Matthew F. Pech

Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells

The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres, a hallmark of dyskeratosis congenita, impair tissue stem cell function in mouse models, indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state, iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT, the telomerase reverse transcriptase, a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast, mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53), telomerase catalytic activity is unperturbed, yet the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal, indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics.

TPP1 OB-Fold Domain Controls Telomere Maintenance by Recruiting Telomerase to Chromosome Ends

Telomere synthesis in cancer cells and stem cells involves trafficking of telomerase to Cajal bodies, and telomerase is thought to be recruited to telomeres through interactions with telomere-binding proteins. Here, we show that the OB-fold domain of the telomere-binding protein TPP1 recruits telomerase to telomeres through an association with the telomerase reverse transcriptase TERT. When tethered away from telomeres and other telomere-binding proteins, the TPP1 OB-fold domain is sufficient to recruit telomerase to a heterologous chromatin locus. Expression of a minimal TPP1 OB-fold inhibits telomere maintenance by blocking access of telomerase to its cognate binding site at telomeres. We identify amino acids required for the TPP1-telomerase interaction, including specific loop residues within the TPP1 OB-fold domain and individual residues within TERT, some of which are mutated in a subset of pulmonary fibrosis patients. These data define a potential interface for telomerase-TPP1 interaction required for telomere maintenance and implicate defective telomerase recruitment in telomerase-related disease.

A platform for rapid exploration of aging and diseases in a naturally short-lived vertebrate.

VIDEO ABSTRACT Aging is a complex process that affects multiple organs. Modeling aging and age-related diseases in the lab is challenging because classical vertebrate models have relatively long lifespans. Here, we develop the first platform for rapid exploration of age-dependent traits and diseases in vertebrates, using the naturally short-lived African turquoise killifish. We provide an integrative genomic and genome-editing toolkit in this organism using our de-novo-assembled genome and the CRISPR/Cas9 technology. We mutate many genes encompassing the hallmarks of aging, and for a subset, we produce stable lines within 2-3 months. As a proof of principle, we show that fish deficient for the protein subunit of telomerase exhibit the fastest onset of telomere-related pathologies among vertebrates. We further demonstrate the feasibility of creating specific genetic variants. This genome-to-phenotype platform represents a unique resource for studying vertebrate aging and disease in a high-throughput manner and for investigating candidates arising from human genome-wide studies.

Reversible cell-cycle entry in adult kidney podocytes through regulated control of telomerase and Wnt signaling

Mechanisms of epithelial cell renewal remain poorly understood in the mammalian kidney, particularly in the glomerulus, a site of cellular damage in chronic kidney disease. Within the glomerulus, podocytes—differentiated epithelial cells crucial for filtration—are thought to lack substantial capacity for regeneration. Here we show that podocytes rapidly lose differentiation markers and enter the cell cycle in adult mice in which the telomerase protein component TERT is conditionally expressed. Transgenic TERT expression in mice induces marked upregulation of Wnt signaling and disrupts glomerular structure, resulting in a collapsing glomerulopathy resembling those in human disease, including HIV-associated nephropathy (HIVAN). Human and mouse HIVAN kidneys show increased expression of TERT and activation of Wnt signaling, indicating that these are general features of collapsing glomerulopathies. Silencing transgenic TERT expression or inhibiting Wnt signaling through systemic expression of the Wnt inhibitor Dkk1 in either TERT transgenic mice or in a mouse model of HIVAN results in marked normalization of podocytes, including rapid cell-cycle exit, re-expression of differentiation markers and improved filtration barrier function. These data reveal an unexpected capacity of podocytes to reversibly enter the cell cycle, suggest that podocyte renewal may contribute to glomerular homeostasis and implicate the telomerase and Wnt–β-catenin pathways in podocyte proliferation and disease.

High telomerase is a hallmark of undifferentiated spermatogonia and is required for maintenance of male germline stem cells

Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of Tert(High) undifferentiated spermatogonia and Tert(Low) differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular features of each compartment. Transplantation studies show that germline stem cell activity is confined to the Tert(High) cKit(-) population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells, thus explaining the broad dependence on telomerase for germline immortality in metazoans.

TRAPping telomerase within the intestinal stem cell niche.

