4D controllable extracellular matrix properties to guide iPSC-derived intestinal organoid fate and form

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.

Grant Number: 5R00DK135907-03
NIH Institute/Center: NIH
Principal Investigator: Michael Blatchley