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

Human embryonic stem cells (hESCs) provide an essential tool to investigate early human development, study disease pathogenesis, and examine therapeutic interventions. Adenomatous polyposis coli (APC) is a negative regulator of Wnt/β-catenin signaling, implicated in the majority of sporadic colorectal cancers and in the autosomal dominant inherited syndrome familial adenomatous polyposis (FAP). Studies into the role of Wnt/β-catenin signaling in hESCs arrived at conflicting results, due at least in part to variations in culture conditions and the use of external inhibitors and agonists. Here, we directly targeted APC in hESCs carrying a germline APC mutation, derived from affected blastocysts following preimplantation genetic diagnosis (PGD) for FAP, in order to answer open questions regarding the role of APC in regulating pluripotency and differentiation potential of hESCs. Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), we generated second hit APC mutations in FAP-hESCs. Despite high CRISPR/Cas9 targeting efficiency and the successful isolation of many clones, none of the isolated clones carried a loss of function mutation in the wild-type (WT) APC allele. Using a fluorescent β-catenin reporter and analysis of mutated-allele frequencies in the APC locus, we show that APC double mutant hESCs robustly activate Wnt/β-catenin signaling that results in rapid differentiation to endodermal and mesodermal lineages. Here, we provide direct evidence for a strict requirement for constant β-catenin degradation through the APC destruction complex in order to maintain pluripotency, highlighting a fundamental role for APC in self-renewal of hESCs. Stem Cells 2019;37:1505-1515.