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Dyskeratosis 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.
Dyskeratosis congenita (DC) is a bone marrow failure (BMF) syndrome whereby patients have telomeres at, or below, the first percentile in length when compared with the rest of the population (Savage and Alter, 2009). Patients with DC usually come to clinical attention during childhood and present with a triad of oral leukoplakia, reticular skin pigmentation, and nail dystrophy. While these patients are at increased risk for developing leukemia and solid tumors, BMF is the major cause of death in DC, commonly in young adulthood (Wilson et al., 2014). While the severity of DC varies across patients, more than 85% of afflicted individuals have cytopenias in one or more lineages in late childhood, and more than 95% will develop pancytopenia by adulthood.
All mutations identified to date in DC occur in genes that regulate different aspects of telomere maintenance (Armanios and Blackburn, 2012). The active telomerase complex, which is responsible for telomere elongation in vertebrates, is composed of the reverse transcriptase TERT, the RNA component of telomerase TERC, the TERC-stabilizing nucleolar protein DKC1, and TCAB1, which is responsible for telomerase biogenesis and trafficking (Schmidt and Cech, 2015). Due to the tight regulation of TERT expression, telomerase is only active in stem and progenitor cells (including hematopoietic stem cells [Morrison et al., 1996, Yui et al., 1998]), which suggests that DC is caused by a stem cell failure in highly proliferative tissues, including the bone marrow.
Although there has been extensive biochemical characterization of telomere maintenance mechanisms and their role in maintaining genomic integrity, the connection between telomere dysfunction and the specific clinical phenotypes of BMF in DC patients remains poorly understood. Patient samples are rare and cannot address the effect of telomere deficiency on the genesis of tissue failure that occurs during hematopoietic development. Here, we characterize the primitive and definitive hematopoietic development of isogenic human embryonic stem cells (hESCs) carrying disease-associated mutations in the telomerase components TERT and DKC1, two of the most commonly mutated genes in DC (Armanios and Blackburn, 2012). We show that telomerase expression levels are highly regulated during hESC differentiation toward hematopoietic progenitors, with reduced TERT expression within mesoderm, but restored TERT levels in hematopoietic progenitors. Telomere shortening causes an increase in primitive hematopoietic potential but significantly ablates definitive hematopoietic potential. Furthermore, we demonstrate that p53 mediates the effects of telomere shortening on definitive hematopoiesis, which can be rescued by inhibition of DNA damage signaling or by reactivation of telomerase. Our findings elucidate telomerase regulation patterns during early blood development, indicate a role of DNA damage-induced p53 signaling in hematopoietic specification, and demonstrate the value of in vitro hematopoietic differentiation to study the pathogenesis of BMF in DC patients.
To study hematopoietic failure in cells harboring common DC-associated mutations, we generated DKC1_A353V and TERT_P704S mutant hESCs using CRISPR/Cas9-mediated genome editing (Figures 1A–1C). Cells retained expression of pluripotency markers (Figure 1D) and had no chromosomal abnormalities (Figure S1A). We then analyzed the consequences of these mutations on the expression of telomerase components. As expected, TERT expression remained the same in both mutant cell lines while TERC expression was significantly reduced in DKC1_A353V hESCs (Figure 1E), as dyskerin is necessary for TERC stabilization (Mitchell et al., 1999). No difference was observed in the expression of dyskerin and shelterin components in both DKC1_A353V and TERT_P704S hESCs (Figure S1B). Importantly, telomere repeat amplification assays (TRAP, Figure 1F) showed that telomerase activity, in comparison with wild-type control, is reduced by approximately 75% in DKC1_A353V and TERT_P704S hESCs. While DC hESCs did exhibit subtle changes in cell-cycle distribution (Figures S1C and S1D), they do not show apoptosis induction (Figures S1E–S1G), in contrast with hESCs to full deletion of telomerase (Sexton et al., 2014), suggesting that hESCs with residual telomerase activity behave differently from those completely lacking telomerase activity. To confirm this hypothesis, we created hESCs completely lacking TERC (TERC−/−; Figure S2H). Accordingly, these cells undergo robust apoptosis induction in culture (Figure S2I), similar to TERT−/− hESCs (Sexton et al., 2014). Combined, these data suggest that phenotypes caused by disease-associated mutations in telomerase cannot be recapitulated by complete knockout of telomerase components.
Importantly, both DKC1_A353V and TERT_P704S hESCs have significantly impaired telomere maintenance (Figure 1G), the major characteristic of telomere syndromes. Telomere length in DKC1_A353V and TERT_P704S hESCS averaged <4 kb at passage 30 (compared with >10 kb in the isogenic, parental wild-type; Figure 1G). Collectively, these results indicate that the DKC1_A353V and TERT_P704S point mutations interfere directly with the catalytic activity of telomerase, similar to what is observed in patients. Thus, hESCs harboring patient-specific mutations allow us to directly interrogate the consequences of telomere dysfunction during human tissue development in vitro.
