Modeling APC mutagenesis and familial adenomatous polyposis using human iPS cells

Abstract

Mutations in the gene Adenomatous Polyposis Coli or APC appear in most sporadic cases of colorectal cancer and it is the most frequent mutation causing hereditary Familial Adenomatous Polyposis. The detailed molecular mechanism by which APC mutations predispose to the development of colorectal cancer is not completely understood. This is in part due to the lack of accessibility to appropriate models that recapitulate the early events associated with APC mediated intestinal transformation. We have established a novel platform utilizing human induced Pluripotent Stem cells or iPSC from normal or FAP-specific APC mutant individuals and evaluated the effect of the mutation in the cells before and after differentiation into intestinal organoids. In order to minimize genetic background effects, we also established an isogenic platform using TALEN-mediated gene editing. Comparison of normal and APC mutant iPSC revealed a significant defect in cell identity and polarity due to the presence of APC in heterozygosity as well as chromosomal aberrations including abnormal anaphases and centrosome numbers. Importantly, upon specification into intestinal progeny, APC heterozygosity was responsible for a major change in the transcriptional identity of the cells with dysregulation of key signaling pathways, including metabolic reprogramming, abnormal lipid metabolism and intestinal-specific cadherin expression. In conclusion, we have developed a novel iPSC/intestinal model of APC mutagenesis and provide strong evidence that APC in heterozygosity imparts a clear phenotypic and molecular defect, affecting basic cellular functions and integrity, providing novel insights in the earlier events of APC-mediated tumorigenesis.

Adenomatous Polyposis Coli as a Major Regulator of Human Embryonic Stem Cells Self-Renewal

“…Therefore, we conclude that monoallelic APC expression is sufficient for the maintenance of self‐renewal of hESCs. This is in contrast to the findings of Sommer et al that showed significant spontaneous differentiation of iPSC already with monoallelic APC expression.…”

“…However, in all isolated clones harboring CRISPR/ Cas9-induced heterozygous frameshift mutations, full length APC protein expression was maintained, indicating that the mutation occurred in the germline-mutant allele rather than the WT allele. Congruently, none of the isolated clones had a homozygous loss of function mutation in both APC alleles, in line with recent results by Sommer et al [50].…”

Generation of mesenchyme free intestinal organoids from human induced pluripotent stem cells

“…Patient HIOs are suitable for disease modeling. While the translational potential of organoids to the ultimate goal of cell or tissue replacement therapy is still years away, they have already demonstrated significant utility in disease modeling, particularly in the context of monogenic disorders such as Familial Adenomatous Polyposis (FAP) 46 and cystic fibrosis (CF) 12,47,48 . In CF, cell-based models have proved invaluable in developing CFTR modulators.…”

Insights Into Human Development and Disease From Human Pluripotent Stem Cell Derived Intestinal Organoids

“…Additionally, they demonstrated that WNT signaling was suboptimal in HIOs derived from DC patient iPSCs and that this could be compensated by the addition of a WNT agonist which resulted in increased telomerase activity, telomere length and telomere capping. Additionally, two different groups generated iPSCs from patients with Familial adenomatous polyposis (FAP) (16,42). Both groups were able to demonstrate that organoids grown from FAP patient-derived iPSCs exhibited increased WNT signaling compared to controls.…”