www.nature.com/scientificreports OPEN Heterozygous APC germline mutations impart predisposition to colorectal cancer Livia Preisler1,2, Aline Habib1,2, Guy Shapira2, Liron Kuznitsov‑Yanovsky1,2, Yoav Mayshar1,5, Ilana Carmel‑Gross1, Mira Malcov1, Foad Azem1, Noam Shomron2, Revital Kariv3, Dov Hershkovitz4 & Dalit Ben‑Yosef1,2* Familial adenomatous polyposis (FAP) is an inherited syndrome caused by a heterozygous adenomatous polyposis coli (APC) germline mutation, associated with a profound lifetime risk for colorectal cancer. While it is well accepted that tumorigenic transformation is initiated following acquisition of a second mutation and loss of function of the APC gene, the role of heterozygous APC mutation in this process is yet to be discovered. This work aimed to explore whether a heterozygous APC mutation induces molecular defects underlying tumorigenic transformation and how diferent APC germline mutations predict disease severity. Three FAP‑human embryonic stem cell lines (FAP1/2/3‑hESC lines) carrying germline mutations at diferent locations of the APC gene, and two control hESC lines free of the APC mutation, were diferentiated into colon organoids and analyzed by immunohistochemistry and RNA sequencing. In addition, data regarding the genotype and clinical phenotype of the embryo donor parents were collected from medical records. FAP‑hESCs carrying a complete loss‑of‑function of a single APC allele (FAP3) generated complex and molecularly mature colon organoids, which were similar to controls. In contrast, FAP‑hESCs carrying APC truncation mutations (FAP1 and FAP2) generated only few cyst‑like structures and cell aggregates of various shape, occasionally with luminal parts, which aligned with their failure to upregulate critical diferentiation genes early in the process, as shown by RNA sequencing. Abnormal disease phenotype was shown also in non‑pathological colon of FAP patients by the randomly distribution of proliferating cells throughout the crypts, compared to their focused localization in the lower part of the crypt in healthy/non‑FAP patients. Genotype/phenotype analysis revealed correlations between the colon organoid maturation potential and FAP severity in the carrier parents. In conclusion, this study suggest that a single truncated APC allele is sufcient to initiate early molecular tumorigenic activity. In addition, the results hint that patient‑specifc hESC‑derived colon organoids can probably predict disease severity among FAP patients. Colorectal cancer (CRC) is the third most frequently diagnosed cancer and the second leading cause of cancer death worldwide1. Colorectal tumorigenesis begins with the transformation of normal epithelium to aberrant crypt foci, which then progress to adenoma and proceed to carcinoma2,3. Tis process is closely linked to the stepwise accumulation of multiple genetic and epigenetic aberrations that promote deregulated diferentia- tion and uncontrolled proliferation4–6. Inactivation of adenomatous polyposis coli (APC), a tumor suppressor gene, has been recognized as a key early event in up to 85% of sporadic CRCs4,7. While most cases of CRC are considered sporadic, hereditary CRC syndromes account for 2–5% of all cases 8. As hereditary CRC syndromes are caused by genes that are also somatically altered in the sporadic form, they have been used as a model to understand the molecular pathogenesis underlying CRC in general 9. Familial adenomatous polyposis (FAP) is an inherited autosomal-dominant syndrome caused by a germline mutation in the APC gene10. In accord- ance with Knudson’s ‘two hit’ hypothesis, FAP patients with a germline APC mutation have a very high risk to acquire an additional somatic APC mutation mainly in the colorectum, that will eventually lead to the develop- ment of multiple adenomas, starting at adolescence, with nearly 100% progression to CRC by the age of 40, if 1Wolfe PGD-Stem Cell Laboratory, Racine IVF Unit, Lis Maternity Hospital, Tel-Aviv Sourasky Medical Center, 64239 Tel-Aviv, Israel. 2Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Sagol School of Neuroscience, Tel-Aviv University, Tel-Aviv, Israel. 