Garrido Flores, Matías; (2024) Mind the Long-Gap: Harnessing the properties of stem/progenitor cells to regenerate human oesophageal epithelium. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Introduction: Long-gap oesophageal atresia (LGOA) is a malformation that lacks a significant oesophageal segment. Current therapies are associated with morbidity and mortality. It has been proposed that a tissue-engineered (TE) oesophagus could overcome these issues. Epithelial regeneration is a critical step in this strategy. However, fundamental aspects of the human oesophageal epithelium are poorly understood. This thesis aimed to identify and locate stem/progenitor human oesophageal epithelial cells, and to scale our TE approach on a large animal model. Methods: Paediatric oesophageal samples were studied. Epithelial cells were sorted and seeded for single-cell clonal analysis to determine their potency. In parallel, culture methods and delivery of pig oesophageal epithelial cells were developed for further use in an orthotopically transplanted TE oesophagus. Results: Thirty paediatric samples were accessed during the PhD period. Additionally, five samples previously received were included. Histological analysis revealed that the human oesophageal epithelium is characterised by a quiescent basal layer, proliferative epibasal layers, and terminally-differentiated suprabasal layers. Single-cell clonal analysis was performed on seven freshly dissociated tissues and three other samples from cultured cells previously derived. It aligned with the heterogeneous model, revealing that holoclones-like cells are present only in the basal and epibasal compartments. Additionally, human oesophageal epithelial cell sheets were generated using temperature-responsive flasks. Regarding the translational aspect, although pig oesophageal epithelial cells were successfully derived and expanded, obtaining epithelial cells from endoscopic biopsies was not possible. Further analysis revealed that these biopsies were sampling only suprabasal layers. Nevertheless, epithelial regeneration was achieved by the animals without the presence of an engineered epithelium. Conclusions: The human oesophageal epithelium contains holoclones in the basal and epibasal layers. Thoracic replacement by TE oesophagus on LGOA patients is possible, but epithelial regeneration by pigs does not guarantee that it may also occur in humans.

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