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Abstract：
Background Human embryo vasculogenesis (blood vessel development starting from endothelial precursors) includes the ability of mesenchymal cells and pluripotent stem cells to differentiate into endothelial cells. Quantification of endothelial progenitor cells is difficult to assess during the early steps of human embryo development due to several factors, especially due to the paucity of human embryo tissue which is usually discarded after early-stage pregnancy abortive methods. CD133 (Promimin-1) is a general marker of progenitor cells, but combined with other endothelial markers such as CD34, it may identify endothelial progenitor cells during embryonic development. CD34 immunohistochemistry was previously performed by our team to identify human embryo capillaries and comparatively assess microvessel density between different human embryonic tissues. TIE2 is an angiopoietin receptor strongly involved in the newly formed blood vessel maturation due to its expression in some mesenchymal precursors for future pericytes. CD34 assesses the presence of endothelial cells but its single use does not evaluate the endothelial progenitor state as CD133 may do nor vessel maturation as TIE2 may do. Data about the dynamics of CD133/TIE2 expression in the early stages of human embryo development are scarce. Hence, in this study, we aimed to comparatively assess the dynamic of CD133+ endothelial precursors and TIE2 expression on five and seven-week-old human embryonic tissues with a special emphasis on their expression on embryonic vascular beds. Methodology CD133 and TIE2 immunohistochemistry was performed on five and seven-week-old human embryonic tissues followed by their quantification using the Qu Path digital image analysis (DIA) automated method. Results CD133 and TIE2 showed divergent patterns of expression during the initial phases of human embryonic development, specifically in the vascular endothelium of tiny capillaries. The expression of CD133 in endothelial cells lining the perfused lumen gradually decreased from five to seven-week-old embryos. It remained expressed with greater intensity in cells located at the tip of the vascular bud that emerged into pre-existing capillaries. TIE2 was much more specific than CD133, being restricted to the level of the vascular endothelium; therefore, it was easier to quantify using digital image analysis. The endothelium of the embryonic aorta was an exception to the divergent expression, as CD133 and TIE2 were consistently co-expressed in the seven-week-old embryo. The Qu Path DIA assessment increased the accuracy of CD133 and TIE2 evaluation, being the first time they were quantified by using automated software and not manually. Conclusions High heterogeneity of CD133 and TIE2 was observed between five and seven-week-old embryonic tissues as well as between different embryonic regions from the same gestational age. The unique finding of CD133/TIE2 co-expression persistence inside aortic endothelium needs further studies to elucidate the role of this co-expression.
摘要：
背景技术人胚胎血管发生(从内皮前体开始的血管发育)包括间充质细胞和多能干细胞分化成内皮细胞的能力。由于多种因素，在人类胚胎发育的早期阶段很难评估内皮祖细胞的定量。特别是由于人类胚胎组织的缺乏，通常在早期妊娠流产方法后被丢弃。CD133（Promimin-1）是祖细胞的一般标记，但结合其他内皮标志物如CD34，它可以识别胚胎发育过程中的内皮祖细胞。我们的团队先前进行了CD34免疫组织化学，以鉴定人类胚胎毛细血管并比较评估不同人类胚胎组织之间的微血管密度。TIE2是一种血管生成素受体，由于其在某些间充质前体中表达，因此强烈参与了新形成的血管成熟。CD34评估内皮细胞的存在，但其单次使用不能评估内皮祖细胞状态，因为CD133可能也不能像TIE2那样评估血管成熟。关于人类胚胎发育早期CD133/TIE2表达动力学的数据很少。因此，在这项研究中，我们旨在比较评估CD133+内皮前体和TIE2在5周龄和7周龄人胚胎组织中的表达动态,并特别关注它们在胚胎血管床上的表达.方法对5周龄和7周龄的人胚胎组织进行CD133和TIE2免疫组织化学，然后使用QuPath数字图像分析（DIA）自动化方法对其进行定量。结果CD133和TIE2在人类胚胎发育的初始阶段显示出不同的表达模式，特别是在微小毛细血管的血管内皮中。从5到7周龄的胚胎，灌注腔内内皮细胞中CD133的表达逐渐降低。它在位于血管芽尖端的细胞中以更大的强度表达，这些细胞出现在预先存在的毛细血管中。TIE2比CD133更具特异性，仅限于血管内皮水平；因此，使用数字图像分析更容易量化。胚胎主动脉的内皮是不同表达的例外，因为CD133和TIE2在7周大的胚胎中一致共表达。QuPathDIA评估提高了CD133和TIE2评估的准确性，这是第一次使用自动化软件而不是手动对它们进行量化。结论在5周龄和7周龄的胚胎组织之间以及相同胎龄的不同胚胎区域之间观察到CD133和TIE2的高度异质性。主动脉内皮内CD133/TIE2共表达持久性的独特发现需要进一步研究以阐明这种共表达的作用。
