Peter Zandstra

Stem cell-derived T-cells have the potential for use in immunotherapies to treat cancer and immunological disorders. However, building in vitro systems to guide stem cell differentiation into T-cells remains challenging. In the thymus, T-cell development is coordinated by unique microenvironments that provide temporal signals to guide their development. Efforts to understand this process have historically employed gain- or loss-of-function experiments in model animal thymus to perturb signaling networks and measure their effect. While invaluable, these studies can be difficult to interpret. Signaling redundancies stabilize T-cell developmental programs and can mask the effects of perturbations; the magnitude of a gene knock-in or knock- out can result in pathological phenotypes that are not physiological; and differences between species often require reexamining results from animal models in a human context. This creates a challenge for designing clinically relevant systems that mimic the thymus’ function but with much lower complexity.A complementary approach involves studying T-cell development in minimalist engineered system and iteratively adding layers of complexity enable new functions. This strategy incorporates knowledge from studies of the thymus but applies engineering principles to rationally design systems for clinical translation. The work described here realizes this approach: beginning with an engineered thymic niche (ETN) that supports T-cell progenitor differentiation from hematopoietic stem and progenitor cells (HSPCs), the extracellular environment was optimized to enable the development of T-cells without the need for xenogeneic supplementation or stroma. Statistical models were used to learn how responses to signalling molecules change over time as T-cells develop and optimized to support cytotoxic T-cell differentiation from umbilical cord blood- and pluripotent stem cell-derived HSPCs. A theme of this work is how developing T-cells integrate extracellular signals differently depending on their stage, and that these signals must be carefully controlled to support those dynamic developmental processes. The resultant ETN provides a platform for future study of human T-cell development and the insights gained represent a starting point for scaled-up bioprocesses for manufacturing stem cell- derived T-cells for clinical immunotherapies.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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