Periodic Reporting for period 1 - OESOPHAGEAL FATE (Epithelial/Mesenchymal Cross-talk in response to injury and early tumorigenesis; a spatiotemporal perspective)
Periodo di rendicontazione: 2019-06-01 al 2021-05-31
Epithelial tissues have evolutionarily adapted to respond to environmental challenges. They must react rapidly to tissue disruption in order to restore the epithelial barrier and ensure survival. Indeed, epithelial stem cells have been shown to present a high degree of fate plasticity in response to tissue perturbation, being this a property by which a cell can take on different and potentially reversible identities. This ability allows a reactivation of their regenerative capacity and ensures rapid restoration of the tissue after damage. However, changes in cell identity require a fine regulation. Unless cell fate dynamics are balanced back to pre-injury levels upon wound healing, inadequate cell activation can lead to epithelial regenerative problems and cancer. Therefore, understanding the molecular pathways governing this finely tuned process will address clinical issues of significant relevance, offering prospects for the development of more adequate targeted therapies.
In this project, we aimed to investigate the mechanisms regulating the dynamic changes in the cell fate programme of epithelial cells during tissue regeneration. To this end, we chose the uncomplicated architecture of the mouse oesophagus as an ideal system to investigate the precise mechanisms regulating squamous epithelial plasticity in adults after tissue perturbation. Given the increasing relevance of mesenchymal cells both in tissue maintenance and disease, we paid particular attention to the contribution of the stromal compartment in the regulation of epithelial fate changes.
On completion of this action, our work has identified distinct changes in epithelial cell behaviour in a model of oesophageal tissue injury, unveiled relevant regulatory mechanisms underlying epithelial cell fate transitions, and described the contribution of the niche in defining epithelial cell plasticity and fate identity.
In this project, we aimed to investigate the mechanisms regulating the dynamic changes in the cell fate programme of epithelial cells during tissue regeneration. To this end, we chose the uncomplicated architecture of the mouse oesophagus as an ideal system to investigate the precise mechanisms regulating squamous epithelial plasticity in adults after tissue perturbation. Given the increasing relevance of mesenchymal cells both in tissue maintenance and disease, we paid particular attention to the contribution of the stromal compartment in the regulation of epithelial fate changes.
On completion of this action, our work has identified distinct changes in epithelial cell behaviour in a model of oesophageal tissue injury, unveiled relevant regulatory mechanisms underlying epithelial cell fate transitions, and described the contribution of the niche in defining epithelial cell plasticity and fate identity.
To define the changes in epithelial and mesenchymal cell behaviour in the events after tissue injury, we developed a 3D heterotypic culture system to recapitulate oesophageal re-epithelialisation. This innovative and versatile model allowed us to investigate the mechanisms regulating epithelial plasticity in adult tissues following tissue perturbation, but also provided us with a highly efficient model of cell identity conversion. We used this system to challenge oesophageal cell progenitors to re-epithelialize the ectopic stromal compartment of the skin, producing an unexpectedly efficient model of stem cell transdifferentiation of oesophageal-to-skin cells. Using this approach, we have been able to transdifferentiate oesophageal epithelial progenitors into cells showing hair follicle identity and architecture. Remarkably, transdifferentiation can also be recapitulated in vivo following heterotypic transplantation experiments, and oesophageal derived hair follicles can be maintained long term. Our work suggests that environmental cues can re-wire cell identity in adult epithelial cells in vitro and in vivo.
We combined this powerful system with genetic cell labelling techniques, lineage tracing experiments, and high-throughput sequencing of single-cell whole transcriptome (single-cell RNA-Seq) to further describe the stages of cellular plasticity during re-epithelialisation. Our results revealed a widespread contribution of the oesophageal epithelial progenitor during tissue identity conversion. More importantly, we identified cells in a highly permissive state to undergo identity changes in response to signals from the foreign stroma of the adult skin. Interestingly, we found that a strong regenerative signature, acquired during early re-epithelialization stages, was retained by those cells undergoing the identity switch at later time points.