While we and others have made great strides in demonstrating that induced pluripotent stem cells (iPSCs) can serve as a valuable resource to study the biochemical regulation of mutant telomerase, intrinsic clonal variability (Gu et al., 2015, Winkler et al., 2013) and conflicting results arising from different reprogramming methodologies (Agarwal et al., 2010, Batista et al., 2011) have precluded the use of these cells to understand hematopoietic disease development. Vertebrate hematopoietic development comprises at least two distinctive hematopoietic programs: a transient, erythro-myeloid restricted primitive hematopoietic program, and the definitive hematopoietic program, which gives rise to all blood cell lineages found in the adult (Medvinsky et al., 2011). Clinical symptoms of DC are usually not present at birth, with patients not having indications of in utero hematopoietic deficiency, suggesting that aplastic anemia in DC clinically manifests as defective postnatal definitive hematopoiesis. However, most hESC/iPSC differentiation strategies to date have yielded hematopoietic progenitors that are heterogeneous for both programs (reviewed in Ditadi et al., 2017), confounding their use in studying the consequences of telomere dysfunction during human definitive hematopoietic development.
We have recently demonstrated that stage-specific manipulation of canonical WNT-β-catenin signaling allows for the generation of exclusively primitive or exclusively definitive hematopoietic progenitors from hESCs (Figure 2A; Sturgeon et al., 2014). This methodology gives rise to multipotent NOTCH-dependent clonal definitive erythro-myelo-lymphoid progenitors (Ditadi et al., 2015), a hallmark of definitive hematopoiesis (Clements and Traver, 2013). With this approach, we asked whether we could dissect the consequences of dysfunctional telomere maintenance in human definitive hematopoietic progenitors, independently from primitive hematopoietic development.
We first asked whether we could detect telomerase expression during hESC hematopoietic differentiation. We analyzed the expression of the telomerase core components TERT and TERC during early primitive and definitive hematopoietic specification. Mesoderm harboring primitive hematopoietic potential, identified by KDR and CD235a expression, was efficiently obtained on day 3 of differentiation (Figure S2A) following WNT inhibition. By day 8 of differentiation, CD43+ primitive hematopoietic progenitors were identified by flow cytometry (Figure S2A). We isolated these different populations by fluorescence-activated cell sorting (FACS) and found that TERC expression was sustained in all populations (Figure 2Bi). Intriguingly, TERT was transiently repressed in primitive hematopoietic mesoderm, but then its expression was restored in CD43+ primitive hematopoietic progenitors (Figure 2Bii).
Similarly, KDR+CD235a− mesoderm harboring definitive hematopoietic potential was efficiently obtained on day 3 of differentiation (Figure S2A) following WNT activation, which then gave rise to a CD34+CD43−CD73−CD184− hemogenic endothelial population (Figure S2A). When isolated and further cultured under hemato-endothelial conditions, this definitive population gives rise to CD45+ definitive hematopoietic progenitors (Choi et al., 2012, Ditadi et al., 2015). After these populations were isolated by FACS, we observed that TERC expression was retained in all populations (Figure 2Biii). Similar to what was observed in primitive mesoderm, TERT levels were strongly reduced in mesoderm harboring definitive hematopoietic potential (Figure 2Biv), indicating that canonical WNT signaling had no effect on TERT expression. However, following this transient repression, TERT expression within hemogenic endothelium, and its resultant CD45+ definitive hematopoietic progenitors, was significantly upregulated (Figure 2Biv). When taken together, these observations indicate that TERT expression is highly regulated during mesoderm/blood development, with robust telomerase expression being a conserved characteristic of both primitive and definitive hematopoietic progenitors, highlighting the importance of telomere maintenance during hematopoiesis.
With this in vitro model to study the role of telomerase during hematopoietic development, we differentiated our DC hESCs toward primitive and definitive hematopoietic progenitors (Figure 2A). Telomere shortening did not affect the specification of mesoderm, as late-passage DKC1_A353V and TERT_P704S hESCs gave rise to KDR+CD235a+ (primitive) and KDR+CD235a− (definitive) mesodermal populations with efficiency similar to that of wild-type hESCs (Figures S2B and S2C). However, we observed striking differences at later times in hematopoietic development. In comparison with wild-type cells and early-passage (longer telomeres) DC hESCs, late passage (shorter telomeres) had significantly enhanced CD43+ primitive hematopoietic progenitor expansion (Figures S2D and S2E) and primitive erythroid colony-forming cell potential (EryP-CFC; Figures 2Ci and 2Di). These observations indicate that telomere shortening does not broadly abrogate hematopoietic differentiation potential, as DC hESCs with short telomeres are still able to efficiently generate primitive hematopoietic progenitors.
