Transplantation of tissue-engineered cell sheets for stricture prevention after endoscopic submucosal dissection of the oesophagus

Abstract

Background and objective: Endoscopic mucosal dissection (ESD) is a treatment option for oesophagus tumours localized to the mucosa enabling en bloc removal of large lesions. The resulting larger mucosal defects have resulted in an increase in the occurrence of post-treatment strictures. Transplantation of autologous cell sheets, cultured from oral mucosa, has been shown to prevent post-ESD strictures. The aim of the study was to assess the efficacy and safety of cell sheet transplantation after oesophageal ESD in a Western patient population where reflux-associated pre-malignant and malignant conditions predominate.

Methods: Patients with Barrett's oesophagus associated high-grade dysplasia or early adenocarcinoma where ESD entailed a resection >3 cm in length and ≥75% of the circumference were eligible for treatment under hospital exemption. Cell sheets were cultured from buccal mucosa according to Good Manufacturing Practice and were endoscopically applied to the post-ESD defect directly after resection. Patients were followed with weekly endoscopy examinations, including confocal laser microscopy, for a total of four weeks.

Results: Five patients were treated. ESD was extensive with resections being circumferential in three patients and 9-10 cm in length in two. The number of transplanted cell sheets ranged from two to six. Three patients developed strictures requiring two to five dilatation sessions.

Conclusions: Cell sheet transplantation shows to be safe and feasible in a Western population. Results suggest that transplantation has a protective effect on the mucosal defect after ESD, decreasing both the risk for and extent of stricture formation.

Keywords: Barrett’s oesophagus; cell sheets; early oesophageal cancer; endoscopic submucosal dissection; oesophagus stricture prevention.

Figures

Figure 1.

Endoscopic images of patient 3 showing a 10 cm circumferential Barrett’s mucosa containing areas of high-grade dysplasia (a), the near-complete endoscopic mucosal dissection (ESD) (b), the mucosal defect after ESD (c), transplanted cell sheets with the support membranes clearly visible (d) and (e), the distal oesophagus at control gastroscopy four months after the procedure (f).

Figure 2.

Histology and protein distribution in cell sheets. Hematoxylin and eosin (H&E) staining showing the oral mucosal epithelial cells detached as a continuous cell layer (a) (scale bar = 200 µm). In higher magnification the sheet is observed having 4–5 layers ((a) insert) (scale bar = 20 µm). Gene expression of cell sheets for keratin 18 (KRT18), Oct-4 and Sox-2 (b). Several extracellular matrix proteins were retained in the detached cell sheets: collagen I (c), collagen IV (d) and elastin (e). Laminin was found expressed on the basal side of the layer (f) (scale bar = 50 µm). Connexin 43 staining is suggesting gap-junctions in the lower parts of the sheet (scale bar = 20 µm) (g). Pluripotency markers were detected in the detached sheets, SSEA4 (h), C-kit (i), Oct-4 (j) and Sox-2 (k) (scale bar = 50 µm).

Figure 3.

Cell viability, metabolism, proliferation and transmission electron microscopy. The detached cell sheet showed a very low number of dead cells ((a) arrows) (scale bar = 200 µm) and the cells were found to be metabolically active (b). 12 ± 7% of cells were proliferating based on Ki-67-staining (c) (scale bar = 50 µm). Transmission electron microscopy reveals a multi-layered cell configuration of up to five layers, with several cell-to-cell contacts ((d) arrows) (scale bar = 20 µm). Higher magnification reveals abundance of desmosomes, indicating healthy epithelial cells (e) (scale bar = 500 nm). The majority of cells are healthy (f) (scale bar = 20 µm) but some are showing enlarged nuclei with loss of structure and cell-to-cell contact, indicating necrosis ((f) insert) (scale bar = 5 µm).

Figure 4.

Confocal laser endomicroscopy of normal squamous epithelium (a) with intrapapillary capillary loops (b). After one week squamous cells are visualized in the transplanted areas ((c) and (d)), whereas non-transplanted surfaces of the post-endoscopic mucosal dissection defect were mostly covered by fibrin and macrophages ((e) and (f)).

Figure 5.

Results of the EORTC QLQ – OES18 questionnaire. Values are shown as medians documented pre-interventionally and at week 1 and week 4 after endoscopic mucosal dissection with cell sheet transplantation.