Adult intestinal stem cells (ISCs) reside at the crypt base, where they continuously proliferate to maintain homeostasis of the intestinal epithelium. As a result of a lifetime of cell division, these LGR5+ ISCs face the combined risk of genomic mutations and telomere attrition. In this issue of EMBOJ , [Schepers et al (2011)][1] examine whether ISCs employ telomerase expression and asymmetric chromosome segregation to protect their genome. [1]: #ref-9

Purification of GFRα1+ and GFRα1– Spermatogonial Stem Cells Reveals a Niche-Dependent Mechanism for Fate Determination

Summary Undifferentiated spermatogonia comprise a pool of stem cells and progenitor cells that show heterogeneous expression of markers, including the cell surface receptor GFRα1. Technical challenges in isolation of GFRα1+ versus GFRα1– undifferentiated spermatogonia have precluded the comparative molecular characterization of these subpopulations and their functional evaluation as stem cells. Here, we develop a method to purify these subpopulations by fluorescence-activated cell sorting and show that GFRα1+ and GFRα1– undifferentiated spermatogonia both demonstrate elevated transplantation activity, while differing principally in receptor tyrosine kinase signaling and cell cycle. We identify the cell surface molecule melanocyte cell adhesion molecule (MCAM) as differentially expressed in these populations and show that antibodies to MCAM allow isolation of highly enriched populations of GFRα1+ and GFRα1– spermatogonia from adult, wild-type mice. In germ cell culture, GFRα1– cells upregulate MCAM expression in response to glial cell line-derived neurotrophic factor (GDNF)/fibroblast growth factor (FGF) stimulation. In transplanted hosts, GFRα1– spermatogonia yield GFRα1+ spermatogonia and restore spermatogenesis, albeit at lower rates than their GFRα1+ counterparts. Together, these data provide support for a model of a stem cell pool in which the GFRα1+ and GFRα1– cells are closely related but show key cell-intrinsic differences and can interconvert between the two states based, in part, on access to niche factors.

Abstract 980: Encoding immortality: Transcriptional control of telomerase in stem cells in vivo

One of the invariant features of human cancer is unlimited proliferation, a hallmark conferred by telomerase in 90% tumors. Somatic mutations in the telomerase reverse transcriptase (TERT) gene promoter are highly recurrent in human cancers. Telomerase is also critically important in human stem cells, as evidenced by mutations in telomerase, which contribute to degenerative diseases. Despite the importance of telomerase in tissue maintenance, the identity of telomerase-positive cells has remained elusive, owing to low levels of the core telomerase components. The ability to isolate TERT-positive cells in vivo would significantly advance our understanding of telomerase regulation, tissue function and carcinogenesis. To address these issues, we created knock-in transcriptional reporters of TERT expression by replacing the TERT open reading frame with the red fluorescent protein, TdTomato. Among mouse tissues, telomerase activity is most strongly expressed in testis, a tissue in which resident stem cells fuel the continuous generation of male gametes. In human sperm, telomere lengths are preserved with age, although how this is achieved, in contrast to the age-dependent telomere shortening seen in somatic tissues, remains unresolved. Using TERTTdTomato/+ knock-in reporter mice, we found that only a rare subset of cells in mouse testis expresses high levels of TERT. By double immunostaining, these TERTHigh cells were synonymous with undifferentiated spermatogonia, the primitive cell population in which male germline stem cells reside. By FACS of the germ cells in testis, TERTHigh cells and TERTLow cells represent discrete populations that were further studied using additional markers. The undifferentiated spermatogonia in the TERTHigh population were further fractionated into GFRalpha+ and GFRalpha- populations. Cells in the TERTLow population were nearly all cKit+, consistent with their identification as differentiated spermatogonia. We characterized these populations in molecular and functional terms. Using RNAseq, we established a hierarchy among these populations according to which the TERTHigh GFRalpha1+ cells give rise to TERTHigh GFRalpha1- cells, which in turn yield TERTLow cKit+ cells. Surprisingly, in transplantation studies, TERTHigh GFRalpha1+ cells and TERTHigh GFRalpha1- cells possess comparable stem cell activity. These data suggest the existence of stem cell plasticity according to which cells in either primitive population retain stem cell potential. In contrast, TERTLow cKit+ cells fail to reconstitute spermatogenesis in transplantation experiments and therefore lack stem cell activity. These studies reveal marked transcriptional regulation of telomerase in vivo and show a strong concordance between stemness and telomerase levels in rare subsets of tissue stem cells in vivo. These findings indicate the existence of innate signaling pathways controlling TERT expression over a surprising dynamic range. Citation Format: Matthew Pech, Alina Garbuzov, Meena Sukhwani, Berenice Benayoun, Shengda Lin, Anne Brunet, Kyle Orwig, Steven E. Artandi. Encoding immortality: Transcriptional control of telomerase in stem cells in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 980. doi:10.1158/1538-7445.AM2015-980