Molecular atlas of fetal and adult human liver sinusoidal endothelial cells: a F8 secreting cell [methylation II]. Molecular atlas of fetal and adult human liver sinusoidal endothelial cells: a F8 secreting cell [methylation II]

In human F8 deficiency leads to hemophilia A and is largely synthesized and secreted by the sinusoidal endothelial cells of the liver (LSECs). However, the specificity and characteristics of these cells is not well known. In this study, we performed genome wide expression and CpG methylation profiling of fetal and adult human LSECs together with other fetal endothelial cells, from lung (micro-vascular and arterial), and heart (micro-vascular). Our results reveal plenty of expression and methylation markers distinguishing LSECs at both fetal and adult stages. Differential gene expression of fetal LSECs in comparison to other fetal endothelial cells pointed to several differential regulated pathways and functions in fetal LSECs. We used targeted bisulfite re-sequencing to confirm selected top differentially methylated regions. We further designed an assay where we used the selected methylation markers to test the degree of similarity of in house iPSs generated vascular endothelial cells to fetal and adult LSECs; a higher similarity was found to the fetal than to adult LSECs. Our molecular profiling study provides a guide to test the effectiveness of production of in vitro differentiated LSECs that could be used in cellular therapies. In-silico analysis to identify molecular signature of liver sinusoidal endothelial cells in comparison to other endohtelial cells Overall design: Commercial endothelial cells were ordered from ScienCell research laboratories via Provitro GmbH. All molecular materials were isolated at passage one as stated by manufacturing certificate.

人凝血因子F8缺陷可导致A型血友病，该因子主要由肝脏血窦内皮细胞（liver sinusoidal endothelial cells, LSECs）合成并分泌。然而，目前对这类细胞的特异性与特征仍知之甚少。
本研究对胎儿及成人来源的人肝脏血窦内皮细胞（LSECs），以及其他胎儿来源的内皮细胞——包括肺组织的微血管与动脉内皮细胞、心脏组织的微血管内皮细胞——开展了全基因组表达谱与CpG甲基化谱分析。
研究结果鉴定出大量可区分胎儿与成人阶段肝脏血窦内皮细胞的表达与甲基化标志物。相较于其他胎儿来源内皮细胞，胎儿肝脏血窦内皮细胞的差异基因表达分析揭示了其存在多条差异调控的通路与功能特征。
我们采用靶向亚硫酸氢盐重测序技术，对筛选出的关键差异甲基化区域进行了验证。此外，我们设计了检测实验，利用筛选得到的甲基化标志物，评估本实验室自主构建的诱导多能干细胞（induced pluripotent stem cells, iPSCs）分化所得血管内皮细胞与胎儿及成人肝脏血窦内皮细胞的相似程度；结果显示其与胎儿肝脏血窦内皮细胞的相似性高于成人肝脏血窦内皮细胞。
本分子谱分析研究为体外分化获得可用于细胞治疗的肝脏血窦内皮细胞的有效性评估提供了参考依据。本研究还开展了计算机模拟（in silico）分析，以鉴定肝脏血窦内皮细胞相较于其他内皮细胞的分子特征谱。
【研究整体设计】：研究人员通过Provitro GmbH向ScienCell研究实验室订购了商用内皮细胞，所有分子材料均按照厂商说明书要求，于第1代传代时完成分离。