In vitro characterization of N-BMSCs and TBD-BMSCs. (A) As N-BMSCs become confluent, they become tightly packed. (B) TBD-BMSCs spontaneously formed adipocytes as demonstrated by staining with oil red O (noted in TBD-BMSC cultures from patients 1 [TERC], 2 [TINF2], 3 [RTEL1], 4 [TERC], 5 [TERT], 6 [RTEL1], 9 [WRAP53], 10 [no mutation in known telomere-related genes found to date], 11 [TINF2]). A representative experiment is shown. (C) TBD-BMSCs express very high levels of the adipogenic master gene, PPARG (measured in triplicate from BMSCs from 2 normal donors and TBD patients 5 [TERT] and 9 [WRAP53]). A representative experiment is shown (*P ≤ .0001). (D) Unlike N-BMSCs, TBD-BMSCs from 2 related patients formed areas of very dense matrix that was contracted by the cells (noted in 2 of 11 TBD-BMSC cultures, TBD patients 7 [TERT], and 8 [TERT]). In vitro, TDB-BMSCs from the other 11 TBD patients showed far less fibrosis. (E) At P4, N-BMSCs maintained their fibroblastic morphology. (F) At ∼P4, TBD-BMSCs became large and flat, failed to achieve confluency, and stopped growing (TBD-BMSCs from all patients). (G) Little staining for SA-β-galactosidase was found in N-BMSC populations. (H) TBD-BMSC populations had substantially higher levels of SA-β-galactosidase at the same passage (TBD patients 1 [TERC], 2 [TINF2], 3 [RTEL1], 4 [TERC], 5 [TERT], 6 [RTEL1]). A representative experiment is shown. The senescent cells, with blue-green staining, were counted (1 field/chamber, 3-4 chambers/group) and the average number of positive cells is indicated (*P ≤ .05).