the routine petri dish culture of naïve pluripotent stem cells it was found that these acquire errors in a group of genes named imprinted. Naïve pluripotent stem cells are cells that represent the very earliest stage of embryonic development, and have the ability to self-replicate exactly or to change into any cell type in the body. Imprinting is a phenomenon that leads to genes being expressed in a parent of origin specific manner. This process involves modifications, DNA methylation and histone methylation, at regulatory regions of imprinted genes that affects their expression. These imprint marks are naturally erased and established in the germline of the parents and are subsequently maintained in all the other cells (soma) from the fertilized egg till adulthood. Abnormally, these imprints are erased in the routine petri dish culture of naïve pluripotent stem cells hindering the potential of these cells.

My group has recently discovered a gene, Impera, that potentially directs the observed removal of imprints at imprinted genes. Now we want to define mechanistically how Impera works and to define strategies that prevent Impera from inducing imprint erasure. Achieving these goals will lead to a fundamental discovery in developmental biology, the first identified gene capable of mediating imprint erasure, and to the prevention of imprint erasure in the cultures of both mouse and human naïve pluripotent stem cells.

In the medical context, appropriate imprinting is important for normal development. Human diseases involving inappropriate/lack of imprinting include Angelman syndrome and Prader-Willi syndrome. Thus, understanding mechanistic how imprint erasure occurs may uncover pathways and processes that will enhance our understanding of pathological processes.

Naïve pluripotent stem cells are also widely used as a platform for early developmental research, with much investment being focussed on both understanding how cells maintain their naïve state and how they commit to becoming specific cell types. The proposed research will provide insights into how we might be able to stabilise stem cells in this naïve state, providing more stable stem cell platforms for use as a research tool, in drug discovery programmes and in regenerative medicine applications.

Our research group has a strong track record in stem cell biology and has the necessary expertise to successfully complete this important project. Further, working within the Cambridge Stem Cell Institute puts us in a strategically strong position, with close collaborators including Dr Kevin Chalut. An expert in the physical biology of pluripotency and differentiation which together with my lab developed a novel hydrogel protocol (manuscript in preparation) which supports naïve pluripotent stem cell self-replication. This may prove a very useful tool in our aim to generate naïve pluripotent stem cells free of imprint errors

We have been investigating mechanistically for many years how the drivers of acquisition of naïve pluripotency work. Interestingly we found Nanog to work in synergy with epigenetic mechanisms and its requirement in reprogramming can be bypassed, although with lower efficiency, by the use of small molecules that impact on the epigenome of the cells.
When comparing the epigenetic profile of Nanog wild type with null naïve pluripotent stem cells (nPSCs) we noticed a difference in the levels of trimethylation at lysine 9 of histone H3 (H3K9me3) at Imprinting Control Regions (ICRs). These were slightly increased in Nanog null compared to wt nPSCs. Following on this observation we looked at the transcriptome of Nanog wt versus null nPSCs in search of candidate genes. Multiple candidate genes showing lower levels of expression in Nanog null nPSC lines compared to control lines were identified. Some were tested by overexpressing it in Nanog null nPSCs. Surprisingly, one of these erased H3K9me3 completely and specifically at a number of ICRs. As a result of this finding we named this gene Impera, for Imprint eraser. With the support from BBSRC we now aim at characterising Impera, the first potential imprint eraser, and in finding strategies to overcome the epigenetic errors at imprinted loci caused by its continuous expression in in vitro nPSC cultures. Specifically we aim at investigating the following:

i- Impera mediated imprint erasure mechanism
ii- Requirement of Impera for Nanog mediated reprogramming and reprogramming in general
iii- Requirement of Impera for human nPSC self-renewal
iv- Generation of human nPSCs with normal Imprints
v- Assess the differentiation potential of newly generated human nPSCs with normal imprints

In conclusion, this work expects to deliver a fundamental discovery in developmental biology and also the elimination of a defect hindering the developmental potential of in vitro cultured human and mouse nPSCs.

Impact summary:

Stem cell biology and regenerative medicine has enormous potential in advancing our fundamental understanding of cell biology and in advancing treatment options for a range of diseases through the ability to repair, replace and regenerate damaged cells, tissues and ultimately organs. As such, stem cell biology is a priority area for investment in the UK. The research proposed in this application has the potential to significantly advance our understanding of stem cell biology, with direct impact on the academic community, biotechnology and pharmaceutical industries, clinicians and patient groups and the wider public more generally.

Academic Community: This project will advance our understanding of the biology of both human and mouse naïve pluripotent stem cells. Deciphering and overcoming observed epigenetic erosion at imprinted loci in mouse and human naïve pluripotent stem cells will both deliver a fundamental discovery in developmental biology and also the elimination of a defect hindering the developmental potential of in vitro cultured human and mouse naïve pluripotent stem cells. For regenerative medicine research, naïve pluripotent stem cells are a fundamental research tool for understanding cell fate decisions, therefore the more detailed understanding we have of these cells, the further we can develop this platform for regenerative therapies. The specific development of technology that leads to the generation of human naïve pluripotent stem cells with normal imprints may generate new Intellectual property and will be made available for use in the wider research community.

Biotechnology and Pharmaceutical Industries: Increasingly, biotechnology and pharmaceutical industries are using stem cell platforms as a research tool in drug discovery and regenerative medicine. The delivery of more in-depth understanding of naïve pluripotent stem cell biology will lead to more control within stem cell platforms and the generation of better pluripotent stem cells feeding in to more reproducible drug discovery systems.

General Public: We will share our research findings with the public who fund our research and with the wider world. Through direct interaction at public events, and via online and print communications, we aim to increase awareness of stem cell research and relay the positive impacts that this research can have on public health and people's lives. Our lab has a strong track record in this area, including the recent delivery of 'Stem Cell Exchanges', a highly successful public engagement project which included a series of stem cell podcasts and a public 'stem cell inspired' art exhibition which ran as part of the MRC Festival 2017.