Alina Garbuzov

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.

Reversibility of Defective Hematopoiesis Caused by Telomere Shortening in Telomerase Knockout Mice.

Telomere shortening is common in bone marrow failure syndromes such as dyskeratosis congenita (DC), aplastic anemia (AA) and myelodysplastic syndromes (MDS). However, improved knowledge of the lineage-specific consequences of telomere erosion and restoration of telomere length in hematopoietic progenitors is required to advance therapeutic approaches. We have employed a reversible murine model of telomerase deficiency to compare the dependence of erythroid and myeloid lineage differentiation on telomerase activity. Fifth generation Tert-/- (G5 Tert-/-) mice with shortened telomeres have significant anemia, decreased erythroblasts and reduced hematopoietic stem cell (HSC) populations associated with neutrophilia and increased myelopoiesis. Intracellular multiparameter analysis by mass cytometry showed significantly reduced cell proliferation and increased sensitivity to activation of DNA damage checkpoints in erythroid progenitors and in erythroid-biased CD150hi HSC, but not in myeloid progenitors. Strikingly, Cre-inducible reactivation of telomerase activity restored hematopoietic stem and progenitor cell (HSPC) proliferation, normalized the DNA damage response, and improved red cell production and hemoglobin levels. These data establish a direct link between the loss of TERT activity, telomere shortening and defective erythropoiesis and suggest that novel strategies to restore telomerase function may have an important role in the treatment of the resulting anemia.

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