Deregulation of the imprinted DLK1-DIO3 locus ncRNAs is associated with replicative senescence of human adipose-derived stem cells

Abstract

Background: Human adult adipose-derived stem cells (hADSCs) have become the most promising cell source for regenerative medicine. However the prolonged ex vivo expansion periods required to obtain the necessary therapeutic dose promotes progressive senescence, with the concomitant reduction of their therapeutic potential.

Aim and scope: A better understanding of the determinants of hADSC senescence is needed to improve biosafety while preserving therapeutic efficiency. Here, we investigated the association between deregulation of the imprinted DLK1-DIO3 region and replicative senescence in hADSC cultures.

Methods: We compared hADSC cultures at short (PS) and prolonged (PL) passages, both in standard and low [O2] (21 and 3%, respectively), in relation to replicative senescence. hADSCs were evaluated for expression alterations in the DLK1-DIO3 region on chromosome 14q32, and particularly in its main miRNA cluster.

Results: Comparison of hADSCs cultured at PL or PS surprisingly showed a quite significant fraction (69%) of upregulated miRNAs in PL cultures mapping to the imprinted 14q32 locus, the largest miRNA cluster described in the genome. In agreement, expression of the lncRNA MEG3 (Maternally Expressed 3; Meg3/Gtl2), cultured at 21 and 3% [O2], was also significantly higher in PL than in PS passages. During hADSC replicative senescence the AcK16H4 activating mark was found to be significantly associated with the deregulation of the entire DLK1-DIO3 locus, with a secondary regulatory role for the methylation of DMR regions.

Conclusion: A direct relationship between DLK1-DIO3 deregulation and replicative senescence of hADSCs is reported, involving upregulation of a very significant fraction of its largest miRNA cluster (14q32.31), paralleled by the progressive overexpression of the lncRNA MEG3, which plays a central role in the regulation of Dlk1/Dio3 activation status in mice.

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 P_L cultures as compared with P_S cultures of hADSCs. (C) Relative quantitation of MEG3 lncRNA in two different hADSC samples (n = 3 technical replicates) cultured for short (P_S) and long (P_L) periods. Data shown represent mean ± SEM. (D) Relative quantitation of MEG3 expression in P_S and P_L 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 (P_S) versus long-term (P_L) 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 P_S and P_L 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] (P_S vs. P_L) 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 P_S and P_L 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]).