Tummala Padmaja, Arnsdorf Emily J, Jacobs Christopher R
Department of Veterans Affairs, Bone and Joint Rehabilitation Center, Palo Alto, CA, USA.
Cell Mol Bioeng. 2010 Sep 1;3(3):207-212. doi: 10.1007/s12195-010-0127-x.
Primary cilia are sensory organelles that have been shown to play a critical role in lineage commitment. It was our hypothesis that the primary cilium is necessary for chemically induced differentiation of human mesenchymal stem cells (MSC). To investigate this, polaris siRNA was used to inhibit the primary cilia and the mRNA levels of transcription factors Runx2, PPARgamma were measured by RT PCR as markers of osteogenic, adipogenic and chondrogenic differentiation, respectively. MSCs with inhibited primary cilia had significantly decreased basal mRNA expression levels of all three lineages specific transcription factors indicating that primary cilia are critical in multiple differentiation pathways. Furthermore, to determine if primary cilia play a role in the differentiation potential of MSCs, progenitor cells transfected with either scrambled or polaris siRNA were cultured in osteo-inductive, chondro-inductive, or adipo-inductive media and lineage commitment was ascertained. Interestingly, within 24 h of culture, cells transfected with polaris siRNA in both osteogenic and adipogenic media lost adhesion and released from the slides; however MSCs in chondrogenic media as well as cells transfected with scrambled siRNA did not. These results suggest that the primary cilium is necessary for the normal progression of chemically induced osteogenic and adipogenic differentiation. As a control, the experiment was repeated with NIH3T3 fibroblasts and none of the effects of inhibited primary cilia were observed indicating that the loss of adhesion may be specific to MSCs. Furthermore after biochemically inducing the cells to differentiate, polaris knockdown resulted in abrogation of both Runx2 and PPARgamma mRNA while SOX9 mRNA expression was significantly lower. These results suggest that primary cilia play an essential role not only in the initiation of both osteogenic and adipogenic differentiation, but also in maintaining the phenotype of differentiated cells. Interestingly, chondrogenic differentiation appeared less dependent on a functional primary cilium.
初级纤毛是感觉细胞器，已被证明在细胞谱系定向中起关键作用。我们的假设是初级纤毛对于化学诱导的人间充质干细胞（MSC）分化是必需的。为了研究这一点，使用北极星小干扰RNA（siRNA）抑制初级纤毛，并通过逆转录聚合酶链反应（RT-PCR）分别测量转录因子Runx2、PPARγ的mRNA水平，作为成骨、成脂和软骨分化的标志物。初级纤毛受到抑制的间充质干细胞中，所有三种谱系特异性转录因子的基础mRNA表达水平均显著降低，表明初级纤毛在多种分化途径中至关重要。此外，为了确定初级纤毛是否在间充质干细胞的分化潜能中发挥作用，将用乱序或北极星siRNA转染的祖细胞在成骨诱导、软骨诱导或成脂诱导培养基中培养，并确定细胞谱系定向。有趣的是，在培养24小时内，在成骨和成脂培养基中用北极星siRNA转染的细胞失去黏附并从玻片上脱落；然而，软骨诱导培养基中的间充质干细胞以及用乱序siRNA转染的细胞并未出现这种情况。这些结果表明初级纤毛对于化学诱导的成骨和成脂分化的正常进程是必需的。作为对照，用NIH3T3成纤维细胞重复该实验，未观察到初级纤毛受抑制的任何效应，表明黏附丧失可能是间充质干细胞特有的。此外，在对细胞进行生化诱导使其分化后，北极星基因敲低导致Runx2和PPARγ mRNA均被消除，而SOX9 mRNA表达显著降低。这些结果表明初级纤毛不仅在成骨和成脂分化的起始中起重要作用，而且在维持分化细胞的表型中也起重要作用。有趣的是，软骨分化似乎对功能性初级纤毛的依赖性较小。
