Trafficking, storage and timely release of lipids: unfolding the fundamental mechanisms underlying metabolic reprogramming in pluripotent stem cells.
Lead Research Organisation:
Imperial College London
Department Name: Surgery and Cancer
Abstract
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.
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 Summary
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.
Planned 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 extra-embryonic 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.
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 extra-embryonic 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.
Organisations
- Imperial College London (Lead Research Organisation)
- UNIVERSITY OF OXFORD (Collaboration)
- Babraham Institute (Collaboration)
- University of Ghent (Collaboration)
- University of Warwick (Collaboration)
- Radboud University Nijmegen (Collaboration)
- IMPERIAL COLLEGE LONDON (Collaboration)
- French National Institute of Agricultural Research (Collaboration)
- KING'S COLLEGE LONDON (Collaboration)
People |
ORCID iD |
| Veronique Azuara (Principal Investigator) |
Publications
Mau KHT
(2022)
Dynamic enlargement and mobilization of lipid droplets in pluripotent cells coordinate morphogenesis during mouse peri-implantation development.
in Nature communications
| Description | Key findings Mammalian pre-implantation embryos accumulate substantial amounts of lipids, which are stored into lipid droplets. Despite the fundamental roles of lipids in many cellular functions, the mechanisms underlying the building-up of stored lipids and its functional significance for the developing embryo and stem cells have remained largely unexplored. We discovered a previously unrecognised role for lipid droplets in orchestrating pluripotent tissue remodelling and revealed under-appreciated facets of lipid metabolism during peri-implantation embryo development. This work has now been published in Nature Communications (2022). • Dynamics of lipid storage coordinate morphogenesis during peri-implantation embryonic development (Nature Communications, 2022 - Dr Azuara's lab). Pluripotency is a transient property of embryonic cells existing from pre- to early post-implantation development. At the blastocyst stage, pluripotent epiblast progenitors (i.e., the future embryo) polarize to form rosette-like epithelial structures enclosing a central (pro-amniotic) lumen upon implantation. This morphogenesis event is fundamental for all subsequent development and hence the success or failure of a pregnancy. Concurrently, epiblast cells adopt distinct molecular states, which delineate the transition from naive (pre-implantation) to primed (post-implantation) pluripotency. While these molecular signatures are thought to prime or capacitate cells for differentiation, associated metabolic switches have long been regarded as survival adaptations prior to the establishment of placental exchange during embryo implantation. Recent studies suggest, however, that metabolism can play active roles in regulating cell fate transitions during early embryogenesis. Notably, we uncovered how intrinsic LD-associated mechanisms fundamentally link lipid metabolism to the control of morphogenesis in mouse peri-implantation embryos. Using three-dimensional (3D) cultures of embryonic stem cells (ESCs), in combination with embryo studies, we found that the sequential storage and mobilization of lipids, mediated by LDs, coordinate the formation of an apical lumen in epiblast rosettes. Mechanistically, we showed that pre-implantation blastocysts and pluripotent ESCs share the ability to transiently fuse and enlarge LDs under the tight control of the LD-surface protein CIDEA. Critically, CIDEA-mediated LD enlargement confers protection against promiscuous degradation by cytosolic lipases upon cell polarization. This permits the hydrolysis of stored lipids into lysosomes at the onset of lumen formation in a time-dependent manner. Abrogating CIDEA's function or lipophagy-dependent mobilization of LDs impairs lumenogenesis, demonstrating the functional significance of lipid storage during peri-implantation development. • Metabolic heterogeneity in embryonic stem cells underlies the exit from naïve pluripotency (manuscript in preparation - Dr Azuara's lab). We observed that ESCs exhibit considerable heterogeneity in size and number of lipid storage organelles (i.e., LDs) when cultured under self-renewing conditions (serum/LIF). To address whether this genuinely delineates distinct cell populations, we isolated ESC clones that stably harbour different LD morphologies and neutral lipid contents referred to as small LDs (s-LD) and large LDs (L-LD) clones. While both s-LD and L-LD clones retain the ability to form germ-layer derivatives in vitro, we found that L-LD clones exhibit higher index of spontaneous differentiation relative to s-LD clones. This corroborates with the declined expression of naïve pluripotency factors and induction of low transcript levels at primed differentiation genes, underlying distinct cellular and transcriptional states. Differential expression was also evident for metabolic genes including genes associated with lipid synthesis and storage, in agreement with the distinct neutral lipid contents of established ESC clones. To establish the existence of distinct metabolic states we used state-of-the-art metabolic assays including GC-MS based profiling, Seahorse, mitochondrial potential, and ROS production measurements in "naive-like" s-LD and "primed-like" L-LD ESC clones. We uncovered an anabolism-driven TCA enhancement in L-LD clones to support the demands of de novo lipid synthesis and building-up of stored lipids at the onset of ESC differentiation. Quantitative LC-MS confirmed rapid turnover of FAs and higher TG levels in L-LDs and furthermore revealed differential CE/TG ratios, suggesting a change in LD composition. Seahorse analyses established s-LD clones primarily utilise pyruvate for biosynthesis while L-LD clones rely on both pyruvate and glutamine, further highlighting metabolic diversity in ESCs. Using multi-photon fluorescence lifetime microscopy, we observed an increase in the fraction of bound NAD(P)H and therefore an increase in mean NAD(P)H lifetime as ESCs commit for differentiation. We confirmed the ability to read out NAD(P)H autofluorescence and GFP reporter simultaneously. This provides us with a useful readout for "metabolic switches" in ESC sub-populations for future investigations. • The interplay between metabolism and transcriptional network impacts on ESC developmental potency (work in progress - Dr Azuara's lab). We mapped the transcriptional profiles of individual s-LD and L-LD clones using bulk and single cell RNA-sequencing. S-LD and L-LD sub-populations could be reliably discriminated based on differential gene expression programs with clone clusters delineated by specific enrichment for pluripotency and cholesterol efflux processes (s-LD), active catabolic and anabolic pathways including Fatty acid oxidation, TCA, glycolysis, lipid synthesis and esterification, and cellular remodelling and differentiation (L-LD). Advanced computational analysis and visual data representation mapped these transcriptional and metabolic signatures onto a stem cell differentiation-trajectory, confirming the existence of metabolic switches at the onset of ESC differentiation. Survey of chromatin-bound proteins by proteomics revealed that detected naive pluripotency factors were not differentially bound in s-LD and L-LD clones. Interestingly, this analysis, however, highlighted factors that enforce a distinct epigenetic axis in L-LD clones. The further exploration of this outcome has been impeded by COVID and enforced lab closures, and thus will require further funding to be secured. Future work will most specifically focus on deciphering the interplay between identified factors and metabolic switches in ESCs with impact on cell fitness and developmental potency. • Investigating LD-associated proteomics in embryonic stem cells (manuscript in preparation - Professor Christian's lab). We have undertaken analysis of the LD proteome in ESCs. This has determined for the first time the key regulators of lipid storage in ESCs. COVID significantly impacted this part of the project. In addition to lab closures, the appointed PDRA was due to travel to our collaborator's lab (Pingsheng Liu) to undergo training in the isolation procedure. This was not possible due to COVID restrictions and with online advice we progressed in investigating the ESC's LS proteome. We fully optimising the protocol and have completed Mass Spectrometry with our collaborator Dr Boocock. The high-quality data has identified up to 1000 proteins associated with LDs in ESCs over several independent samples. This has led to the identification of novel LD-associated proteins as well as previously reported factors. One protein METTL3 has been detected in three separate preparations. This protein has not been reported on LDs previously but has been found to have essential roles in modifying RNA affecting the pluripotent status of ESCs. We have investigated the role of LDs in providing a site for inactive storage of METTL3 to maintain pluripotency. We also identified TPD family members, and this has been further investigated with a new collaboration. We have also used a new label-free technology to investigate the morphology and dynamics of lipid droplets in stem cells. The proteome profile also points to LDs functioning as a repository for proteins such as histones in ESCs. |
