Amerion Monire, Valojerdi Mojtaba Rezazadeh, Abroun Saeid, Totonchi Mehdi
Department of Anatomy, Faculty of Medical Science, Tarbiat Modares University, P.O.Box. 14115-331, Tehran, Islamic Republic of Iran.
Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Islamic Republic of Iran.
Cytotechnology. 2018 Feb;70(1):397-413. doi: 10.1007/s10616-017-0155-7. Epub 2017 Dec 20.
The procedure of obtaining qualified endothelial progenitor cells (EPCs) is still unclear and there has been some controversy on their biological properties and time of emergence. In this study, we used long-term culture of Adipose Derived Stem Cells (ADSCs) in an endothelial induction medium to obtain endothelial progenitor-like cells, and investigated the features of a few surface markers and the physiologic functions of the cells produced. In order to achieve our aim, rat ADSCs were isolated and cultured in an endothelial basal medium (EBM2), supplemented with an endothelial growth medium (EGM2). The cells were cultured 1 week for short-time, 2 weeks for mid-time, and 3 weeks for long-time cultures. Morphological changes were monitored by phase contrast and electron microscopy. The expressions of a few endothelial progenitor cells markers were analyzed by real-time RT-PCR. Low-density lipoprotein uptake and lectin binding assay were also performed for functional characterization. After induction, ADSCs showed changes in morphology from spindle-shaped in the first week to cobblestone-shaped during the next 2 weeks. Then, endothelial cell phenotype was defined by the presence of Weibel-Palade bodies in the cytoplasm and tube formation, without the use of Matrigel in the third week. In keeping with gene expression analysis, VEGFR-2 showed significant expression during early stages of endothelial differentiation for up to 3 weeks. A significantly increased expression of Tie2 was observed on day 21. Likewise, VE-Cadherin, CD34, CD133, WVF and CD31 were not significantly expressed within the same period of time. Endothelial differentiated cells also showed little LDL uptake and little to no lectin binding during the first 2 weeks of induction. However, high LDL uptake and lectin binding were observed in the third week. It appears that long term culture of ADSCs in EGM2 leads to significantly increased expression of some endothelial progenitor cells markers, strong DiI-ac-LDL uptake, lectin binding and tube-like structure formation in endothelial differentiated cells. Therefore, selection of an appropriate culture time and culture medium is crucial for establishing an efficient route to obtain sufficient numbers of EPCs with optimized quantity and quality.
获取合格内皮祖细胞（EPCs）的程序仍不明确，关于其生物学特性和出现时间也存在一些争议。在本研究中，我们在内皮诱导培养基中对脂肪来源干细胞（ADSCs）进行长期培养以获得内皮祖样细胞，并研究了所产生细胞的一些表面标志物特征和生理功能。为实现我们的目标，分离大鼠ADSCs并在内皮基础培养基（EBM2）中培养，添加内皮生长培养基（EGM2）。细胞分别进行短期培养1周、中期培养2周和长期培养3周。通过相差显微镜和电子显微镜监测形态变化。通过实时RT-PCR分析一些内皮祖细胞标志物的表达。还进行了低密度脂蛋白摄取和凝集素结合试验以进行功能表征。诱导后，ADSCs的形态从第一周的纺锤形变为接下来两周的鹅卵石形。然后，在第三周，通过细胞质中存在Weibel-Palade小体和形成管状物来定义内皮细胞表型，且未使用基质胶。与基因表达分析一致，VEGFR-2在内皮分化的早期阶段直至3周均有显著表达。在第21天观察到Tie2表达显著增加。同样，VE-钙黏蛋白、CD34、CD133、WVF和CD31在同一时期内未显著表达。在内皮分化细胞诱导的前2周，其对低密度脂蛋白的摄取很少，凝集素结合也很少或没有。然而，在第三周观察到高的低密度脂蛋白摄取和凝集素结合。似乎在EGM2中对ADSCs进行长期培养会导致内皮分化细胞中一些内皮祖细胞标志物的表达显著增加、强烈的DiI-乙酰化低密度脂蛋白摄取、凝集素结合和管样结构形成。因此，选择合适的培养时间和培养基对于建立一条有效途径以获得足够数量的、具有优化数量和质量的EPCs至关重要。
