Epigenetic regulation of lineage competence in human pluripotent stem cells

The human body consists of more than 250 specialised cell types. During embryo development, this diversity originates from a small group of about 10-20 cells named "epiblast", which are initially equivalent, but will then continue development and form the whole body. This ability to produce all cells of the body is called "pluripotency". Pluripotent cells will take diverse pathways in order to eventually become different cell types (a process referred to as "differentiation"): neurons of the brain, heart muscle cells, among many others.

All specialised cells (with very few exceptions) have the same genes, but only a specific subset of genes is active in each cell type - this is what makes the cells unique. There are special mechanisms to switch genes "on" and "off". Many of them are related to the fact that our genes are not simply naked DNA molecules, but rather represent DNA in complex with many different proteins. Specific molecular tags can be placed onto these proteins or onto DNA itself. These so-called "epigenetic" modifications do not alter our DNA sequence, they are reversible and employed to turn genes "on" and "off".

One of the most fascinating questions in developmental biology is how pluripotent cells of an embryo make their first decisions what specialised cells to become. Remarkably, when the epiblast first emerges in the embryo, these cells are not sensitive to the signals that would induce them to specialise (we call this stage "naïve"). Only after 8-10 days, during which the embryo implants in the uterus, the epiblast cells will gain the capacity to respond to these signals ("primed" stage) and go on to develop further. Our project aims to understand the mechanism of how pluripotent cells become competent to differentiate.

There are major limitations to study development of human embryos directly, due to ethical considerations. The solution in many cases is to use so called "pluripotent stem cells" (PSC), which are derived from embryos. Using special growth factors and chemicals allows for trapping PSC so that they remain very similar to epiblast cells. In these conditions, PSC can be grown in a dish practically indefinitely and remain unspecialised. If PSC are exposed to factors that stimulate development, they can produce mature cells as if they were in the embryo. Besides their use for fundamental research, PSC is a promising tool for regenerative medicine, as a source of mature cells for transplantation.

Previously, we developed conditions in which human PSC reproduce development of the epiblast from the naïve to primed stage. This takes about 10 days, which is very similar to the length of this process in the actual embryo. We plan to use this unique model to understand the very beginnings of human embryo development. Our hypothesis is that epigenetic mechanisms operate not only simply to turn genes "on" and "off" but also before that, in order to prepare genes for activation. We predict that during transition from naive to primed pluripotency, some genes for differentiation become "pre-activated" by epigenetic modifications. This makes cells sensitive to differentiation signals. Once these signals appear, these genes will turn on and pluripotent cells will produce many different specialised cells.

In this project, we will identify "pre-activated" genes and the epigenetic modifications that "pre-activate" them. Then, we will apply modern genetic engineering tools to understand which factors "pre-activate" the genes in the first place, and how these genes become activated later during differentiation. Altogether, our results will reveal how a small group of 10-20 epiblast cells make their first decisions in order to generate complex organisms like ourselves. Furthermore, this insight will advance our abilities to employ PSC for biomedical science and potential clinical application.

Pluripotency is the ability of single cells to differentiate into all somatic lineages and the germline. Two forms of pluripotency have been defined, naïve and primed, that correspond to the pre-implantation and pre-gastrulation stage epiblast, respectively. We have developed a system for conversion of naïve human pluripotent stem cells (hPSC) to the primed state in vitro termed formative transition. Remarkably, this formative transition in vitro recapitulates features of primate embryo development in vivo. During this transition hPSC acquire the ability to respond to differentiation signals, first to endoderm and then to neuroectoderm. Thus differentiation competences to these lineages are separable in time and potentially mechanistically. The molecular basis of differentiation competence is unknown; it could rely on the expression of specific transcription factors, or the epigenetic marks of regulatory elements, or a combination of both. Here, we will explore the epigenetic component of the mechanism of lineage competence in hPSC.
Hypothesis: Competence for differentiation relies on certain epigenetic marks in promoters and enhancers of lineage-specific genes, which enables their activation in response to differentiation signals, once those signals appear ("epigenetic lineage priming"). We predict that epigenetic lineage priming occurs during the formative transition in hPSC.
Specific aims and questions:
1) Identify the epigenetic lineage priming programmes to endoderm and neuroectoderm.
2) How are epigenetic lineage priming programmes established during the formative transition?
3) How are epigenetic lineage priming programmes activated during differentiation?
The project will address the molecular mechanism of the transition from pluripotency to differentiation. Insight from our research will have far reaching implications, as it will provide a paradigm of epigenetic regulation and cell fate choice fundamental to understanding of all developing organisms.