Engineering 3D Human Neural Tissue Models with Bio-Instructive Hydrogels

Abstract

Neurodegenerative diseases are increasing in prevalence as the population of the United States ages. There are currently no cures or effective treatments for many of these diseases, likely in part due to the lack of predictive in vitro human models of the brain that can bridge the gap between in vivo animal models and human patients. The use of human induced pluripotent stem cells (iPSCs) as a source for neural cells has emerged as a promising tool for modeling the brain; however, it remains difficult to create mature, synaptically connected networks of iPSC-derived neurons in 3D assemblies that mimic the complexity of the human brain.

This dissertation focuses on the development of a novel hydrogel in which N-cadherin peptides are conjugated to methacrylated gelatin (termed GelMA-Cad) and how this bio-instructive hydrogel improves 2 different 3D iPSC-derived neuronal models. The first application of GelMA-Cad in our 3D models focuses on supporting synaptically connected single-cell suspensions of cortical glutamatergic neurons. We demonstrated that the addition of N-cadherin biological cues encourages the survival maturation of the neurons through synaptic tracing assays, electric field potential, and immunofluorescent staining. The embedded neurons in GelMA-Cad were able to form functional synapses after 2 weeks unlike neurons embedded in Matrigel. We then explore how N-cadherin density and the length of time GelMA-Cad was exposed to UV light (UV crosslinking time) affect these 3D glutamatergic neuron cultures. While neurite extensions are longer in lower densities of N-cadherin, we found that synaptic connectivity is not significantly affected. Similar results were observed in the UV crosslinking time conditions. Finally, we demonstrated that gelatin hydrogels functionalized with N-cadherin peptides improves the reproducibility and maturation of iPSC-derived cortical brain organoids, addressing some of the major limitations preventing widespread use of this model.

These studies demonstrate that bio-instructive hydrogel scaffolds can be used to improve iPSC-derived 3D models of the human brain. These two model systems produced through this proposal will serve as vital resources for understanding neurodegenerative disease mechanisms and for developing future therapeutics.