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    • br Results To develop iPSC

    2018-11-08


    Results To develop iPSC-based model systems of disease, we first prepared a bank of >60 iPSC clones (ten clones per donor; partial set and reprogramming methodology described previously [Mills et al., 2013; Somers et al., 2010]) derived from the dermal fibroblasts of three control individuals without any known disease and three recruited volunteers previously diagnosed with AATD due to homozygous inheritance of mutant Z SC 144 encoding the AAT protease inhibitor (PiZZ genotype, a common monogenic cause of hepatic cirrhosis [Eriksson et al., 1986]). Pluripotency of the resulting cells was confirmed in teratoma assays (Somers et al., 2010), as well as by Pluritest global transcriptomic analysis (Müller et al., 2011; Figure S1). We previously published microarray profiling of the global transcriptomes of nine iPSC lines made from three normal donors (three iPSC lines/donor) and three embryonic stem cell (ESC) lines. We determined that donor-to-donor genetic variability in stem cell phenotype (defined as the global transcriptome expressed in the pluripotent/undifferentiated state) rather than clone-to-clone variability per donor was most responsible for variance in gene expression among cell lines (Mills et al., 2013). In addition to gene expression, we found the propensity to differentiate into a particular fate was also due mainly to genetic background as opposed to clonal variability, consistent with others’ work focused on the relative contribution of donor, cell type of origin, and reprogramming approach to differentiation efficiency (Kajiwara et al., 2012). These data prompted us to select only a single clone per donor for all disease-modeling experiments, as individual clones of a given genetic background tend to behave similarly.
    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 cells 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.