Telomere shortening occurs in the TINF2-DC stem cells and reduces the proliferative capacity of differentiated cells. (A) IF staining of TRF1 (red) and γ-H2A.X (green) of WT, heterozygous, and homozygous hESCs. TIF (+): positive control. DNA was stained with DAPI (blue). Scale bar, 10 μm. (B) Quantification of the number of TIFs detected per telomeres per cell. Mann-Whitney test. NS: P > .05. (C) Telomerase repeat amplification assay of the indicated cell lines as hESCs or fibroblasts 30 days after differentiation. IC, internal control. (D) Experimental overview of fibroblast differentiation and growth curves of cumulative population doublings (PDs) over days after differentiation. The isogenic fibroblasts lines were serially passaged until they reached replicative senescence. (E) Quantification of the size of TeSLA PCR products (n = 8) of fibroblasts at indicated time points after differentiation and telomere shortening rate in base pairs per PD (bp/PD).