Department of Cell Biology and Anatomy, Institute of Biomedicine, University of Turku, Turku, Finland.
J Tissue Eng Regen Med. 2018 Mar;12(3):775-783. doi: 10.1002/term.2496. Epub 2017 Sep 25.
Endothelial progenitors found among the peripheral blood (PB) mononuclear cells (MNCs) are interesting cells for their angiogenic properties. Mesenchymal stromal cells (MSCs) in turn can produce proangiogenic factors as well as differentiate into mural pericytes, making MSCs and MNCs an attractive coculture setup for regenerative medicine. In this study, human bone marrow-derived MSCs and PB-derived MNCs were cocultured in basal or osteoblastic medium without exogenously supplied growth factors to demonstrate endothelial cell, pericyte and osteoblastic differentiation. The expression levels of various proangiogenic factors, as well as endothelial cell, pericyte and osteoblast markers in cocultures were determined by quantitative polymerase chain reaction. Immunocytochemistry for vascular endothelial growth factor receptor-1 and α-smooth muscle actin as well as staining for alkaline phosphatase were performed after 10 and 14 days. Messenger ribonucleic acid expression of endothelial cell markers was highly upregulated in both basal and osteoblastic conditions after 5 days of coculture, indicating an endothelial cell differentiation, which was supported by immunocytochemistry for vascular endothelial growth factor receptor-1. Stromal derived factor-1 and vascular endothelial growth factor were highly expressed in MSC-MNC coculture in basal medium but not in osteoblastic medium. On the contrary, the expression levels of bone morphogenetic protein-2 and angiopoietin-1 were significantly higher in osteoblastic medium. Pericyte markers were highly expressed in both cocultures after 5 days. In conclusion, it was demonstrated endothelial cell and pericyte differentiation in MSC-MNC cocultures both in basal and osteoblastic medium indicating a potential for neovascularization for tissue engineering applications.
在外周血（PB）单个核细胞（MNC）中发现的内皮祖细胞因其血管生成特性而备受关注。间充质基质细胞（MSCs）反过来也可以产生促血管生成因子，并分化为壁周细胞，这使得 MSCs 和 MNC 成为再生医学中很有吸引力的共培养体系。在这项研究中，将人骨髓来源的 MSCs 和 PB 来源的 MNC 在基础或成骨培养基中进行共培养，没有外源性生长因子，以证明内皮细胞、周细胞和成骨细胞分化。通过定量聚合酶链反应测定共培养物中各种促血管生成因子以及内皮细胞、周细胞和成骨细胞标志物的表达水平。在第 10 和 14 天时进行血管内皮生长因子受体-1 和α-平滑肌肌动蛋白的免疫细胞化学染色以及碱性磷酸酶染色。在共培养 5 天后，内皮细胞标志物的信使核糖核酸表达在基础和成骨条件下均高度上调，表明发生了内皮细胞分化，这得到了血管内皮生长因子受体-1 的免疫细胞化学染色的支持。基质细胞衍生因子-1 和血管内皮生长因子在基础培养基中的 MSC-MNC 共培养中高度表达，但在成骨培养基中不表达。相反，骨形态发生蛋白-2 和血管生成素-1 的表达水平在成骨培养基中显著升高。在共培养物中，周细胞标志物在 5 天后均高度表达。总之，在基础和成骨培养基中，MSC-MNC 共培养中均显示出内皮细胞和周细胞分化，表明其在组织工程应用中具有促进血管生成的潜力。
