Telomere Dysfunction Activates P53 And Represses Hnf4 Alpha Expression Leading To Impaired Human Hepatocyte Development And Function

Background and Aims Telomere attrition is a major risk factor for end-stage liver disease. Due to a lack of adequate models and intrinsic difficulties in studying telomerase in physiologically relevant cells, the molecular mechanisms responsible for liver disease in patients with telomere syndromes remain elusive. To circumvent that, we used genome editing to generate isogenic human embryonic stem cells (hESCs) harboring clinically relevant mutations in telomerase and subjected them to anin vitro, stage-specific hepatocyte differentiation protocol that resembles hepatocyte developmentin vivo. Approach and Results Using this platform, we observed that while telomerase is highly expressed in hESCs, it is quickly silenced, specifically due to telomerase reverse transcriptase component (TERT) down-regulation, immediately after endoderm differentiation and completely absent inin vitro-derived hepatocytes, similar to what is observed in human primary hepatocytes. While endoderm derivation is not impacted by telomere shortening, progressive telomere dysfunction impaired hepatic endoderm formation. Consequently, hepatocyte derivation, as measured by expression of specific hepatic markers as well by albumin expression and secretion, is severely compromised in telomerase mutant cells with short telomeres. Interestingly, this phenotype was not caused by cell death induction or senescence. Rather, telomere shortening prevents the up-regulation and activation of human hepatocyte nuclear factor 4 alpha (HNF4 alpha) in a p53-dependent manner. Both reactivation of telomerase and silencing of p53 rescued hepatocyte formation in telomerase mutants. Likewise, the conditional expression (doxycycline-controlled) ofHNF4 alpha, even in cells that retained short telomeres, accrued DNA damage, and exhibited p53 stabilization, successfully restored hepatocyte formation from hESCS. Conclusions Our data show that telomere dysfunction acts as a major regulator ofHNF4 alpha during hepatocyte development, pointing to a target in the treatment of liver disease in telomere-syndrome patients.