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

Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, 50 South Drive, Bethesda, MD 20892, USA.
Stem Cell Research & Therapy (Impact Factor: 4.63). 06/2010; 1(2):18. DOI: 10.1186/scrt18
Source: PubMed

ABSTRACT Mesenchymal 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.
Human 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)-beta3) 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-beta1 and TIMP1(tissue inhibitor of metalloproteinases-1)).
Significantly higher glycosaminoglycan accumulation was seen in NCCM treated pellets than in CM or TGFbeta groups. With NCCM treatment, increased gene expression of collagen III, and a trend of increasing expression of laminin-beta1 and decreased expression of sox-9 and collagen II relative to the TGFbeta group was observed.
Together, results suggest NCCM stimulates mesenchymal stem cell differentiation toward a potentially NP-like phenotype with some characteristics of the developing IVD.


Available from: James C Iatridis, Oct 15, 2014
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Notochordal cells (NC) remain in the focus of research for regenerative therapy for the degenerated intervertebral disc (IVD) due to their progenitor status. Recent findings suggested their regenerative action on more mature disc cells, presumably by the secretion of specific factors, which has been described as notochordal cell conditioned medium (NCCM). The aim of this study was to determine NC culture conditions (2D/3D, fetal calf serum, oxygen level) that lead to significant IVD cell activation in an indirect co-culture system under normoxia and hypoxia (2% oxygen).
    BMC Musculoskeletal Disorders 12/2014; 15(1):422. DOI:10.1186/1471-2474-15-422 · 1.90 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Embryonic notochordal disc nucleus cells (NC) have been identified to protect disc tissue against disc degeneration but in human beings NC phenotype gets lost with aging and the pathophysiological mechanisms are poorly understood. NC may stimulate other cells via soluble factors, and NC-conditioned medium can be used to stimulate matrix production of other disc cells and mesenchymal stem cells and thus may be of special interest for biological disc repair. As this stimulatory effect is associated with the NC phenotype, we investigated how cell morphology and gene-expression of the NC phenotype changes with time in 3D-cell culture.
    Archives of Orthopaedic and Trauma Surgery 10/2014; 134(12). DOI:10.1007/s00402-014-2097-2 · 1.31 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Early degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term 3D co-culture with canine NPCs or MSCs. Canine NCs and canine NPCs or MSCs were co-cultured in alginate beads for 28 days under hypoxic and high osmolarity conditions. The cell viability, cell morphology and DNA content, the extracellular matrix production, and the expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15, and 28 days of culture. NCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In co-cultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared to monoculture groups, while co-cultures of MSCs and NCs showed increased glycosaminoglycan (GAG)/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC co-culture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa. No regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium (NCCM) may be better than co-culture for future studies to the regenerative potential of NCs.
    Arthritis Research & Therapy 12/2015; 17(1):569. DOI:10.1186/s13075-015-0569-6 · 4.12 Impact Factor