PROJECT SUMMARY/ABSTRACT Our understanding of human intestinal development is limited by a lack of tissue accessibility and limitations to existing benchtop models. These laboratory models have advanced with the emergence of organoids, which can better recapitulate the cellular composition and spatial organization of tissue-specific cells than classical in vitro models. Further, induced pluripotent stem cell (iPSC)-derived intestinal organoids (HIOs) are particularly relevant in modeling human intestinal development. However, state-of-the-art protocols fail to account for all relevant niche cues that may influence cell fate, maturation, and morphogenesis, yielding organoids with an immature (i.e., fetal-like) gene signature that limits their relevancy in modeling human disease. Crucially, the timing of exposure to niche cues is vital for proper fate specification. Additionally, we hypothesize that niche cues, beyond the traditionally studied soluble biochemical factors, namely the dynamic properties of the surrounding extracellular matrix (ECM), can and will alter cell signaling and subsequent changes to cell fate. We propose to use a reductionist approach to study the role of the ECM on HIO-derived epithelial organoids (HDEs) and design “blank-slate” biomaterials to precisely and specifically match the properties of the niche that are amenable to organoid growth, then globally and locally alter these properties to understand their role in cell fate decisions, maturation state, and the generation of biomimetic intestinal crypt-villus architecture. We posit that by using advanced imaging techniques, including expansion microscopy and metabolic labeling of nascent proteins, we will be able to further characterize how the ECM changes globally and locally as HDEs grow. In Aim 1, we will investigate how phototunable changes to matrix stiffness (by controlled softening or stiffening) change HDE cellular composition and maturation state over time. In Aim 2, we will spatiotemporally alter local matrix mechanics by photoinduced matrix softening to coax architectural changes to growing HDEs to match in vivo crypt dimensions. We will then study how these changes influence cell fate and maturation. In the K99 phase of the award, Prof. Kristi Anseth, a luminary in using dynamic PEG-based hydrogel materials for manipulating cellular phenotypes, and Prof. Peter Dempsey, a world-leading expert in intestinal biology, will serve as my co- mentors. I will consult my mentoring team, including Prof. Jason Spence (iPSC-derived organoids, scRNA-seq), Prof. Richard Benninger (imaging and image analysis), Dr. Joseph Dragavon (imaging and image analysis), and Prof. Jay Hesselberth (scRNA-seq and bioinformatics analysis). My K99 training will consist of learning key iPSC- derived organoid techniques, advanced imaging and image analysis methods, and scRNA-seq analysis and interpretation to propel me towards developing better models of human development to understand the role ECM niche cues during the independent investigator R00 phase. In sum, the proposed research will address an unmet need to specifically study the role of the ECM in intestinal development and controllably tune properties of the ECM to build better models of the intestine towards improved translational efficacy in future studies.

PROJECT NARRATIVE Organoids are powerful laboratory models of functional organs in the human body. However, their clinical utility is limited because they don’t fully mimic their full-scale counterparts. Here, we seek to address limitations with current methods for growing intestinal organoids in the lab by using engineered biomaterials to characterize and guide growth to build better mini-guts with increased translational potential.