p53 Mediates Failure of Human Definitive Hematopoiesis in Dyskeratosis Congenita

Abstract

SummaryDyskeratosis congenita (DC) is a bone marrow failure syndrome associated with telomere dysfunction. The progression and molecular determinants of hematopoietic failure in DC remain poorly understood. Here, we use the directed differentiation of human embryonic stem cells harboring clinically relevant mutations in telomerase to understand the consequences of DC-associated mutations on the primitive and definitive hematopoietic programs. Interestingly, telomere shortening does not broadly impair hematopoiesis, as primitive hematopoiesis is not impaired in DC cells. In contrast, while phenotypic definitive hemogenic endothelium is specified, the endothelial-to-hematopoietic transition is impaired in cells with shortened telomeres. This failure is caused by DNA damage accrual and is mediated by p53 stabilization. These observations indicate that detrimental effects of telomere shortening in the hematopoietic system are specific to the definitive hematopoietic lineages. This work illustrates how telomere dysfunction impairs hematopoietic development and creates a robust platform for therapeutic discovery for treatment of DC patients.

Telomere dysfunction represses HNF4α leading to impaired hepatocyte development and function

“…These include Endoderm, Hepatic Endoderm, Immature Hepatocytes and Mature Hepatocytes. Hepatocyte differentiation protocol adapted from 10 . (B) Relative gene expression analysis by real-time quantitative PCR of different markers specific for each cellular population obtained during hepatic differentiation: OCT4 and SOX2 , hESCs (Day 0); CXCR4 and SOX17 , Endoderm (Day 6); HNF4α , Hepatic Endoderm (Day 11); AFP , Immature Hepatocytes (Day 16); FGA, FGG , Albumin and CYP1A1 , Mature Hepatocytes (Day 21).…”

“…We therefore decided to probe if the observed p53 up-regulation during the hepatocyte differentiation of DKC1_A353V_LP hESCs cells (Figure 3A) was a determinant factor in the reduced efficiency of hepatocyte development observed in cells with short telomeres. For that we used DKC1_A353V_LP hESCs where we ablated p53 through genome engineering (CRISPR/cas9) 10 . These DKC1_A353V_p53 −/− hESCs retain short telomeres, as telomerase is still defective, but have no activation of p53-dependent signaling 10 .…”

“…For that we used DKC1_A353V_LP hESCs where we ablated p53 through genome engineering (CRISPR/cas9) 10 . These DKC1_A353V_p53 −/− hESCs retain short telomeres, as telomerase is still defective, but have no activation of p53-dependent signaling 10 . Interestingly, although retaining short telomeres, these cells have normal hepatocyte differentiation capacity, with restored levels of HNF4α expression during the hepatic endoderm stage (Figure 3D) and restored expression of hepatocyte markers (Figure 3E).…”

“…Differentiation of hESCs into hepatocyte-like cells was performed according to 10 . Briefly, hESCs were transferred to a 100 mm plate coated with matrigel and incubated at 37°C in 5% CO 2 and 5% O 2 for 3 days or until 90-95% confluent.…”

“…To overcome this limitation, we used the targeted differentiation of human embryonic stem cells (hESCs) into mature, hepatocyte-like cells. We used wild-type (H1 hESC line) and CRISPR/cas9 isogenic engineered hESCs harboring a clinically relevant mutation in telomerase (DKC1_A353V mutants created by introduction of a 1058C>T point mutation in the DKC1 gene 10 ). The engineered cells are free of detectable chromosomal abnormalities by G-band analysis and are pluripotent, but have reduced telomerase activity and progressive telomere shortening due to low levels of TERC expression (Supplemental Figure 1A-G).…”