(567d) Gradient Hydrogel System to Decode Paracrine Signaling Between Hematopoietic Stem Cells and Their Bone Marrow Niche

We have previously shown that opposing gradients of multiple cell types (i.e. HSCs, osteoblast niche cells) can be created within individual multi-gradient hydrogels (‘microgels’). The local ratio of HSCs to niches cells was determined via populational (fluorescent slide scanner) and individual (FACS, confocal imaging of local cell microenvironment) cell measurements. These microgels support cell culture with no observed increase in cell death due to the microfluidic mixing. Ongoing work is determining local small molecule biotransport properties within the microgels using fluorescently labeled Dextran molecules for estimating the efficiency of paracrine signaling processes. We have also created preliminary HSC:niche cell cultures to assess changes in the frequency of key HSC fates in response to defined microenvironments. By varying the type and density of niche cells as well as the density of the hydrogel, we can control the kind of paracrine signals the HSCs are exposed to and their diffusion characteristics. Here, HSCs are isolated as Lin^-Sca-1⁺c-Kit⁺ (LSK) from murine bone marrow; niche cell populations chosen for this assay are un-enriched Lin+ bone marrow cells, CXCL12+ bone marrow stromal cells, and murine osteoblasts. We are determining the relative frequency of HSC viability/proliferation (MTT assay) vs. differentiation (surface antigen expression, MethoCult functional assays) within these defined heterotypic cell microenvironments. Improved understanding of HSC fate decision is critical for optimizing biomaterial systems for ex vivo expansion of clinically relevant hematopoietic cells and for studying the etiology, niche regulation and treatment of hematopoietic pathologies.