| Exploitation Route | Our project aims to advance fundamental understanding of how cellular metabolism controls cell state transitions in pluripotent stem cells and in the developing embryo with immediate relevance in regenerative and reproductive medicine. This project thus fulfils the remit of UKRI's mission to support basic research underpinning health. Our cohesive research program contributes towards maintaining world class UK bioscience by supporting Systems and multidisciplinary approaches that will enable others to develop innovative ideas beyond their specific area of scientific expertise. As part of our work was performed through international collaborations, the proposal also supports UKRI's strategic priority "International Partnerships". To maximise the impact of our research we will continue to engage and disseminate information to stakeholders across the full spectrum of relevant activities: professionals (scientists, graduate, and undergraduate students), enthusiastic public (self-motivated lay enthusiasts) and, general public (without a regular interest in life sciences). |
| Sectors | Education,Healthcare |
| URL | https://www.nature.com/articles/s41467-022-31323-2 |
| Description | UK economic competitiveness is facilitated by training and skills acquisition. The project has succeeded in providing an excellent training of the appointed PDRAs, KHTM and MPB, and a computational biologist (RdS, Imperial President's PhD scholar) working on this project. These researchers together with colleagues and collaborators developed the cellular, molecular and bioinformatic tools and methods used in the study. The wide range of skills acquired from the project (e.g., time management, problem solving, information technology, presentation skills, record keeping, budgeting, innovative thinking, and mentoring) are applicable to career progression both within and outside academia. Thus, these researchers have benefitted as individuals from the training, and the UK and international institutions where they apply their skills and training will benefit. KHTM is continuing his scientific career at the Francis Crick Institute, and MPB is working within science at Micregen's Secretomix. RdS is continuing to work in Dr Azuara's lab and developing outcomes from this work. With this project, we have contributed to increase understanding of science through engagement with intercalated medical BSc and postgraduate students in reproductive and developmental sciences. |
| First Year Of Impact | 2020 |
| Sector | Education,Manufacturing, including Industrial Biotechology |
| Impact Types | Economic |
| Description | A digital light-sheet microscope for the analysis of biological processes under almost natural conditions |
| Amount | £484,480 (GBP) |
| Funding ID | BB/T017929/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 11/2020 |
| End | 03/2021 |
| Description | Departmental Studentship (SL) - Functional Interplay between Lipid Trafficking and mTORC1 Signalling during Peri-implantation Development. |
| Amount | £100,000 (GBP) |
| Funding ID | F30503 |
| Organisation | Imperial College London |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 11/2021 |
| End | 10/2025 |
| Description | Faculty Strategic Research Funding Application Form - Collaboration Kick-Start |
| Amount | £20,000 (GBP) |
| Organisation | Imperial College London |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 12/2017 |
| End | 03/2018 |
| Description | Functional Interplay between Lipid Trafficking and mTORC1 Signalling during Peri-implantation Development |
| Amount | £10,600 (GBP) |
| Funding ID | GEN126-PSM158 |
| Organisation | Genesis Research Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 08/2021 |
| End | 07/2022 |
| Description | Genesis Research Trust - Supplement Studentship Funding 2017 |
| Amount | £7,500 (GBP) |