We then explored the mechanisms regulating the identity switch of oesophageal cells into skin cells when exposed to the skin stroma during re-epithelialisation. Single-cell RNA sequencing and histological analysis, capturing the temporality of this process, revealed that most oesophageal cells switching towards skin identity remain in an intermediate state marked by a transient regenerative signature, with a particularly strong hypoxic profile. Interestingly, recent work has shown that the hypoxic niche of the upper hair follicle promotes the stem cell state, raising the question as to whether this environment may be playing a role during the oesophageal cell fate conversion. Finally, our validation experiments inhibiting the hypoxia signalling pathway unveiled the relevance of this pathway for this plasticity process, driving cells away from their transition state and ultimately favouring cell fate conversion.
These results have been shared to the scientific community in renowned conferences, including an oral presentation at the International Society for Stem Cell Research Annual meeting (ISSCR 2021) and an invited talk at the opening session of the Epithelial Differentiation and Keratinization Gordon Research Conference meetings (2021), where the researcher was able to meet and discuss the results with prominent scientists of the field. The results of this project have also been shared as an open access preprint in bioRxiv. The novelty and interest of the outputs and activities of this action have been widely acknowledged with recognition and awards in international conferences, but also reflected by the metrics of their impact on the social media.
We combined this powerful system with genetic cell labelling techniques, lineage tracing experiments, and high-throughput sequencing of single-cell whole transcriptome (single-cell RNA-Seq) to further describe the stages of cellular plasticity during re-epithelialisation. Our results revealed a widespread contribution of the oesophageal epithelial progenitor during tissue identity conversion. More importantly, we identified cells in a highly permissive state to undergo identity changes in response to signals from the foreign stroma of the adult skin. Interestingly, we found that a strong regenerative signature, acquired during early re-epithelialization stages, was retained by those cells undergoing the identity switch at later time points.
We then explored the mechanisms regulating the identity switch of oesophageal cells into skin cells when exposed to the skin stroma during re-epithelialisation. Single-cell RNA sequencing and histological analysis, capturing the temporality of this process, revealed that most oesophageal cells switching towards skin identity remain in an intermediate state marked by a transient regenerative signature, with a particularly strong hypoxic profile. Interestingly, recent work has shown that the hypoxic niche of the upper hair follicle promotes the stem cell state, raising the question as to whether this environment may be playing a role during the oesophageal cell fate conversion. Finally, our validation experiments inhibiting the hypoxia signalling pathway unveiled the relevance of this pathway for this plasticity process, driving cells away from their transition state and ultimately favouring cell fate conversion.
These results have been shared to the scientific community in renowned conferences, including an oral presentation at the International Society for Stem Cell Research Annual meeting (ISSCR 2021) and an invited talk at the opening session of the Epithelial Differentiation and Keratinization Gordon Research Conference meetings (2021), where the researcher was able to meet and discuss the results with prominent scientists of the field. The results of this project have also been shared as an open access preprint in bioRxiv. The novelty and interest of the outputs and activities of this action have been widely acknowledged with recognition and awards in international conferences, but also reflected by the metrics of their impact on the social media.
The OESOPHAGEAL FATE project presents an innovative broad perspective to study, all at once, the changes in proliferation, gene expression, and cellular communication of cells involved in epithelial wound repair and identity conversion. The excellence of this project is reflected in the combination of cutting-edge techniques used including – but not limited to – precise lineage tracing, single-cell whole transcriptomics and transgenic animal lines. The outputs of our interdisciplinary approach led to the identification of key players governing the dynamic behaviour of epithelial cells with temporal resolution during tissue repair.
This work will lay the foundation to understand how the microenvironmental cues orchestrate cell fate plasticity in epithelial tissues and to help develop novel clinical therapies to improve wound healing, an unresolved aspect of modern medicine. Furthermore, future studies will unveil whether the molecular changes identified using our 3D culture system are important for other clinically relevant models, such as tumorigenesis, where plasticity is known to operate.
This work will lay the foundation to understand how the microenvironmental cues orchestrate cell fate plasticity in epithelial tissues and to help develop novel clinical therapies to improve wound healing, an unresolved aspect of modern medicine. Furthermore, future studies will unveil whether the molecular changes identified using our 3D culture system are important for other clinically relevant models, such as tumorigenesis, where plasticity is known to operate.