Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes

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.

Identification of a primitive intestinal transcription factor network shared between oesophageal adenocarcinoma and its pre-cancerous precursor state

“…S4C). CLRN3 has been identified as a HNF4A transcriptional target in iPSC-derived hepatocytes (Mallanna et al, 2016) and CLDN18 has been confirmed as a GATA6 target in gastric cancer (Sulahian et al 2014), therefore we used these likely targets to test the regulatory potential of HNF4A and GATA6 in OE19 cells. Both of these genes exhibit multiple regions of increased chromatin accessibility in both tumour tissue and OE19 cells, several of which contain motifs for HNF4A or GATA6 binding ( Supplementary Fig.…”

A Drug Screen using Human iPSC-Derived Hepatocyte-like Cells Reveals Cardiac Glycosides as a Potential Treatment for Hypercholesterolemia

“…Recently, technologies have been developed to produce induced pluripotent stem cells (iPSCs) from patients (Takahashi et al, 2007; Yu et al, 2007). Since patient-specific iPSCs can be induced to differentiate into a variety of cell types, including cells with hepatocyte characteristics, investigators can now model heritable human diseases in culture and screen for therapeutics (Lee et al, 2009; Ebert et al, 2009; Rashid et al, 2010; Matsa et al, 2014; Robinton and Daley, 2012; Choi et al, 2013; Szkolnicka et al, 2016; Cameron et al, 2015; Lang et al, 2016; Schwartz et al, 2012; Mallanna et al, 2016). We have previously described the generation of iPSCs from a hoFH patient (Cayo et al, 2012).…”

SurfaceGenie: a web-based application for prioritizing cell-type specific marker candidates

“…Lymphocyte samples were prepared as 50 ng/µL total sample peptide concentration with Pierce Peptide Retention Time Calibration Mixture (PRTC, Thermo) spiked in at a final concentration of 2 fmol/µL and queued in blocked and randomized order with two technical replicates analyzed per sample. CSC samples of mouse islet cell types were analyzed as described (38,39). MS data were analyzed using Proteome Discoverer 2.2 (Thermo Fisher Scientific) and SkylineDaily (v4.2.1.19095) (40).…”