Biomaterial-based notch signaling for the differentiation of hematopoietic stem cells into T cells.

Thymocyte development takes place in a complex milieu of supportive cells and ECM that are responsible for the proliferation, adhesion, migration, and selection processes these cells undergo before reaching maturity. In recent years, the role of notch signaling in lymphocyte development, specifically T-cell development, has been extensively characterized. Although notch ligand mediated signals have been shown to be a necessary component of T-cell generation from stem cells, high-throughput, synthetic biomaterial-based systems for notch-directed stem-cell differentiation into lymphocytes are yet to be reported. Here, we present a microbead-based, artificial notch signaling system to study stem-cell differentiation into the T-cell lineage. Magnetic microbeads were functionalized with the notch ligand DLL4 using streptavidin-biotin binding and antibody-antigen coupling. Immunohistochemistry and flow cytometry analysis indicated approximately 65% conjugation efficiency. Efficient notch signaling through these functionalized microbeads was demonstrated through a myotube inhibition assay in C2C12 myoblasts. Thy1.2(+) early T cells were successfully generated from mouse bone marrow hematopoietic stem cells (BMHSCs) using DLL4 functionalized beads using both insert-based and mixed stromal cell (OP9) coculture conditions, indicating that stem cell-stromal cell physical contact is not necessary for DLL4 directed T-cell differentiation. Coculture studies with bead-to-cell ratios of 1:1 generated higher T-cell differentiation efficiencies, compared to bead-to-cell ratios of 5:1. These data demonstrate the promising potential of this biomaterial-based notch signaling system to generate T cells from stem cells and to elucidate the molecular interactions in T-cell development.