Prolonged in vitro expansion partially affects phenotypic features and osteogenic potential of ovine amniotic fluid-derived mesenchymal stromal cells

Background aims. Ovine amniotic fluid mesenchymal stromal cells (oAFMSCs) are an emerging alternative source of stemcells to develop pre-clinical cell replacement protocols. For tissue engineering purposes, oAFMSCs can be used eitherimmediately after isolation or after in vitro expansion. However, detailed studies are still required to investigate theadvantages and drawbacks of their in vitro expansion. Methods. The phenotype and osteogenic differentiation potential ofoAFMSCs were analyzed in relation to in vitro expansion that was carried out for 20 consecutive passages. ExpandedoAFMSCs were analyzed for proliferation index, expression profiles of several surface, pluripotency-associated and HLAantigens, global DNA methylation, telomere length and karyotype. The osteogenic differentiation ability of expandedoAFMSCs was assessed by qualitative and quantitative methods. Results. Expanded oAFMSCs reduced their proliferativeactivity after 10 passages and partially modified the expression of surface antigens and the intracellular distribution ofpluripotency-associated markers (NANOG, SOX2 and TERT) after 20 passages. The phenotypic alteration of culturedoAFMSCs was associated with a reduction of in vitro osteogenic plasticity. In detail, after 20 passages of cellular expansion,oAFMSCs lost the ability to increase osteocalcin and decreased collagen type I messenger RNA expression. Also, a lowerpercentage of cells displayed intracellular calcium release after stimulation with salmon calcitonin. Conclusions. The resultspresented here suggest that long-term in vitro expansion may cause significant alterations in phenotypic features and plasticityof oAFMSCs, suggesting a careful re-evaluation of in vitro cultural and temporal conditions before employingexpanded oAFMSCs for therapeutic purposes.[...]