Intestinal epithelial crypt cells with low basal autophagy use a YAP-mediated fetal regeneration program

Abstract

Background and rationale: The intestinal epithelium is a layer of cells that creates a protective barrier separating immune cells from the luminal contents of the gut. Homeostasis of the intestinal epithelium is maintained by actively-dividing intestinal stem cells (aISC). When aISCs are depleted via tissue damaging agents such as chemotherapy, irradiation, inflammation, other cell types aid in epithelial regeneration. We call these other cell types facultative stem cells because they can arise from differentiated cells that repurpose themselves as stem cells via unknown mechanisms. Previous work from our lab showed that cells with relatively high autophagy levels can act as facultative stem cells. Specifically, we used a lineage-agnostic autophagy dye called CytoID that marks autophagic vesicles and showed that CytoID-High differentiated cells grew organoids at a higher effciency compared to CytoID-Low cells. The mechanism by which differentiated cells de-differentiate is unknown. Fetal gene expression programs have been observed in intestinal regeneration and is suggested to enable differentiated cells to revert to aISCs. Multiple signals have been shown to regulate fetal-like reversion including YAP/TAZ signaling. We hypothesized that CytoID-High cells exhibit relatively high fetal-like reversion programs and that CytoID-Low cells can be enlisted to contribute to regeneration via stimulation of a fetal-like reversion gene expression program. The objective of this study is to define the signals that regulate the difference in organoid formation between cells with high versus low autophagy. Methods: We used organoid formation assays as a proxy for aISC activity by plating a defined number of single cells purified from the murine jejunum and calculating organoid formation effciency 5 days later. Lgr5-eGFP+ sorted cells- a gold standard in the field- were used as a control for aISC organoid formation. We used fluorescence-activated cell sorting (FACS) of CytoID-stained, EpCAM+ single epithelial cells to distinguish CytoID-High versus CytoID-Low cells and evaluated baseline fetal-like reversion gene expression by qPCR. Sorted cells were plated in Matrigel and supplied with aISC niche factors (EGF, R-spondin, Noggin, CHIR) in the presence of a large tumor suppressor (LATS) kinase inhibitor to activate YAP signaling followed by evaluation of organoid formation effciency. Results and Conclusions: CytoID-High cells sorted directly from tissue exhibited relatively lower expression of fetal-like reversion genes Ly6a and Tacstd2 compared to CytoID Low cells ( Ly6a: -0.2887 ± 0.07813, p= 0.0102, Tacstd2: -0.3580 ± 0.03722, p<0.0001) despite having higher organoid formation effciency. Treatment with a LATS inhibitor to activate YAP-mediated fetal-like reversion genes increased organoid formation in both CytoID-High and -Low cells, but the percent increase in organoid formation between control and LATS inhibitor-treated cells was significantly higher in CytoID-Low cells (% difference: 74.26 ± 27.76, p= 0.0181). Taken together, our data suggest that YAP activation broadly enhances organoid formation regardless of autophagic state, but that CytoID-Low cells exhibit a greater sensitivity to YAP-mediated increases in organoid formation than CytoID-High cells. Since CytoID-High cells exhibit relatively less fetal-like reversion gene expression and less sensitivity to LATS inhibitor-mediated organoid formation, we preliminarily conclude that CytoID low cells represent previously described regenerative cells that utilize YAP-mediated fetal-like reversion to aISCs, whereas facultative stem cell mechanisms in CytoID-High cells remain unknown. NIH T32GM007229 and NIH R01DK124369. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.