There is a critical need to move advanced Central Nervous System (CNS) models into screening applications for drug discovery and toxicology applications. Current in vitro models do not accurately reflect the complexity of cell types and important cell-cell interactions and animal models fail to recapitulate the human condition. There is also a great need for more accurate and scalable models for developmental neurotoxicity screenings as there are 86,405 compounds listed on the Toxic Substance Control Act inventory17 with little biological data to understand their risks. Recent advances in stem-cell derived neural organoids have led to use of these models to study developmental mechanisms, infectious diseases, and toxicology applications (18-26 and reviewed in27-29), but their cost, complexity, and workflow requirements make them challenging to transition to screening applications. Work performed in our successful Phase I activities at Stem Pharm with iPSC-derived precursor and differentiated cells has demonstrated that complex neural organoids containing a variety of neural subtypes can be developed reproducibly in a 96-well plate on engineered hydrogel substrates. Unlike organoids cultured in suspension systems, these organoids can be formed, cultured, and assayed in multi-well plates. RNA-seq analysis demonstrated high intraclass correlation and low coefficients of variation. Importantly, we demonstrated incorporation of microglia into the organoids and demonstrated their activation as a model of neural inflammation as well as their activation or depletion in response to compound treatment. In order to bring this novel model to the market we propose the following specific aims for the Phase II proposal: 1) To optimize timing and seeding densities with cells derived from a single iPSC-donor source, optimize incorporation of microglia to maintain robust activation signatures but decrease cost and maintain data integrity. To compare a less-costly transcriptional read-out, the TempO-Seq S1500 human panel, to our RNA-seq data obtained in Phase I activities and to validate a qPCR panel for product release quality control. 2) To validate organoids generated on our thin hydrogel coatings to enable better imaging options, microelectrode array analysis and liquid handling automation and 3) Validate multiplexed assays to assess multiple responses in single wells including MEA analysis, cytokine and LDH release and harvest for transcript or protein analysis. This work will lead to the first commercially available neural organoid containing vascular cells and microglia with broad applicability in both toxicology and drug discovery markets. .

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
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Special Emphasis Panel (ZRG1)
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Ravichandran, Lingamanaidu V
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Stem Pharm, Inc.
United States
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