The ability to use cell surface proteins to specifically sor

The ability to use cell-surface proteins to specifically sort iPSC-derived hepatocyte-like cells is currently limited. The liver-specific cell-surface protein ASGR1 has been used to sort human ESC- and iPSC-derived hepatocyte-like cells (Basma et al., 2009; Peters et al., 2016). These studies demonstrated enrichment of hepatic phenotype in ASGR1-positive cells by performing transcriptional analysis and functional assays. Although we identified ASGR1 on the cell surface of primary hepatocytes, its transcript expression pattern during hPSC differentiation into hepatocyte-like cells did not meet the stringent enrichment criteria we required to select the final candidate proteins for our studies. More recently, microRNA switches have been used to enrich for a desired population of hepatocyte-like cells generated by directed differentiation (Miki et al., 2015). In this approach, reporter mRNAs containing target sequences for miRNAs expressed in the desired cell type are transfected, and diminished reporter expression enables sorting of the target cell population. Although useful, transfection adds technical variability, and differences in transfection efficiency of reporter mRNAs could contribute to differences in the cell types sorted from distinct biological samples. With this in mind, we believe the use of endogenous proteins that are accessible to affinity reagents for live cell sorting will prove most apt for the selection of homogeneous cell populations suitable for research and clinical applications. Finally, we provide evidence that cell sorting using Bleomycin Sulfate to either SLC10A1, CLRN3, or AADAC can improve the quality of hepatocyte-like cells generated from iPSCs. Cell populations resulting from positive selection based on these markers displayed higher levels of characteristic hepatic mRNAs. Importantly, a comparison of marker expression among four independent differentiations revealed that the cells sorted based on the presence of SLC10A1 had substantially lower variation in expression of characteristic mRNAs than the unsorted population. This implies that sorting cells using SLC10A1, CLRN3, or AADAC will improve comparisons among control and patient iPSC-derived hepatocytes. Minimizing heterogeneity within cultures of iPSC-derived cells should facilitate our ability to compare and interpret data resulting from hepatocytes generated from iPSCs that model metabolic liver disease. Until now, because of variations with differentiation, analyses of disease phenotypes have been limited to patients with Mendelian disorders. However, the ability to generate homogeneous populations of iPSC-derived hepatocytes should facilitate analyses of complex phenotypes. For example, several common allelic variations that associate with lipid traits have been identified by genome-wide association studies (Teslovich et al., 2010). Producing homogeneous populations of iPSC-derived hepatocytes from either patient iPSCs or iPSCs modified using genome editing approaches could facilitate direct testing of a specific variant\'s contribution to levels of secreted lipid. Use of recombinant laminins as substrates have recently been shown to improve cell organization, cell function, and phenotype of human ESC-derived hepatocyte-like cells (Cameron et al., 2015). Using data published by Cameron et al. (2015), we compared the liver-enriched gene regulatory network (GRN) of SLC10A1-positive cells with hepatocyte-like cells differentiated on recombinant laminin substrates. The GRN of SLC10A1-positive cells appeared to be comparable with hepatocyte-like cells differentiated on recombinant laminin substrates, although we acknowledge that differences in experimental protocol may introduce some error into this comparative analysis (Figure S6B). Importantly, however, SLC10A1-positive cells from independent differentiations exhibited less variation in the liver GRN compared with independent differentiations of hepatocyte-like cells on recombinant laminin substrates, which is consistent with the belief that sorting using such cell-surface markers can enhance the homogeneity of the hepatocyte-like cell population (Figure S6B). These analyses also suggest that by combining approaches, further advances in the quality and homogeneity of iPSC-derived hepatocytes when compared with fresh hepatocytes could be obtained (Figure S6C).