Lifting regenerative barriers promotes epithelial cell fate plasticity supporting lineage conversion.

The ability of adult epithelial cells to rewire their cell fate programme in response to injury has emerged as a new paradigm in stem cell biology. This plasticity supersedes the concept of strict stem cell hierarchies, granting cells access to a wider repertoire of fate choices. Yet, in order to prevent a disordered cellular response, this process must be finely regulated. Here we investigate the little-known regulatory processes that restrict fate permissibility in adult cells, and keep plasticity in check. Using a 3D regenerative culture system, that enables co-culturing epithelium and stroma of different origins, we demonstrate that oesophageal cells exposed to the ectopic signals of the dermis are capable of switching their identity towards skin. Lineage tracing experiments and histological analysis, however, reveal that the oesophageal-to-skin lineage conversion process is highly inefficient, pointing to the existence of barriers limiting cell fate re-specification. Single-cell RNA sequencing capturing the temporality of this process shows that cells transitioning towards skin identity resist the natural progression towards tissue maturation by remaining in a persistent regenerative state marked by a particularly strong hypoxic signature. Gain and loss of function experiments demonstrate that the HIF1a-SOX9 axis acts as a key modulator of epithelial cell fate plasticity, restricting changes in identity during tissue regeneration. Taken together, our results reveal the existence of lineage conversion barriers that must be resolved for cells to respond to signals instructing alternative fate choices, shedding light on the principles underlying the full regenerative capacity of adult epithelial cells.