The regenerative potential of young versus senescent rabbit adipose-derived mesenchymal stem cells and their impact on the treatment of intervertebral disc degeneration.

Abstract

Implantation of mesenchymal stem cells (MSCs) appears to be a promising choice for intervertebral disc (IVD) regeneration. Among MSCs, adipose-derived MSCs (ADSCs) have shown stronger differentiation ability towards a nucleus pulposus (NP)-like phenotype. The aim of the current study was to assess the IVD regenerative potential of rabbit ADSCs (rADSCs) in vitro and in vivo. Given that preceding propagation by serial in vitro subculturing is often required to achieve high numbers of viable cells necessary for tissue regeneration applications (which could result in senescence induction), we explored the effect of senescence on rADCSs' molecular profile and function in vitro, as well as its direct effect on the final outcome of IVD tissue quality for the first time, when injected intradiscally in a rabbit model. The molecular profile of isolated young and senescent rADSCs was assessed in vitro, before their delivery into rabbit lumbar IVDs to explore differences regarding their effect on IVD tissue quality. Alcian Blue, Oil Red O and Alizarin Red S staining procedures were performed for the in vitro determination of young and senescent rADSCs' chondrogenic, adipogenic and osteogenic capacity, respectively. Assessment of rADSCs' viability, cell cycle progression and activation of selected biochemical pathways after hyperosmotic treatment was performed by the MTT assay, by flow cytometric analysis and immunofluorescence experiments for the estimation of bromodeoxyuridine (BrdU) incorporation and by western blot analysis, respectively. Senescent rADSCs were characterized in vitro based on their positive SA-β-Gal staining, inability for nuclear BrdU incorporation and catabolic transcriptional profile, the latter assessed by quantitative RT-PCR. The effect of senescence on IVD tissue quality in vivo was explored by histochemical analysis of isolated rabbit lumbar IVDs 8 weeks post-injection with young or senescent rADSCs. Young rADSCs showed a high chondrogenic differentiation potential and were found to generally respond in a similar way with NP IVD cells to high osmolality in vitro, both traits being beneficial for their subsequent use in IVD regenerative applications. Senescent rADSCs displayed a complete loss of their chondrogenic potential and a highly catabolic molecular profile in vitro (up-regulation of MMP1, MMP3 and MMP13 and down-regulation of the proteoglycan aggrecan), which foreboded a negative impact on IVD tissue quality if used in a cell-based therapy. Indeed, delivery of young rADSCs into the NP of rabbit IVDs in vivo resulted in higher staining intensities of collagen type II and aggrecan, in contrast to senescent rADSCs which had the opposite effect. While injection of young rADSCs is beneficial for IVD regeneration in a rabbit model in vivo, here we provide evidence for the first time that senescent rADSCs are not only ineffective when used in IVD therapy, but they could also worsen IVD histological features.