Notch ligand functionalized microbead system for T cell differentiation

Hematopoietic stem cells (HSC) are cells that can self-replicate and give rise to a wide array of different cell types through the process of differentiation. HSC are found in blood and bone marrow, and they are responsible for the daily production of blood and immune cells essential to humans’ survival. HSCs’ multipotency makes it a powerful candidate for new, revolutionary treatments for diseases. However, in order to harness its multpotency, we must first understand what instructs HSC to become very specific cell types. In particular, we are interested in the role of the Notch signaling pathway in HSC differentiation into T-cells, white blood cells that regulate the immune system and destroy tumor and virus-infected cells. The Notch signaling pathway been shown to be necessary and sufficient for directing HSC differentiation into T-cells. Specifically we limit our studies to two proteins in the pathway: Notch1 receptor on the signal receiving cell’s membrane and delta-like ligand 4, a protein that activates Notch1. However, scalable and efficient biomaterial-based systems for this differentiation process have yet to be reported. Such a system would allow unprecedented temporal and ligand density control, prerequisites for the ultimate goal of mass producing T-cells “on demand” for therapeutic treatments for immunodeficient or cancer patients. Here we describe an artificial Notch signaling system using magnetic microbeads coated with DLL4 to activate Notch1 receptors. The DLL4 have been shown to be capable of directing HSC differentiation to T cells. The aim of my project is to characterize the cell-bead interaction to better understand how Notch1 activation varies with DLL4 density and temporal presentation. Mouse embryonic stem cells and myoblasts (muscle progenitor cells) were used as model cell lines since HSCs are difficult to maintain in culture. Although RT-PCR results indicated that DLL4 beads induced a Pg. 4 3-5 fold increase in HES1 (a downstream target gene of Notch1 signaling) gene expression, fluorescent labeling of activated Notch1 was unable to distinguish basal level of Notch activity with activation induced by the DLL4 beads. Although my studies were not able to confirm DLL4 bead activation of Notch1 with fluorescent labeling, the results will aid in developing the parameters for using murine ES cells and myoblasts as model systems to test the functionality of beads and bead-cell interaction. Knowledge gained from this study and the proposed future experiments will enhance our understanding of in vitro T cell generation. Moreover, continuation of the cell-bead interaction studies would further contribute to the development of a biomaterials-based system for high throughput T cell production for novel therapies.