Hanyang Med Rev.  2015 Nov;35(4):196-206. 10.7599/hmr.2015.35.4.196.

Recent Advances for Enhancing Drug Metabolizing Functions of Hepatocyte-like Cells Derived from Human Pluripotent Stem Cells

• Han J
• Kim JH

• KMID: 2129472
• DOI: http://doi.org/10.7599/hmr.2015.35.4.196

Abstract

Hepatocyte-like cells (HLCs) derived from human pluripotent stem cells are a promising cell source for drug screening and toxicity tests. Thus, various hepatic differentiating protocols have been developed, leading to a hepatic differentiation efficiency of approximately 90%. However, HLC drug metabolizing ability remains very low compared to human primary hepatocytes. In order to overcome this problem, several alternative methods, such as, co-culture, three-dimensional (3D) culture, bioreactor, nanochip-based, etc., have been developed, but optimization to produce fully functional HLCs is ongoing. Recently, our group reported that repeated exposure of HLCs to xenobiotics can improve the expression of hepatic metabolizing enzymes such as cytochrome P450s (CYPs) and glutathione S-transferases (GSTs). These data suggest that we should develop strategies for differentiating cells into mature HLCs by more closely mimicking in vivo fetal and postnatal liver development. Here, we review the current development of alternative methods for enhancing the drug metabolizing functions of HLCs derived from human embryonic stem cells, human-induced pluripotent stem cells, and mesenchymal stem cells as used for drug screening and toxicity tests.

Keyword

Bioreactors; Coculture Techniques; Hepatocytes; Stem Cells

MeSH Terms

Bioreactors
Coculture Techniques
Cytochrome P-450 Enzyme System
Drug Evaluation, Preclinical
Embryonic Stem Cells
Glutathione
Hepatocytes
Humans*
Liver
Mesenchymal Stromal Cells
Pluripotent Stem Cells*
Stem Cells
Toxicity Tests
Xenobiotics
Cytochrome P-450 Enzyme System
Glutathione
Xenobiotics

Figure

• 

  Fig. 1 (A) Characterization of 2D and 3D cultured HLCs. Phase contrast images of HLCs and (B) immunofluorescent images of hepatic markers (ALB: red and CK18: green) in HLCs at the final stage III of hepatic differentiation. Note that 3D hepatic spheroids were formed and further differentiated from single-cell dissociated 2D hepablast. HLCs, hepatocyte-like cells; 3D, three-dimensional.

• 

  Fig. 2 Enhanced hepatic metabolizing HLCs using 3D culture systems and repeated exposure to xenobiotics. (A) Schematic representation of repeated exposure of 2D-cultured HLCs and 3D hepatic spheroids to xenobiotics. Note that the HLCs and hepatic spheroids derived from BGO1 hESCs were exposed to each xenobiotic concentration at the end of stage III. The xenobiotic treatment was then withdrawn for 2 days followed by a second exposure for 2 days. (B) qPCR analysis of ALB and three transcription factors known for their expression in mature hepatocytes (PROX1, C/EBPa, and ATF5) and phase I enzymes (CYP1A2, CYP2D6, CYP2C9, CYP3A4, and CYP3A7) in 2D-cultured HLCs after repeated exposure to xenobiotics. The 'a' denotes statistical significance. Control (C, untreated HLCs); the 'b' denotes statistical significance, compared to the first exposure of its own group. Phenobarbital (PB, widely prescribed as an anti-seizure medication in children); Acetaminophen (AP, a common cold medication); rifampicin (RIF, an antibiotic used to treat infections). OSM, oncostatin M and DEX, dexamethasone. Ref. 2 with permission from Oxford University Press.

• 

  Fig. 3 Comparison of hepatic gene expression among 2D-cultured HLCs (2D), 3D-cultured hepatic spheroids at day 6 of stage III (3D), and three different human primary hepatocytes 24 hours after in vitro culture (2D) using qPCR analysis. Each of the 2D-cultured human primary hepatocytes (green bars) were obtained from Caucasian (C), African American (AA), and Asian (A) subjects. Statistical significance is reported as 'a' for statistical significance of gene expression between 2D HLCs and 3D hepatic spheroids, 'b' for statistical significance of gene expression between 3D hepatic spheroids and 2D human primary hepatocytes, and 'c' for statistical significance of gene expression between 2D HLCs and 2D human primary hepatocytes. (A) Expression of ALB (albumin), PROX1, and AFP. (B) Expression of nuclear receptor (NR), which regulate the expression of key hepatic CYP genes. (C) Gene expression of various isoform GSTs, which are part of the GST phase II metabolic enzyme family. (D) Gene expression of hepatic CYPs. Data are presented as the means 6SD of 4 separate experiments. Ref. 2 with permission from Oxford University Press.

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