A LGR5 reporter pig model closely resembles human intestine for improved study of stem cells in disease

FASEB J. 2023 Jun;37(6):e22975. doi: 10.1096/fj.202300223R.

Abstract

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.

Keywords: APC mutation; Lgr5; colorectal cancer; intestine; porcine; stem cell; transgenic large animal.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Colon
  • Green Fluorescent Proteins / genetics
  • Humans
  • Ileum
  • In Situ Hybridization, Fluorescence
  • Intestines*
  • Mice
  • Receptors, G-Protein-Coupled / genetics
  • Stem Cells
  • Swine

Substances

  • Green Fluorescent Proteins
  • LGR5 protein, human
  • Receptors, G-Protein-Coupled
  • Lgr5 protein, mouse