Synthetic hydrogels for biomanufacturing of iPSC-derived neural cells for precision medicine

Human neural cells manufactured using patient-derived induced pluripotent stem cells (iPSCs) hold great promise for modeling neurodevelopmental disorders, discovering new precision therapies, and screening for potential risks from environmental toxins 1-4. There have been significant advances in the last decade in protocols and commercial media systems developed for differentiation into specific neural cell types 5-8. However, there remain significant technical challenges to overcome in their generation, manufacturing and assay workflows. iPSCs are typically differentiated on animal-derived substrates that introduce intrinsic variability and lack control over mechanical stiffness and biochemical composition. This often results in low yields and high variability, which may be more pronounced when generating cellular models of diseases. There is a critical need to develop commercial tools that promote differentiation of iPSCs into mature neural cells in a controlled, efficient, and reproducible fashion and that eliminate animal derived products. The resulting cells, associated cell-based assays and cellular therapeutics will have a transformative impact on neural disease modeling, drug and therapeutic discovery and toxin screening.Our Phase I study identified chemically defined and robust synthetic hydrogels for efficient differentiation of iPSC-derived neural progenitor cells (NPCs) into cortical neurons and subsequent maturation to post-mitotic, functionally mature neurons. The highly innovative aspects of this work are that the substrates are employed as thin hydrogel coatings using our proprietary surface-localized polymerization methods which provides several technical and commercialization advantages. In order to bring these novel substrates to market we propose the following specific aims for our Phase II proposal: Specific Aim 1 will further validate the work that demonstrated our optimized synthetic thin hydrogel coatings support neural differentiation and maturation. Including further functional characterization of cells cultured on the substrates by employing microelectrode array analysis and differential transcriptional analysis to compare cells cultured on the substrate. We will characterize of the physical and mechanical properties of the optimized thin hydrogels and develop methods for coating plates using automated systems. Specific Aim 2 will apply the substrates in a Proof-of-Concept demonstration utilizing the substrates to assess cortical neurons from Major Depressive Disorder patient-derived samples compared with controls. Specific Aim 3 will expand the technology platform by optimizing coating techniques on microcarriers suitable for bioreactor scaling, which is a critical step to demonstrate these substrates are applicable to biomanufacturing applications. This work is significant, as there is a critical need for better tools to optimize yields and reduce variability in the differentiation of iPSCs to defined neural subtypes, support their long-term culture, reduce the time needed to reach functional maturity and eliminate animal-derived products in the workflow.Public Health Relevance The use of induced pluripotent stem (iPSC) technologies for disease modeling of neurodevelopmental disorders holds great promise for developing treatment strategies, understanding disease etiologies and screening for potential risks from environmental exposures. However, there are current limitations in the differentiation and maturation protocols that need to be improved including growing and differentiating the cells on animal-derived, hard substrates. We aim to develop novel, soft synthetic hydrogels that will provide a defined substrate which is more amenable to the culture and differentiation of neural cells.