Development of a peristaltic tissue-engineered oesophagus

Supervisors: Dr Paolo De Coppi, Dr Simon Eaton, Dr Nikhil Thapar and Dr Alan Burns

This project aims to build and test tissue-engineered oesophagus as alternative treatment for congenital gastrointestinal defects. Artificial tissues will be built using decellularised oesophagus, subsequently re-populated with stem and specialized cells using dynamic cell seeding techniques to create a functional tissue. Engineered tissues will be transplanted in murine, rabbit and porcine animal models.

Aims and timeline:
• Year 1. Develop a natural acellular oesophagus with mechanical properties, engineered possibly with bioabsorbable nanocomposite material to modulate mechanical/structure features.
• Year 1-2. Re-populate the matrix with different cell types (smooth muscle, epithelium, endothelium, neural crest stem cells) to re-create the original tissue organization and function. For this purpose, dynamic culture methods (bioreactor) will be applied and tested.
• Year 2-3. Transplant of tissue-engineered oesophagus in mouse and rabbit models using cell tracking techniques. Post-transplantation analysis will be performed to assess tissue and cell engraftment, function and matrix remodeling.
• Year 3. The most promising combination of cells and acellular matrix will be developed in GMP?? and tested in a porcine animal model as preclinical model.

Background:
Oesophageal atresia is a congenital defect of the digestive system in which a variable length of the oesophagus does not develop properly. Oesophageal replacement severely impairs the quality of life of recipient children and adults and presents problems related to donor site morbidity.

The use of a tissue engineered oesophageal conduit could dramatically improve the management of these conditions. In particular, we propose that decellularised cadaveric oesophagus could provide an alternative approach, with a lower complication rate than the present treatments.

Within regenerative medicine, tissue engineering is concerned with the manufacturing of tissue by combining appropriate cells with a scaffold that can be either synthetic or naturally-derived [1,2]. The use of extracellular matrix (ECM)-derived scaffolds obtained from decellularised tissues is increasingly frequent in regenerative medicine. This attractive technique allow the generation of a scaffold retaining the architecture of the native tissue including vasculature, biofactors and ECM components, providing good biomechanical characteristic and permissiveness to foster efficient reseeding.

Engineered tissues, especially whether of substantial dimensions, need to be functionalised with committed and specialised cells. In this project, smooth muscle cells will be used to repopulate the muscle part of the acellular tissue. Oesophageal specific epithelial cells will be isolated from animal and human biopsies to repopulate the internal part, as well as endothelial cells for the vascular tree. In addition, stem cells derived from the fetus could be used for tissue regeneration approaches. In particular, amniotic fluid stem (AFS) cells can be easily collected from amniotic fluid from amniocentesis. Our group and others have shown that AFS cells are able to differentiate in vitro and in vivo into different lineages [3], they express both embryonic and adult stem cell markers, expand extensively, they are not tumorigenic when injected in vivo and does not involve ethical issues. AFS cell population can be isolated from the affected fetus and expanded to be engineered to produce the gastrointestinal tract patch before the baby is born allowing the repair in early neonatal life of the congenital defect with autologous tissue.

For cell seeding, growth, differentiation and cell-matrix interaction, a bioreactor will be employed to perform dynamic culture. Such devices are indispensable when cells are cultured in 3D scaffolds to achieve tissue uniformity and avoid widespread necrosis in the inner regions [4].

Project interface between basic and clinical science
Successful engineering of oesophagus with decellularized matrices and subsequent re-cellularization have not been established yet. We believe that this is the limiting step for their application in human. This project covers several areas of investigation, including biological matrices development and characterization, isolation and culture of several cell populations, potential understanding of molecular basis of cell-matrix interaction, in vivo experiments in small and large animal models and post-transplantation analysis.

References:
1) Baiguera S, Jungebluth P, Burns A, Mavilia C, Haag J, De Coppi P, et al. Tissue engineered human tracheas for in vivo implantation. Biomaterials 2010; 31:8931e8.
2) Macchiarini P, Jungebluth P, Go T, Asnaghi MA, Rees LE, Cogan TA, et al. Clinical transplantation of a tissue-engineered airway. Lancet 2008; 372:2023e30.
3) De Coppi P, Bartsch G Jr, Siddiqui MM, Xu T, Santos CC, Perin L et al. Isolation of amniotic stem cell lines with potential for therapy. Nature Biotechnology 2007; 25:100.
4) Flaibani M, Luni C, Sbalchiero E, Elvassore N. Flow cytometric cell cycle analysis of muscle precursor cells cultured within 3D scaffolds in a perfusion bioreactor. Biotechnol Prog. 2009 Jan-Feb;25(1):286-95.