Similarly, early stages of definitive hematopoietic specification were not affected by telomere shortening, as early- and late-passage DKC1_A353V and TERT_P704S hESCs generated a CD34+CD43− population on day 8 of differentiation with efficiency similar to that of control hESCs (Figure S3A). From these, we isolated the definitive CD34+CD43− population and cultured them under hemato-endothelial conditions, to assess their ability to undergo the endothelial-to-hematopoietic transition and yield definitive erythro-myeloid progenitors (Ditadi et al., 2015). Strikingly, late-passage DKC1_A353V and TERT_P704S hESCs exhibited a severe defect in definitive hematopoietic potential, giving rise to approximately 5-fold less definitive erythroid burst-forming units (BFU-E), and 3-fold less myeloid colony-forming units (CFU-M) when compared with wild-type or DC hESCs in early passage (Figures 2Cii and 2Dii). Furthermore, while these cells still retained the ability to give rise to CD4+CD8+ T cell progenitors (Figure 2E), late-passage DC hESCs exhibited a severe reduction in cellularity, suggesting that definitive hematopoiesis is impaired with progressive telomere shortening. The definitive hematopoietic profile of our differentiations was further confirmed by their hemoglobin expression profile (Figure S3B). Importantly, the difference observed is not caused by differential regulation of WNT canonical activity between wild-type and DC mutant cells, as treatment with CHIR and IWP2 led to identical levels of activation/repression of the WNT target gene AXIN2 at day 3 of differentiation, between the wild-type and different isogenic lines (Figure S3C). Combined, these observations suggest that progressive telomere shortening in telomerase-mutant hESCs causes a specific impairment of definitive hematopoietic potential, which may share similar mechanism(s) with the severe pancytopenia observed in DC patients.
Next, we interrogated the mechanism behind the differential regulation of hematopoiesis in hESCs with dysfunctional telomeres. Telomere uncapping due to progressive telomere shortening is a potent inducer of DNA damage. Accordingly, we detected phosphorylated H2AX (γH2AX) accumulation in DKC1_A353V and TERT_P704S hESCs in late passage (Figure 3A). Additionally we observed a cumulative increase in p53 levels in DC-mutant hESCs with progressively shorter telomeres (Figures 3B and S4A), a hallmark of cells harboring dysfunctional telomeres (Palm and de Lange, 2008). As p53-dependent DNA damage response causes BMF in murine models of Fanconi anemia (Ceccaldi et al., 2012), we hypothesized that telomere dysfunction-mediated p53 stabilization could also regulate the specification of hematopoiesis in hESCs. To test this hypothesis, we ablated p53 in our DKC1_A353V hESCs (DKC1_A353V_p53−/−; Figures S4B–S4D). Similar to those with functional p53, these hESCs continue to exhibit progressive telomere shortening with extended passage, as telomerase activity remains impaired (Figure 3C).
We observed that primitive hematopoietic potential was restored to normal levels following p53 deletion, as late-passage DKC1_A353V_p53−/− hESCs exhibited significantly less CD43+ primitive hematopoietic progenitors (Figures 3D and 3E) and concomitant EryP-CFC potential (Figure 3F). Conversely, when we specified these hESCs toward definitive hematopoiesis, we observed that definitive hematopoietic potential was restored in late-passage DKC1_A353V_p53−/− hESCs, as they gave rise to definitive erythro-myeloid and lymphoid progenitors (Figures 3G and S4E) with efficiency similar to that of controls. Furthermore, this functional increase in definitive hematopoiesis was not due to an increase in phenotypic hemogenic endothelium specification, as there was no increase in the observed CD34+CD43−CD73−CD184− population in DKC1_A353V_p53−/− cells compared with DKC1_A353V cells (Figures 3H and 3I). Taken together, these data establish p53 as a regulator of human hematopoietic potential, eliciting positive and negative effects on primitive and definitive hematopoiesis, respectively.
Currently, DC has no cure. However, murine models have shown that reactivation of telomerase is able to rescue homeostasis across different tissues in telomerase-mutant mice (Bar et al., 2016, Jaskelioff et al., 2011). We therefore asked whether telomerase reactivation via genetic correction could restore human definitive hematopoietic potential in DC hESCs. Moreover, by specifically restoring TERC levels in hESCs that retain mutant dyskerin, defects caused specifically by telomere dysfunction can be isolated from defects potentially caused by dyskerin's role in ribosomal biology (Meier, 2005). We therefore inserted a U3-driven TERC within the AAVS1 “safe-harbor” locus (Hockemeyer et al., 2009, Sim et al., 2015), creating DKC1_A353V + TERC hESCs (Figure 4A). Both TERC expression levels (Figure 4B) and TRAP analysis (Figure 4C) indicate restored telomerase activity, which is corroborated by a significant increase of telomere length in DKC1_A353V + TERC hESCs (Figure 4D). We next differentiated these hESCs toward primitive and definitive hematopoiesis. As observed with p53 deletion, telomerase reactivation decreased primitive hematopoietic potential (Figures 4E–4G) and successfully restored definitive erythro-myeloid potential in late-passage DKC1_A353V hESCs (Figure 4H). Thus, reactivation of telomerase can restore normal primitive and definitive hematopoietic output in DC hESCs.
