(258b) Hydrogels Containing Gradients in Vascular Density Reveal Dose-Dependent Role of Angiocrine Cues on Stem Cell Behavior

Problem: The use of stem cells for regenerative medicine holds enormous potential, but widespread translation of stem cell therapies is currently limited by inefficient methods of expanding large quantities of functional stem cells and the inability to precisely control stem cell fate. Biomaterial platforms are seen as attractive options for encapsulating, expanding, and delivering stem cells due to the ability to engineer the biomaterial microenvironment to mimic the native stem cell niche. Inspired by the influence that the extracellular matrix exerts on stem cell behavior, efforts have largely focused on defining the structure-function relationships between biochemical and biophysical biomaterial properties and stem cell behavior. Moreover, recent emphasis has been placed on the development of combinatorial or screening platforms to efficiently explore the large design space generated by material parameters. In addition to biochemical and biophysical properties, the niche microenvironment also regulates stem cell behavior via signaling from resident cells (e.g. vascular, immune). In particular, several types of stem cells are known to reside in perivascular niches and receive angiocrine signals from endothelial and perivascular stromal cells. However, the relationship between vascular complexity and stem cell behavior is not well-understood, in part due to the lack of methodologies to generate series of biomaterial microenvironments with varying extents of vascularization. Here, we utilized a microfluidic mixing platform to create hydrogels containing gradients in vascularization. Vascular complexity can be tuned via gradients in cell density and material properties. We used these gradient hydrogels to reveal the dose-dependent relationship between vascular complexity and hematopoietic stem cell (HSC) behavior.

Methods: We used a staggered herringbone microfluidic mixer to generate methacrylamide-functionalized gelatin (GelMA) hydrogels containing gradients in vascular density. Gradients were achieved by mixing GelMA solutions containing different amounts of GelMA, acrylated growth factors, or vascular cell densities. Vascular networks across lateral hydrogel regions were stained using CD31, imaged, and quantified for metrics of complexity (e.g. length, number of branches). To define the effect of vascular complexity on HSC behavior, we generated hydrogels containing a lateral gradient of vascular cell density and a constant presence of HSCs. After seven days, hydrogels were sectioned into four regions, and HSCs from each region were analyzed for stem versus differentiation markers using flow cytometry. Finally, Western blot and secretome analysis were used to identify differences in angiocrine signaling as a function of vascular density.

Results: We generated hydrogels containing regional differences in vascular density via gradients in GelMA matrix density or matrix-bound VEGF. Regional differences in vascular architecture also arose from gradients in vascular cell density, in which the ratio of endothelial to perivascular stromal cells was critical for maintaining gradient resolution across the hydrogels. Increasing vascular density promoted the expansion of differentiated hematopoietic cells while maintaining a small population of quiescent stem cells. Decreased Akt signaling and increased ANG2 secretion were affiliated with hematopoietic expansion at higher vascular densities.

Implications: We developed a methodology for screening the synergistic contributions of matrix properties and resident niche cells on stem cell behavior. We demonstrated that the addition of vascular signaling to biomaterial microenvironments enables the simultaneous expansion of hematopoietic cells for short-term immune reconstitution and maintenance of quiescent stem cells for long-term recovery of the bone marrow. This platform can be used to optimize the inclusion of resident niche cells into biomaterial-based stem cell therapies, thereby enabling an additional avenue with which to modulate stem cell behavior.