Periodic Reporting for period 4 - MetEpiStem (Dissecting the crosstalk between metabolism and transcriptional regulation in pluripotent stem cells.)
Reporting period: 2022-04-01 to 2024-03-31
Pluripotent stem cells (PSCs) have the capacity to give rise to all cells of our body, such as neurons or cells of our skin or blood. They also expand easily in our laboratories and it is possible, via a process named “reprogramming”, to make personalised PSCs.
From a small skin biopsy or a blood sample of a patient, affected, for instance, by a neurodegenerative disease, it is now possible to obtain PSCs and to differentiate them into neurons that will display defects found also in the patient. In other words, PSCs give the unprecedented possibility to study human diseases and test drugs in a dish.
For all these reasons, PSCs have the potential to revolutionise modern medicine.
However, our understanding of the behaviours of human PSCs is partial, we need to better understand how they differentiate, in order to make the process more efficient.
For example, in some cases, the neurons obtained are not fully matured as those found in the adult brain.
The process of reprogramming could also be optimised, both in terms of efficiency and costs and in terms of obtaining PSCs of better quality.
Metabolism is defined as a large set of chemical reactions that maintain a cell alive, or an entire organism, by providing energy, by providing the “building blocks” of the cell and by eliminating the waste products.
Metabolism has been for a long time considered as a static component of cellular physiology, a sort of “cellular housekeeping”. However, we increasingly appreciate how metabolism has the capacity to dynamically affect the behaviour of the cell.
Thus, our overall objectives have been understanding the role of metabolism in the process of PSC differentiation and in reprogramming, in order to make these processes more efficient.
In the context of PSC differentiation, we started from the observation that inactivation of some metabolic pathways affected the rate of differentiation. We then identified specific metabolites, whose levels control the differentiation process, making it faster or slower. We also investigated the molecular mechanism by which these metabolites act, which is by making some portions of the genome more or less active.
We also investigated the process of reprogramming, starting from the observation that a mild inhibition of energy production was enough to completely block the generation of PSCs. We have been able to significantly increase reprogramming efficiency, by using computational tools, which allowed us to simulate reprogramming under several different conditions and then perform real experiments only under the most promising conditions. We further optimised reprogramming by miniaturising the process, increasing the efficiency while reducing the amount of reagents needed.
Finally, we demonstrated that the mechanisms we are studying in PSCs are also relevant for other stem cells. The stem cells that constantly renew our intestine are under the same metabolic control we identified in PSCs, further demonstrating how metabolism has the potential to control key aspects of our physiology.
From a small skin biopsy or a blood sample of a patient, affected, for instance, by a neurodegenerative disease, it is now possible to obtain PSCs and to differentiate them into neurons that will display defects found also in the patient. In other words, PSCs give the unprecedented possibility to study human diseases and test drugs in a dish.
For all these reasons, PSCs have the potential to revolutionise modern medicine.
However, our understanding of the behaviours of human PSCs is partial, we need to better understand how they differentiate, in order to make the process more efficient.
For example, in some cases, the neurons obtained are not fully matured as those found in the adult brain.
The process of reprogramming could also be optimised, both in terms of efficiency and costs and in terms of obtaining PSCs of better quality.
Metabolism is defined as a large set of chemical reactions that maintain a cell alive, or an entire organism, by providing energy, by providing the “building blocks” of the cell and by eliminating the waste products.
Metabolism has been for a long time considered as a static component of cellular physiology, a sort of “cellular housekeeping”. However, we increasingly appreciate how metabolism has the capacity to dynamically affect the behaviour of the cell.
Thus, our overall objectives have been understanding the role of metabolism in the process of PSC differentiation and in reprogramming, in order to make these processes more efficient.
In the context of PSC differentiation, we started from the observation that inactivation of some metabolic pathways affected the rate of differentiation. We then identified specific metabolites, whose levels control the differentiation process, making it faster or slower. We also investigated the molecular mechanism by which these metabolites act, which is by making some portions of the genome more or less active.
We also investigated the process of reprogramming, starting from the observation that a mild inhibition of energy production was enough to completely block the generation of PSCs. We have been able to significantly increase reprogramming efficiency, by using computational tools, which allowed us to simulate reprogramming under several different conditions and then perform real experiments only under the most promising conditions. We further optimised reprogramming by miniaturising the process, increasing the efficiency while reducing the amount of reagents needed.
Finally, we demonstrated that the mechanisms we are studying in PSCs are also relevant for other stem cells. The stem cells that constantly renew our intestine are under the same metabolic control we identified in PSCs, further demonstrating how metabolism has the potential to control key aspects of our physiology.
We have identified two mechanisms whereby a single metabolite controls the activity of the DNA of the stem cell, ultimately affecting how fast stem cells proliferate and also their differentiation propensity.
