BACKGROUND:Human adult adipose-derived stem cells (hADSCs) have become the most promising cell source for athletic-shirts 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% Plush [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.