Novel Application of Flow Cytometry Side Population Identifies Intestinal Stem Cells in Wild-type Pigs

Severe intestinal ischemia is a critical emergency diagnosis in both human and veterinary medicine, with mortality rates up to 80%. Its underlying pathophysiology is multifactorial but fundamentally linked to the preservation or loss of the intestine’s epithelial lining – a crucial barrier separating the gut contents from the sterile body. This barrier is maintained by proliferative intestinal stem cells (ISCs) that continuously replace the epithelial lining, renewing it entirely approximately every six days. Given ISCs’ essential role in maintaining intestinal barrier integrity, their function following injury merits deeper investigation. However, animal studies of ISCs have been limited to murine models, which have demonstrated poor translatability to human application. Thus, there is a pressing need for improved animal models to advance our understanding of the reparative processes driven by ISCs. Pigs closely approximate human gastrointestinal anatomy and physiology, offering a promising alternative as a large animal model. However, studies of porcine ISCs (pISCs) have been limited by an absence of known external markers for identification. Current methods rely on internal cellular markers, necessitating fixation and precluding in vitro analyses, or the use of an expensive and singularly available transgenic pig model. Altogether, these limitations have hindered progress in leveraging pISCs as a translational study tool. The objective of this study was to implement an alternative method for identifying pISCs using a flow cytometry (FCM)-based approach known as "side population" (SP) analysis. SP uses a cell-permeable dye that is actively effluxed by the membrane-bound transporters in cycling cells such as ISCs and has been effectively used in identifying both mouse and human ISCs. In contrast, non-cycling cells ( e.g., epithelial cells) retain the dye, creating a distinct FCM fluorescent signature. Thus, we hypothesized that SP analysis could be used to identify live pISCs in a wild-type pig model. To this end, we first optimized both chemical and mechanical dissociation protocols to produce a single-cell suspension that, upon FCM analysis, contained a significantly greater SP fluorescent signature compared to traditional dissociation techniques. We then validated the SP specificity by using a Ca 2+ channel blocker control (Verapamil) to inhibit transporter-mediated dye efflux, effectively preventing the SP. These findings are consistent with SP analysis identifying cycling cells, including ISCs. Ongoing analyses aim to exclude CD45+ leukocytes and CD166+ Paneth and goblet cells to further purify this SP. Additionally, 3D cell culture will be used to confirm ISC identity and their proliferative capacity. Furthermore, concurrent evaluations of novel surface markers such as CD24 and CD44 are pending, which may provide additional alternatives for histological pISC identification. Future directions will include fluorescence-activated cell sorting (FACS) for confirmatory downstream qPCR analysis of ISC markers such as LGR5, HopX, and Olfm4. These data represent the first implementation of SP analysis for identifying ISCs in a porcine model. Establishing a method for live pISC isolation is foundational for broader investigations into their regenerative potential. Thus, this work has the potential to broaden our understanding of ISCs, ultimately leveraging their reparative capabilities to advance therapeutic strategies in human and veterinary medicine alike. NIH T35OD011070 Interdisciplinary Biomedical Research Training Program; The Discovery Fund This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.