Nuclear mechanics of intestinal cells determine cellular composition in 3D tissue models and in vivo

Introduction: The crypt-villi architecture of mammalian gut epithelial lining is essential for tissue functions and can be modeled in vitro using intestinal stem cells (ISCs) derived organoids. During homeostasis, the ISC niche exists together with differentiated cells at the crypt base [1]. Anomalies in this homeostatic regulation can cause diseases like colorectal cancer. While molecules secreted by the surrounding mesenchymal cells constitute vital biochemical signals in guiding ISC differentiation and organization, the role of mechanical cues in the process has only been recently identified [2]. However, the spatiotemporal role of the cell nuclei during crypt formation and cellular fate determination is largely unexplored.

Materials and

Methods: We employed an interdisciplinary approach integrating murine ISC derived organoids, in vivo tissue sections, novel hydrogels and sophisticated imaging techniques like light-sheet and expansion microscopy. The hydrogel system consists of poly(ethylene glycol) (PEG) chains functionalized with nitrobenzyl-azide and dibenzylcyclooctyne (DBCO). The two PEG macromers polymerize by strain promoted alkyne-azide cycloaddition (SPAAC) bio-click reaction providing a synthetic platform to encapsulate and study organoids. Uniquely, the ortho-nitrobenzyl crosslinks can be cleaved with a 405nm confocal laser, softening the hydrogel at predefined regions, thus directing crypt formation with spatial and temporal control [3]. Nuclear mechanics was probed by immunostaining of nuclear envelope markers and 3D shape analysis routines. To track evolution of cell population in crypts generated in vitro, organoids bearing fluorescent reporters were used.

Results, Conclusions, and Discussions: We discovered noticeable changes in the nuclear envelope architecture and composition as ISCs differentiate. Alterations in nuclear morphology is associated with changes in the chromatin accessibility and methylation levels. Differentiated cells demonstrate altered localization of the nuclei in the epithelium, which is also observed in vivo. Moreover, variations in initial epithelial shape can dictate nuclear forces, ultimately leading to differences in cell fate. Our results indicate that the nuclei might play an active role in the process of crypt formation and differentiation. Cumulatively, we showcase the design and utilization of a novel biomaterial platform together with 3D organoid systems for studying hitherto unknown nuclear mechano-transduction during intestinal crypt homeostasis (Fig. 1). Our findings can be further extended to decipher the role of cancer stem cells and their regulation during development and progression of colorectal cancer.

Acknowledgements (Optional): This research was supported by the National Science Foundation grant RECODE-2033723, the National Institutes of Health grant R01-DK120921 and the Helen Hay Whitney Foundation award F-1339.

References: [1] M.R. Blatchley & K.S. Anseth, 2023, Nature Reviews Bioengineering. [2] F.M. Yavitt et al., 2023, Science Advances. [3] N. Gjorevski et al., 2022, Science