Abstract
Human hepatocytes show marked differences in cell size, gene expression, and function throughout the liver lobules, an arrangement termed liver zonation. However, it is not clear if these zonal size differences, and the associated phenotypic differences, are retained in isolated human hepatocytes, the "gold standard" for in vitro studies of human liver function. Here, we therefore explored size differences among isolated human hepatocytes and investigated whether separation by size can be used to study liver zonation in vitro. We used counterflow centrifugal elutriation to separate cells into different size fractions and analyzed them with label-free quantitative proteomics, which revealed an enrichment of 151 and 758 proteins (out of 5163) in small and large hepatocytes, respectively. Further analysis showed that protein abundances in different hepatocyte size fractions recapitulated the in vivo expression patterns of previously described zonal markers and biological processes. We also found that the expression of zone-specific cytochrome P450 enzymes correlated with their metabolic activity in the different fractions. In summary, our results show that differences in hepatocyte size matches zonal expression patterns, and that our size fractionation approach can be used to study zone-specific liver functions in vitro.
Highlights
Hepatocytes are the main cell type of the liver, constituting around 80% of its volume (Kmiec, 2001)
The expression patterns of liver zonation have been extensively characterized on both the messenger RNA and protein levels (Ben‐Moshe et al, 2019; Berndt et al, 2020; Brosch et al, 2018; Halpern et al, 2017; MacParland et al, 2018; McEnerney et al, 2017), even though human data is still lacking at the protein level
To rule out contamination by nonparenchymal cells (NPCs), we examined the expression of reliably quantified markers for the major NPC types, that is, liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC), and hepatic stellate cells (HSC; Ölander et al, 2020)
Summary
Hepatocytes are the main cell type of the liver, constituting around 80% of its volume (Kmiec, 2001). The expression patterns of liver zonation have been extensively characterized on both the messenger RNA (mRNA) and protein levels (Ben‐Moshe et al, 2019; Berndt et al, 2020; Brosch et al, 2018; Halpern et al, 2017; MacParland et al, 2018; McEnerney et al, 2017), even though human data is still lacking at the protein level Some of these studies used computational methods to separate cells from different zones (Halpern et al, 2017; MacParland et al, 2018), while others used laser capture microdissection (Brosch et al, 2018; McEnerney et al, 2017) or fluorescence‐activated cell sorting (FACS; Ben‐Moshe et al, 2019; Berndt et al, 2020) to perform actual physical separation of cells. Our results indicate that differences in cell size among isolated human hepatocytes are related to zonal origin, and that elutriation‐based size fractionation enables studies of zonated human liver functions in vitro
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