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

Abstract

Introduction: 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.

Methods: 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)).

Results: 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.

Conclusions: Together, results suggest NCCM stimulates mesenchymal stem cell differentiation toward a potentially NP-like phenotype with some characteristics of the developing IVD.

Figure 1

Schematic of experimental design to produce NCCM. NP tissue from the porcine IVD was harvested and digested to obtain a mixture of large NC and small NP cells. Large NC cells were isolated from the mixture by FACS (top FACS image is combined NC/NP cells with the reference gate to select NC cells, bottom FACS image is the isolated cells from the gate (NC cells). NC cells were embedded in alginate, and cultured for four days to produce conditioned medium (NCCM).

Figure 2

Viability and morphology of NC cells embedded in alginate and cultured for four days. Cell viability (a, c) was assessed using LIVE/DEAD staining and morphology (b, d) was observed by means of phase contrast microscopy. Cells retained viability and morphology after FACS (a, b) and after being embedded in alginate for four days (c, d). Cell viability dyes are calcein AM (green) and ethidium homodimer red (red). Scale bar = 200 microns.

Figure 3

Morphology and sGAG production in MSC pellet cultures treated with NCCM and TGFβ. Cultures were examined after seven days of treatment. A) Significantly greater GAG production was noted in the NCCM than TGFβ group (* indicates significance, P = 0.04). Values are expressed as average ±SEM. sGAG/DNA normalized to CM group. B) Pellets stained with alcian blue and imaged at 20× show an increase in sGAG content with NCCM treatment. (L to R: CM, TGFβ medium, NCCM) Scale bar = 200 microns. C) NCCM and TGFβ treated pellets were larger than control pellets as analyzed by gross morphology (L to R: CM, TGFβ medium, NCCM) scale bar = 1.8 mm.

Figure 4

qRT-PCR analysis of MSC pellet cultures treated with NCCM or TGFβ. Expression of genes associated with chondrocyte or NP phenotypes were measured. Data were averaged over three patients, normalized to GAPDH and expressed relative to CM values (average +/- SEM). * indicates significantly greater collagen III expression in NCCM relative to TGFβ medium (P < 0.05).