There is a well-recognized need for more accurate and cost-effective toxicology screening for therapeutic and environmental compounds. Current methods using cell cultures or animal models lack predictability and can be costly. Recent advances in the development of stem-cell derived brain-organoids have led to increasing interest in these models for neural toxicology screening as well as for drug screening and the study of neural developmental mechanisms. However, because of their cost, complexity, and workflow requirements, these methods have yet to be effectively implemented beyond research applications. Currently neural organoids are typically formed on a complex extracellular-matrix derived from tumors cultured in rodents (commonly known as Matrigel). The process is cumbersome, makes interrogation difficult, and lacks reproducibility. More recently, investigators, including a Stem Pharm cofounder, have demonstrated that complex neural organoids can be formed, cultured, and assayed reproducibly in a plate-based system on engineered hydrogel substrates with human embryonic stem (ES) cell-derived precursor cells. Work in this proposal will further that development and lead to the validation of an iPSC-derived neural organoid that can be self-assembled and cultured on Stem Pharm's specialized synthetic hydrogel in a 96-well plate format that is amenable to automated screening applications.
Specific Aims will 1) validate iPSC-derived human neural organoids formed on Stem Pharm synthetic hydrogels utilizing neural progenitors as well as vascular and microglial cells; 2) transition the organoid formation, growth, and interrogation to a 96-well plate format. This will require experimentation and optimization of hydrogel characteristics, cell seeding densities, and media replenishment. Organoid size, morphology, neural maturation, and health will be assessed using microscopy and immunologic and molecular methods and will yield potential biomarkers for future studies; and 3) compare the inflammatory cytokine response to lipopolysaccharide (LPS) microglial activation in the neural organoid system to the response generated by microglia in isolation in a 2-dimensional (2D) culture system. Completion of this study will result in a hydrogel composition and culturing protocols to support the formation of reproducible brain organoids in medium-throughput toxicology screening applications. Phase II studies will utilize data from Phase I transcript expression analyses to identify quantitative molecular panels to assess toxicity and develop phenotypic screens that will be used to validate these assays with known developmental neural toxins and non-toxic controls. This work will develop more accurate models for neural health and pathogenesis and ultimately lead to commercially available assays for screening applications.
This project will develop human neural organoid models that are amenable to toxicology screening in 96-well formats, addressing the well-recognized need for more accurate and cost- effective methods. The study will use Stem Pharm's specialized hydrogel material for the formation of neural organoids from induced pluripotent stem cell-derived cells. Validation of this model and development of biomarkers associated with toxicity will lead to commercially available accurate assays for screening chemicals and drug candidates for neurotoxicity.
