Peter Zandstra

The field of regenerative medicine and bioengineering presents an exciting opportunity to harness the potential of hematopoietic stem cells (HSCs) through stem cell niche and cellular engineering. These approaches aim to recreate the natural environment of the stem cells and modify protein expression in the cells to enhance the differentiation and therapeutic capabilities of HSCs. This makes enhanced HSCs valuable for treating a range of blood-related diseases and genetic conditions. Our first study is focused on developing a hydrogel-based system that provides a serum-free, feeder-free thymic-like niche for HSCs to develop into T cells. We synthesized hydrogel containing thymic-associated components including immobilized Notch ligand Delta-like-4 (DLL4), hyaluronic acid, vascular cell adhesion molecule 1 (VCAM1) and collagen-derived gelatin. DLL4-Fc immobilized using conjugated protein G resulted in T cell differentiation. Gelatin addition in the engineered 3D system enhanced T cell differentiation, while VCAM1 had no effect on cell development. The second study is focused on using the platform engineered in the first study to develop a system for generation of T cells from induced pluripotent stem cells (iPSCs) derived HSCs. We created the first feeder-free serum-free 3D system for T cell generation from PSCs-derived cells. Finally, in the third study, we explored the use of lipid nanoparticles (LNPs) loaded with mRNA to modify phenotype of megakaryocyte (MK) progenitors derived from HSCs. We found an optimal formulation of LNPs for MK progenitor transfection and engineered the cells to produce coagulation factor VII, FVII, which decreased clot time in the presence of FVII-deficient plasma. Our study highlights the significant potential of biomaterials to engineer HSCs into functional T cells and megakaryocytes, thus opening up new avenues in therapy development.

View record

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.

View record

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

View record