These differentiated HLCs retain a strong fetal

These differentiated HLCs retain a strong fetal and immature phenotype, as demonstrated by the persistence of alpha-fetoprotein (AFP) and diminished hepatic functions when compared to primary adult hepatocytes (PHHs). Strategies to improve the differentiation protocol are being explored. For example, treatment of differentiated Ro 3306 with small-molecule compounds may hold the key to improving hepatic maturation or functionality (Shan et al., 2013). However, in order to screen for small molecules, the differentiation protocol needs to be miniaturized to 384-well plates. Thus far, the efficient differentiation of hPSCs has relied on a preliminary colony-type culture of hPSCs, directly treated to produce hepatocytes without passage at any stage of the hepatic differentiation (Hannan et al., 2013; Mallanna and Duncan, 2013). Using this approach, efficient differentiation of hPSC-derived HLCs is usually limited to 6-, 12- or 24-well plates, curtailing the usefulness of this approach in generating cell populations suitable for high-throughput assay (Reviewed in Schwartz et al., 2014). Some published protocols suggested passing definitive endoderm (DE) cells (Hay et al., 2008; Agarwal et al., 2008; Liu et al., 2010; Duan et al., 2010), to allow a more homogeneous and flatter cell population (Inamura et al., 2011), but none of them took advantage of this approach to miniaturize the format of plates. Furthermore the HLCs tend to lose their differentiated phenotype with time without special culturing condition (Khetani and Bhatia, 2008). Here we describe a protocol allowing efficient and consistent differentiation of hESC and hiPSC into functional hepatocyte-like cells in 96- and 384-well plates. We also describe a new HLC culturing condition, allowing maintenance of SC-derived HLCs for more than 3weeks in these high-throughput formats, with a gradual improvement of their hepatic phenotype and functions.
Results
Discussion Here we described for the first time the efficient and reproducible differentiation of human pluripotent stem cells into functional hepatocyte-like cells in 384-well plates. We identified here several critical factors that ensure efficiency and reproducibility of hepatic differentiation of hPSCs (Fig. 7): While the HLCs generated from this new protocol do not exhibit a significantly more mature hepatic phenotype than what has been described before, we described here for the first time the consistent and reproducible differentiation of HLCs in 384-well plates, which opens the door to their use for high throughput assay. Importantly, we showed here that results for both phenotypic (expression of hepatic markers by IFA) and functional (secretion of ALB assessed by AlphaLISA) characterization of the 384w-HLCs show coefficients of variation below 15%, making them suitable as readout for high-throughput screening. In this context, the screening for compounds that could improve the level of in vitro maturation of HLCs is currently in progress. Recently, a study used micro patterned PHHs (MCPPs) on feeder cells to perform a high-throughput screen in order to identify drugs capable of improving functionality in vitro of PHHs and potentially promoting maturation of SC-derived HLCs (Shan et al., 2013). However this screen was not performed directly on HLCs, because they could not be efficiently differentiated in wells smaller than a 24-well plate. Only candidate drugs selected on adult PHHs were then validated on HLCs, suggesting that drugs specifically acting on early stages of hepatic maturation could have been missed. In addition, we described a new long-term culture medium allowing maintenance and further maturation of HLCs for more than 3weeks in 384-well plates. During maintenance in WEM medium, HLCs exhibited improved phenotypic stability, increased secretion and higher percentage of cells positive for adult hepatic markers. Importantly, CV% of expression of hepatic markers, like AFP and ALB, was maintained below 15% throughout the maintenance in WEM medium, compatible with use for high-throughput assay.