Trafficking, storage and timely release of lipids: unfolding the fundamental mechanisms underlying metabolic reprogramming in pluripotent stem cells.
Lead Research Organisation:
Nottingham Trent University
Department Name: School of Science & Technology
Abstract
Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.
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 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.
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.
People |
ORCID iD |
| Mark Christian (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 | 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 an unexpected role for lipid droplets in orchestrating embryonic tissue remodelling and revealed underappreciated facets of lipid metabolism in peri-implantation development. This work is has now been published in Nature Communications. • The dynamics of lipid storage coordinate morphogenesis during peri-implantation embryonic development (revised manuscript under review - 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 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 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 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 "naïve-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 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. • 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 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 naive pluripotency factors were not differentially bound in s-LD and L-LD clones. Interestingly, this analysis highlighted that a new factor enforcing a distinct epigenetic axis in L-LD clones. Progress in this analysis has been largely delayed by COVID and enforced lab closures. Future work will focus on exploring the interplay between this epigenetic regulator and metabolic switches in ESCs. • Investigating LD-associated proteomics in embryonic stem cells (manuscript in preparation - Dr 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 the 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,Pharmaceuticals and Medical Biotechnology |
| Description | UK economic competitiveness is facilitated by training and skills acquisition. The project has succeeded in providing an excellent training of the appointed Post Docs Jinous Samavat, Rafaella Lucciola, and Merell Billacura. These researchers developed the molecular techniques 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. Rafaella is continuing her scientific career in the USA and Jinous is has commenced working within science at Orbit Discovery. Merell worked in my lab and developed outcomes from this work. With this project, we have contributed to increase understanding of science through engagement with postgraduate students. The international multidisciplinary collaboration network (UK-China) established through this project has served to enhance the research capacity and skills base. The management skills involved in coordinating a multicentre collaboration are important on a personal development perspective and are applicable to economic advancement. The network has served to raise the profile of UK research with a group of influential scientists and policy makers within their different fields. The impact of COVID has led to new approaches to address research questions. |
| First Year Of Impact | 2021 |
| Sector | Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Economic |
| Title | Lipid droplet isolation from stem cells |
| Description | We have applied nitrogen cavitation cell disruption in the lysis of cultured mouse embryonic stem cells in conjunction with serial ultracentrifugation to isolate and purify the lipid droplet fraction. |
| Type Of Material | Biological samples |
| Year Produced | 2019 |
| Provided To Others? | No |
| Impact | This method allows us to generate lipid droplet fractions for analysis and identification of the lipid droplet proteome. |
| Description | Determination of the stem cell lipid droplet proteome |
| Organisation | Chinese Academy of Sciences |
| Country | China |
| Sector | Public |
| PI Contribution | Experimental planning for investigating the lipid droplet proteome in mouse embryonic stem cells |
| Collaborator Contribution | Pingsheng Liu and his team will purify lipid droplets from cells and mass spectrometry will be performed. |
| Impact | None |
| Start Year | 2017 |
| Description | Investigation of Mettl3 in lipid droplets |
| Organisation | National Institutes of Health (NIH) |
| Country | United States |
| Sector | Public |
| PI Contribution | Identification of Mettl3 as a lipid droplet-associated protein in mouse embryonic stem cells. |
| Collaborator Contribution | Access to expertise and research tools to assess the subcellular localisation of Mettl3 provided by Dr Pedro Batista. |
| Impact | N/A |
| Start Year | 2021 |
| Description | Investigation of TPD proteins in Lipid droplets with Steve Royle |
| Organisation | University of Warwick |
| Department | Warwick Medical School |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Investigated the location TPD52, TPD53, and TPD54 on lipid droplets in mouse embryonic stem cells |
| Collaborator Contribution | Steve Royle provided expression constructs and investigated location of TPD52, TPD53, and TPD54 on lipid droplets in Hela cells |
| Impact | Identified association of TPD52 and TPD54 on lipid droplets. This finding has not been published. |
| Start Year | 2021 |
| Description | Lipidomics |
| Organisation | Babraham Institute |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | We have generated the samples from ESCs for lipidomic analysis |
| Collaborator Contribution | The late Michael Wakelam and his group, including Bebiana Da Costa Sousa and Andrea Lopez, undertook quantitative LC-MS of ESCs to determine differences in lipid profiles associated with different lipid droplet size. This is an ongoing collaboration. |
| Impact | The data from this work has revealed that there are key differences in the levels of triacylglycerol and degree of double bond saturation in cells that have small compared to large lipid droplets. In addition, the levels of key phosphlipids, including phosphatidylcholine were increased in stem cells presenting small lipid droplets. |
| Start Year | 2019 |
| Description | Mass Spectrometry of Stem Cell Lipid Droplets |
| Organisation | Nottingham Trent University |
| Department | School of Science and Technology |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | My researcher has prepared a protein sample from cultured mouse embryonic stem cells. |
| Collaborator Contribution | Dr David Boocock has processed the samples for Mass Spectrometry and analysed the data. This is enabling us to understand which proteins are associated with the lipid droplets found in mouse embryonic stem cells. |
| Impact | N/A |
| Start Year | 2020 |
| Description | Single cell RNAseq of ESCs with small and large lipid droplets |
| Organisation | Radboud University Nijmegen |
| Country | Netherlands |
| Sector | Academic/University |
| PI Contribution | Selection and culture of ESC population with different lipid droplet morphologies |
| Collaborator Contribution | Undertook single cell RNAseq to determine different transcriptional heterogeneity of stem cell populations. |
| Impact | Datasets for single cell transcriptomes for distinct embryonic stem cell populations |
| Start Year | 2021 |
| Description | Presentation and Discussion of the technologies used to investigate lipid droplets |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | The staining and visualisation of lipid droplets in mouse embryonic stem cells was demonstrated to Postgraduate students. This included practical and analytical investigations. The student cohort showed great interest in the procedure and the importance of lipid droplets in stem cell biology. Students followed up with further discussions related to potential new investigations. |
| Year(s) Of Engagement Activity | 2020 |
| Description | Student Engagement and Training Activity |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Microscopy of lipid droplets. Students undertook hands-on training in the lipid droplet staining. This led to increased student engagement and interest in the importance of lipid droplets in developmental events. The technican team supporting this training also were highly engaged in this event. |
| Year(s) Of Engagement Activity | 2021 |