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 Res Ther. 2010 Jun 16;1(2):18. doi: 10.1186/scrt18.
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.
间充质干细胞（MSCs）为椎间盘（IVD）修复和再生提供了希望，因为它们易于分离和扩增，并能分化为几种间充质组织。脊索细胞（NC）有助于椎间盘的发育，并入髓核（NP），并刺激成熟的椎间盘细胞。然而，目前还没有研究探讨 NC 细胞对成体干细胞分化的影响。本研究的前提是，IVD 的再生更类似于 IVD 的发育，而不是 IVD 的维持，我们假设 NC 细胞的可溶性因子在发育过程中使 MSCs 分化为具有 NP 细胞表型的特征。最终的临床目标将是分离或化学/重组产生活性物质来诱导治疗效果，并将其用作早期干预椎间盘疾病的注射治疗，或在组织工程 NP 构建体中预先分化 MSC 细胞。
从骨髓中扩增和沉淀人 MSC 以形成高密度培养物。MSC 沉淀暴露于对照培养基（CM）、软骨形成培养基（含地塞米松和转化生长因子-β3 的 CM）或脊索细胞条件培养基（NCCM）中。NCCM 是从在无血清培养基中维持 4 天的 NC 细胞中制备的。培养 7 天后，分析 MSC 沉淀的外观、生化成分（糖胺聚糖和 DNA）和基因表达谱（Sox-9、胶原 II 型和 III 型、层粘连蛋白-β1 和 TIMP1（金属蛋白酶抑制剂-1））。
与 CM 或 TGFβ 组相比，NCCM 处理的沉淀中糖胺聚糖的积累明显更高。用 NCCM 处理后，观察到胶原 III 的基因表达增加，与 TGFβ 组相比，层粘连蛋白-β1 的表达增加趋势和 Sox-9 和胶原 II 的表达减少。
综上所述，结果表明 NCCM 刺激间充质干细胞向具有潜在 NP 样表型的分化，具有一些发育中 IVD 的特征。
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