Colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing

Abstract

With the goal of modeling human disease of the large intestine, we sought to develop an effective protocol for deriving colonic organoids (COs) from differentiated human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). Extensive gene and immunohistochemical profiling confirmed that the derived COs represent colon rather than small intestine, containing stem cells, transit-amplifying cells, and the expected spectrum of differentiated cells, including goblet and endocrine cells. We applied this strategy to iPSCs derived from patients with familial adenomatous polyposis (FAP-iPSCs) harboring germline mutations in the WNT-signaling-pathway-regulator gene encoding APC, and we generated COs that exhibit enhanced WNT activity and increased epithelial cell proliferation, which we used as a platform for drug testing. Two potential compounds, XAV939 and rapamycin, decreased proliferation in FAP-COs, but also affected cell proliferation in wild-type COs, which thus limits their therapeutic application. By contrast, we found that geneticin, a ribosome-binding antibiotic with translational 'read-through' activity, efficiently targeted abnormal WNT activity and restored normal proliferation specifically in APC-mutant FAP-COs. These studies provide an efficient strategy for deriving human COs, which can be used in disease modeling and drug discovery for colorectal disease.

Figure 1.. A defined strategy to derive colonic organoids from human embryonic stem cells (hESCs).

(a) Scheme of the stepwise differentiation strategy. The cell number at each differentiation stage is estimated based on a starting population of 1 million hPSCs. (b) Representative bright field images of developing hESC-derived COs. Scale bars = 100 μm. (c) Representative bright field image of a hESC-derived CO. Scale bar = 1 mm. (d) qRT-PCR analysis of colonic cell markers in hESC derived D38 small intestinal spheroids (SISs), D38 colonic spheroids (CSs), D48 small intestinal organoids (SIOs) and COs. (e) Immunohistochemistry analysis of hESC-derived SISs, CSs, SIOs and COs. E14.5 mouse embryonic SI and colon, and adult human duodenum tissue samples were used as the controls. Scale bars = 200 μm. (f) Immunocytochemistry analysis in sections of COs showing the presence of CDX2⁺, Ki67⁺, LGR5⁺, EPHB2⁺, KRT20⁺, VILLIN⁺, MUC2⁺, TPH1⁺, CHGA⁺, 5HT⁺, and GLP1⁺ cells. Scale bars = 100 μm. (g) The percentage of major cell types was quantified using MetaMorph Image Analysis Software (Molecular Devices). HE: hindgut endoderm, SISs, small intestinal spheroids, CSs, colonic spheroids, SIOs, small intestinal organoids, COs, colonic organoids. n=3 independent experiments for each condition. P values by unpaired two-tailed student t-test were *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The center value is “median”. Error bar is S.D.

Figure 2.. Global gene expression analysis of hESC-derived COs or SIOs.

(a) Heatmaps derived from RNA-seq profiles of small intestine or colon marker genes in human duodenum, distal small intestine, colon (E-MTAB-1733) and hESC-derived COs and SIOs. (b) Heatmap showing sample-to-sample distances. Distance between two samples was calculated by comparing the normalized expression data with Euclidean distances. The darker color indicates no difference while the lighter color indicates larger distances. (c) Heatmap showing the expression data of marker genes of human duodenum, distal small intestine, and colon (E-MTAB-1733). Marker genes of each sample type were selected such that a gene has at least 3 times higher expression in that sample type compared to the other two sample types. (d) GSEA indicates that hESC-derived COs are significantly enriched for transcripts that are also highly expressed in human colon while hESC-derived SIOs are significantly enriched for transcripts that are also highly expressed in human duodenum. (e) GSEA indicates that an adult gene set (genes >50 fold upregulated in adult human colon/GSM1698576 vs. fetal human colon/GSM1698561) is significantly upregulated in hESC-derived COs and the fetal gene set (genes >50 fold upregulated in fetal human colon/GSM1698561 vs. adult human colon/GSM1698576) is significantly downregulated in hESC-derived COs. Enriched gene sets were selected based on statistical significance (FDR q value <0.25 and/or NOM p value <0.05).

Figure 3.. FAP colonic organoids show enhanced WNT activity and increased cell proliferation.

(a) Mutations in the APC locus in FAP iPSC lines detected by genomic DNA sequencing. (b) Western blotting demonstrates reduced APC protein expression levels in FAP iPSCs. (c) Representative bright field images of wildtype (wt) and FAP COs. Scale bar= 1 mm. (d) Western blotting demonstrates the reduced APC protein expression levels in iPSC-FAP COs. (e) 3’3’-diaminobenzidine immunohistochemical staining and quantification of the percentage of nuclear β-catenin of wildtype and FAP iPSC-derived COs. β-catenin is enriched in nuclei only in the mutant lines (insets). Scale bar= 100 μm. (f, g) Gene ontology pathway analysis of genes that are at least two-fold higher expressed in FAP8 COs (f) or FAP9 COs (g) than wildtype COs. (h) qRT-PCR analysis of WNT pathway target genes in wildtype, FAP8 and FAP9 COs. (i) Maximal intensity projection images and (j) quantification of wildtype and FAP COs immunostained for CDX2 and CD1. (k) Maximal intensity projection images and (l) quantification of cells comparing wildtype and FAP COs immuno-stained for CDX2 and Ki67. Scale bar= 200 μm. n=3 independent experiments for each condition. P values by unpaired two-tailed student t-test were *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The center value is “median”. Error bar is S.D.

Figure 4.. A platform to evaluate drug candidates that can rescue APC mutation-induced hyper-proliferation in human colonic organoids.

(a) Maximal intensity projection images and (b) quantification of cells in wildtype and FAP COs treated with DMSO, 10 μM rapamycin or 5 μM XAV939, after being immuno-stained for CDX2 and Ki67. (c) Maximal intensity projection images and (d) quantification of cells in wildtype and FAP COs treated with DMSO, rapamycin or XAV939, after being immuno-stained for CDX2 and CD1. (e) qRT-PCR analysis of WNT pathway target genes comparing wildtype and FAP9 COs treated with DMSO, rapamycin or XAV939. (f-g) Representative western blot assay comparing wildtype and FAP iPSCs (f) and wildtype and FAP COs (g) treated with different concentrations of geneticin (Gen). (h) Maximal intensity projection images and (i) quantification of cells from wildtype and FAP COs treated with DMSO and geneticin immuno-stained for CDX2 and CD1. (j) Maximal intensity projection images and (k) quantification of cells from wildtype and FAP COs treated with DMSO and geneticin immunostained for CDX2 and Ki67. (l) qRT-PCR analysis of WNT pathway target genes in wildtype and FAP COs treated with DMSO or geneticin. wt: wildtype; rapa: rapamycin; XAV: XAV939; Gen: Geneticin. n=3 independent experiments for each condition. P values by unpaired two-tailed student t-test were *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The center value is “median”. Error bar is S.D. Scale bar = 200 μm.