Isolation and expansion of endothelial progenitor cells derived from mouse embryonic stem cells.
Isolation and expansion of endothelial progenitor cells derived from mouse embryonic stem cells.
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DOI：
10.1007/978-1-61779-980-8_7
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发表时间：
2012-01-01
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影响因子：
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通讯作者：
Boudreau, Nancy J
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文献类型：
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作者：
Bahrami, S Bahram;Veiseh, Mandana;Boudreau, Nancy J
胚胎干细胞的无限分化和增殖能力是再生医学的巨大资源。在这里，我们描述了一种从小鼠胚胎干细胞(MESCs)中分化、分离和扩增内皮细胞(ECs)的方法。首先，在小鼠胚胎成纤维细胞(MEF)饲养层上扩增MESCs，并通过在超低贴壁平板中培养长达5天的细胞来部分分化为类胚体(EBS)。然后使用添加40 ng/mL血管内皮生长因子(VEGF)的内皮生长介质沿内皮细胞谱系分化EBS。分化后的内皮细胞在细胞表面同时表达胎肝激酶1(Flk-1)和VE-钙粘附素，可用荧光激活细胞分选(FACS)系统进一步纯化，然后在0.1%明胶涂层平板上扩增。分化后的细胞可以通过实时定量聚合酶链式反应和流式细胞仪进行分析，以确认EC特异性基因和蛋白的浓缩。
The unlimited differentiation and proliferation capacity of embryonic stem cells represents a great resource for regenerative medicine. Here, we describe a method for differentiating, isolating, and expanding endothelial cells (ECs) from mouse embryonic stem cells (mESCs). First, mESCs are expanded on a mouse embryonic fibroblast (mEF) feeder layer and partially differentiated into embryoid bodies (EBs) by growing the cells in an ultra-low attachment plate for up to 5 days. The EBs are then differentiated along the endothelial lineage using endothelial growth medium supplemented with 40 ng/mL vascular endothelial growth factor (VEGF). The differentiated endothelial population expresses both Fetal Liver Kinase 1 (Flk-1) and VE-Cadherin on the cell surface which can be further purified using a fluorescence-activated cell sorting (FACS) system and subsequently expanded on 0.1 % gelatin-coated plates. The differentiated cells can be analyzed by real-time PCR and flow cytometry to confirm enrichment of EC-specific genes and proteins.