Project title

Developing a bioengineered human oesophagus: advancing towards clinical application 

Supervisors


Background

Long-Gap Oesophageal Atresia (LGOA) is a severe birth defect where the oesophagus doesn’t connect to the stomach, requiring complex surgery and leading to long-term issues like reflux and malignancy risk. Building on success with paediatric tracheal transplants produced with patient-derived cells on cadaveric scaffolds, our team is developing a tissue-engineered (TE) oesophagus using ecellularized porcine scaffolds seeded with autologous Mesoangioblasts (MABs) and Fibroblasts (FBs). Preclinical studies in minipigs show promising results, with grafts supporting growth, peristalsis, and neovascularisation driven by a pro-angiogenic, hypoxic cell phenotype established during in vitro graft maturation. However, key translational hurdles remain: GMP-compliant production, a deeper understanding of oxygen dynamics during graft manufacture, and a robust system for in vivo tracking of transplanted cells to confirm their contribution to regeneration.

Aims and objectives

This PhD project aims to overcome critical manufacturing and tracking challenges to advance the clinical translation of tissue-engineered oesophageal grafts:

  1. Developing GMP-compliant protocols for human cell derivation, expansion, and automated seeding onto ecellularised porcine oesophageal scaffolds, leading to the production of clinical-grade TE oesophageal grafts. Integrate metagenomic screening throughout production to detect and monitor microbial contamination, ensuring graft sterility and safety.
  2. Characterising human cells’ behaviour, focusing on pro-angiogenic and hypoxic signatures established during graft production, and monitoring oxygen dynamics during in vitro maturation.
  3. Establishing an innovative cell tracking system using clinically approved markers to assess transplanted cell survival, migration, and integration in vivo.

 

Methods

  1. Human MABs and FBs will be derived from rectus abdominis biopsies, expanded and characterised using GMP-compliant reagents. A robot stereotaxic microinjector will be adapted to automate and standardise cell delivery for uniform scaffold seeding. Quality control will include a novel metagenomic screening method at multiple production stages (from oesophagus harvesting to post-bioreactor maturation) using shotgun sequencing to detect microbial contaminants (bacteria, viruses, fungi).
  2. Single-nucleus RNAseq will assess transcriptomic changes in human cells pre- and post-bioreactor incubation, focusing on pro-angiogenic and hypoxic markers. An oxygen sensing system will be integrated into the bioreactor for real-time monitoring of pO2 fluctuations during graft maturation.
  3. Lentiviral vectors encoding non-functional truncated membrane receptors will be generated and validated for labelling human and porcine MABs and FBs. Autologous labelled porcine cells will be used for engineered oesophagus transplantation into minipigs. Post-mortem analysis will evaluate transplanted cell persistence, migration, and gene expression.

Timeline

  • Year 1 – Establish GMP-compliant human cell derivation and expansion protocols. Begin integrating and testing the oxygen sensing system. Initiate lentiviral vector generation and in vitro validation of cell labelling. Collection of samples for metagenomics analysis.
  • Year 2 – Produce GMP-compliant human cell-seeded grafts with automated cell delivery. Conduct oxygen-sensing experiments with human grafts. Execute the in vivo transplantation of labelled porcine grafts in minipigs and commence initial post-mortem analyses.
  • Year 3 –  Complete snRNAseq analysis for both human cells and in vivo labelled cells. Finalise data interpretation; thesis writing.

 

References

  1. Durkin et al. 2024 (under revision).
  2. Elliott, M. J. et al. 2012 The Lancet (PMID:22841419).
  3. Urbani, L., et al. (2018). Nature Communications (PMID:30327457).
  4. Hannon E., Pellegrini M., et al, 2022 iScience (PMID: 36217545).


Who should students contact?

Marco Pellegrini (marco.pellegrini@ucl.ac.uk).

Research topic

Paediatric surgery