| Funding ID | P54998 (DK) |
| Organisation | Genesis Research Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 10/2017 |
| End | 09/2018 |
| Description | Genesis Research Trust - Supplement Studentship funding (SL) |
| Amount | £58,000 (GBP) |
| Funding ID | GEN136- PA3494 |
| Organisation | Genesis Research Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 03/2022 |
| End | 10/2025 |
| Description | PhD studentship for KHTM |
| Amount | £113,000 (GBP) |
| Funding ID | DPCHP3440 |
| Organisation | Genesis Research Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 10/2016 |
| End | 04/2020 |
| Description | President PhD Scholarship |
| Amount | £100,000 (GBP) |
| Funding ID | RdS |
| Organisation | Imperial College London |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 08/2018 |
| End | 07/2022 |
| Title | Elucidating the role of CIDEA in mouse embryonic stem cells |
| Description | * Generation of ESCs (1) overexpressing CIDEA and (2) depleted for CIDEA using (2.1) shRNA vectors and (2.2) CRISPR-Cas9 gene editing. * Generation of CIDEA knockout (KO) and control ESCs stably expressing LifeACT-RFP. * Generation of "rescued" CIDEA (KO) with wild-type and point-mutated CIDEA forms. |
| Type Of Material | Cell line |
| Year Produced | 2022 |
| Provided To Others? | Yes |
| Impact | We established that the protein CIDEA, found abundantly in adipocytes, is required to fulfil the full process of lipid droplet fusion and enlargement in mouse embryonic stem cells under self-renewing and differentiation conditions (Nature Communications, 2022). |
| URL | https://www.nature.com/articles/s41467-022-31323-2 |
| Title | Investigating the role of AMPK in modulating the metabolic state of pluripotent embryonic stem cells |
| Description | Generation of mouse embryonic stem cells that stably expressed FRET AMPK biosensors. |
| Type Of Material | Cell line |
| Year Produced | 2019 |
| Provided To Others? | No |
| Impact | Multi-disciplinary approach - embryonic stem cell (ESC) biology and FRET-based biosensors (imaging). We established the feasibility of quantitatively monitoring in real time AMPK activity in ESCs under different treatments. Future planning is to combine this approach with newly described 3D stem cell-based organoid systems to explore the activity of AMPK (and other key signalling pathways) during the transition from pre- to post-implantation embryo development in vitro (3R replacement methods). |
| Title | Investigating the role of lipid droplets in embryonic stem cells |
| Description | * Isolation of mouse embryonic stem cell clones stably harbouring different neutral lipid contents. * Generation of ESCs over-expressing ZSCAN4. |
| Type Of Material | Cell line |
| Year Produced | 2014 |
| Provided To Others? | No |
| Impact | We observed that mouse embryonic stem cells (ESCs) exhibit considerable heterogeneity in size and number of lipid storage organelles (i.e., lipid droplets) when cultured under self-renewing conditions (serum/LIF). To address whether this genuinely delineates distinct cell populations, we isolated ESC clones that stably harbour different LD morphologies and neutral lipid content referred to as small LDs (s-LD) and large LDs (L-LD) clones. We observed that ZSCAN4 is preferentially induced in ESCs harbouring higher capacity to synthesise and store lipids as lipid droplets. In contrast, ectopically expressing ZSCAN4 does not impact on the metabolic state of ESCs. |
| Title | Profiling of lipids and metabolites collected from embryonic stem cell populations harbouring different neutral lipid contents and differentiation status.. |
| Description | * Generation and analysis of GC-MS based profiling of metabolites in embryonic stem cell populations harbouring different neutral lipid contents and differentiation status as well as C13 glucose GC-MS based analysis of metabolic fluxes (e.g., de novo lipid synthesis) in the same populations. * Generation and analysis of LC-MS based profiling of triglycerides, cholesterol esters, free fatty acids and free cholesterol in the same populations. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2018 |
| Provided To Others? | No |
| Impact | Our GC-MS analyses combined with Seahorse measurements and other assays uncovered the existence of an anabolism-driven TCA enhancement in embryonic stem cells at the onset of differentiation. This work forms part of a study being currently completed and organised for publication. Additionally, our quantitative LC-MS based measurements of triglycerides and cholesterol esters highlighted differences in the composition of lipid droplets amongst embryonic stem cells grown under serum and LIF conditions. |
