SBIR-STTR Award

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.

Public Health Relevance Statement:
Project Narrative 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.

Project Terms:
Address; Animal Model; Animals; Astrocytes; base; Biological; Biological Assay; Biological Models; biomarker development; Bioreactors; Blinded; Blood Vessels; Brain; Calcium; Cell Culture Techniques; Cells; Cerebrovascular system; Characteristics; Chemicals; Clinical; Coculture Techniques; Communicable Diseases; Complement; Complex; cost; cost effective; cytokine; Data; data archive; density; Development; developmental neurotoxicity; Down-Regulation; drug candidate; Drug Screening; embryonic stem cell; Endothelial Cells; Endothelium; Engineering; Enzyme-Linked Immunosorbent Assay; Expression Profiling; Extracellular Matrix; Failure; FOXG1B gene; Future; gamma-Aminobutyric Acid; Gene Expression Profiling; Glial Fibrillary Acidic Protein; Goals; Growth; Health; Human; Human Development; human embryonic stem cell; Hydrogels; Immunologics; In Vitro; induced pluripotent stem cell; Inflammation; Inflammatory Response Pathway; interest; International; Laboratories; Lead; Letters; Lipopolysaccharides; matrigel; Methods; Microglia; Microscopy; Mitochondria; model development; Modeling; Molecular; Morphology; nerve stem cell; neural model; Neuraxis; neurodevelopment; Neurons; neurotoxicity; Nuclear Pore Complex; Organoids; Output; Pathogenesis; PECAM1 gene; Pericytes; Phagocytosis; Pharmaceutical Preparations; Phase; phase 2 study; Phenotype; Poison; potential biomarker; pre-clinical; precursor cell; Process; Protocols documentation; Publishing; relating to nervous system; Reproducibility; Research; Research Personnel; response; Risk; Rodent; Safety; Sampling; screening; small molecule; SOX1 gene; stem; Stem Cell Development; Stem cells; Suspensions; System; Testing; Therapeutic; Toxic effect; toxicant; Toxicology; Toxin; Transcript; transcriptome; transcriptome sequencing; Tumor-Derived; two-dimensional; Universities; Validation; Wisconsin; Work

There is a critical need to move advanced Central Nervous System (CNS) models into screeningapplications for drug discovery and toxicology applications. Current in vitro models do notaccurately reflect the complexity of cell types and important cell-cell interactions and animalmodels fail to recapitulate the human condition. There is also a great need for more accurate andscalable models for developmental neurotoxicity screenings as there are 86,405 compoundslisted on the Toxic Substance Control Act inventory17 with little biological data to understand theirrisks. Recent advances in stem-cell derived neural organoids have led to use of these models tostudy developmental mechanisms, infectious diseases, and toxicology applications (18-26 andreviewed in27-29), but their cost, complexity, and workflow requirements make them challenging totransition to screening applications. Work performed in our successful Phase I activities at StemPharm with iPSC-derived precursor and differentiated cells has demonstrated that complex neuralorganoids containing a variety of neural subtypes can be developed reproducibly in a 96-wellplate on engineered hydrogel substrates. Unlike organoids cultured in suspension systems, theseorganoids can be formed, cultured, and assayed in multi-well plates. RNA-seq analysisdemonstrated high intraclass correlation and low coefficients of variation. Importantly, wedemonstrated incorporation of microglia into the organoids and demonstrated their activation asa model of neural inflammation as well as their activation or depletion in response to compoundtreatment. In order to bring this novel model to the market we propose the following specific aimsfor the Phase II proposal: 1) To optimize timing and seeding densities with cells derived from asingle iPSC-donor source, optimize incorporation of microglia to maintain robust activationsignatures but decrease cost and maintain data integrity. To compare a less-costly transcriptionalread-out, the TempO-Seq S1500 human panel, to our RNA-seq data obtained in Phase I activitiesand to validate a qPCR panel for product release quality control. 2) To validate organoidsgenerated on our thin hydrogel coatings to enable better imaging options, microelectrode arrayanalysis and liquid handling automation and 3) Validate multiplexed assays to assess multipleresponses in single wells including MEA analysis, cytokine and LDH release and harvest fortranscript or protein analysis. This work will lead to the first commercially available neural organoidcontaining vascular cells and microglia with broad applicability in both toxicology and drugdiscovery markets..

Public Health Relevance Statement:


Project narrative:
This Phase II SBIR project will continue development of 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. The distinguishing characteristics of the model is the ability to incorporate vascular and immune components into the organoid. This allows interrogation of neuroinflammatory mechanisms which are dysregulated by toxin exposure or disease pathologies. Validation of this model will lead to a commercially available accurate assays for screening chemicals and drug candidates for neurotoxicity and neuroinflammation.

Project Terms: