Decoupling Hydrogel Stiffness and Diffusivity for Hematopoietic Stem Cell Culture and Differentiation

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PROJECT ABSTRACT Hematopoietic stem cells (HSCs) in the bone marrow receive mechanical signals via adhesions to the extracellular matrix and chemical signals from cytokines diffusing through the extracellular matrix. These two signaling methods influence HSC survival, proliferation, and differentiation, with critical long-term effects on the immune system and the body’s ability to maintain homeostasis. While in vitro studies using synthetic hydrogels as artificial matrices have led to new insights on environmental control of HSC behavior, stiffness and solute transport are highly correlated in synthetic hydrogels, so the mechanisms behind HSC responses to these environments remain uncertain. The resulting uncertainty and limited control of hydrogel physical properties may explain why robust methods for in vitro HSC expansion have not been established. Therefore, the studies proposed here will fill a critical gap in hydrogel design capabilities and apply that new knowledge to in vitro HSC culture. First, we will create a library of forty-five unique hydrogel formulations by simultaneously manipulating three structural hydrogel synthesis parameters that our fundamental models have predicted to create robust, independent variations in stiffness and solute transport. Second, we will culture HSCs in a smaller, nine-formulation square matrix of hydrogel formulations with independently tuned stiffnesses and solute transport profiles to decouple how the two physical hydrogel properties affect HSC survival, proliferation, and differentiation. These studies will provide fundamental insight into HSC interactions with their physical environment and identify physically optimized conditions for in vitro HSC culture.