| Title | RNA-sequencing datasets collected from CIDEA over-expressing and control embryonic stem cells. |
| Description | Transcriptional profiling of CIDEA over-expressing and control mouse embryonic stem cells under self-renewing conditions (serum/LIF). |
| Type Of Material | Database/Collection of data |
| Year Produced | 2021 |
| Provided To Others? | No |
| Impact | Data are being analysed now. |
| Title | RNA-sequencing datasets collected from CIDEA-depleted and control embryonic stem cells prior to and upon 3D differentiation. |
| Description | Transcriptional profiling of CIDEA-depleted and control embryonic stem cells prior to and upon 3D differentiation. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2022 |
| Provided To Others? | Yes |
| Impact | Our results revealed that CIDEA-depleted ESCs transit normally from naive to primed pluripotency as examined at the transcriptional level (revised manuscript under review with Nature Communications). Additional data mining still on-going. |
| URL | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE165563 |
| Title | Transcriptomics and proteomics datasets collected from embryonic stem cell populations harbouring different neutral lipid contents and differentiation status. |
| Description | * Generation of bulk and single-cell RNA-sequencing datasets from embryonic stem cell clones harbouring different lipid contents and differentiation status. Datasets have been generated; analyses are still on-going. * Generation and analysis of CheP datasets from the same populations. |
| Type Of Material | Database/Collection of data |
| Year Produced | 2018 |
| Provided To Others? | No |
| Impact | Our RNA-sequencing analyses uncovered the existence of metabolic switches at the onset of embryonic stem cell differentiation. Data mining still ongoing. Our CheP analyses identified a distinct metabolic-epigenetic axis in embryonic stem cells harbouring higher lipid droplet content coinciding with the onset of differentiation. |
| Description | Advanced computational analysis and visual data representation |
| Organisation | Imperial College London |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | * Investigating the mechanisms regulating the emergence of different functional metabolic states in pluripotent stem cells; BBSRC grant application awarded in October 2016. * Generation of RNA-seq datasets collected from embryonic stem cell (ESC) subpopulations harbouring different lipid droplet morphologies (small and large). * Generation of RNA-seq datasets collected from ESC-based 3D spheroids at different differentiation time-points. |
| Collaborator Contribution | Biomathematics support; integrative analysis of population-based RNA-seq datasets; named as collaborator on BBSRC grant application investigating metabolic switches at the exit of pluripotency - awarded in October 2016. |
| Impact | Multi-disciplinary collaboration - embryonic stem cell (ESC) biology and biomathematics. Advanced computational analysis and visual data representation allowed us to map the transcriptional and metabolic signatures of ESC sub-populations onto a differentiation-trajectory, confirming the existence of metabolic switches at the onset of ESC differentiation. This work forms part of a study being currently completed and organised for publication. |
| Start Year | 2017 |
| Description | Establishing embryonic stem cell lines with an AMPK FRET biosensor. |
| Organisation | Imperial College London |
| Department | MRC London Institute of Medical Sciences |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Investigating the mechanisms regulating the emergence of different functional metabolic states in pluripotent embryonic stem cells. |
| Collaborator Contribution | Provision of plasmids coding for T2AMPKAR-NES and T2AMPKAR-T391A-NES; expertise in exploiting genetically expressed FRET biosensors including the AMPK biosensor. |
| Impact | Multi-disciplinary collaboration - embryonic stem cell (ESC) biology and FRET-based biosensors (imaging). We established the feasibility of quantitatively monitoring in real time AMPK activity in ESCs. |
| Start Year | 2017 |
| Description | Establishing embryonic stem cell lines with an AMPK FRET biosensor. |