We have also verified that such metabolic control of stem cell proliferation occurs also in stem cells of the intestine.
We have identified an additional mechanism linking metabolism to oxygen-sensing, showing its involvement in the formation of new blood vessels and activity of blood cells.
We generated 3D models of PSC differentiation and observed coherent changes in cell metabolism.
We have generated more efficient protocols for reprogramming by using computational approaches.
We also used novel bioengineering approaches for the generation of stem cells from human cells, called microfluidics, making the process more efficient also when starting from just a few hundred cells.
We have developed a novel reprogramming protocol, based on epigenetic and metabolic perturbations, conferring extended differentiation capacity to PSCs, allowing them to form cells of the human placenta.
We have developed a set of computational tools that can be used to make reprogramming and differentiation more efficient, to test the quality of PSCs generated and for the analysis of the gene activity of single PSCs.
Overall, our results identified new links between metabolism and DNA activity (i.e. epigenetics).
At the same time, our understanding of the PSC differentiation process was significantly expanded.
We also made significant improvements in the generation of PSCs via reprogramming and their characterisation, thanks to a set of novel experimental and computational approaches.
These results have several implications for society, as a better understanding of PSC differentiation will favour their use in translation settings.
Similarly, generating PSCs of higher quality, with expanded differentiation capacities, will profoundly help using them in translational and commercial settings.
The dissemination of results is crucial to me and it is achieved by the organisation of several meetings (national and international) about metabolism and stem cell research, but also via a lab website and social media like Twitter/X, Facebook and LinkedIn. All our results are also poster on pre-prints servers like bioRxiv and made open-access after publication.
The exploitation of our results includes new research projects on the role of metabolism in the formation of human placenta, which has been recently supported by a Marie Skłodowska-Curie Actions Postdoctoral fellowship. We have developed new 3D models of differentiation which we intend to use to test the impact of metabolism, nutrients and chemicals on stem cell differentiation. Finally, several results obtained in PSCs might be relevant also in the context of adult stem cells, both under physiological and pathological conditions, so new lines of investigation will be opened.
We have also verified that such metabolic control of stem cell proliferation occurs also in stem cells of the intestine.
We have identified an additional mechanism linking metabolism to oxygen-sensing, showing its involvement in the formation of new blood vessels and activity of blood cells.
We generated 3D models of PSC differentiation and observed coherent changes in cell metabolism.
We have generated more efficient protocols for reprogramming by using computational approaches.
We also used novel bioengineering approaches for the generation of stem cells from human cells, called microfluidics, making the process more efficient also when starting from just a few hundred cells.
We have developed a novel reprogramming protocol, based on epigenetic and metabolic perturbations, conferring extended differentiation capacity to PSCs, allowing them to form cells of the human placenta.
We have developed a set of computational tools that can be used to make reprogramming and differentiation more efficient, to test the quality of PSCs generated and for the analysis of the gene activity of single PSCs.
Overall, our results identified new links between metabolism and DNA activity (i.e. epigenetics).
At the same time, our understanding of the PSC differentiation process was significantly expanded.
We also made significant improvements in the generation of PSCs via reprogramming and their characterisation, thanks to a set of novel experimental and computational approaches.
These results have several implications for society, as a better understanding of PSC differentiation will favour their use in translation settings.
Similarly, generating PSCs of higher quality, with expanded differentiation capacities, will profoundly help using them in translational and commercial settings.
The dissemination of results is crucial to me and it is achieved by the organisation of several meetings (national and international) about metabolism and stem cell research, but also via a lab website and social media like Twitter/X, Facebook and LinkedIn. All our results are also poster on pre-prints servers like bioRxiv and made open-access after publication.
The exploitation of our results includes new research projects on the role of metabolism in the formation of human placenta, which has been recently supported by a Marie Skłodowska-Curie Actions Postdoctoral fellowship. We have developed new 3D models of differentiation which we intend to use to test the impact of metabolism, nutrients and chemicals on stem cell differentiation. Finally, several results obtained in PSCs might be relevant also in the context of adult stem cells, both under physiological and pathological conditions, so new lines of investigation will be opened.
Compared to the state of the art at the beginning of the project 2017, we have identified several novel mechanisms whereby metabolism controls pluripotent stem cell behaviour, demonstrating their relevance also for adult stem cells.
We also generated novel 3D models for the differentiation of pluripotent stem cells and significantly improved the reprogramming process, by making it more efficient and by generating stem cells with expanded differentiation potential.
We also generated novel 3D models for the differentiation of pluripotent stem cells and significantly improved the reprogramming process, by making it more efficient and by generating stem cells with expanded differentiation potential.