Molecular Profiling of Multiplexed Gene Markers to Assess Viability of Ex Vivo Human Colon Explant Cultures

Abstract

Human colon tissue explant culture provides a physiologically relevant model system to study human gut biology. However, the small (20-30 mg) and complex tissue samples used present challenges for monitoring tissue stability, viability, and provision of sufficient tissue for analyses. Combining molecular profiling with explant culture has potential to overcome such limitations, permitting interrogation of complex gene regulation required to maintain gut mucosa in culture, monitor responses to culture environments and interventions. Human ex vivo colon explant gene expression profiles were assayed using an in-house custom-designed hCellMarkerPlex assay at culture time points 0, 1, 2, 4, and 14 h. Characteristic profiles of epithelial cell markers linked to differentiation, cellular polarization, and apoptosis were correlated with visible histochemical changes in explant epithelium during culture and tissue donors. The GenomeLab System provides effective assay of multiple targets not possible from small tissue samples with conventional gene expression technology platforms. This is advantageous to increase the utility of the ex vivo human colon model in applications to interrogate this complex and dynamic tissue environment for use in analytical testing.

Keywords: cell marker; gut explant; microanatomy; molecular profile; multiplex.

FIG. 1.

Histological features of normal colon tissue in explant culture at 0 h (A), 1 h (B), 2 h (C), 4 h (D), and 14 h (E). Frozen tissue is Hematoxylin and eosin stained. Scale bar = 100 μm. (F) Biplot of the first two principle components (PCA plot) of hCellMarkerPlex gene expression data. The hCellMarkerPlex was applied to total RNA from colon explants cultured at 0 h (E0), 1 h (E1), 2 h (E2), 4 h (E4), and 14 h (E14) and compared with hCellMarkerPlex data from a previous study of human colon biopsy tissues, normal (N), adenomatous polyp (P), and carcinoma (T). Data have been normalized to UBE2D2. The PCA plot reveals clustering of colon explants and normal tissue indicating a greater similarity in gene expression profiles compared with the greater divergence in gene expression in adenomatous polyp and carcinoma. Gene targets are identified by name and location designated by (▲). The position of the gene targets signifies levels of expression characterizing the tissue types. e, epithelium; lp, lamina propria; m, muscularis mucosa; PCA, principal component analysis.

FIG. 2.

(A) Relative gene expression levels in human colon explants (n = 14) generated using the hCellMarkerPlex assay. The hCellMarkerPlex assay was applied to assess gene expression profiles of colon explant total RNA (50 ng in triplicate) samples extracted from cultured colon explants at 0, 1, 2, 4, and 14 h. The percentage of CV for each gene was calculated. The percentage of CV of 10% or less was achieved consistently for 20 of the genes within the hCellMarkerPlex (ACTG2, VWF, EZR, NOX1, HDAC1, UBE2D2, CCND1, B4GALNT2, SLC9A2, DES, LGR5, COL1A1, PCNA, CDX1, MS4A12, KRT18, FSP1, B2M, CDX2, and CASP3) as well as for the internal reference marker Kan(r). Low expressers (CNN1, MUC2, and NTN1) exhibited more variable percentage of CV ranging from 10% to 25%. GeNorm (http://medgen.ugent.be/genorm/) identified UBE2D2 as a consistently stable reference gene and data were thus normalized to UBE2D2. ANOVA blocked for tissue donor, with time points as treatment factors, was applied. A post hoc Bonferroni correction was applied and significant differences in the expression of each gene target between the culture time points tested is indicated by unique letters above each bar (p < 0.05). (B) B4GALNT2 expression in colon explants dissected from right and left colon. CV, coefficient of variation.