Mouse embryonic fibroblast (MEF)/BMP4-conditioned medium enhanced multipotency of human dental pulp cells
Mouse embryonic fibroblast (MEF)/BMP4-conditioned medium enhanced multipotency of human dental pulp cells
复制标题
小鼠胚胎成纤维细胞（MEF）/BMP4条件培养基增强人牙髓细胞的多能性
DOI：
10.1007/s10735-017-9743-2
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发表时间：
2018-02-01
影响因子：
3.2
通讯作者：
Wei, Xi
中科院分区：
文献类型：
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作者：
Liu, Lu;Peng, Zhengjun;Wei, Xi
牙髓细胞（DPC）是牙齿再生的重要细胞来源，但由于目前培养条件的限制，其多能性受到限制。小鼠胚胎成纤维细胞（MEFs）作为饲养层，通过长期体外培养来维持iPSC和ESCs的未分化状态。骨形态发生蛋白4（BMP 4）通过调节微环境，在调节细胞未分化状态和谱系定型中发挥重要作用。然而，目前对微环境对牙源性细胞多能性的影响还知之甚少。为了验证优化培养条件对DPC多能性的影响，采用CCK 8、端粒酶活性和流式细胞术检测了MEF和/或rhBMP 4-CM对DPC增殖和衰老的影响。免疫荧光染色、Real-time PCR和western blot检测细胞的多向分化。ELISA检测共培养液中BMP 4/NFATc 1/LIF的表达，Real-time PCR检测共培养液中Oct-4/Sox 2/c-Myc/NFATc 1的表达。用NFATc 1抑制剂印加-6处理含MEF和/或rhBMP 4-CM的DPC，Realtime PCR和western blot检测NFATc 1/Oct-4/Sox 2/c-Myc的表达。结果表明，MEF和/或rhBMP 4-CM可促进DPC的增殖、端粒酶活性和多向分化能力。MEF-CM通过分泌BMP-4/NFATc 1，促进Oct-4/Sox 2/c-Myc/NFATc 1的表达。印加-6可有效抑制MEF/BMP 4-CM诱导的DPC中Oct-4/Sox 2/c-Myc/NFATc 1的上调。结果表明，MEF-CM和BMP 4-CM均能提供相似的培养体系，提高DPC的多能性，为DPC在牙体再生中的应用奠定了基础。
Dental pulp cells (DPCs) are valuable cell source for dental regeneration, albeit their application is restricted by limited pluripotency due to current culture condition. Mouse embryonic fibroblasts (MEFs) are served as feeder layer to maintain undifferentiated state of iPSCs and ESCs with long-term in vitro culture. Bone morphogenetic protein 4 (BMP4) plays an important role in the regulation of undifferentiated state and lineage commitment of cells through modulation of microenvironment. However, so far little was known how micro environment affect the multipotency of dental derived cells. To demonstrate the effect of optimized culture condition on multipotency of DPCs, cell proliferation and senescence of DPCs with MEF and/or rhBMP4-CM were examined by CCK8, telomerase activity and flow cytometry. Multilineage differentiation was detected by immunofluorescent staining, Real-time PCR and western blot. Expression of BMP4/NFATc1/LIF in the co-culture medium was evaluated by ELISA and expression of Oct-4/Sox2/c-Myc/NFATc1 in co-cultured DPCs was detected by Real-time PCR. NFATc1 inhibitor INCA-6 was applied to DPCs with MEF and/or rhBMP4-CM, expression of NFATc1/Oct-4/Sox2/c-Myc was examined by Realtime PCR and western blot. Our results demonstrated that DPCs cultured with MEF and/or rhBMP4-CM showed increased cell proliferation, telomerase rate and multilineage differentiation capability. MEF-CM enhanced expression of Oct-4/Sox2/c-Myc/NFATc1 in co-cultured DPCs through secretion of BMP4/NFATc1 in the culture medium. INCA-6 effectively restrained the MEF/BMP4-CM induced upregulation of Oct-4/Sox2/c-Myc/NFATc1 in DPCs. These resuts indicate that both MEF-CM and BMP4-CM provided similar efficient culture system to improve the multipotency of DPCs, which might contribute to the application of DPCs in dental regeneration.