Lessons learned from pre-clinical testing of xenogeneic decellularized esophagi in a rabbit model

Summary Decellularization of esophagi from several species for tissue engineering is well described, but successful implantation in animal models of esophageal replacement has been challenging. The purpose of this study was to assess feasibility and applicability of esophageal replacement using decellularized porcine esophageal scaffolds in a new pre-clinical model. Following surgical replacement in rabbits with a vascularizing muscle flap, we observed successful anastomoses of decellularized scaffolds, cues of early neovascularization, and prevention of luminal collapse by the use of biodegradable stents. However, despite the success of the surgical procedure, the long-term survival was limited by the fragility of the animal model. Our results indicate that transplantation of a decellularized porcine scaffold is possible and vascular flaps may be useful to provide a vascular supply, but long-term outcomes require further pre-clinical testing in a different large animal model

Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds.

The thymus is a primary lymphoid organ, essential for T cell maturation and selection. There has been long-standing interest in processes underpinning thymus generation and the potential to manipulate it clinically, because alterations of thymus development or function can result in severe immunodeficiency and autoimmunity. Here, we identify epithelial-mesenchymal hybrid cells, capable of long-term expansion in vitro, and able to reconstitute an anatomic phenocopy of the native thymus, when combined with thymic interstitial cells and a natural decellularised extracellular matrix (ECM) obtained by whole thymus perfusion. This anatomical human thymus reconstruction is functional, as judged by its capacity to support mature T cell development in vivo after transplantation into humanised immunodeficient mice. These findings establish a basis for dissecting the cellular and molecular crosstalk between stroma, ECM and thymocytes, and offer practical prospects for treating congenital and acquired immunological diseases

Investigating Human Oesophageal Epithelial Stem-Progenitor Cells and their Ability to Reconstruct a Functional Epithelium within a Tissue Engineered Oesophagus

Reconstructing an oesophagus through tissue engineering might be beneficial for patients suffering from a broad spectrum of oesophageal diseases, both congenital and acquired. ‘Long-gap’ oesophageal atresia is a congenital malformation, where the oesophagus does not properly develop, creating a gap between the proximal and distal parts. Regenerative medicine may offer a therapeutic alternative to these patients by combining biomaterials with bona fide stem cells to create a suitable oesophageal replacement. In order to rebuild a functional oesophageal replacement, all the structural features of the oesophageal layers need to be regenerated in vivo. This study focuses on the underlying biology of human oesophageal epithelial cells (HuOEC) and their ability to reconstruct a functional stratified epithelium within a tissue engineered oesophagus. In particular, I have (i) established the conditions necessary for HuOEC to extensively expand in vitro, while maintaining their capacity to differentiate, (ii) assessed HuOEC clonogenicity using single-cell analysis and identified different clonogenic populations within the oesophageal epithelium, (iii) studied the development of human foetal oesophageal epithelium to understand its morphogenesis and maintenance and (iv) succeeded in the reconstitution of an epithelium barrier ex vivo and in vivo using cultivated oesophageal epithelial cells, derived both from rat and human samples, over a decellularised extracellular matrix. Importantly, oesophageal epithelial cells are able to give rise to a squamous stratified epithelium on a xenogeneic extracellular matrix that resembles the native oesophageal epithelium. The newly reconstructed epithelium maintains a subpopulation of epithelial cells with proliferation capacity and clonogenic potential, thus supporting their stem/progenitor identity. The results that will be presented include part of the extensive characterisation of HuOEC, the feasibility of engineering a human oesophageal replacement with mucosal barrier function and suggest the existence of oesophageal epithelial clonogenic and self-renewing stem cells

Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds.

The thymus is a primary lymphoid organ, essential for T cell maturation and selection. There has been long-standing interest in processes underpinning thymus generation and the potential to manipulate it clinically, because alterations of thymus development or function can result in severe immunodeficiency and autoimmunity. Here, we identify epithelial-mesenchymal hybrid cells, capable of long-term expansion in vitro, and able to reconstitute an anatomic phenocopy of the native thymus, when combined with thymic interstitial cells and a natural decellularised extracellular matrix (ECM) obtained by whole thymus perfusion. This anatomical human thymus reconstruction is functional, as judged by its capacity to support mature T cell development in vivo after transplantation into humanised immunodeficient mice. These findings establish a basis for dissecting the cellular and molecular crosstalk between stroma, ECM and thymocytes, and offer practical prospects for treating congenital and acquired immunological diseases

Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds

The thymus is a primary lymphoid organ, essential for T cell maturation and selection. There has been long-standing interest in processes underpinning thymus generation and the potential to manipulate it clinically, because alterations of thymus development or function can result in severe immunodeficiency and autoimmunity. Here, we identify epithelial-mesenchymal hybrid cells, capable of long-term expansion in vitro, and able to reconstitute an anatomic phenocopy of the native thymus, when combined with thymic interstitial cells and a natural decellularised extracellular matrix (ECM) obtained by whole thymus perfusion. This anatomical human thymus reconstruction is functional, as judged by its capacity to support mature T cell development in vivo after transplantation into humanised immunodeficient mice. These findings establish a basis for dissecting the cellular and molecular crosstalk between stroma, ECM and thymocytes, and offer practical prospects for treating congenital and acquired immunological diseases

Multi-stage bioengineering of a layered oesophagus with in vitro expanded muscle and epithelial adult progenitors

A tissue engineered oesophagus could overcome limitations associated with oesophageal substitution. Combining decellularized scaffolds with patient-derived cells shows promise for regeneration of tissue defects. In this proof-of-principle study, a two-stage approach for generation of a bio-artificial oesophageal graft addresses some major challenges in organ engineering, namely: (i) development of multi-strata tubular structures, (ii) appropriate re-population/maturation of constructs before transplantation, (iii) cryopreservation of bio-engineered organs and (iv) in vivo pre-vascularization. The graft comprises decellularized rat oesophagus homogeneously re-populated with mesoangioblasts and fibroblasts for the muscle layer. The oesophageal muscle reaches organised maturation after dynamic culture in a bioreactor and functional integration with neural crest stem cells. Grafts are pre-vascularised in vivo in the omentum prior to mucosa reconstitution with expanded epithelial progenitors. Overall, our optimised two-stage approach produces a fully re-populated, structurally organized and pre-vascularized oesophageal substitute, which could become an alternative to current oesophageal substitutes