Controlled Generation of Progenitor T-cells from Hematopoietic Stem Cells and Pluripotent Stem Cells

The thymus integrates multiple niche molecules including cytokines, ligands and extracellular matrix to drive T-cell development in vivo. We hypothesized that engineering technologies to integrate essential thymic niche molecules required for progenitor T-cell development would enable defined in vitro generation and quantification of T-cells. We optimized a defined 2D serum-free culture system using Notch ligand Delta-like-4 (DL4) for producing progenitor T-cells from mouse Sca-1+cKit+ hematopoietic stem and progenitor cells (HSPCs). Serum-free culture enabled us to define assay design criteria such as input cell density, DL4 presentation and cytokine concentrations. The fully defined engineered in vitro niche revealed synergistic interactions between DL4 and vascular cell adhesion molecule-1 (VCAM-1), and its role in enhancing downstream Notch pathway activation, progenitor T-cell migration and differentiation. The engineered niche also enabled scalable differentiation of human umbilical cord blood-derived CD34+ HSPCs to CD7+ progenitor T-cells, yielding 25-fold total cell expansion with 59.3±10.8% CD7+ T-cells. CD7+ progenitor T-cells generated in the engineered niche were capable of thymic engraftment in lymphodeficient SIRPa-/- Rag2-/- gc-/- mice and generated mature functional CD3+ T-cells in vivo. Building on this system, we next immobilized DL4 in a 3D methylcellulose hydrogel (DL4-MC) using thiol-maleimide chemistry. DL4-MC hydrogels exhibited a dose-dependent increase in Notch pathway activation. We demonstrated that production of progenitor T-cells from HSPCs encapsulated in DL4-MC hydrogels could be enhanced when VCAM-MC was incorporated, creating a 3D engineered hydrogel thymus-like niche. Thus, controlled and reproducible differentiation of progenitor T-cells from stem cells can be achieved by engineering a thymus-like niche in vitro using minimum essential cues required for T-cell development. This provides an important step towards systematically engineering the niche for progenitor T-cell development with the ultimate goal of generating histocompatible “off-the-shelf” T-cells from normal or gene-edited stem cells for T-cell immunotherapeutic applications.Ph.D.2019-07-11 00:00:0