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.
Highlights
Mesenchymal stem cells (MSCs) offer promise for intervertebral disc (IVD) repair and regeneration because they are isolated and expanded, and can differentiate into several mesenchymal tissues
The annulus fibrosus (AF) cells were used as a reference gate population where pulse width and forward scatter (FSC) were used to set the gate, followed by introduction of the nucleus pulposus (NP)/NC cell population and separation of the small NP cells and the large NC cells (Figure 1)
This study demonstrates that NC cells secrete soluble factor(s) that stimulate MSC differentiation down a lineage different from standard mesenchymal chondrogenesis
Summary
Mesenchymal stem cells (MSCs) offer promise for intervertebral disc (IVD) repair and regeneration because they are 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. 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. The ability to separate the NC and NP cells allowed for the first accurate comparisons of their respective gene expression profiles, and comparison to AF cells from the same animals [7]
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