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

Abstract

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.

Generation of Functional, Antigen-Specific CD8+ Human T Cells from Cord Blood Stem Cells Using Exogenous Notch and Tetramer-TCR Signaling

“…14–17 Our group has previously shown that culturing mouse Lin-c-kit+Sca-1+ HSCs with DLL4-functionalized microbeads in an insert co-culture system using OP9-DL1 cells can induce early T lineage commitment and differentiation without direct stromal cell contact. 18 However, generation of mature, functional SP cells from these in vitro culture systems has not been reported extensively. Recently, a bulk population of OP9-DL1-derived mouse T cells were successfully expanded into antigen-specific, functional CD8+ T cells using bone marrow-derived dendritic cells (DCs) induced to express various antigen epitopes.…”

Biomaterial Strategies for Immunomodulation

“…The lymphocytes that were generated expressed diverse T cell receptor-α and -β chains and responded appropriately to mitogenic stimuli in vitro. In pursuit of strategies to generate T cells in the absence of xenogeneic stromal cells, Taqvi et al (140) reported using synthetic microspheres functionalized with recombinant notch ligands, which mimic key signals provided by stromal cells during thymocyte development. This approach may be able to be combined with other biomaterials-based approaches to create fully synthetic thymic microenvironments.…”

“…In a mouse tumor model, a single injection at day 10 after tumor implantation of loaded particles releasing IL-2 and displaying anti-CD3 and anti-CD28 significantly delayed the kinetics of tumor growth (175). A final approach to immune stimulation, based on immobilization of surface ligands, has been developed by Roy and coworkers (140). They differentiated stem cells into immunocompetent T cells using beads displaying synthetic notch ligands.…”

“…For such patients, robust and reproducible in vitro generation of functional, transplantable T cells from embryonic stem or adult stem cells will be necessary (176). With this in mind, Roy and coworkers (140) functionalized magnetic microbeads with the notch ligand DLL4 using streptavidin--biotin binding and antibody--antigen coupling. Thy1.2 + early T cells were successfully generated from mouse bone marrow hematopoietic stem cells using DLL4 functionalized beads.…”

Challenges and Opportunities to Harnessing the (Hematopoietic) Stem Cell Niche

“…Presentation of extracellular components or growth factors via surface immobilization of material substrates have revealed that HSC-matrix interactions significantly influence HSC adhesion [59], spreading [31], proliferation [60, 61], and differentiation [62]. HSCs show preferential adhesion to several extracellular matrix (ECM) proteins and proteoglycans [28, 59].…”