Joshua Snyder

Intestinal epithelial stem cells (ISCs) are responsible for intestinal epithelial barrier renewal; thereby, ISCs play a critical role in intestinal pathophysiology research. While transgenic ISC reporter mice are available, advanced translational studies lack a large animal model. This study validates ISC isolation in a new porcine Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5) reporter line and demonstrates the use of these pigs as a novel colorectal cancer (CRC) model. We applied histology, immunofluorescence, fluorescence-activated cell sorting, flow cytometry, gene expression quantification, and 3D organoid cultures to whole tissue and single cells from the duodenum, jejunum, ileum, and colon of LGR5-H2B-GFP and wild-type pigs. Ileum and colon LGR5-H2B-GFP, healthy human, and murine biopsies were compared by mRNA fluorescent in situ hybridization (FISH). To model CRC, adenomatous polyposis coli (APC) mutation was induced by CRISPR/Cas9 editing in porcine LGR5-H2B-GFP colonoids. Crypt-base, green fluorescent protein (GFP) expressing cells co-localized with ISC biomarkers. LGR5-H2B-GFPhi cells had significantly higher LGR5 expression (p < .01) and enteroid forming efficiency (p < .0001) compared with LGR5-H2B-GFPmed/lo/neg cells. Using FISH, similar LGR5, OLFM4, HOPX, LYZ, and SOX9 expression was identified between human and LGR5-H2B-GFP pig crypt-base cells. LGR5-H2B-GFP/APCnull colonoids had cystic growth in WNT/R-spondin-depleted media and significantly upregulated WNT/β-catenin target gene expression (p < .05). LGR5+ ISCs are reproducibly isolated in LGR5-H2B-GFP pigs and used to model CRC in an organoid platform. The known anatomical and physiologic similarities between pig and human, and those shown by crypt-base FISH, underscore the significance of this novel LGR5-H2B-GFP pig to translational ISC research.

<jats:p>&lt;p&gt;mRNA FISH of cell state markers&lt;/p&gt;</jats:p>

<jats:p>&lt;p&gt;Supplementary Table 1: Cluster-specific markers from all cells (8,486 cells) Supplementary Table 2: Cluster-specific markers from all epithelial cells (3,843 cells) Supplementary Table 3: Cluster-specific markers from all immune cells (1,803 cells) Supplementary Table 4: Cluster-specific markers from all fibroblast cells (922 cells) Supplementary Table 5a: State-specific markers from all d16:HBOW epithelial cells (1,421 cells) Supplementary Table 5b: State-specific markers from all p95:HBOW epithelial cells (1,258 cells)&lt;/p&gt;</jats:p>

<jats:p>&lt;p&gt;MaXFISH coregistry of sequenced tumors&lt;/p&gt;</jats:p>

<jats:p>&lt;p&gt;UMAP clustering of sequenced cells and cluster identification&lt;/p&gt;</jats:p>