The Telomerase Complex Directly Controls Hematopoietic Stem Cell Differentiation and Senescence in an Induced Pluripotent Stem Cell Model of Telomeropathy.

Shyam Sushama Jose,Tomas Kepak,Jan Fric,Federico Tidu,Petra Burilova,Kamila Bendickova

doi:10.3389/fgene.2018.00345

Abstract

Telomeropathies are rare disorders associated with impaired telomere length control mechanisms that frequently result from genetic mutations in the telomerase complex. Dyskeratosis congenita is a congenital progressive telomeropathy in which mutation in the telomerase RNA component (TERC) impairs telomere maintenance leading to accelerated cellular senescence and clinical outcomes resembling premature aging. The most severe clinical feature is perturbed hematopoiesis and bone-marrow failure, but the underlying mechanisms are not fully understood. Here, we developed a model of telomerase function imbalance using shRNA to knockdown TERC expression in human induced pluripotent stem cells (iPSCs). We then promoted in vitro hematopoiesis in these cells to analyze the effects of TERC impairment. Reduced TERC expression impaired hematopoietic stem-cell (HSC) differentiation and increased the expression of cellular senescence markers and production of reactive oxygen species. Interestingly, telomere length was unaffected in shTERC knockdown iPSCs, leading to conclusion that the phenotype is controlled by non-telomeric functions of telomerase. We then assessed the effects of TERC-depletion in THP-1 myeloid cells and again observed reduced hematopoietic and myelopoietic differentiative potential. However, these cells exhibited impaired telomerase activity as verified by accelerated telomere shortening. shTERC-depleted iPSC-derived and THP-1-derived myeloid precursors had lower phagocytic capacity and increased ROS production, indicative of senescence. These findings were confirmed using a BIBR1532 TERT inhibitor, suggesting that these phenotypes are dependent on telomerase function but not directly linked to telomere length. These data provide a better understanding of the molecular processes driving the clinical signs of telomeropathies and identify novel roles of the telomerase complex other than regulating telomere length.

Highlights

Telomeres are repetitive nucleotide sequences found on the ends of chromosomes that ensure chromosome integrity by protecting chromosome ends from degradation or fusion
The capacity to differentiate into CD34+ cells, as measured by fluorescence-activated cell sorting (FACS), was strongly affected in shTERC-induced pluripotent stem cells (iPSCs) compared to controls (Figures 1C,D)
Others have reported of crosstalk between KLF4 and telomerase; for example, the telomerase reverse transcriptase (TERT) promoter is activated by KLF4 and KLF4 has an important role in maintaining telomerase activity (Wong et al, 2010; Hoffmeyer et al, 2012)

Summary

Introduction

Telomeres are repetitive nucleotide sequences found on the ends of chromosomes that ensure chromosome integrity by protecting chromosome ends from degradation or fusion. The telomere complex machinery is highly active in proliferating and differentiating stem cells (Hiyama and Hiyama, 2007). Telomeres progressively shorten with each cell division as a consequence of the “end replication problem” that telomeres themselves evolved to prevent occurring on the chromosome (Blackburn, 1991; Cawthon et al, 2003). This shortening impairs cellular proliferation and overall cellular regenerative capacity (Flores et al, 2005; Sharpless and DePinho, 2007), and is a useful indicator of cellular senescence. Transgenic TERT-deficient mice exhibit accelerated telomere shortening associated with pathological abnormalities in the gut, extramedullar hematopoiesis in the spleen and liver and a skewed myeloid/erythroid ratio in the bone marrow (Strong et al, 2011)

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