| Organisation | Imperial College London |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Investigating the mechanisms regulating the emergence of different functional metabolic states in pluripotent embryonic stem cells. |
| Collaborator Contribution | Provision of plasmids coding for T2AMPKAR-NES and T2AMPKAR-T391A-NES; expertise in exploiting genetically expressed FRET biosensors including the AMPK biosensor. |
| Impact | Multi-disciplinary collaboration - embryonic stem cell (ESC) biology and FRET-based biosensors (imaging). We established the feasibility of quantitatively monitoring in real time AMPK activity in ESCs. |
| Start Year | 2017 |
| Description | Exploiting photonics imaging approaches to monitor metabolic switches in embryonic stem cells. |
| Organisation | Imperial College London |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Investigating the mechanisms regulating the emergence of different functional metabolic states in pluripotent embryonic stem cells; BBSRC grant application awarded in October 2016. |
| Collaborator Contribution | The Photonics group at Imperial College has long-standing expertise in fluorescence microscopy including automated multi-well plate FLIM of cellular autofluorescence to read out metabolic stem cell states; named collaborator on a BBSRC grant application awarded in October 2016. |
| Impact | Multi-disciplinary collaboration - embryonic stem cell biology and physics (imaging). Using multiphoton fluorescence lifetime microscopy, we observed an increase in the fraction of bound NAD(P)H and therefore an increase in mean NAD(P)H lifetime as ESCs commit for differentiation. We confirmed the ability to read out NAD(P)H autofluorescence and GFP reporter simultaneously. This provides us with a useful readout for "metabolic switches" in ESC sub-populations for future investigations. |
| Start Year | 2017 |
| Description | Investigating the functional importance of CIDEA-mediated lipid droplet enlargement in the developing early embryo |
| Organisation | French National Institute of Agricultural Research |
| Country | France |
| Sector | Academic/University |
| PI Contribution | Generation and characterisation of CIDEA knockout (KO) ESCs via CRISPR-Cas9 gene editing. Generation of CIDEA KO and control ESCs stably expressing LifeACT-RFP. |
| Collaborator Contribution | Generation and analysis of chimaeric embryos; providing training in manipulating and culturing pre-implantation embryos. |
| Impact | Multi-disciplinary collaboration - developmental biology and lipid metabolism. We established that abrogating CIDEA's function in the developing pluripotent epiblast leads to morphogenesis defects in early post-implantation chimeric embryos (Nature Communications, 2022). |
| Start Year | 2018 |
| Description | Lipid droplet dynamics in pluripotent stem cells |
| Organisation | University of Warwick |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Joint BBSRC grant application investigating metabolic switches at the exit of pluripotency - awarded in October 2016. |
| Collaborator Contribution | Provide expertise in lipid droplet biology in adipocytes; exchange of reagents and technologies. |
| Impact | Multi-disciplinary collaboration - embryonic stem cell and lipid droplet biology. We demonstrated that the protein CIDEA, found abundantly in adipocytes, is required to fulfil the full process of lipid droplet enlargement in embryonic stem cells under self-renewing and differentiation conditions. We have established that CIDEA-mediated LD enlargement confers protection against promiscuous degradation by cytosolic lipases, permitting the lysosomal hydrolysis of the organelles. This process has a key role in early development at the onset of lumen formation in the implanting epiblast tissue. Impairing the function of CIDEA or inhibiting lipophagy-dependent LD mobilisation impairs morphogenesis of pluripotent cells, demonstrating the significance of lipid storage and release from LDs during peri-implantation development (Nature Communications, 2022). We isolated ESC clones that stably harbour different LD morphologies and neutral lipid contents referred to as small LDs (s-LD) and large LDs (L-LD) clones. While both s-LD and L-LD clones retain the ability to form germ-layer derivatives in vitro, we found that L-LD clones exhibit lower plating efficiencies (self-renewal) relative to s-LD clones. This corroborates with the declined expression of naive pluripotency factors and induction of low transcript levels at primed differentiation genes, underlying distinct cellular and transcriptional states. Differential expression was also evident for metabolic genes associated with lipid synthesis and storage, in agreement with the distinct neutral lipid content of ESC clones (manuscript in preparation). We have undertaken analysis of the LD proteome in ESCs with different metabolic and differentiation status, and identified proteins that have been further investigated (manuscript in preparation). |
