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

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