Spatial Sorting of mouse hepatocytes

The mammalian liver is composed of repeating hexagonal units termed lobules. Spatially resolved single-cell transcriptomics revealed that about half of hepatocyte genes are differentially expressed across the lobule. In order to examine additional cellular features of the different lobule layers, there is a need for an approach which will enable isolation of bulk hepatocytes from distinct lobule layers.To this end, we developed a spatial sorting approach. We perfused livers of five ad-libitum-fed mice to dissociate single cells and performed fluorescence-activated cell sorting (FACS) of isolated hepatocytes stained with antibodies against the pericentral CD73 labeled with APC and periportal E-cadherin labeled with PE. We filtered hepatocytes by size and selected cells that were negative for the endothelial cell marker CD31 and the immune cell marker CD45, to avoid pairs of hepatocytes and non-parenchymal cells. We further filtered out non-viable cells and selected tetraploid hepatocytes using Hoechst staining. We sorted 8 population based on the intensities of CD73 and E-cadherin, representing the centro-portal lobule axis (1- pericentral, 8-periportal). Used total 5 mice. mRNA was extracted for each of the 40 populations and prepared SCRB-seq libraries.

哺乳动物肝脏由重复排布的六边形结构单元——肝小叶（lobules）——构成。空间分辨单细胞转录组学研究显示，近半数肝细胞基因在肝小叶内呈现差异表达。为探究不同肝小叶层的额外细胞特征，亟需一种可从不同肝小叶层分离大量肝细胞的方法。
为此，我们开发了一种空间分选方法。我们对5只自由采食（ad-libitum-fed）的小鼠肝脏进行灌注以解离单细胞，并对经APC标记的中央静脉旁CD73抗体、PE标记的门静脉旁E-钙粘蛋白抗体染色的分离肝细胞开展荧光激活细胞分选（fluorescence-activated cell sorting, FACS）。
我们通过细胞大小对肝细胞进行初步筛选，并选取内皮细胞标志物CD31与免疫细胞标志物CD45阴性的细胞，以排除肝细胞团块与非实质细胞。我们进一步通过Hoechst染色剔除死细胞，并筛选出四倍体肝细胞。我们依据CD73与E-钙粘蛋白的荧光强度，将细胞分为8个群体，以代表中央-门静脉肝小叶轴（1为中央静脉旁群体，8为门静脉旁群体）。本实验共使用5只小鼠，为全部40个群体提取mRNA并制备了SCRB-seq文库。