The results presented here demonstrate the essential role of telomerase during human hematopoietic development. Our stage-specific directed differentiation approach demonstrated that telomere shortening elicits a differential effect on primitive and definitive hematopoiesis, mediated by p53.
While a positive role of p53 in erythropoietic differentiation has been observed during malignant hematopoietic differentiation (Molchadsky et al., 2010), our results indicate that p53 can also regulate non-malignant hematopoietic development. Our observed near-complete failure of definitive hematopoietic output from phenotypic hemogenic endothelium, which is restored by p53 deletion, suggests that p53 directly regulates definitive hematopoietic potential in DC cells. Hemogenic endothelium is the direct precursor to the hematopoietic stem cell (Julien et al., 2016), raising the possibility that our observations and clinical BMF may both be due to conserved mechanism(s) governed by p53. As our understanding of human definitive hemogenic endothelium improves, we will be able to better define the role of p53 in this complex developmental process.
Importantly, while telomerase-deficient mice have abnormal blood phenotypes, overt cytopenias due to telomere dysfunction have never been fully recapitulated in murine models (Calado and Young, 2008), which suggests that our observed pan-hematopoietic defect specifically in definitive derived lineages more broadly recapitulates that found in patients. While the exact mechanism(s) exerted by p53 in the regulation of definitive hematopoiesis remains elusive, this in vitro platform of hematopoietic differentiation can be used to address this question. Importantly, however, DC patients are clinically diverse in terms of genetic mutations, inherited telomere length, age of onset, and disease severity. Therefore, it remains to be demonstrated that p53 activation is a universal marker for hematopoietic failure in this disease.
The ability to restore hematopoietic output by reactivation of telomerase lends further support for recent clinical trials demonstrating that increased telomerase function has a positive effect on hematopoiesis in adult aplastic anemia patients (Townsley et al., 2016). Our observations suggest that similar approaches may be used in DC children suffering from hypoplastic marrow. Moreover, the restoration of hematopoietic potential in DKC1 mutant hESCs by rescuing TERC levels, while not interfering with dyskerin's role in ribosomal biology (Meier, 2005), conclusively establishes X-linked DC as a telomere-dysfunction disease.
Together, these results provide evidence that human BMF syndromes can be modeled in vitro, by directed differentiation strategies of human PSCs that recapitulate in vivo development. This opens the exciting possibility of identifying novel therapeutics against BMF in a high-throughput, controlled manner.
H1 (WA01) hESCs were acquired from the WiCell Research Institute (Madison, WI), following all institutional guidelines determined by the Embryonic Stem Cell Research Oversight Committee (ESCRO) at Washington University. hESCs were routinely cultured with mouse embryonic fibroblasts (MEFs) that had been previously γ-irradiated (30 Gy). The cellular passages described as “early” represent the shortest telomeres that still allow efficient hematopoietic differentiation.
DKC1_A353V (X-linked), TERT_P704S (homozygous mutation), DKC1_A353V_p53−/−, and TERC−/− were generated using CRISPR/Cas9 genome-editing technology. CRISPR guides were designed using the Massachusetts Institute of Technology (MIT) CRISPR Design tool (http://crispr.mit.edu/) and generated as described previously (Ran et al., 2013).
W.C.F., E.L.d.O.N., C.D., C.M.S., and L.F.Z.B. designed the experiments and analyzed the data; W.C.F., E.L.N., C.D., and K.A.B. performed the experiments; W.C.F., C.M.S., and L.F.Z.B. wrote the manuscript.
W.C.F. and C.D. were supported by an NHLBI T32 Training Grant in Molecular Hematology (HL007088-41). E.L.d.O.N. was supported by CNPq, Brazil. K.A.B. was supported by the National Fellowship Foundation. L.F.Z.B. is supported by the NHLBI (4R00HL114732-03) and grants from the V Foundation for Cancer Research, Edward Mallinckrodt Jr. Foundation, the Concern Foundation, and the AA&MDS International Foundation. Part of this project was also supported by the Washington University DDRCC grant (NIDDK P30 DK052574) to L.F.Z.B.
Published: July 27, 2017
Supplemental Information includes Supplemental Experimental Procedures, four figures, and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.stemcr.2017.06.015.