br Discussion In these studies

Discussion In these studies, we included multiple donors in each cohort, rather than multiple isogenic clones, in an approach intended to mimic the design of human clinical trials. This allowed us to determine whether, within a genetically diverse cohort, we could detect disease-specific differences emerging above the known phenotypic variation among normal individuals. Indeed, we were able to detect disease-state-specific differences in gene expression among cohorts, in addition to some differences in levels of intracellular protein accumulation among clones, that could represent genetic differences in protein processing or the cellular response to misfolded proteins that has been postulated to exist among individuals (Pan et al., 2009; Wu et al., 1994). Human iPSC-hepatic Tozadenant derived using our differentiation protocol were similar to primary human fetal hepatocytes in terms of expression levels of a subset of hepatic genes. These results are consistent with other published protocols (Rashid et al., 2010; Si-Tayeb et al., 2010) demonstrating differentiation of hepatic cells that were incomplete in their maturity, as evidenced by persistent, high levels of AFP expression that were similar to fetal levels in our experiments. This hurdle in directing differentiation of pluripotent stem cells to fully mature differentiated cells has been seen across germ layers and cell types (Baxter et al., 2015; Smith et al., 2013), reflects the general state of the field, and is the focus of a growing number of investigators (Ogawa et al., 2013; Shan et al., 2013). As our data and the published literature suggest (Leung et al., 2013; Rashid et al., 2010; Suzuki et al., 2014), however, the ability to fully mature a cell in vitro might not be necessary to model and study key disease features when disease-causative genes are expressed at high levels. We used our iPSC human disease model to assess both well-accepted and controversial pathways for handling protein misfolding that have been interrogated using other approaches. Our report utilizes the classical pulse-chase labeling technique to quantify the kinetics of AAT processing and secretion using human patient-derived hepatocyte-like cells, demonstrating the ability of iPSC-hepatic cells to model a key feature of Z AAT protein-driven cellular dysfunction. In cells accumulating misfolded, insoluble Z AAT protein polymers, the autophagy pathway is activated in an attempt to deal with this toxic protein accumulation. Our studies document increased formation as well as increased clearance of autophagosomes in PiZZ iPSC-hepatic cells, consistent with augmented autophagic flux. These findings are in accord with those previously observed in mouse embryonic fibroblasts (MEFs), cell lines, and transgenic mice overexpressing human Z AAT. Increased autophagosome numbers have been observed in liver biopsy specimens from PiZZ individuals (Teckman and Perlmutter, 2000), but it has not been possible previously to measure flux in their tissues. Our report extends to human hepatic cells the observation made in PiZ transgenic mice (Hidvegi et al., 2010) that further CBZ-induced augmentation of this flux ameliorates intracellular accumulation of mutant protein. A second cellular stress pathway implicated in the setting of accumulated intracellular Z AAT protein is the UPR, postulated to link Z AAT polymer-induced cellular injury and downstream development of liver disease, a poorly understood progression (Lawless et al., 2004; Perlmutter et al., 2007). Previous studies of PiZZ liver disease in other model systems have not detected a UPR, despite its known role in the cellular response to high volumes of protein misfolding (Hidvegi et al., 2005). Our detection of a UPR, early during the hepatic differentiation of PiZZ iPSCs, thus contrasts with these studies, though it is in keeping with recent patient-based findings in circulating monocytes from PiZZ individuals (Carroll et al., 2010).