A microgel bone marrow model of mesenchymal stromal 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 ex vivo 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 ex vivo. Here, we describe a granular biomaterial approach to create a multicellular platform for HSC culture. 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 strategy 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. STATEMENT OF SIGNIFICANCE: Hematopoietic stem cells (HSCs) give rise to the entire blood and immune systems and are clinically relevant in the treatment of hematologic disorders. These stem cells reside primarily within bone marrow, the heterogeneity of which is difficult to capture without advances in tissue modeling approaches. HSCs are supported by a plethora of colocalized "niche" cells, including mesenchymal stromal cells (MSCs). In vitro culture of HSCs has primarily used 2D substrates, large (bulk) hydrogels, or scaffolds. This manuscript reports the use of gelatin-maleimide microgels to create a granular hydrogel co-culture to regulate multicellular interactions between HSCs and marrow-derived MSCs. This work contrasts with most granular hydrogel studies, which seed cells only within the interstitial space between particles. We show that encapsulation of MSCs within gelatin microgels forms a mosaic culture that enhances maintenance of co-cultured hematopoietic stem cells, forming a prototypical granular model of bone marrow paracrine signaling.