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.