Periodic Reporting for period 1 - PLURImet (Building a metabolic atlas of human pluripotency: dissecting the interplay between pluripotency and metabolism in human stem cells.)
Período documentado: 2023-04-01 hasta 2025-03-31
The naive inner cell mass of the human embryo is characterised by unlimited developmental plasticity as it gives rise to all embryonic and extraembryonic lineages. This state can be recapitulated in vitro using naive human pluripotent stem cells (hPSCs), a new prominent tool for basic and translational research with the potential for significant impact on health and economics. This state differs from the well-known and widely used conventional pluripotent state (primed hPSCs), which resembles a later stage of development primed toward the embryonic lineages. In vitro models are allowing significant progress in understanding the mechanistic roles of transcriptional factors and signalling pathways in pluripotency. However, we still do not know how naive hPSCs’ striking plasticity is regulated, also due to technical limitations in the manipulation of this recently discovered cell type.
Understanding naive hPSCs requirements has significant implications for biological and medical research, improving:
i) the awareness of similarities/differences with animal models;
ii) the implementation of optimised and standardised protocols for the generation and manipulation of this cell type and its derivatives, with consequent increase in a) stability of the cultures, b) efficiency in the generation of the desired cell types and c) comparison and integration of inter-laboratories/research institutions/companies’ results.
The JAK/STAT3 pathway has been demonstrated to play a fundamental role in maintaining murine naive pluripotency by i) expressional regulation and inhibition of early differentiation markers, and ii) metabolic reconfiguration. JAK/STAT3 activator LIF is routinely used in naive hPSCs culture and derivation media, but the molecular impact of JAK/STAT3 in human pluripotency was still not known. To understand if the pathway plays a role in naive pluripotency maintenance and/or acquisition, I unravelled the contribution of the JAK/STAT3 pathway to the transcriptional program of naive hPSCs in maintenance and during their derivation from primed hPSCs via chemical resetting, a non-genetic alterations-based protocol to produce naive hPSCs from the vast plethora of already available primed hPSC lines. Moreover, I am generating the first computational model of hPSCs metabolism to identify metabolic changes associated with the two states of human pluripotency, for further dissection of human developmental mechanisms and comparison with mouse data. This bioinformatic approach has been chosen and used as a deep metabolic investigation in naive hPSCs is at the moment prevented by technical limitations linked to the standard culture system of naive hPSCs, which relies on a feeder layer of cells on which naive hPSCs grow. This setting impacts different research and applied aspects in the use of naive hPSCs, such as confounding experimental results or non-implementable analyses, marked culture variability, high resource consumption, and limited scalability. Therefore, this project also aimed to establish and validate a new feeder-free culture system, and to propose it to the scientific community as a new standard for the cultivation of naive hPSCs to overcome all listed limitations.
Understanding naive hPSCs requirements has significant implications for biological and medical research, improving:
i) the awareness of similarities/differences with animal models;
ii) the implementation of optimised and standardised protocols for the generation and manipulation of this cell type and its derivatives, with consequent increase in a) stability of the cultures, b) efficiency in the generation of the desired cell types and c) comparison and integration of inter-laboratories/research institutions/companies’ results.
The JAK/STAT3 pathway has been demonstrated to play a fundamental role in maintaining murine naive pluripotency by i) expressional regulation and inhibition of early differentiation markers, and ii) metabolic reconfiguration. JAK/STAT3 activator LIF is routinely used in naive hPSCs culture and derivation media, but the molecular impact of JAK/STAT3 in human pluripotency was still not known. To understand if the pathway plays a role in naive pluripotency maintenance and/or acquisition, I unravelled the contribution of the JAK/STAT3 pathway to the transcriptional program of naive hPSCs in maintenance and during their derivation from primed hPSCs via chemical resetting, a non-genetic alterations-based protocol to produce naive hPSCs from the vast plethora of already available primed hPSC lines. Moreover, I am generating the first computational model of hPSCs metabolism to identify metabolic changes associated with the two states of human pluripotency, for further dissection of human developmental mechanisms and comparison with mouse data. This bioinformatic approach has been chosen and used as a deep metabolic investigation in naive hPSCs is at the moment prevented by technical limitations linked to the standard culture system of naive hPSCs, which relies on a feeder layer of cells on which naive hPSCs grow. This setting impacts different research and applied aspects in the use of naive hPSCs, such as confounding experimental results or non-implementable analyses, marked culture variability, high resource consumption, and limited scalability. Therefore, this project also aimed to establish and validate a new feeder-free culture system, and to propose it to the scientific community as a new standard for the cultivation of naive hPSCs to overcome all listed limitations.
