Nonspecific targeting of highly proliferative, non-cancerous cell types during chemotherapy highlights the limitations of these treatment modalities. In the intestinal tract, chemotherapeutics target highly proliferative intestinal stem cells (ISCs). ISCs are responsible for maintaining homeostasis in the intestinal epithelium, and their loss results in detrimental side effects that limit the efficacy of treatment and affect patient quality of life, often many years after treatment. Following injury, regeneration of the ISC compartment is driven by dedifferentiation of various committed lineages, including secretory Paneth cells, leading to recovery from detrimental side effects. As such, there is interest in understanding the molecular mechanisms that influence regeneration. Such knowledge would motivate the development of novel therapeutics to enhance the rate of regeneration, reducing the time and cost associated with chemotherapeutic induced side effects. While in vivo mouse models have been used to study intestinal regeneration following injury, they afford no evaluation of dynamic and transient processes, due to the inability to conduct live cell imaging. In vitro cultures of intestinal organoids, which recapitulate the structure and function of the intestinal epithelium, allow for real time tracking of cell populations in order to study the dynamic interactions between cell populations. However, the heterogeneity and stochastic growth of intestinal organoid cultures often limits their advantage when imaging. Photodegradable poly(ethylene glycol) (PEG) hydrogels can be used to pattern regions of localized softening to direct the formation of intestinal crypt in vitro, resulting in the reproducible formation of uniform crypts. We propose that this material platform can be used to probe the rapidly changing cell interactions and mechanisms that drive regeneration following injury.
In Aim 1, the formation of mature intestinal crypts in vitro is validated under homeostatic conditions. Directed light exposure is used to degrade regions adjacent to 3D encapsulated intestinal organoids, resulting in crypt formation into the degraded regions. Organoids with live cell markers for ISC and Paneth cells will be tracked by live confocal microscopy and custom MATLAB scripts will be used to quantify the migration and interactions of these cell types in real time. Immunostaining for markers of other committed lineages will define the distribution of cell types during homeostasis.
In Aim 2, injury is induced by applying doxorubicin, a chemotherapeutic agent, which eliminates the ISC population. Following injury, the drug will be withdrawn, allowing dedifferentiation of remaining cells and the regeneration of the ISC population. During injury and regeneration, live confocal imaging will be used to track and quantify the ISC and Paneth cell populations, affording insight into their real time dynamic behavior. Single cell transcriptome analysis during injury and regeneration will be used as an unbiased assessment to identify novel pathways that influence Paneth cell dedifferentiation and regeneration. Localization of gene expression will be coupled to real time cell tracking data to further understand the spatiotemporal contributions of essential pathways to intestinal regeneration.
Chemotherapeutic treatment depletes proliferating cells, such as intestinal stem cells, resulting in the development of gastrointestinal related side effects that hinder the efficacy of treatment. The goal of this project is to develop a platform that enables the study of intestinal cells in a controlled environment, allowing for the identification of signaling pathways that influence injury. Identification of such pathways that modulate the injury response and contribution to regeneration following injury could lead to advancement of technologies to reduce detrimental side effects following chemotherapy induced intestinal injury.
