Control of Astrocyte Quiescence and Activation in a Synthetic Brain Hydrogel

Abstract

Bioengineers designed numerous instructive brain extracellular matrix (ECM) environments that have tailored and tunable protein composition and biomechanics in vitro to study astrocyte reactivity during trauma and inflammation. However, a major limitation of both protein-based and model microenvironments is that astrocytes within fail to retain their characteristic stellate morphology and quiescent state without becoming activated under "normal" culture conditions. Here we introduce a synthetic hydrogel, that for the first time demonstrates maintenance of astrocyte quiescence, and control over activation on demand. With this synthetic brain hydrogel, we show the brain-specific integrin-binding and matrix metalloprotease (MMP)degradable domains of proteins control astrocyte star-shaped morphologies, and we can achieve an ECM condition that maintains astrocyte quiescence with minimal activation. In addition, we can induce activation in a dose-dependent manner via both defined cytokine cocktails and low molecular weight hyaluronic acid. We envision this synthetic brain hydrogel as a new tool to study the physiological role of astrocytes in health and disease.Astrocytes constitute approximately 30% 1 of the cells within the mammalian brain and function as key producers and maintainers of the brain extracellular matrix (ECM) during brain tissue homeostasis. 2,3 During brain trauma 4 and inflammation 1,5 , changes in the ECM composition 6-10 , ECM stiffness 11 , and the introduction of cytokine molecules 12 transform astrocytes from a quiescent to a reactive state. This reactive state is typically characterized by the upregulation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) 1-3 and vimentin 13 . Recent studies 1 have sought to understand the profile and origination of reactive and quiescent astrocytes 12,14 toward developing therapeutics that inhibit astrocyte activation 1 . Yet, these studies are hindered due to the large complexity and lack of control over the in vivo astrocyte microenvironment. Thus, researchers have increasingly sought in vitro models in which to study astrocyte activation. However, a major limitation to the study of normal and healthy astrocytes in vitro is that there is no reproducible system that can maintain astrocytes in a quiescent state to study activation.For this reason, bioengineers have developed defined and controllable cell culture environments in which to study cells in more native-like conditions. Relevant to the brain, astrocytes grown as three-dimensional (3D) organoids 15,16 provide aspects of their native phenotype, such as a stellate morphology and astrocyte cell-to-cell heterogeneity. However, the close cell-cell contact in these organoid culture is an important drawback, as astrocyte processes only overlap in vivo during their reactive state. 2,17 Additionally, this culturing cells as organoids is time consuming 15,18 and does not allow for the customization of ECM cues like stiffness 6,19,20 and ligand density 7,20 present in real tissue. Prot...

Improved Neural Inductivity of Size-Controlled 3D Human Embryonic Stem Cells Using Magnetic Nanoparticles

“…In order to investigate the improvement of hESC neural inductivity in the form of MNP-hEBs, experimental groups were compared based on addition of 3 variables for neural differentiation: NIM, MNPs, and 3D. Since the hEBs mimic the stiffness of the native tissue [ 53 , 54 ], uniformly sized hEB generation is considered to be a worthwhile strategy to facilitate neuronal differentiation, providing a similar physical environment to brain tissue. In addition, MNP-hEB production could be an efficient approach enhancing cell-to-cell interactions through magnetic force, resulting in improved neural induction.…”