Fig 1.
Replicative senescence is associated with upregulated lncRNA MEG3 expression.
(A) Model of hADSC proliferation kinetics. From the cumulative population doubling data for different hADSC cultures, we obtained a curve fitted to a polynomic function that modeled hADSC proliferation kinetics. The various hADSC cultures were used to establish a mean proliferation curve for 150 days. Each dot represents the mean ± SEM of data obtained by modeling the different hADSC cultures. (B) Representative images indicating loss of differentiation potential (adipogenic and osteogenic) in PL cultures as compared with PS cultures of hADSCs. (C) Relative quantitation of MEG3 lncRNA in two different hADSC samples (n = 3 technical replicates) cultured for short (PS) and long (PL) periods. Data shown represent mean ± SEM. (D) Relative quantitation of MEG3 expression in PS and PL hADSC cultures from a different source, Inbiobank (aADSC, n = 4 biological replicates; with overexpressed hTERT; +hTERT, n = 2 biological replicates); data represent mean ± SEM (* p <0.05, ** p <0.01, *** p <0.001; two-tailed paired t-test). (E) Relative quantitation of MEG3 expression in different cell models (hFB, human fibroblasts; hES-H9, human embryonic stem cell H9; hES-HS181, human embryonic stem cell HS181; hNPC 9.5w, human neural precursor cells from a 9.5-week fetus; hNPC 10w, human neural precursor cells from a 10-week fetus); shown is a representative experiment with 3 technical replicates. (F) Relative quantitation of Meg3 and Dlk1 expression in mouse ADSCs (mADSCs) at various passages; shown is a representative experiment with 3 technical replicates. Data represent mean ± SEM.
Fig 2.
miRNA 14q32.31 cluster is deregulated in association with replicative senescence.
(A) Bar graph shows log fold-change expression for the miRNAs in the 14q32 chromosome region that were deregulated in short-term (PS) versus long-term (PL) cultured adult ADSC samples (n = 2 biological replicates in both cases; mean ± SEM). (B) Relative quantitation (see extended methods in Supporting information for details) of selected miRNAs for array validation in adult (n = 3 biological replicates) and pediatric (n = 2 biological replicates) hADSCs; data represent mean ± SEM (* p <0.05; one-tailed paired t-test).
Fig 3.
DNA methylation analysis of DLK1-DIO3 IG- and MEG-DMR regions.
(A) Percentage of DNA methylation in hADSC samples; x-axes indicate the CpG analyzed in the IG-DMR (1–15) and in the MEG-DMR region (1–8). Data represent mean ± SEM for PS and PL cultures. (B) Bisulfite genomic sequencing of the MEG-DMR regions. CpG dinucleotides are represented as lollipops; methylated cytosines, black; unmethylated cytosines, white. Cultures grown at 21% or 3% [O2] (PS vs. PL) were compared for the indicated hADSC samples.
Fig 4.
Epigenetic analysis of DLK1-DIO3 DMR regions.
(A) Relative quantitation of DNMT1, DNMT3a and DNMT3b (hADSCs; n = 5 biological replicates). Data represent mean ± SEM (* p <0.05; two-tailed paired t-test). (B) Fold enrichment of AcK16H4 relative to total H3 in hADSC05 cultured at 3% [O2], comparing PS and PL samples. Enriched DNA was analyzed by qPCR using primers specific for the different regions (D1, M1–M3; see S1 Fig and S4 Table). Nonspecific adjustment (dCq) was calculated by (dCq = Cq[IP] − Cq[IgG]). Fold enrichment was calculated as 2^(-ddCq) where ddCq is (ddCq = Cq[PL] − Cq[PS]).