Defective telomere elongation and hematopoiesis from telomerase-mutant aplastic anemia iPSCs

Abstract

Critically short telomeres activate p53-mediated apoptosis, resulting in organ failure and leading to malignant transformation. Mutations in genes responsible for telomere maintenance are linked to a number of human diseases. We derived induced pluripotent stem cells (iPSCs) from 4 patients with aplastic anemia or hypocellular bone marrow carrying heterozygous mutations in the telomerase reverse transcriptase (TERT) or the telomerase RNA component (TERC) telomerase genes. Both mutant and control iPSCs upregulated TERT and TERC expression compared with parental fibroblasts, but mutant iPSCs elongated telomeres at a lower rate compared with healthy iPSCs, and the deficit correlated with the mutations' impact on telomerase activity. There was no evidence for alternative lengthening of telomere (ALT) pathway activation. Elongation varied among iPSC clones derived from the same patient and among clones from siblings harboring identical mutations. Clonal heterogeneity was linked to genetic and environmental factors, but was not influenced by residual expression of reprogramming transgenes. Hypoxia increased telomere extension in both mutant and normal iPSCs. Additionally, telomerase-mutant iPSCs showed defective hematopoietic differentiation in vitro, mirroring the clinical phenotype observed in patients and demonstrating that human telomere diseases can be modeled utilizing iPSCs. Our data support the necessity of studying multiple clones when using iPSCs to model disease.

Bone marrow mesenchymal stem cells from patients with aplastic anemia maintain functional and immune properties and do not contribute to the pathogenesis of the disease

“…3,32 Previous studies demonstrated the defective HSC as well as aberrant T-cell immunity in AA. 3,[33][34][35] Immunosuppressive therapy and allogeneic BM transplantation are the initial treatments of choice for newly diagnosed patients with severe AA. 1,36 On the one hand, the good responses to immunosuppressive treatments such as antithymocyte globulin and cyclosporine A support the belief that pathological T-cell-mediated autoimmune responses are a cause of the BM failure in AA.…”

Acquired TERT promoter mutations stimulate TERT transcription in mantle cell lymphoma

“…Mean telomere length was measured by qPCR based on a modification of the method described by Cawthon [15], as we previously described [16,17]. Briefly, qPCR was conducted in triplicate and all qPCR reactions were prepared on a QIAgility automated pipettor (Qiagen, California).…”

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers

“…GFP-or hDCD19-positive colonies were mechanically transferred onto new MEFs and expanded for further analyses. PCR and Southern blot analysis were performed as previously described 12,44 in order to screen for targeted integration and exclude clones with random integration. Sequences for the primers we utilized are listed in Table S2.…”