Abstract

Hematopoietic stem cells (HSCs) reside in the bone marrow within discrete niches defined by a complex milieu of external signals including biophysical cues, bound and diffusible biomolecules, and heterotypic cell-cell interactions. Recent studies have shown the importance of autocrine-mediated feedback of cell-secreted signals and the interplay between matrix architecture and biochemical diffusion on hematopoietic stem cell activity. Autocrine and paracrine signaling from HSCs and niche-associated mesenchymal stromal cells (MSCs) have both been suggested to support HSC maintenance in vivo and in vitro. Here we report the development of a library of methacrylamide-functionalized gelatin (GelMA) hydrogels to explore the balance between autocrine feedback and paracrine signals from co-encapsulated murine bone marrow MSCs on murine HSCs. The use of a degradable GelMA hydrogel enables the possibility for significant MSC-mediated remodeling, yielding dynamic shifts in the matrix environment surrounding HSCs. We identify a combination of an initially low-diffusivity hydrogel and a 1:1 HSPC:MSC seeding ratio as conducive to enhanced HSC population maintenance and quiescence. Further, gene expression and serial mechanical testing data suggests that MSC-mediated matrix remodeling is significant for the long-term HSC culture, reducing HSC autocrine feedback and potentially enhancing MSC-mediated signaling over 7-day culture in vitro. This work demonstrates the design of an HSC culture system that couples initial hydrogel properties, MSC co-culture, and concepts of dynamic reciprocity mediated by MSC remodeling to achieve enhanced HSC maintenance. One Sentence SummaryCoupling effects of hydrogel biotransport, heterotypic cell culture, and matrix remodeling enhances hematopoietic stem cell culture and quiescence.