Gene Modification and Three-Dimensional Scaffolds as Novel Tools to Allow the Use of Postnatal Thymic Epithelial Cells for Thymus Regeneration Approaches

Abstract

Defective functionality of thymic epithelial cells (TECs), due to genetic mutations or injuring causes, results in altered T‐cell development, leading to immunodeficiency or autoimmunity. These defects cannot be corrected by hematopoietic stem cell transplantation (HSCT), and thymus transplantation has not yet been demonstrated to be fully curative. Here, we provide proof of principle of a novel approach toward thymic regeneration, involving the generation of thymic organoids obtained by seeding gene‐modified postnatal murine TECs into three‐dimensional (3D) collagen type I scaffolds mimicking the thymic ultrastructure. To this end, freshly isolated TECs were transduced with a lentiviral vector system, allowing for doxycycline‐induced Oct4 expression. Transient Oct4 expression promoted TECs expansion without drastically changing the cell lineage identity of adult TECs, which retain the expression of important molecules for thymus functionality such as Foxn1, Dll4, Dll1, and AIRE. Oct4‐expressing TECs (iOCT4 TEC) were able to grow into 3D collagen type I scaffolds both in vitro and in vivo, demonstrating that the collagen structure reproduced a 3D environment similar to the thymic extracellular matrix, perfectly recognized by TECs. In vivo results showed that thymic organoids transplanted subcutaneously in athymic nude mice were vascularized but failed to support thymopoiesis because of their limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of 3D biomimetic scaffolds, may represent a novel approach allowing the use of postnatal TECs for thymic regeneration. Stem Cells Translational Medicine 2019;8:1107–1122

Thymus Extracellular Matrix‐Derived Scaffolds Support Graft‐Resident Thymopoiesis and Long‐Term In Vitro Culture of Adult Thymic Epithelial Cells

“…The resorption of these tissues under in vivo conditions prevented sustained thymopoiesis, as previously observed with an analogous approach. [ 24 ] A future task will therefore be to tune the physical properties of the scaffold to change the degradation kinetics of the TS whilst still ensuring efficient in situ growth and preservation of functional TEC. Moreover, the approach chosen will allow to render the size and shape of the scaffold bespoke to individual needs, as well as to optimize the microstructure of the scaffold to better mimic the native tissue microenvironment.…”

“…[ 23 ] Recently, collagen I was used to produce scaffolds in an attempt to mimic the thymic extracellular matrix (ECM) microstructure. [ 24 ] These scaffolds sustained TEC survival, but failed to support thymopoiesis when grafted in vivo. Although these methods did provide a 3D environment closer to the organ's physiological architecture, none could robustly support lasting thymopoiesis—likely because the resultant microenvironments lacked the complex molecular cues conveyed by the native thymic ECM.…”

Current and Future Therapeutic Approaches for Thymic Stromal Cell Defects

“…For clinical translation, it is essential to develop new tools that can deliver TECs within an appropriate 3D structure, capable of supporting thymopoiesis. One group used artificial collagen scaffolds seeded with postnatal, gene-modified murine TECs, able to transiently express Oct4 to promote TEC expansion in vitro and in vivo ( 161 ). However, these artificial collagen scaffolds did not show efficacy in supporting thymopoiesis when transplanted into mice.…”

The contribution of thymic tolerance to central nervous system autoimmunity

“…More specifically, artificially bioengineered thymic epithelial tissue can be combined with biocompatible 3D scaffolds that mimic the organisation of thymic extracellular matrix to support ex vivo or even in situ generation of artificial thymic organoids (ATOs) [231][232][233][234].…”

“…An alternative and technologically more complex approach to modulate thymopoiesis is the in vitro differentiation from host-derived human induced pluripotent stem cells (iPSCs) of thymic epithelial progenitors (TEPs), which mature into functional TECs upon transplantation into the recipient. More specifically, artificially bioengineered thymic epithelial tissue can be combined with biocompatible 3D scaffolds that mimic the organisation of thymic extracellular matrix to support ex vivo or even in situ generation of artificial thymic organoids (ATOs) [ 231 – 234 ].…”