In this project, I evaluated the contribution of the JAK/STAT3 pathway in naive hPSCs maintenance and derivation from primed hPSCs in vitro. I treated naive hPSCs with pathway activators (LIF and IL6) or inhibitors (JAKi) and genetically knocked down the pathway, analysing different features such as proliferation rate, cologenicity, expression of lineage-specific markers by citofluorimetry, qRT-PCR and RNA sequencing. These experiments allowed the identification of a role of the JAK/STAT3 pathway in the maintenance of the naive pluripotency profile, in particular in blocking the expression of early differentiation markers. After demonstrating that the JAK/STAT3 pathway does not influence the primed hPSCs state, I performed similar experiments to evaluate the effect of the modulation of the pathway during the derivation of naive hPSCs from primed ones, highlighting a fundamental role of the pathway for the acquisition of naive fate. Thus, similarly to what happens in mouse PSCs, the JAK/STAT3 pathway substantially contributes to the human naive pluripotency profile.
Together with expressional shifts, a metabolic reconfiguration characterises the two known human pluripotent states. In order to obtain a detailed view of the naive and primed hPSCs metabolism, I am integrating omics data, such as RNA sequencing and metabolomics, together with experimental data like proliferation rates and amount of proteins and media composition, in a scaffold core model of metabolism and a bioinformatic pipeline that allows to generate feasible metabolic fluxes of naive and primed hPSCs that can take into account and show the differences between the two cell types. This approach will generate the first comprehensive computational model of hPSCs metabolism to identify metabolic changes associated with human pluripotency transitions and differences with mouse.
As the culture system for naive hPSCs prevents reliable experimental measurements of metabolism via standard methodologies, I set up and validated a new culture system for naive hPSCs to bypass these kinds of issues in the future and, more generally, open new experimental possibilities for this cell type. The layer of feeder cells could be efficiently replaced with a foetal bovine serum-based coating, which allows the long-term maintenance of all naive hPSCs features and differentiation potential, eliminating feeder-related confounding factors.
Together with expressional shifts, a metabolic reconfiguration characterises the two known human pluripotent states. In order to obtain a detailed view of the naive and primed hPSCs metabolism, I am integrating omics data, such as RNA sequencing and metabolomics, together with experimental data like proliferation rates and amount of proteins and media composition, in a scaffold core model of metabolism and a bioinformatic pipeline that allows to generate feasible metabolic fluxes of naive and primed hPSCs that can take into account and show the differences between the two cell types. This approach will generate the first comprehensive computational model of hPSCs metabolism to identify metabolic changes associated with human pluripotency transitions and differences with mouse.
As the culture system for naive hPSCs prevents reliable experimental measurements of metabolism via standard methodologies, I set up and validated a new culture system for naive hPSCs to bypass these kinds of issues in the future and, more generally, open new experimental possibilities for this cell type. The layer of feeder cells could be efficiently replaced with a foetal bovine serum-based coating, which allows the long-term maintenance of all naive hPSCs features and differentiation potential, eliminating feeder-related confounding factors.
JAK/STAT3 activator LIF is routinely used in naive hPSCs culture and derivation media, but the molecular impact of the JAK/STAT3 pathway in human pluripotency was still not known. In this project, I identified the pathway as essential to maintain and acquire naive pluripotency by blocking the emergence of differentiation cues. With this project, I therefore highlighted a conserved role of JAK/STAT3 in mice and humans in sustaining naive pluripotency, which will be useful as a background for developmental comparisons between species.
Moreover, metabolic maps are being produced to provide a computational model of human metabolism and its reconfiguration in hPSC states, which can be used to dissect metabolic shifts during development and for further comparison with the already developed mouse map to gain new insightful information on the conservation or differences between human and mouse pluripotency.
Finally, the successful establishment of a feeder-free culture system for naive hPSCs with the requisites to become the new culture standard expands the range of molecular investigations implementable, increases the consistency of obtained results and eases their routine culture and manipulation for applied research.
In sum, this project allowed to:
- highlight similarities between mouse and human pluripotency in terms of JAK/STAT3 involvement in naive pluripotency expression profile;
- produce the first comprehensive metabolic model of naive and primed hPSCs and get new insightful information on the metabolic changes during human developmental time course and the conservation or differences between human and mouse pluripotency.
- develop protocols for the culture and manipulation of naive hPSCs as new international standards, expanding the reliable use and application of naive hPSCs in basic and applied research.
Moreover, metabolic maps are being produced to provide a computational model of human metabolism and its reconfiguration in hPSC states, which can be used to dissect metabolic shifts during development and for further comparison with the already developed mouse map to gain new insightful information on the conservation or differences between human and mouse pluripotency.
Finally, the successful establishment of a feeder-free culture system for naive hPSCs with the requisites to become the new culture standard expands the range of molecular investigations implementable, increases the consistency of obtained results and eases their routine culture and manipulation for applied research.
In sum, this project allowed to:
- highlight similarities between mouse and human pluripotency in terms of JAK/STAT3 involvement in naive pluripotency expression profile;
- produce the first comprehensive metabolic model of naive and primed hPSCs and get new insightful information on the metabolic changes during human developmental time course and the conservation or differences between human and mouse pluripotency.
- develop protocols for the culture and manipulation of naive hPSCs as new international standards, expanding the reliable use and application of naive hPSCs in basic and applied research.