Spheroid culture enhances osteogenic potential of periodontal ligament mesenchymal stem cells
Spheroid culture enhances osteogenic potential of periodontal ligament mesenchymal stem cells
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DOI：
10.1111/jre.12577
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发表时间：
2018-10-01
影响因子：
3.5
通讯作者：
Nakashima, K.
中科院分区：
文献类型：
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作者：
Moritani, Y.;Usui, M.;Nakashima, K.
目的与背景人牙周膜间充质干细胞(HPDLMSCs)被认为是促进牙周组织动态平衡和再生的细胞。尽管hPDLMSCs通常在单层培养，但据报道，单层培养不如三维培养，例如球体，球体是通过自组装形成的球状细胞簇。本研究的目的是检测hPDLMSCs球体的成骨表型，并与hPDLMSC单层培养的成骨表型进行比较。材料和方法用聚乙二醇包被的微孔芯片形成球体。用流式细胞仪检测hPDLMSC球体中的间充质干细胞(MSC)标志物。实时定量聚合酶链式反应检测单层和球形培养的hPDLMSCs的干性标志物和成骨相关基因的表达。免疫荧光分析证实PDLMSC球体中的茎标记蛋白的表达。在小鼠颅骨缺损模型上进行结节形成实验、碱性磷酸酶(ALP)活性测定和移植实验，以证实hPDLMSC球体的成骨能力。为了阐明球形培养促进hPDLMSCs在骨诱导介质(OIM)作用下的成骨作用机制，采用以分泌型卷曲相关蛋白3(SFRP3)为靶点的小干扰RNA(SiRNA)分析方法。采用实时定量聚合酶链式反应和免疫印迹法检测单层和球状培养的人PDLMSCs中sFRP3的表达水平。结果hPDLMSC球体表达MSC标志物，与单层培养的hPDLMSCs相似。有趣的是，与单层培养的hPDLMSCs相比，hPDLMSC球体中调节茎的转录因子的蛋白质和mRNA表达显著增加。与单层培养的hPDLMSCs相比，加入OIM的球形培养的hPDLMSCs结节形成显著增加。与单层培养相比，hPDLMSC球体中碱性磷酸酶的活性和成骨相关基因的表达也显著增强。与单层培养的hPDLMSCs相比，hPDLMSC球体处理显著促进了小鼠颅骨缺损模型中的新骨形成。最后，为了阐明球形培养促进OIM培养的hPDLMSCs碱性磷酸酶激活的机制，采用siRNA分析方法调控Wnt信号拮抗剂sFRP3的表达。SFRP3基因的敲除抑制了OIM培养的hPDLMSCs中ALP基因的表达，进而抑制了球形培养中的ALP活性。这些结果提示hPDLMSC球体成骨能力的增强是通过sFRP3介导的碱性磷酸酶激活来调节的。结论hPDLMSCs球体培养可能是一种新的、有用的再生医学工具。
Objective and BackgroundHuman periodontal ligament mesenchymal stem cells (hPDLMSCs) are reported to be responsible for homeostasis and regeneration of periodontal tissue. Although hPDLMSCs are commonly cultured in monolayers, monolayer cultures have been reported as inferior to 3-dimensional cultures such as spheroids, which are spherical clusters of cells formed by self-assembly. The aim of this study was to examine the osteogenic phenotype of spheroids of hPDLMSCs, compared with monolayer cultures of hPDLMSC, in vitro and in vivo.Material and MethodsSpheroids were formed using microwell chips that were tagged with polyethylene glycol. Mesenchymal stem cell (MSC) markers in hPDLMSC spheroids were examined by flow cytometer. Real-time polymerase chain reaction analysis was examined to measure the expressions of stemness markers and osteogenesis-related genes in monolayer and spheroid-cultured hPDLMSCs. Immunofluorescence analysis was performed to confirm protein expressions of stemness markers in PDLMSC spheroids. Nodule formation assay, alkaline phosphatase (ALP) activity assay and transplantation assay in a mouse calvarial defect model were performed to confirm the osteogenic potential of hPDLMSC spheroids. To elucidate the mechanism of spheroid culture enhanced osteogenesis in hPDLMSCs with osteoinductive medium (OIM), a small interfering RNA (siRNA) assay targeted with secreted frizzled-related protein 3 (SFRP3) was examined. The levels of SFRP3 expression in monolayer and spheroid-cultured hPDLMSCs with OIM were measured by real-time polymerase chain reaction and western blotting analysis. ALP gene expression and ALP activity were examined in SFRP3-deficient hPDLMSC spheroids.ResultsThe hPDLMSC spheroids expressed MSC markers, which were similar to hPDLMSCs grown in monolayer cultures. Intriguingly, the protein and mRNA expressions of transcription factors that regulate stemness were significantly increased in hPDLMSC spheroids, compared with hPDLMSCs in monolayer cultures. Nodule formation by hPDLMSCs was significantly increased in spheroid cultures grown with OIM, compared with monolayer-cultured hPDLMSCs. ALP activity and expression of osteogenesis-related genes were also significantly enhanced in hPDLMSC spheroids, compared with monolayer cultures. Treatment with hPDLMSC spheroids significantly enhanced new bone formation in a murine calvarial defect model, compared with hPDLMSCs in monolayer culture. Finally, to elucidate mechanisms by which spheroid culture enhances ALP activation in hPDLMSCs grown with OIM, an siRNA assay was used to manipulate expression of SFRP3, a Wnt signaling antagonist. Knockdown of SFRP3 suppressed ALP gene expression in hPDLMSCs grown in OIM; further, it suppressed ALP activity in spheroid culture. These data suggest that the enhancement of osteogenic potential in hPDLMSC spheroids is regulated through SFRP3-mediated ALP activation.ConclusionSpheroid cultures of hPDLMSCs may be a novel and useful tool in regenerative medicine.