Multisite studies for optimization of a highly efficient culture assay used for in vitro detection of residual undifferentiated human pluripotent stem cells intermingled in cell therapy products.

Introduction: MEASURE2 (Multisite Evaluation Study on Analytical Methods for Non-clinical Safety Assessment of HUman-derived REgenerative Medical Products 2) is a Japanese experimental public-private partnership initiative that aims to standardize testing methods for tumorigenicity evaluation of human pluripotent stem cell (hPSC)-derived cell therapy products (CTPs). MEASURE2 organized multisite studies to optimize the methodology of the highly efficient culture (HEC) assay, a sensitive culture-based in vitro assay for detecting residual undifferentiated hPSCs in CTPs. Methods: In these multisite studies, 1) the efficiency of colony formation by human induced pluripotent stem cells (hiPSCs) under two different culture conditions and 2) the sorting efficiency of microbeads conjugated to various anti-hPSC markers during hiPSC enrichment were evaluated using samples in which hiPSCs were spiked into hiPSC-derived mesenchymal stem cells. Results: The efficiency of colony formation was significantly higher under culture conditions with the combination of Chroman 1, Emricasan, Polyamines, and Trans-ISRIB (CEPT) than with Y-27632, which is widely used for the survival of hPSCs. Between-laboratory variance was also smaller under the condition with CEPT than with Y-27632. The sorting efficiency of microbeads conjugated with the anti-Tra-1-60 antibody was sufficiently higher (>80%) than those of the other various microbeads investigated. Conclusions: Results of these multisite studies are expected to contribute to improvements in the sensitivity and robustness of the HEC assay, as well as to the future standardization of the tumorigenicity risk assessment of hPSC-derived CTPs.

Collaborative Study of Thresholds for Mutagens: Adaptive Responses in the Micronucleus Test and Gene Induction by Mutagenic Treatments.

Background: We have been conducting a collaborative study on the thresholds of mutagens. In our previous examinations of cell activity and cell proliferation as endpoints, both displayed hormesis. This time, we conducted experiments to determine thresholds using the micronucleus test as an endpoint. Methods: The micronucleus test was conducted using Chinese hamster CHL/IU cells and mouse lymphoid L5178Y cells. Additionally, we conducted preliminary investigations into the gene expression using human TK6 cells. Results: When adhesive CHL/IU cells were treated with mitomycin C (MMC), and the hormetic response was examined, hormesis was not observed clearly. When L5178Y cells were treated with methyl methanesulfonate (EMS), AF-2, MMC, and colchicine, all of them exhibited an adaptive response. Additionally, cross-adaptive responses using AF-2 and MMC or EMS and MMC were conducted, both combinations showed a cross-adaptive response. When the gene expression patterns of six genes were investigated by RT-PCR after treatment with MMC, EMS, and H2O2 using TK6 cells, two genes, GADD45 A and P21, were induced in a dose- and time-dependent manner. Conclusion: Adaptive responses arise from preconditioning. As hormesis is inherently linked to preconditioning, adaptive responses observed in this study strongly suggest that hormesis was induced, hence existence of thresholds.

Collaborative Study of Thresholds for Mutagens: Hormetic Responses in Cell Proliferation Tests Using Human and Murine Lymphoid Cells.

BACKGROUND: We previously showed that hormetic responses can be established in cell activity tests using human and murine adherent cells. This time, we examined whether hormetic responses can be established in cell proliferation tests using suspended human and murine lymphoid cells. METHODS: Human lymphoblastoid cells (TK6) and mouse lymphoma cells (L5178Y) were cultured in multi-well culture plates and treated with mitomycin C, ethyl methansulfonate, hygromycin B, aclarubicin or colchicine at various dose levels and the number of cells was measured at varied times using a flow cytometer. RESULTS: When the ratio of the number of cells treated with a test chemical to those in the negative control was plotted, the dose-response relationship typically showed a reverse U-shaped curve, indicating the occurrence of hormesis and existence of thresholds in cell toxicity. The hormetic responses depended largely on the test chemical, dose level and exposure time. When examining responses over the course of time, a J-shaped or fallen S-shaped curve was also observed. CONCLUSIONS: The dose-response relationship showed a reverse U-shaped curve, a hallmark of hormesis, at least some time points for all chemicals tested here, indicating that chemical hormesis can be established in in vitro cell proliferation tests.

Comparison of the inducibility of CYP mRNA exposed to typical inducers in fresh and cryopreserved cynomolgus monkey hepatocytes.

