Human Embryonic Stem Cell Derived Hepatocyte-Like Cells as a Tool for In Vitro Hazard Assessment of Chemical Carcinogenicity

Abstract

Hepatocyte-like cells derived from the differentiation of human embryonic stem cells (hES-Hep) have potential to provide a human relevant in vitro test system in which to evaluate the carcinogenic hazard of chemicals. In this study, we have investigated this potential using a panel of 15 chemicals classified as noncarcinogens, genotoxic carcinogens, and nongenotoxic carcinogens and measured whole-genome transcriptome responses with gene expression microarrays. We applied an ANOVA model that identified 592 genes highly discriminative for the panel of chemicals. Supervised classification with these genes achieved a cross-validation accuracy of > 95%. Moreover, the expression of the response genes in hES-Hep was strongly correlated with that in human primary hepatocytes cultured in vitro. In order to infer mechanistic information on the consequences of chemical exposure in hES-Hep, we developed a computational method that measures the responses of biochemical pathways to the panel of treatments and showed that these responses were discriminative for the three toxicity classes and linked to carcinogenesis through p53, mitogen-activated protein kinases, and apoptosis pathway modules. It could further be shown that the discrimination of toxicity classes was improved when analyzing the microarray data at the pathway level. In summary, our results demonstrate, for the first time, the potential of human embryonic stem cell--derived hepatic cells as an in vitro model for hazard assessment of chemical carcinogenesis, although it should be noted that more compounds are needed to test the robustness of the assay.

Comparative transcriptomics of hepatic differentiation of human pluripotent stem cells and adult human liver tissue

“…hPSC-HEP have the potential to be such a source (1,7,15,18,55,58) and possibly replace current hepatic models lacking adequate properties. However, for some applications the functionality of hPSC-HEP still needs to be further improved (15,25,37,42,48,56).…”

“…HPSCs have successfully been differentiated into hepatocytes (hPSC-HEP), sharing many features with in vivo hepatocytes, such as cytochrome P450 enzyme activity and glycogen storage, in addition to expression of several drug transporters (1,17,18,52,58). Furthermore, they are applicable for long-term toxicity studies (21) and can accurately classify toxic agents (55). HPSC-HEP have also been successfully and functionally engrafted in actual liver injured immune-compromised mice (37).…”

Hepatic Differentiation and Maturation of Human Embryonic Stem Cells Cultured in a Perfused Three-Dimensional Bioreactor

“…Several studies report on hESC differentiation to functional HEP by using directed developmental signaling [6,8,9,26,27], but up to recent years, the hepatocyte-directed differentiation of hESC has mainly been done in 2D culture, and so far, no one has produced highly functional hepatocytes. Embryonic stem cells are derived from the inner mass of blastocyst-stage embryos [28,29], and when disrupted from their complex microenvironment and plated in 2D culture, the loss of structural architecture will affect the cell differentiation and maturation process [10].…”

“…By directing the hESC toward specific cell types, they have the potential to constitute an almost endless supply of different functional cells types. During the last decades, much effort has been made to derive hepatocytes from hESC, and numerous studies report on hepatocyte-like cells (HEP) that in many aspects resemble their in vivo counterparts [6][7][8][9]. However, the hESC-derived HEP have in some aspects demonstrated reduced hepatic functionality when compared to hPH.…”

Long-Term Chronic Toxicity Testing Using Human Pluripotent Stem Cell–Derived Hepatocytes

“…Protocols to differentiate human embryonic stem cells and human induced pluripotent stem cells into hepatocytes have improved immensely in recent years, and now hepatocyte cultures with high purity and many characteristics of functional hepatocytes can be obtained from hPSC (Brolen et al, 2010;Si-Tayeb et al, 2010;Touboul et al, 2010;Yildirimman et al, 2011;Hannan et al, 2013;Ulvestad et al, 2013;Szkolnicka et al, 2014). The fact that these cells can be derived in a robust and reproducible fashion from a well-characterized source makes them very attractive as a toxicity assessment model.…”