A reductionist organoid-based model to study how matrix remodelling and soluble signals impact cell fate decisions in human pancreas development

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The interplay between the pancreatic epithelium, its surrounding extracellular matrix, andcell-derived chemical cues are pivotal for pancreas formation and beta cell differentiation.Organoids allow for molecular analyses of complex biological phenomena; however, there arevery few tools available to visualise, modulate or probe matrix/chemical cues and understandtheir impact on cellular responses.iPSC-derived pancreas progenitor organoids (iPPO) can be expanded in Matrigel as itsexternal signals (chemical and mechanical) are sufficient to support iPPO prior to theirdifferentiation to beta cells. We have developed fully synthetic PEG-based hydrogels that cansimilarly support iPPOs.Here, we aim to use PEG-based hydrogels to understand the fundamental molecular, solubleand physical cues required to drive iPSCs differentiation to beta cells that could be used as atherapy in diabetes. We will pursue the following aims:1. Map matrix remodelling around iPPOs.We have established an "embryonic pancreas development" model whereby iPPOs areencapsulated within synthetic hydrogels. We will characterize matrix remodelling aroundorganoids using AFM force spectroscopy to measure peri-organoid stiffness, and multipleparticle tracking microrheology to monitor local degradation.2. Model the diffusion of soluble signals from iPPOs in static and dynamic hydrogels thatmimic peri-organoid stiffness changes. We will use COMSOL to simulate how key solublesignals between the epithelium and mesenchyme are affected by cell-mediated matrixremodelling around organoids. Models will be validated with experimental work usinghydrogels that controllably soften (hydrolysis) or stiffen (secondary radical cross-linking) tomatch dynamics observed in Aim 1.3. Harness the platform to identify the role of specific matrix cues in iPPOs differentiation. Wewill culture iPPOs in softening/stiffening hydrogels to determine if mechanical modulation issufficient to induce differentiation into beta cells. We will also inhibit/overexpress candidatematrix-modulating proteins, allowing us to relate specific mechanistic cues within the matrixto cell fate.