An official website of the United States government
The cellular organization within organoid models is important to regulate tissue-specific function, yet few engineering approaches can control or direct cellular organization. Here, a photodegradable hydrogel is used to create softened regions that direct crypt formation within intestinal organoids, where the dimensions of the photosoftened regions generate predictable and defined crypt architectures. Guided by in vivo metrics of crypt morphology, this photopatterning method is used to control the width and length of in vitro organoid crypts, which ultimately defines the curvature of the epithelium. By tracking expression of differentiated Paneth-cell markers in real time, we show that epithelial curvature directs the localization of Paneth cells within engineered crypts, providing user-directed control over organoid functionality. We anticipate that our improved control over organoid architecture and thus Paneth-cell localization will lead to more consistent in vitro organoid models for both mechanistic studies and translational applications.
more »
« less
Synthetic Photoresponsive Hydrogels Enable In Situ Control Over Murine Intestinal Monolayer Differentiation and Crypt Formation
Abstract As a model of the intestinal epithelium, intestinal stem cells (ISCs) are grown and differentiated as monolayers on materials where stochastic organization of the crypt and villi cells occurs. An allyl sulfide crosslinked photoresponsive hydrogel with a shear modulus of 1.6 kPa is developed and functionalized with GFOGER, Bm‐binder peptide ligands for monolayer growth of ISCs. The allyl sulfide chemistry allows in situ control of mechanics in the presence of growing ISC monolayers and structured irradiation affords spatial regulation of the hydrogel properties. Specifically, ISC monolayers grown on 1.6 kPa substrates are in situ softened to 0.29 kPa, using circular patterns 50, 75, and 100 µm in diameter, during differentiation, resulting in control over the size and arrangement of de novo crypts and monolayer cellularity. These photoresponsive materials should be useful in applications ranging from studying crypt evolution to drug screening and transport across tissues of changing cellular composition. Spatiotemporal softening enables control over the size and arrangement of de novo crypts within intestinal monolayers.
more »
« less
- Award ID(s):
- 2033723
- PAR ID:
- 10555339
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Functional Materials
- Volume:
- 35
- Issue:
- 3
- ISSN:
- 1616-301X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract Intracellular pH dynamics is increasingly recognized to regulate myriad cell behaviors. We report a finding that intracellular pH dynamics also regulates adult stem cell lineage specification. We identify an intracellular pH gradient in mouse small intestinal crypts, lowest in crypt stem cells and increasing along the crypt column. Disrupting this gradient by inhibiting H+efflux by Na+/H+exchanger 1 abolishes crypt budding and blocks differentiation of Paneth cells, which are rescued with exogenous WNT. Using single-cell RNA sequencing and lineage tracing we demonstrate that intracellular pH dynamics acts downstream of ATOH1, with increased pH promoting differentiation toward the secretory lineage. Our findings indicate that an increase in pH is required for the lineage specification that contributes to crypt maintenance, establishing a role for intracellular pH dynamics in cell fate decisions within an adult stem cell lineage.more » « less
-
The gut epithelium is subject to constant renewal, a process reliant upon intestinal stem cell (ISC) proliferation that is driven by Wnt/b-catenin signaling. Despite the importance of Wnt signaling within ISCs, the relevance of Wnt signaling within other gut cell types and the underlying mechanisms that modulate Wnt signaling in these contexts remain incompletely understood. Using challenge of the Drosophila midgut with a non-lethal enteric pathogen, we examine the cellular determinants of ISC proliferation, harnessing kramer, a recently identified regulator of Wnt signaling pathways, as a mechanistic tool. We find that Wnt signaling within Prospero-positive cells supports ISC proliferation and that kramer regulates Wnt signaling in this context by antagonizing kelch, a Cullin-3 E3 ligase adaptor that mediates Dishevelled polyubiquitination. This work establishes kramer as a physiological regulator of Wnt/b-catenin signaling in vivo and suggests enteroendocrine cells as a new cell type that regulates ISC proliferation via Wnt/b-catenin signaling.more » « less
-
Abstract 3D organoid models have recently seen a boom in popularity, as they can better recapitulate the complexity of multicellular organs compared to other in vitro culture systems. However, organoids are difficult to image because of the limited penetration depth of high‐resolution microscopes and depth‐dependent light attenuation, which can limit the understanding of signal transduction pathways and characterization of intimate cell‐extracellular matrix (ECM) interactions. To overcome these challenges, phototransfer by allyl sulfide exchange‐expansion microscopy (PhASE‐ExM) is developed, enabling optical clearance and super‐resolution imaging of organoids and their ECM in 3D. PhASE‐ExM uses hydrogels prepared via photoinitiated polymerization, which is advantageous as it decouples monomer diffusion into thick organoid cultures from the hydrogel fabrication. Apart from compatibility with organoids cultured in Matrigel, PhASE‐ExM enables 3.25× expansion and super‐resolution imaging of organoids cultured in synthetic poly(ethylene glycol) (PEG) hydrogels crosslinked via allyl‐sulfide groups (PEG‐AlS) through simultaneous photopolymerization and radical‐mediated chain‐transfer reactions that complete in <70 s. Further, PEG‐AlS hydrogels can be in situ softened to promote organoid crypt formation, providing a super‐resolution imaging platform both for pre‐ and post‐differentiated organoids. Overall, PhASE‐ExM is a useful tool to decipher organoid behavior by enabling sub‐micrometer scale, 3D visualization of proteins and signal transduction pathways.more » « less
-
We previously demonstrated that insulin secreting cells (ISCs) accelerate healing of chronic wounds, and it is known that mesenchymal stem cells (MSCs) also accelerate wound healing. Here, we report that the combination of both cell types coencapsulated into a synthetic hydrogel dressing accelerates chronic wound healing 3x faster than control and 2x faster than each cell type delivered singly. Specifically, insulin released by ISCs activates the PI3/Akt pathway, which is vital to the function and survival of MSCs. MSCs in turn improve the viability and function of ISCs.more » « less
