Application of synthetic polymer matrices to develop an in vitro thymus

Abstract

The thymus is the primary lymphoid organ responsible to generating a self-restricted and self-tolerant repertoire of peripheral T-cell receptors. This process is collectively called thymopoiesis and depends on dynamic interactions between the developing T-cells (thymocytes) and the thymic stroma. The main functional element of the thymic stroma is the thymic epithelial cell (TEC), which mediate T-cell lineage development and T cell repertoire selection. Modelling thymopoiesis in vitro is currently limited to two techniques. The original technique is termed reaggregate thymic organ culture (RTOC) and is the only method of culturing ex vivo TEC. Unfortunately, the scarcity of TEC makes RTOC very low throughput. To overcome this limitation OP9 cells were engineered to ectopically express the Notch ligand DLL1. OP9-DLL1 cells recapitulate the initial phase of thymopoiesis, T-cell lineage commitment, but not central tolerance induction. In 2014, our lab published a direct lineage reprogramming strategy that was able to generate cells that could mediate all stages of thymopoiesis from an abundant cell type. The strategy reprograms murine embryonic fibroblasts into induced thymic epithelial cells (iTEC). This thesis outlines experiments through which I optimised the iTEC protocol into a standardised process suitable for scale-up and uses novel synthetic polymer matrices to culture iTEC in both two- and three-dimensions. Synthetic polymers emulate native extra cellular matrices (ECMs) but have key advantages such as tuneable physical and chemical properties and controllable degradation. This thesis identifies polyacrylates that are capable of supporting both ex vivo TEC and iTEC in culture in vitro. Investigation into the behaviour of iTEC on the selected polymers demonstrate more consistent behaviours than had previously been achieved and therefore this is the first step in creating a defined protocol for iTEC manufacture that reduces the inherent variablity of reprogramming strategies. This thesis also proposed and tested a new method for the culture of thymic stroma, which aims to occupy an underdeveloped technical niche. Miniaturisation of the reaggregate thymic organ culture process starting from both native TEC and iTEC allowed production of hundreds micro-physiological thymi able to support T cell development. This provides proof-of- principle for a new in vitro thymic organ culture technology compatible with high-throughput screening technologies, whilst requiring a small fraction for the total tissue requirement of RTOC. Evidence is presented that this reductionist system will be able to query specific hypotheses by producing precise observations, in higher throughput, than currently used techniques.