| Start Year | 2013 |
| Description | Lipidomics in embryonic stem cells (ESCs) and ESC-derived 3D spheroids. |
| Organisation | King's College London |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Investigating lipid dynamics in pluripotent stem cells upon formation of 3D spheroids and morphogenesis. |
| Collaborator Contribution | LC-MS profiling of lipids in embryonic stem cells (ESCs) and upon differentiation of ESC-derived 3D spheroids at different time-points. |
| Impact | Our results complement previous analysis of the dynamics of lipid droplets in ESCs transitioning from naive to primed pluripotency upon differentiation and morphogenesis of ESC-derived 3D spheroids. |
| Start Year | 2021 |
| Description | Lipidomics in pluripotent embryonic stem cells. |
| Organisation | Babraham Institute |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Mapping of metabolic profiles in mouse embryonic stem cells with different lipid content and differentiation status. |
| Collaborator Contribution | LC-MS profiling of triglycerides, cholesterol esters, fatty acids, cholesterol, phospholipids (phosphatidylcholine) in embryonic stem cell subpopulations with different metabolic states. Partners provide us with expertise in LC-MS-based lipidomics; data generation and analysis. |
| Impact | Multi-disciplinary collaboration - embryonic stem cell biology and lipid metabolism (lipidomics). Quantitative LC-MS confirmed rapid turnover of free fatty acid and higher triglyceride levels in embryonic stem cells harbouring high neutral lipid content and furthermore revealed differential cholesterol ester/triglyceride ratios when compared to cells with lower neutral lipid contents, suggesting a change in LD composition. This work forms part of a study being currently completed and organised for publication. |
| Start Year | 2020 |
| Description | Mass Spectrometry based profiling of histone modifications in pluripotent stem cells. |
| Organisation | University of Ghent |
| Country | Belgium |
| Sector | Academic/University |
| PI Contribution | Investigating the interplay between metabolic and epigenetic regulation in pluripotent stem cells. |
| Collaborator Contribution | Proteomic profiling of histone post-translational modifications in ESCs showing different metabolic and differentiation status. Collaborators provide us with expertise in hPTM profiling; data generation and analysis. |
| Impact | Data being now analysed. |
| Start Year | 2022 |
| Description | Metabolomics in pluripotent stem cells |
| Organisation | Imperial College London |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Mapping of metabolic flux in mouse embryonic stem cells with different lipid content and differentiation status; co-supervision of PhD student (DK, ICL President PhD Scholar); BBSRC grant application investigating metabolic switches at the exit of naive pluripotency- awarded in October 2016. |
| Collaborator Contribution | Co-supervision of PhD student (DK, ICL President PhD scholar); exchange of reagents and technologies; named as collaborator on BBSRC grant application investigating metabolic switches at the exit of naive pluripotency- awarded in October 2016. |
| Impact | Multi-disciplinary collaboration - stem cell biology and metabolism. Our collaborative GC-MS analyses combined with Seahorse measurements and other assays uncovered the existence of an anabolism-driven TCA enhancement in embryonic stem cells at the onset of differentiation. This work forms part of a study being currently completed and organised for publication. |
| Start Year | 2014 |
| Description | Monitoring lipid trafficking and storage in pluripotent embryonic stem cells |
| Organisation | Imperial College London |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Evaluating de novo triglyceride esterification activity in embryonic set cell (ESC) subpopulations with different metabolic states using BODIPY 558/668 fatty acid probe and thin layer chromatography (TLC). Setting up pulse-chase labelling protocol using BODIPY 558/668 fatty acid probe in ESCs under self-renewing and differentiation conditions. |
