Objective

Reactive oxygen species (ROS) are important signal transducers in several cell types: in some tissues, like the developing Zebrafish retina, ROS control the balance between stem cell expansion and differentiation.

Based on these findings, we characterized the potential role of ROS in regulating the transition from fetal progenitors to adult stem cells in the mouse intestine, and in further controlling the choice between stem cell self-renewal and differentiation at homeostasis.

Methods

We employed small intestinal organoids derived from adult mice and from E13.5 embryos, grown in Matrigel to recapitulate in vivo-like cell composition and architecture. ROS levels and localization were visualized by live imaging with CellRox and HyPer7 sensors. Menadione and NAC were used to up- and down-modulate ROS levels, respectively. Cell identity was assigned based on Ker20 (enterocytes, ECs), EdU (stem/progenitors) and UEA (Paneth cells) staining.

Results

We found that stem cells are enriched in ROS in adult intestinal organoids, whereas differentiated lineages show low ROS levels. We next probed the functional role of ROS in organoids: increased ROS triggered an expansion of both proliferative and Paneth cells, with ECs loss; in contrast, quenching ROS levels pushed proliferative cells into EC differentiation. Moreover, when we stimulated Paneth cell-enriched organoids with Menadione, we detected reentry into the cell cycle of some terminally differentiated, G₀ Paneth cells, suggesting dedifferentiation.

Fetal organoids are made of homogeneous cycling progenitors and, accordingly, they present uniform high ROS levels. To define how ROS heterogeneity is established throughout intestinal development, we administered pulses of high ROS with Menadione: this triggered a symmetry breaking event in fetal organoids, which acquired adult cell types, architecture and ROS compartmentalization. It has been reported that YAP pharmacological activation with Truli prevents symmetry breaking in organoids; interestingly, Menadione's effect was not impacted by Truli coadministration, suggesting a dominant role of ROS over YAP in the fetal-to-adult transition.

Conclusion

Overall, we show that ROS levels regulate cell fate decisions in mouse intestinal stem cells and trigger mechanisms of plasticity and regeneration in differentiated cells. In parallel, ROS contribute to the fetal-to-adult transition during intestinal development and to the establishment of the adult stem cell pool.