Pluripotent embryonic stem cells (ESCs) develop into all cell types of our bodies. Understanding the properties of ESCs that enable them to maintain their pluripotency as well as commit to differentiation into distinct cell types is a vital research question. There is increasing evidence that the metabolic profile of stem cells is critical to divide, self-renew or differentiate. A fundamental rewiring of cellular metabolism and many cellular adaptations are needed to facilitate the changes in substrate usage from glucose as the primary energy source of ESCs to using fat as they progress through the very early steps of differentiation. Elucidating how this metabolic reprogramming of stem cells impacts on the regulation of cell fate decisions is highly relevant to regenerative medicine and cancer.

Switches in energy metabolism by ESCs are underpinned by profound changes in gene and protein expression, and the number, size and shape of key organelles such as mitochondria and lipid droplets (LDs). Recognized since the early days of cell biology, LDs have long been ignored as passive, uninteresting blobs of fat. However, pioneering research in recent years firmly established them as dynamic organelles that are central, active players in regulating energy storage and generation of signalling molecules. Remarkably, we have discovered that ESCs contain 'supersized' LDs that resemble those present in fat cells in contrast to most cells in the body that store small amounts of fat in many tiny LDs. The presence of large LDs coincides with tight regulation of glucose and fat usage as energy sources, and specifically delineates discrete ESC populations on the verge of differentiation.

In this research, we will unravel how maintenance and fine-tuning of the pluripotent state towards differentiation is coupled to changing metabolic profiles by investigating the functional importance of LDs and lipid metabolism in ESCs. We will identify the molecular interplay between key pluripotency and metabolic regulators to coordinate lipid metabolism and storage with developmental potency during ESC differentiation. We will visualise in individual cells the trafficking of lipids from enlarged LDs to mitochondria that ultimately orchestrates metabolic switches and differentiation. The structure of LDs consists of a neutral lipid core covered with a phospholipid monolayer membrane and proteins. The many proteins that coat the surface of LDs can modulate LD size, stability, and inter-droplet interaction as well as regulate lipid storage and release. We have extensively studied these mechanisms in the specialised energy-burning brown fat cells. Notably, we elucidated how LD-associated protein Cidea promotes LD enlargement to facilitate delivery of stored fat into mitochondria where they are burnt (or oxidised) on demand. Recently, we uncovered that Cidea is highly expressed in ESCs and in the early developing embryo. This finding was unexpected, as Cidea was previously considered to be almost exclusively expressed in specialised fat cells. We will now identify the full set of proteins that coat LDs in discrete ESC populations possessing 'supersized' or small LDs and different differentiation status to unveil the identity of molecules and sensing mechanisms that regulate lipid usage and functional LD-mitochondria interactions as ESCs progress towards differentiation.

Stem cells have unique regenerative abilities and now offer potential for treating diseases such as diabetes, heart disease and Alzheimer's disease. This study will bring exceptional insight into the unique metabolic properties of stem cells. This fundamental knowledge can be harnessed to develop novel stem-cell-targeted therapies, in which stem cells are selectively directed to self-renew or differentiate by manipulating their metabolic needs.

Technical Abstract:
Switches in energy metabolism have emerged as controllers of stem cell activity. In the blastocyst and derived pluripotent embryonic stem cells (ESCs), we uncovered that lipids accumulate in 'supersized' lipid droplets (LDs), which are mobilised upon implantation and differentiation. In this research, we will elucidate how lipid storage and timely release from LDs are regulated to impact on metabolic reprogramming and ESC developmental potency. How metabolic flux in stem cells directs cell fate transitions is a vital question in regenerative medicine that we will address by developing and applying advanced metabolomics, genomics and computational analysis together with innovative proteomic and imaging approaches through interdisciplinary collaboration. Remarkably, ESCs exhibit cell-to-cell heterogeneity in LD size and number. We isolated several clones that, under identical culture conditions, stably harbour small or large LDs, and possess distinct differentiation status (i.e. self-renewal vs. differentiation). Using these clones, we will investigate the existence of distinct metabolic states underlying ESC differentiation pathway. By studying the interplay between pluripotency transcriptional factors and metabolic regulators we will define the mechanisms regulating de novo lipid synthesis, storage and degradation via functional LD-mitochondria interactions eliciting a switch in lipid metabolism upon ESC differentiation. Ultimately we will validate the importance of these lipid signatures by combining gene editing and ESC functional assays both in vitro and in vivo. This fundamental research will uncover key principles governing pluripotency and metabolism plasticity to be applied in better control of differentiation and somatic cell reprogramming. We expect the impact of our findings to reach beyond the stem cell field, as tight coordination of stem cell activity is essential for successful development, organ homeostasis, tissue repair and disease including cancer.

Potential Impact:
The impact of this research will come from the advancement of knowledge in mechanisms of metabolic remodelling coordinating stem cell pluripotency and differentiation. This will have a major impact for diverse groups and applications in several ways:

1-Basic research underpinning health: the proposed research has potential medical implications in two main fields aimed at enhancing the quality of life and nation's health. (1) Regenerative medicine: the research will advance our understanding of how lipid storage impacts on pluripotent cell 'fitness' and developmental potency. Metabolic remodelling is directly relevant to reprogramming of somatic cells from patients and re-directing their fate to a desired cell type for stem cell-based therapies. (2) Reproductive medicine: elucidating how metabolic rewiring impacts the formation of pluripotent and extraembryonic tissues in the implanting embryo is of high significance. Understanding early changes in energy substrate usage will impact on standard culture procedures to maximise embryos viability for assisted reproduction. Maternal nutrition also has the potential to impact the foetus through changes in stem cell fate. Although the effects of maternal starvation on foetal stem cells are not known, in utero changes in metabolism are likely to impact on these cells and tissues that develop from them.

2-Innovative healthcare solutions: There are evident parallels in the metabolic pathways utilised by pluripotent and human cancer cells. Increased lipid droplet numbers have been described in tumours, however its functional significance remains to be determined. Our research will provide novel insights into the biology and dynamics of lipid droplets that will facilitate the future development of novel lipid droplet-targeted therapies applicable to cancer treatment and other diseases related to lipid storage (e.g. obesity).

3-Biotechnology and Industry: The proposed experimental approaches integrating information from metabolomics, genomics, proteomics and high-throughput functional screenings will disclose key metabolic pathways in stem cells that are potentially druggable, opening possibilities for collaboration with industrial partners.

4-Replacement of animal model: The stem cell systems and basic knowledge developed by this research will enable study of the metabolic rewiring implications in development in vitro and thus could be used to replace conventional animal-based models.

5-UK international competitiveness: this program of research will contribute to deliver the BBSRC's mission especially the strategic research priority 3 - Bioscience for Health as well as supporting the general UK strategy for excellence in stem cell research.

6-Education and training: This research contributes towards maintaining the standards of academic excellence at Imperial and Warwick. It will impact on our departments to offer educational opportunities for undergraduate and post-graduate student training. This is a multidisciplinary project involving groups with renowned expertise in developmental and stem cell biology, gene regulation, metabolism, lipid droplet proteomics, metabolic profiling, photonic microscopy and in vivo physiology. Researchers will receive specific scientific and technical training in partner labs as well as foster transferable professional, analytical and communication skills, facilitating their development and future prospects.

7-Science communication: the conceptual advances and material (e.g. pictures and illustrations) generated to present results will be used during outreach and fund raising activities with charities such as Genesis Research Trust. We will raise awareness of advances in the fields of reproductive biology, cell metabolism and regenerative medicine amongst diverse audiences. The pathway towards academic impact will be based on publications in open access high impact journals and presentations at international scientific meetings.