Nikolova-Krstevski Vesna, Bhasin Manoj, Otu Hasan H, Libermann Towia, Oettgen Peter
Division of Cardiology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Institutes of Medicine, Boston, USA.
BMC Genomics. 2008 May 22;9:240. doi: 10.1186/1471-2164-9-240.
Endothelial differentiation occurs during normal vascular development in the developing embryo. This process is recapitulated in the adult when endothelial progenitor cells are generated in the bone marrow and can contribute to vascular repair or angiogenesis at sites of vascular injury or ischemia. The molecular mechanisms of endothelial differentiation remain incompletely understood. Novel approaches are needed to identify the factors that regulate endothelial differentiation.
Mouse embryonic stem (ES) cells were used to further define the molecular mechanisms of endothelial differentiation. By flow cytometry a population of VEGF-R2 positive cells was identified as early as 2.5 days after differentiation of ES cells, and a subset of VEGF-R2+ cells, that were CD41 positive at 3.5 days. A separate population of VEGF-R2+ stem cells expressing the endothelial-specific marker CD144 (VE-cadherin) was also identified at this same time point. Channels lined by VE-cadherin positive cells developed within the embryoid bodies (EBs) formed by differentiating ES cells. VE-cadherin and CD41 expressing cells differentiate in close proximity to each other within the EBs, supporting the concept of a common origin for cells of hematopoietic and endothelial lineages.
Microarray analysis of >45,000 transcripts was performed on RNA obtained from cells expressing VEGF-R2+, CD41+, and CD144+ and VEGF-R2-, CD41-, and CD144-. All microarray experiments were performed in duplicate using RNA obtained from independent experiments, for each subset of cells. Expression profiling confirmed the role of several genes involved in hematopoiesis, and identified several putative genes involved in endothelial differentiation.
The isolation of CD144+ cells during ES cell differentiation from embryoid bodies provides an excellent model system and method for identifying genes that are expressed during endothelial differentiation and that are distinct from hematopoiesis.
内皮细胞分化发生在发育中胚胎的正常血管发育过程中。当成年个体骨髓中产生内皮祖细胞，且这些细胞能够在血管损伤或缺血部位参与血管修复或血管生成时，这一过程得以重现。内皮细胞分化的分子机制仍未完全明确。需要新的方法来鉴定调控内皮细胞分化的因子。
利用小鼠胚胎干细胞（ES细胞）进一步明确内皮细胞分化的分子机制。通过流式细胞术，早在ES细胞分化2.5天后就鉴定出一群VEGF-R2阳性细胞，以及在3.5天时CD41阳性的VEGF-R2+细胞亚群。在同一时间点还鉴定出了另一群表达内皮特异性标志物CD144（血管内皮钙黏蛋白）的VEGF-R2+干细胞。由分化的ES细胞形成的胚状体（EBs）内出现了由血管内皮钙黏蛋白阳性细胞排列而成的通道。在EBs内，表达血管内皮钙黏蛋白和CD41的细胞彼此紧邻分化，这支持了造血和内皮谱系细胞具有共同起源的概念。
对从表达VEGF-R2+、CD41+和CD144+以及VEGF-R2-、CD41-和CD144-的细胞中获得的RNA进行了超过45,000个转录本的微阵列分析。所有微阵列实验均使用从独立实验中获得的RNA对每个细胞亚群进行了重复操作。表达谱分析证实了几个参与造血的基因的作用，并鉴定出了几个参与内皮细胞分化的假定基因。
在ES细胞从胚状体分化过程中分离CD144+细胞，为鉴定在内皮细胞分化过程中表达且与造血不同的基因提供了一个优秀的模型系统和方法。
