Telomere Dysfunction Activates p53 and Represses HNF4α Expression Leading to Impaired Human Hepatocyte Development and Function

Abstract

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 an in vitro, stage-specific hepatocyte differentiation protocol that resembles hepatocyte development in 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 in in 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 (HNF4α) 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) of HNF4α, 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 of HNF4α during hepatocyte development, pointing to a target in the treatment of liver disease in telomere-syndrome patients. (Hepatology 2020;72:1412-1429). T elomeres are repetitive DNA sequences (TTAGGG in humans) that prevent degradation and fusion of chromosomal ends. (1) These structures are largely double-stranded but end in a short single-stranded, G-rich 3′ overhang that spans

Patient induced pluripotent stem cell-derived hepatostellate organoids establish a basis for liver pathologies in telomeropathies

“…Taken together, these results indicate that telomerase dysfunction results in disrupted hepatic development and abnormal hepatocyte proliferation. Our findings are largely consistent with a recent study modeling DC phenotypes in human ES-derived hepatocytes 7 .…”

Telomere erosion in human pluripotent stem cells leads to ATR-mediated mitotic catastrophe

“…While most data generated to date detailing human cells’ response to genetic instability has been derived from terminally differentiated cells, patients with reduced DNA repair capability or reduced telomere maintenance potential commonly show defects in stem or progenitor cells ( Armanios and Blackburn, 2012 ; Fok et al, 2017 ; Munroe et al, 2020 ; Shukla et al, 2020 ). Successful approaches to restore tissue function in these patients must therefore derive from studies that take into consideration the intrinsic differences between stem cells and their differentiated progenies.…”

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