3Department of Gastroenterology, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel. 4Institute of Pathology, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel. 5Present address: Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. *email: [email protected] Scientifc Reports | (2021) 11:5113 | https://doi.org/10.1038/s41598-021-84564-4 1 Vol.:(0123456789) www.nature.com/scientificreports/ lef untreated10–12. Te site of the ’frst hit’ in the APC tumor suppressor gene has an impact on the type of the ’second hit’ that will either cause APC loss of heterozygosity or a truncated protein 13–17. Moreover, the type and location of this germline mutation are associated with colonic polyp burden, surgical outcome, and the presence of extra-colonic FAP manifestations 13,18. Although the role of APC in CRC initiation has been extensively studied19–24, it is not yet clear whether a heterozygous APC mutation is sufcient to cause the earliest molecular changes leading to colon carcinogenesis in human. It also remains to be determined if various APC germline mutations diferentially afect the initiation and the severity of the developing disease. In the current study, three FAP-human embryonic stem cell (hESCs) lines carrying various human APC germline mutations that were derived in our lab from donated embryos 25,26, were exploited to study whether a mutation in a single allele of APC is sufcient to alter the molecular and cellular phenotype of colon epithelial cells in the in vitro derived colon organoids. Moreover, this unique research model enabled the study of the correlation of specifc mutations to FAP manifestations in vivo in the donor parents. Materials and methods Ethics approvals. Te use of spare in-vitro fertilization (IVF) embryos following preimplantation genetic diagnosis (PGD) for the derivation of hESC lines and for the study of genetic disease was approved by the Israeli National Ethics Committee (7/04-043) and was conducted in accordance with the guidelines of the Bioethics Advisory Committee of the Israel Academy of Sciences and Humanities. Clinical data analysis of the FAP fami- lies was performed under IRB 675/18. Cell lines. Tree FAP-hESC lines were examined in this study: Lis25_FAP1 (FAP1), Lis30_FAP2 (FAP2)25, and Lis34_FAP3 (FAP3; NIH hESC line registry). All experiments were conducted using FAP-hESCs at passage 35–45. Two non-mutated APC hESC lines were used as controls: H9 and Hues13 (MTA with WiCell Inc.). We have previously reported full characterization of the FAP1 and FAP2-hESC lines 25. Te pluripotency of FAP3 was verifed by analysis of the pluripotent stem cell nuclear marker OCT4 and the cell surface marker SSEA4 expression profles (Suppl. Figure 1A). FAP3-hESCs also exhibited typical hESCs morphology and had a normal karyotype, as shown by chromosomal microarray analysis (CMA; Suppl. Figure 1B). Cell culture. hESCs were cultured on Geltrex-coated (TermoFisher) plates in mTeSR1 medium (STEM- CELL Technologies), supplemented with 100 µg/ml primocin (InvivoGen). hESCs were cultured in standard hESC medium (DMEM/F12, 20% KOSR (Invitrogen), 10 ng ml−1 basic fbroblast growth factor (FGF) (Pepro- tech), 1% MEM non-essential amino acids, 0.1 mM β-mercaptoethanol and 1 mM glutamine), were grown on a feeder layer of mouse embryonic fbroblast (MEF) cells. hESCs were passaged using accutase (Merck Biological Industries) and medium was supplemented with 10 µM ROCK inhibitor Y-27632 (Axon Medchem) for the frst 24 h of culture, to inhibit apoptosis. Chromosomal microarray analysis. Genomic DNA was isolated from samples using the DNeasy Blood & Tissue Kit (Qiagen). Te DNA was amplifed, labeled, and hybridized to a 24sure V3 microarray (Illumina), according to the manufacturer’s protocol. Scanning was performed using an Agilent G2565CA scanner and the arrays were analyzed using the BlueFuse Multi sofware. Te detected copy number variants (CNVs) were inter- preted by referring to key public databases (ISCA, DGV, Ensembl, Decipher). Colonic epithelial diferentiation. Diferentiation of FAP- and WT-hESC lines into colon organoids was induced using a published protocol 27. Briefy, to generate defnitive endoderm, hPSCs were treated with 3 μM CHIR99021 (CHIR, Stem-RD) and 100 ng/ml activin A (R&D systems) in RPMI (Cellgro), for one day, and then with 100 ng/ml activin A in RPMI supplemented with 0.2% BSA (Gibco) for three days. Tese cells were than subjected to hindgut diferentiation by treatment with 3 μM CHIR99021 and 500 ng/ml FGF4 (Peprotech) in RPMI supplemented with 1X B27 (Gibco) for four days. From day 8, cells were cultured in colonic medium comprised of advanced DMEM F12 (Invitrogen) supplemented with 1X B27 (Gibco), 3 μM CHIR99021, 300 nM LDN193189 (Axon) and 100 ng/mL EGF (R&D). Te medium was refreshed every two days. On day 20, the cells were disaggregated to a single cell suspension and then re-suspended in Matrigel (BD Biosciences). RNA extraction and quantitative real‑time PCR (qRT‑PCR). qRT-PCR and Western blot analysis were conducted as described previously26. Primer sequences are listed in Supplementary Table 1. Immunofuorescence. For hESC staining, cells were grown on MEF feeder cells in 24-well plates and fxed with 4% paraformaldehyde (PFA). For intra-cellular staining, cells were incubated in blocking solution (2.5% BSA in PBS) with 0.1% Triton, followed by