Institute for Regenerative Medicine at Scott and White Hospital, Texas A&M Health Science Center, Temple, TX 76502, USA.
Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4147-52. doi: 10.1073/pnas.0914360107. Epub 2010 Feb 11.
Human mesenchymal stem cells (hMSCs) from bone marrow are regarded as putative osteoblast progenitors in vivo and differentiate into osteoblasts in vitro. Positive signaling by the canonical wingless (Wnt) pathway is critical for the differentiation of MSCs into osteoblasts. In contrast, activation of the peroxisome proliferator-activated receptor-gamma (PPARgamma)-mediated pathway results in adipogenesis. We therefore compared the effect of glycogen-synthetase-kinase-3beta (GSK3beta) inhibitors and PPARgamma inhibitors on osteogenesis by hMSCs. Both compounds altered the intracellular distribution of beta-catenin and GSK3beta in a manner consistent with activation of Wnt signaling. With osteogenic supplements, the GSK3beta inhibitor 6-bromo-indirubin-3'-oxime (BIO) and the PPARgamma inhibitor GW9662 (GW) enhanced early osteogenic markers, alkaline phosphatase (ALP), and osteoprotegerin (OPG) by hMSCs and transcriptome analysis demonstrated up-regulation of genes encoding bone-related structural proteins. At higher doses of the inhibitors, ALP levels were attenuated, but dexamethasone-induced biomineralization was accelerated. When hMSCs were pretreated with BIO or GW and implanted into experimentally induced nonself healing calvarial defects, GW treatment substantially increased the capacity of the cells to repair the bone lesion, whereas BIO treatment had no significant effect. Further investigation indicated that unlike GW, BIO induced cell cycle inhibition in vitro. Furthermore, we found that GW treatment significantly reduced expression of chemokines that may exacerbate neutrophil- and macrophage-mediated cell rejection. These data suggest that use of PPARgamma inhibitors during the preparation of hMSCs may enhance the capacity of the cells for osteogenic cytotherapy, whereas adenine analogs such as BIO can adversely affect the viability of hMSC preparations in vitro and in vivo.
骨髓间充质干细胞（hMSCs）被认为是体内成骨细胞的前体细胞，并在体外分化为成骨细胞。经典的 Wnt 信号通路的阳性信号对于 MSC 分化为成骨细胞至关重要。相比之下，过氧化物酶体增殖物激活受体-γ（PPARγ）介导的途径的激活导致脂肪生成。因此，我们比较了糖原合成酶激酶-3β（GSK3β）抑制剂和 PPARγ 抑制剂对 hMSC 成骨作用的影响。这两种化合物改变了β-catenin 和 GSK3β的细胞内分布，与 Wnt 信号的激活方式一致。在成骨补充剂的作用下，GSK3β抑制剂 6-溴靛红-3'-肟（BIO）和 PPARγ 抑制剂 GW9662（GW）增强了早期成骨标志物碱性磷酸酶（ALP）和骨保护素（OPG）的表达，并通过转录组分析证明了编码与骨相关的结构蛋白的基因上调。在更高剂量的抑制剂下，ALP 水平降低，但地塞米松诱导的生物矿化加速。当 hMSC 用 BIO 或 GW 预处理并植入实验性诱导的非自我愈合的颅骨缺损时，GW 处理显著增加了细胞修复骨病变的能力，而 BIO 处理则没有显著影响。进一步的研究表明，与 GW 不同，BIO 在体外诱导细胞周期抑制。此外，我们发现 GW 处理显著降低了趋化因子的表达，这些趋化因子可能会加剧中性粒细胞和巨噬细胞介导的细胞排斥。这些数据表明，在 hMSC 制备过程中使用 PPARγ 抑制剂可能会增强细胞成骨细胞治疗的能力，而嘌呤类似物如 BIO 可能会对 hMSC 制剂的体外和体内活力产生不利影响。
