A microgel bone marrow model of mesenchymal stem cell paracrine signaling supporting hematopoietic stem cell retention

Hematopoietic stem cells (HSCs) housed within the bone marrow give rise to the full complement of blood and immune cells. Methods to expand HSCs <i>ex vivo</i> have traditionally relied on two-dimensional or liquid culture, but hydrogel approaches have been hypothesized to provide three-dimensional bone marrow-associated biophysical and biomolecular signals that may improve HSC expansion and maintenance <i>ex vivo</i>. Here, we describe a granular biomaterial approach to create a multicellular model of the bone marrow. By seeding HSCs amongst mesenchymal stromal cell (MSC)-laden hydrogel microspheres (microgels), we establish paracrine-mediated interactions between HSCs and hydrogel encapsulated MSCs. We provide support for the importance of microgel encapsulation for the emergence of niche-favorable MSC transcriptional profiles. We identify a common cell culture media that accommodates MSC activity while avoiding the use of serum that typically induces differentiation of HSCs. We observe an MSC-density-dependent increase in maintenance of long-term repopulating HSCs in granular co-culture, and we identify significant depletion of long-term repopulating HSCs when both HSCs and MSCs are interstitially seeded in the granular matrix. Together, these findings establish a granular hydrogel co-culture model to examine the influence of MSC-HSC interactions on maintenance and expansion of HSCs in a defined three-dimensional engineered tissue.