Notochordal cell conditioned medium stimulates mesenchymal stem cell differentiation toward a young nucleus pulposus phenotype

Abstract

IntroductionMesenchymal stem cells (MSCs) offer promise for intervertebral disc (IVD) repair and regeneration because they are easily isolated and expanded, and can differentiate into several mesenchymal tissues. Notochordal (NC) cells contribute to IVD development, incorporate into the nucleus pulposus (NP), and stimulate mature disc cells. However, there have been no studies investigating the effects of NC cells on adult stem cell differentiation. The premise of this study is that IVD regeneration is more similar to IVD development than to IVD maintenance, and we hypothesize that soluble factors from NC cells differentiate MSCs to a phenotype characteristic of nucleus pulposus (NP) cells during development. The eventual clinical goal would be to isolate or chemically/recombinantly produce the active agent to induce the therapeutic effects, and to use it as either an injectable therapy for early intervention on disc disease, or in developing appropriately pre-differentiated MSC cells in a tissue engineered NP construct.MethodsHuman MSCs from bone marrow were expanded and pelleted to form high-density cultures. MSC pellets were exposed to either control medium (CM), chondrogenic medium (CM with dexamethasone and transforming growth factor, (TGF)-β3) or notochordal cell conditioned medium (NCCM). NCCM was prepared from NC cells maintained in serum free medium for four days. After seven days culture, MSC pellets were analyzed for appearance, biochemical composition (glycosaminoglycans and DNA), and gene expression profile (sox-9, collagen types-II and III, laminin-β1 and TIMP1(tissue inhibitor of metalloproteinases-1)).ResultsSignificantly higher glycosaminoglycan accumulation was seen in NCCM treated pellets than in CM or TGFβ groups. With NCCM treatment, increased gene expression of collagen III, and a trend of increasing expression of laminin-β1 and decreased expression of sox-9 and collagen II relative to the TGFβ group was observed.ConclusionsTogether, results suggest NCCM stimulates mesenchymal stem cell differentiation toward a potentially NP-like phenotype with some characteristics of the developing IVD.

The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study

“…Future experiments will test the influence of cell-cell contact by seeding pNCs and bNPCs into alginate beads enabling cell-cell interaction and also the importance of hypoxic conditions. Gene expression revealed a significant up-regulation of Col 1 and Col 2 by the pNCs in the presence of bNPCs, which confirms previous findings of Boyd [3] and Aguiar et al [1] that NCs express certain stimulating cytokines, which seems evident and has been demonstrated using conditioning media [3,9,22]. Korecki et al also detected that SOX-9 (transcription factor for chondrogenesis) was down-regulated if human mesenchymal stem cells (hMSCs) are exposed to notochordal conditioned medium (NCM) as compared to a chondrogenic medium containing TGF-b.…”

“…Previous study on co-culture of non-chondrodystrophoid dog cells (e.g., Greyhound) with bovine NPCs seems to point toward regulatory mechanism and positive cell-cell interaction [1,3,22]. It has been speculated that these cells have precursor character and might belong to the exact same cell lineage as the disc cells since there were not too many differences reported between these two lineages [27,32].…”

The effects of dynamic loading on the intervertebral disc

“…A very low initial MSC survival inside the IVD has been reported [17,20]. On the other hand, in vivo studies have shown that MSCs inside the IVD can differentiate into matrix producing cells [59,99]. The response of the MSC in each specific mechanical condition of the IVD has been studied.…”

Soluble and pelletable factors in porcine, canine and human notochordal cell-conditioned medium: implications for IVD regeneration

“…This process precedes the onset of IVD degeneration, suggesting that NCs play a role in maintaining tissue health. The regenerative effect of NC-secreted factors present in NC-conditioned medium (NCCM) was demonstrated previously on CLCs (Abbott et al, 2012;Potier et al, 2014) and mesenchymal stem cells (MSCs) (de Vries et al, 2015;Korecki et al, 2010;Purmessur et al, 2011), with clear cross-species activity (Bach et al, 2015). Furthermore, porcine NCCM exerted a regenerative effect on bovine NP explants (de Vries et al, 2016), suggesting that NCCM can also stimulate cells in their native tissue.…”

“…Porcine, canine and human NC-secreted factors have already shown potential for IVD regeneration (Abbott et al, 2012;Bach et al, 2015;de Vries et al, 2016;Gantenbein et al, 2014;Korecki et al, 2010;Purmessur et al, 2011). With a clinical directive in mind, the aim of this study was to delineate underlying bioactive NC-secreted substances (with the focus on proteins and EVs) resulting in IVD regeneration.…”