Tolloid-Like 1 Negatively Regulates Hepatic Differentiation of Human Induced Pluripotent Stem Cells Through Transforming Growth Factor Beta Signaling.

Single nucleotide polymorphisms in Tolloid-like 1 (TLL1) and the expression of TLL1 are known to be closely related to hepatocarcinogenesis after hepatitis C virus elimination or liver fibrosis in patients with nonalcoholic fatty liver disease. TLL1 is a type of matrix metalloprotease and has two isoforms in humans, with the short isoform showing higher activity. However, the functional role of TLL1 in human liver development is unknown. Here, we attempted to elucidate the function of human TLL1 using hepatocyte-like cells generated from human pluripotent stem cells. First, we generated TLL1-knockout human induced pluripotent stem (iPS) cells and found that hepatic differentiation was promoted by TLL1 knockout. Next, we explored TLL1-secreting cells using a model of liver development and identified that kinase insert domain receptor (FLK1)-positive cells (mesodermal cells) highly express TLL1. Finally, to elucidate the mechanism by which TLL1 knockout promotes hepatic differentiation, the expression profiles of transforming growth factor beta (TGFß), a main target gene of TLL1, and its related genes were analyzed in hepatic differentiation. Both the amount of active TGFß and the expression of TGFß target genes were decreased by TLL1 knockout. It is known that TGFß negatively regulates hepatic differentiation. Conclusion: TLL1 appears to negatively regulate hepatic differentiation of human iPS cells by up-regulating TGFß signaling. Our findings will provide new insight into the function of TLL1 in human liver development.

Modeling of Hepatic Drug Metabolism and Responses in CYP2C19 Poor Metabolizer Using Genetically Manipulated Human iPS cells.

Cytochrome P450 family 2 subfamily C member 19 (CYP2C19), in liver, plays important roles in terms of drug metabolism. It is known that CYP2C19 poor metabolizers (PMs) lack CYP2C19 metabolic capacity. Thus, unexpected drug-induced liver injury or decrease of drug efficacy would be caused in CYP2C19 substrate-treated CYP2C19 PMs. However, it is difficult to evaluate the safety and effectiveness of drugs and candidate compounds for CYP2C19 PMs because there is currently no model for this phenotype. Here, using human induced pluripotent stem cells (human iPS cells) and our highly efficient genome-editing and hepatocyte differentiation technologies, we generated CYP2C19-knockout human iPS cell-derived hepatocyte-like cells (CYP2C19-KO HLCs) as a novel CYP2C19 PM model for drug development and research. The gene expression levels of hepatocyte markers were similar between wild-type iPS cell-derived hepatocyte-like cells (WT HLCs) and CYP2C19-KO HLCs, suggesting that CYP2C19 deficiency did not affect the hepatic differentiation potency. We also examined CYP2C19 metabolic activity by measuring S-mephenytoin metabolites using ultra-performance liquid chromatography-tandem mass spectrometry. The CYP2C19 metabolic activity was almost eliminated by CYP2C19 knockout. Additionally, we evaluated whether clopidogrel (CYP2C19 substrate)-induced liver toxicity could be predicted using our model. Unexpectedly, there was no significant difference in cell viability between clopidogrel-treated WT HLCs and CYP2C19-KO HLCs. However, the cell viability in clopidogrel- and ketoconazole (CYP3A4 inhibitor)-treated CYP2C19-KO HLCs was significantly enhanced as compared with that in clopidogrel- and DMSO-treated CYP2C19-KO HLCs. This result suggests that CYP2C19-KO HLCs can predict clopidogrel-induced liver toxicity. We succeeded in generating CYP2C19 PM model cells using human iPS cells and genome-editing technologies for pharmaceutical research. SIGNIFICANCE STATEMENT: Although unexpected drug-induced liver injury or decrease of drug efficacy would be caused in CYP2C19 substrate-treated CYP2C19 poor metabolizers, it is difficult to evaluate the safety and effectiveness of drugs and candidate compounds for CYP2C19 poor metabolizers because there is currently no model for this phenotype. Using human iPS cells and our highly efficient genome editing and hepatocyte differentiation technologies, we generated CYP2C19-knockout human iPS cell-derived hepatocyte-like cells as a novel CYP2C19 poor metabolizer model for drug development and research.

Highly efficient biallelic genome editing of human ES/iPS cells using a CRISPR/Cas9 or TALEN system.

Genome editing research of human ES/iPS cells has been accelerated by clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALEN) technologies. However, the efficiency of biallelic genetic engineering in transcriptionally inactive genes is still low, unlike that in transcriptionally active genes. To enhance the biallelic homologous recombination efficiency in human ES/iPS cells, we performed screenings of accessorial genes and compounds. We found that RAD51 overexpression and valproic acid treatment enhanced biallelic-targeting efficiency in human ES/iPS cells regardless of the transcriptional activity of the targeted locus. Importantly, RAD51 overexpression and valproic acid treatment synergistically increased the biallelic homologous recombination efficiency. Our findings would facilitate genome editing study using human ES/iPS cells.