| Collaborator Contribution | Provide expertise in thin layer chromatography (TLC) and data analysis. |
| Impact | Multi-disciplinary collaboration - embryonic stem cell biology and lipid metabolism. Analysis provided evidence for active fatty acid esterification into neutral lipids in embryonic stem cells. |
| Start Year | 2019 |
| Description | Monitoring the behaviour, size and number of lipid droplets in peri-implantation embryos |
| Organisation | University of Oxford |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | BBSRC grant application investigating metabolic switches in stem cells and in the embryo at implantation time - awarded October 2016. |
| Collaborator Contribution | exchange of reagents and technologies; training on pre and post-implantation embryo dissection and handling (e.g. embryo staining and imaging data treatment); Named collaborator on a Named collaborator on BBSRC grant application investigating metabolic switches in stem cells and in the embryo at implantation time - awarded October 2016. |
| Impact | Multi-disciplinary collaboration - developmental biology and lipid metabolism (Nature Communications, 2022). We established that (1) Cidea expression is induced during the transition from the morula to the blastocyst stage, coinciding with maximum activity of lipid droplet fusion and enlargement as observed both in cultured and freshly harvested blastocysts. (2) Cidea transcript sharply declined in the implanting blastocyst (E4.5-4.75) and early post-implantation embryo (E5.5), prompting us to extend our analysis of lipid droplet sizes and numbers in peri-implantation embryos. (3) Enlarged lipid droplets are selectively mobilised in the epiblast of the implanting blastocyst (E4.5-4.75) and are not regained post-implantation (E5.5). At this stage, lipid droplets are smaller and more homogeneous in size, in agreement with Cidea silencing. |
| Start Year | 2015 |
| Description | Monitoring the dynamics of lipid droplet enlargement and mobilisation in pre-implantation embryos. |
| Organisation | Imperial College London |
| Department | MRC London Institute of Medical Sciences |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Examining the clustering and fusion of lipid droplets from the morula to the blastocyst stage upon embryo culturing; evaluating the importance of autophagosome/lipophagy in the process of lipid droplet mobilization in pluripotent cells. |
| Collaborator Contribution | Provided training on morula culture up to the blastocyst stage; performed staining (1) for BODIPY/E-Cadherin/DAPI, (2) BODIPY/LC3/DAPI and (3) ZSCAN4/BODIPY/DAPI on pre-implantation embryos. |
| Impact | Multi-disciplinary collaboration - developmental biology and lipid metabolism . Results showed that mouse early embryos intrinsically acquire the ability to enlarge lipid droplet during the transition from the morula to the early blastocyst stage, and further indicated a critical role for lipophagy-mediated mobilization of stored lipids in the late blastocyst at the onset of implantation (Nature Communications, 2022). |
| Start Year | 2019 |
| Description | Single cell RNA-sequencing and proteomics in pluripotent mouse embryonic stem cells. |
| Organisation | Radboud University Nijmegen |
| Country | Netherlands |
| Sector | Academic/University |
| PI Contribution | Investigating transcriptional and metabolic cross-talks at the exit from naive pluripotency using ESC subpopulations showing different metabolic and differentiation status. |
| Collaborator Contribution | Single cell RNA-sequencing (sc-RNA-seq) and proteomic profiling of the chromatin landscape by chromatin enrichment for proteomics (ChEP) in ESC subpopulations showing different metabolic and differentiation status. Collaborators provide us with expertise in sc-RNA-seq and ChEP; data generation and analysis. |
| Impact | Multi-disciplinary collaboration - embryonic stem cell biology, transcriptomics, and proteomics. Data are being analysed. |
| Start Year | 2020 |
| Description | Genesis Research Trust |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | Yes |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | Talks/presentations sparked questions and discussion afterwards; information provided on GRT website; acknowledgment of the GRT in scientific publications. Increased awareness of our research efforts and impact. |
| Year(s) Of Engagement Activity | 2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019 |