Fig 2: A Subpopulation of iPSC-Derived Hepatocyte-like Cells that Express SLC10A1 Is Enriched for Several Liver-Specific MarkersiPSC-derived hepatocyte-like cells were sorted by FACS using an antibody against SLC10A1 (see also Figure S5A). Bar graphs show qRT-PCR analysis to determine the levels of mRNAs encoding the liver-enriched proteins (HNF4A, albumin, ASGR1, APOA1, AFP) and cell-surface proteins (SLC10A1, CLRN3, AADAC) in the SLC10A1-positive and -negative cell fractions. Levels of mRNAs were compared with the pre-sorted cell population that is set to 1.0 (see also Figures S5B–S5D). To avoid any bias introduced by the sorting process, the pre-sort population was collected as the gated viable cell fraction after passing through the FACS. Data are represented as means ± SEM from sorting performed on three independent differentiations (n = 3). Significance of enrichment in the SLC10A1-positive population compared with the pre-sorted population was determined by Student's t test, ∗p < 0.05.

Fig 3: A Subpopulation of iPSC-Derived Hepatocyte-like Cells Express SLC10A1, CLRN3, and AADAC(A) Confocal micrographs showing the results of immunocytochemistry to detect expression of cell-surface proteins SLC10A1, CLRN3, and AADAC in iPSC-derived hepatocyte-like cells (green). Nuclei are identified by DAPI staining (blue) (see also Figures S3B and S3C). Scale bars, 100 μm.(B) FACS histogram plots of iPSC-derived hepatocyte-like cells stained with primary antibodies against proteins SLC10A1, CLRN3, and AADAC and corresponding phycoerythrin (PE) conjugated secondary antibody are shown. Cells stained with PE conjugated secondary antibody only are used as control.(C) Confocal micrographs showing the results of immunocytochemistry to detect expression of HNF4A (red) and cell-surface proteins SLC10A1, CLRN3, and AADAC (green) in iPSC-derived hepatocyte-like cells (see also Figure S4). Nuclei are identified by DAPI staining (blue). Scale bars, 100 μm.

Fig 4: SLC10A1-Positive iPSC-Derived Hepatocyte-like Cells Are Enriched for Hepatic Gene Regulatory Genes and Sorting Them Minimizes Variability Associated with Differentiation(A) Microarray analysis was performed on different cell fractions of iPSC-derived hepatocyte-like cells sorted for expression of SLC10A1. CellNet classification heatmap showing enrichment of liver-specific GRN genes and depletion of ESC-specific GRN genes in the positive sort cell fraction compared with the pre-sort and negative sort cell fractions. Classification scores corresponding to the ESC- and liver-specific GRN genes from the heatmap are presented below the heatmap. Classification scores for different target tissue GRNs represented in the heatmap are provided in Table S2. Microarray data on the pre-sort, positive sort, and negative sort cell fractions from four independent differentiations were used for CellNet analysis (n = 4) (see also Figure S6).(B) Heatmap showing expression levels of 39 liver signature genes in pre-sort, positive sort, and negative sort cell fractions. Probe sets of 39 liver signature genes and the corresponding raw signal values in different cell fractions are provided in Table S3 in the same order as in the heatmap. Microarray data on the pre-sort, positive sort, and negative sort cell fractions from four independent differentiations were used to generate the heatmap (n = 4).(C) Line diagram showing raw signal values of the top ten abundant transcripts selected from 39 liver signature genes in pre-sort and SLC10A1-positive sort cell fractions of hepatocyte-like cells. Signal values of GAPDH transcript served as internal control. Microarray data from four independent differentiations were used to generate the line diagram (n = 4) (see also Figure S6).

Fig 5: Identification of N-Linked Glycoproteins in Primary Hepatocytes by Cell Surface Capture Technology(A) Illustration outlining the CSC-Technology.(B) Left: Annotated MS/MS spectrum of a representative peptide from SLC10A1 that was identified in primary hepatocytes by CSC-Technology. Two identified sites of N-glycosylation (N115) are indicated. Right: An illustration of the transmembrane topology of SLC10A1 highlighting the extracellular peptide identified by CSC-Technology, including sites of N-glycosylation (also see Figure S2). Image generated using Protter (Omasits et al., 2014).(C) Graphical representation of the distribution of proteins identified in primary hepatocytes. Each column represents an individual cell type documented in the Cell Surface Protein Atlas, which consists of 49 different cell types. The last column shows proteins identified on the surface of primary hepatocytes described in the current study. Each row represents a single protein. A yellow bar indicates the protein was reported as present in the cell line, whereas blue indicates that it was not observed. The frequency of distribution of each protein is lowest at the top and highest at the bottom. The identity of each protein in the order presented is provided in Table S1.