Reciprocal induction of human dermal microvascular endothelial cells and human mesenchymal stem cells: time-dependent profile in a co-culture system
Reciprocal induction of human dermal microvascular endothelial cells and human mesenchymal stem cells: time-dependent profile in a co-culture system
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DOI：
10.1111/j.1365-2184.2012.00822.x
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发表时间：
2012-08-01
影响因子：
8.5
通讯作者：
Monteiro, F. J.
中科院分区：
文献类型：
--
作者：
Laranjeira, M. S.;Fernandes, M. H.;Monteiro, F. J.
目的 血管生成与骨生成密切相关，内皮细胞和成骨细胞之间的相互作用在骨再生中发挥重要作用。出于这些原因，这项工作的目的是开发一种共培养系统，以详细研究人间充质干细胞 (HMSC) 和人真皮微血管内皮细胞 (HDMEC) 之间任何时间依赖性相互作用，在 2D 系统中共培养 35 天。材料和方法 HMSC 和 HDMEC 分别以 1:4 的比例共培养。单细胞培养物用作对照。使用 MTT、DNA 定量和钙黄绿素-AM 测定评估细胞活力/增殖。使用共聚焦显微镜监测细胞形态，并进行实时 PCR。评估碱性磷酸酶活性和组织化学染色。还进行了基质矿化测定。结果与 HMSC 和 HDMEC 单一培养物相比，细胞能够以特征模式生长，在整个培养期间保持其活力和表型表达。 HMSC 分化似乎在共培养条件下得到增强，因为观察到成骨相关基因和 ALP 活性的过度表达。此外，还证实了磷酸钙沉积物的存在。结论 这项工作详细报告了长期共培养 2D 系统中 HMSC 和 HDMEC 之间的相互作用。在本共培养条件下培养的内皮干细胞和间充质干细胞确保了同一培养系统中细胞类型的增殖和表型分化、成骨刺激和血管生成相关基因的过度表达。人们相信，目前的工作可以导致骨组织再生和细胞生物学研究的重大发展。
Objectives Angiogenesis is closely associated with osteogenesis where reciprocal interactions between endothelial and osteoblast cells play an important role in bone regeneration. For these reasons, the aim of this work was to develop a co-culture system to study in detail any time-dependent interactions between human mesenchymal stem cells (HMSC) and human dermal microvascular endothelial cells (HDMEC), co-cultured in a 2D system, for 35days. Materials and methods HMSC and HDMEC were co-cultured at a ratio of 1:4, respectively. Single-cell cultures were used as controls. Cell viability/proliferation was assessed using MTT, DNA quantification and calcein-AM assays. Cell morphology was monitored using confocal microscopy, and real time PCR was performed. Alkaline phosphatase activity and histochemical staining were evaluated. Matrix mineralization assays were also performed. Results Cells were able to grow in characteristic patterns maintaining their viability and phenotype expression throughout culture time, compared to HMSC and HDMEC monocultures. HMSC differentiation seemed to be enhanced in the co-culture conditions, since it was observed an over expression of osteogenesis-related genes, and of ALP activity. Furthermore, presence of calcium phosphate deposits was also confirmed. Conclusions This work reports in detail the interactions between HMSC and HDMEC in a long-term co-culture 2D system. Endothelial and mesenchymal stem cells cultured in the present co-culture conditions ensured proliferation and phenotype differentiation of cell types, osteogenesis stimulation and over-expression of angiogenesis-related genes, in the same culture system. It is believed that the present work can lead to significant developments for bone tissue regeneration and cell biology studies.