Watson, Sara Ann; (2023) Thymus-on-a-chip: Development of a microfluidic model of human T cell development via culture and differentiation of human thymus stromal cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The thymus is the site of differentiation of mature, non-self-reactive T cells. Thymus seeding progenitors (TSP) are recruited from the bone marrow to the post-natal thymus; primed towards a lymphoid fate by Notch1 signalling (Brandstadter and Maillard 2019); and instructed to mature into functional, tolerant, naïve T-cells via interactions with thymic epithelial cells (TEC). Recent works (Campinoti et al. 2020; James, William E. Jenkinson, and G. Anderson 2021); have highlighted the role of other thymus stromal cells (TSC), including interstitial cells (TIC), in thymocyte development and thymus homeostasis. Characterising the interactions between thymocytes and TSC has traditionally been complex due to difficulties accessing human tissue and a lack of good in vitro models. Research to date has necessarily utilised rodent models. However, distinct differences between the mouse and human thymus highlight the need for better, human-specific, ex vivo thymus models. In this work, we sought to develop in vitro models using microfluidic methods to create a thymus-on-a-chip to recapitulate the human thymus microenvironment. To exemplify the recruitment of TSP to the thymus, we developed a microfluidic model of the human thymus using well-characterised, donor-derived TIC and TEC stem cells (Campinoti et al. 2020; Ragazzini et al. 2023). Results of this assay showed that TSC could recruit haematopoietic stem cells (HSC), and that these HSC could extravasate through a 3D endothelium, reflecting the migration of TSP through post-capillary venules in vivo. Results also showed that the endothelium, which expressed the key Notch1 receptor DLL4, could prime HSC towards a T cell fate, as evidenced by the upregulation of CCR7 and CD7. The assay also identified CCL5 as a potent chemoattractant cytokine expressed by thymic stromal cells. Blocking CCL5 in the microdevice reduced HSC extravasation, highlighting the assay’s potential for drug screening. These data were peer-reviewed and published in the Journal of Bioengineering and Translational Medicine (Watson et al. 2022). Subsequent refinement of the microfluidic model by manipulating the microdevice hydrogel and optimising culturing conditions induced TEC and TIC differentiation in 3D. By integrating TSC and endothelial cells, the assay pushed HSC towards the T cell fate. Viable preT cells could be recovered from the device and assessed by flow cytometry and qPCR. The cells cultured in the the thymus-on-a-chip device highly upregulated CD7 and expressed makers of T lineage commitment, including GATA3, TCF7, and BCL11B. This thymus-on-a-chip will be an important tool for researchers to decipher the paracellular signals that drive thymocyte differentiation and maturation.

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