Using a large-scale quantitative mesenchymal stem cells (MSCs) membrane proteomics analysis, we identified a large group of ciliary proteins in the MSCs membrane fraction, which prompted us to examine the cilia, intricate organelles that were originally discovered approximately 100 years ago. Here we characterize their primary structure and function in MSCs. We first characterized the primary cilia on undifferentiated human MSCs by immunostaining and verified these observation with scanning and 3D electronic microscopy. To investigate the function of the primary cilia of the MSCs we induced loss of function by means of siRNA knockdown (targeted to two known ciliary proteins: IFT172 and KIF3A). After either of these two proteins was knocked down by the respective siRNA, the MSCs showed fewer and shortened primary cilia. The MSCs proliferation assays showed increased cell proliferative activity under confluent conditions after the siRNA knockdown of IFT172 or KIF3A; among these MSCs, the proportion in S phase was increased in the IFT172 siRNA knockdown group. The expression of stem cell markers on the MSCs, namely, Oct4, Nanog and Sox2, were downregulated after the siRNA-induced knockdown of IFT172 or KIF3A, and the gene expression upregulation of ectoderm lineage markers was notable. Furthermore, manipulation of the primary cilia-dependent Shh pathway, using the Shh activator SAG (smoothened agonist), upregulated the gene expression of pluripotency markers, including Nanog and Oct4, and transcriptional target genes in the Shh pathway. This study confirms that MSCs have primary cilia and provides evidence that primary cilia-dependent signaling pathways play functional roles in MSCs proliferation and stemness maintenance.

中文翻译：

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原发纤毛依赖性信号传导参与调节间充质干细胞的增殖和多能性维持。

使用大规模定量间充质干细胞（MSCs）膜蛋白质组学分析，我们在MSCs膜级分中鉴定出一大批纤毛蛋白，这促使我们检查最初在大约100年前发现的纤毛，复杂细胞器。在这里，我们描述了它们在MSC中的主要结构和功能。我们首先通过免疫染色对未分化人类MSC的原发纤毛进行了表征，并通过扫描和3D电子显微镜验证了这些观察结果。为了研究MSC的初级纤毛的功能，我们通过敲低siRNA（靶向两种已知的纤毛蛋白：IFT172和KIF3A）诱导了功能丧失。在这两种蛋白质中的任何一种被各自的siRNA敲除后，MSC的原发纤毛变少且变短。MSCs增殖试验显示，在融合条件下，IFT172或KIF3A的siRNA敲除后，细胞增殖活性增加；在这些MSC中，IFT172 siRNA敲低组的S期比例增加。siRNA诱导的IFT172或KIF3A基因敲低后，MSCs上的干细胞标志物Oct4，Nanog和Sox2的表达下调，外胚层谱系标志物的基因表达上调显着。此外，使用Shh激活剂SAG（平滑激动剂）操纵主要的纤毛依赖性Shh途径，可上调多能性标志物的基因表达，包括Nanog和Oct4，以及Shh途径中的转录靶基因。