Identification, isolation, and characterization of human LGR5-positive colon adenoma cells

Abstract

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 () and SPARC-related modular calcium binding 2 (). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis.

Patient-Derived Colorectal Cancer Organoids Upregulate Revival Stem Cell Marker Genes following Chemotherapeutic Treatment

“…This is consistent with previous studies conducted on PDCOs which suggest most epithelial features of the primary tumours are present in vitro [31][32][33][34]. All of the PDCOs were positive for the definitive intestinal marker CDX2 [28,35] and the expression of the CBC stem cell marker, LGR5 [36,37] and the cytokeratin marker CK20 was similar in the primary tissue and organoids. Some organoids displayed more widespread expression and this was reflected in the corresponding primary tissue, while others showed little expression of LGR5.…”

LGR5 and BMI1 Increase Pig Intestinal Epithelial Cell Proliferation by Stimulating WNT/β-Catenin Signaling

“…The WNT target genes cyclin D1 and C-MYC have been confirmed as critical downstream signals promoting cell proliferation [ 33 , 34 ], supporting our results that LGR5 or BMI1 overexpression increased cell proliferation. Similarly, recent studies indicate that LGR5 and BMI1 expression in intestinal epithelia is correlated with WNT/β-catenin signaling [ 35 , 36 ].…”

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape

“…The adult jejunum J2 line was kindly provided by Dr. M. Estes (Baylor College of Medicine). A summary of all the HIE lines used in the study and characteristics are listed in S5 Table. 3D Enteroids were derived as previously described [71][72][73], suspended in Matrigel (Corning) and maintained in complete L-WRN medium [74]. The Complete L-WRN medium contained 50% WRN conditioned media (ATCC), Advanced DMEM/F-12 (Gibco), 2mM Glu-taMax (Invitrogen), 10mM HEPES (Invitrogen), 1% penicillin/streptomycin (Gibco), 1X N-2 media supplement (Invitrogen), 1X B-27 supplement minus vitamin A (Invitrogen), 100 μg/ ml Primocin (InvivoGen), 1 mM N-Acetyl-L-cysteine (Sigma-Aldrich), 50 ng/ml EGF (Invitrogen), 10 μM SB202190 (Sigma-Aldrich), 500 nM A83-01 (Tocris), 10 mM nicotinamide (Sigma-Aldrich), 10 nM [Leu15]-Gastrin I (Sigma-Aldrich) and 10μM Y27632 (R&D Systems).…”