Abstract
SummaryWhen comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.
Highlights
Directed differentiation of pluripotent stem cells (PSCs) to cells of a specific fate holds promise to study a wide variety of human diseases (Robinton and Daley, 2012)
Using a combination of proteomic and transcriptional profiling along with antibody-based sorting, we revealed that SLC10A1, CLRN3, and AADAC are cell-surface N-glycoproteins that are present on a common population of induced PSCs (iPSCs)-derived hepatocyte-like cells that express elevated levels of several hepatic markers
A Subset of Hepatocyte-Enriched N-Glycoproteins Are Induced during the Differentiation of Human Pluripotent Stem Cells into Hepatocyte-like Cells To further narrow the 40 candidates down to those with the most potential for sorting iPSC-derived hepatocyte-like cells, we focused on those N-glycoproteins for which mRNA levels are most robustly expressed in iPSC-derived hepatocyte-like cells and induced during the final stages of differentiation when the hepatocytes reach relative maturity (Figure 1)
Summary
Directed differentiation of pluripotent stem cells (PSCs) to cells of a specific fate holds promise to study a wide variety of human diseases (Robinton and Daley, 2012). Examples include familial hypercholesterolemia and a-1-antitrypsin deficiency, which are caused by mutations in the LDLR (Low-Density Lipoprotein Receptor) and SERPINA1 (Serpin Peptidase Inhibitor, Clade A [Alpha-1 Antiproteinase, Antitrypsin], Member 1) genes, respectively (Rashid et al, 2010; Cayo et al, 2012; Tafaleng et al, 2015; Choi et al, 2013) While such successes are encouraging, it has become clear that complex traits and subtle phenotypes are more challenging to reproduce, primarily due to variations in the efficiency of differentiation among cells. If not all, differentiation protocols generate iPSC-derived hepatocyte-like cells with heterogeneous expression of mature hepatic markers
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