Herein, we evaluated CYPs and their nuclear receptor mRNA induction by exposure to typical inducers, omeprazole, rifampicin, and phenobarbital in cynomolgus monkey hepatocytes. Six freshly-isolated hepatocytes and 6 cryopreserved hepatocytes from cynomolgus monkey liver were prepared for a 14-day monolayer culture, 28-day co-culture with feeder cells, and 28-day 3D spheroid culture with feeder cells. Omeprazole and rifampicin respectively induced CYP1A1 and CYP3A8 mRNAs, while phenobarbital induced CYP2C43, CYP2C75, and CYP3A8, and slightly induced CYP2B6. The nuclear receptors AHR, PXR, and CAR mRNA levels, which were activated by omeprazole, rifampicin, and phenobarbital, respectively, tended to decrease via exposure to inducers despite the increase in CYP mRNA levels. These trends were similar for all three culture methods. No evident difference was observed in CYP mRNA induction between fresh and cryopreserved hepatocytes. Based on mRNA levels, the co-culture and 3D spheroid culture methods are more reasonable than monolayer culture for CYP evaluation, because the use of feeder cells can reduce the number of hepatocytes, improve the cell adhesion, and maintain the mRNA expression levels. In addition, co-culture method is more cost-effective, as common culture plates can be used.

Comparison of mRNA expression profiles of drug-metabolizing enzymes and transporters in fresh and cryopreserved cynomolgus monkey hepatocytes.

In this study, freshly isolated and cryopreserved cynomolgus monkey hepatocytes were seeded on Cell-able® plates with feeder cells to form spheroids and were cultured for 28 days. As a control, hepatocytes were also cultured with or without feeder cells on collagen-coated plates. We verified the mRNA expression levels of drug-metabolizing enzyme-related genes and the leakage of enzymes (AST, ALT, LDH, and γ-GTP) as indicators of cell survival. As a result, the patterns of target mRNA expression in fresh and cryopreserved hepatocytes were very similar during the culture period between culture methods. mRNA expression levels were highly maintained at day 28 using the 3D spheroid and co-culture methods, demonstrating that these methods are useful for maintenance of liver function. Leakage of AST and ALT was higher at day 3 but decreased at day 14. LDH was not detected, suggesting that the cell viability was also maintained during the culture period. Furthermore, the functional differences between fresh and cryopreserved hepatocytes were not clearly detected. The co-culture method was useful for long-term culture not requiring 3D structure, and the 3D spheroid culture method was effective as well. With these techniques, cynomolgus monkey hepatocytes are expected to exhibit smaller individual differences and high reproducibility.

Collaborative study of thresholds for mutagens: proposal of a typical protocol for detection of hormetic responses in cytotoxicity tests.

BACKGROUND: According to the linear no-threshold model (LNT), even the smallest amount of radiation is hazardous. Although the LNT is not based on solid data, this hypothesis has been applied to mutagens and carcinogens. As a result, it has been postulated that there are no thresholds for these chemicals. To demonstrate negativity by experiments is practically impossible, because negative data may leave behind the possibility that additional data might make the resolution power high enough to change negativity to positivity. Furthermore, additional data collection may be endless and we may be trapped in agnosticism. When hormesis is established, in which biological responses are higher at low-doses and lower at high-doses than the control, thresholds could be established between the low- and high-doses. Before examination of thresholds in chemical mutagenesis, hormetic responses in cytotoxicity were tested using cultured mammalian cells. METHOD: Human cells (HeLa S3 and TK6) or Chinese hamster cells (CHL/IU) were cultured in 96-well plates and treated with mitomycin C (MMC) or ethyl methanesulfonate (EMS) at various dose levels and optical density was measured after addition of a reagent to detect cellular activity. In hormetic responses, data might fluctuate to and fro; therefore, experimental conditions were examined from various aspects to eliminate confounding factors including cell numbers, detection time, the edge effect of 96-well plates, and measurement time after addition of the reagent for detection. RESULTS: The dose response relationship was never linear. Cellular activities after treatment with MMC or EMS were generally higher at lower doses levels and lower at higher doses than the control, showing hormesis and allowing the establishment of thresholds. Dose response curves sometimes showed two or three peaks, probably reflecting different cellular responses. CONCLUSION: Hormetic responses in cytotoxicity tests were observed and thresholds could be established. Based on the results of this investigation, we put forward a tentative protocol to detect chemical hormesis in cytotoxicity tests, i.e., inoculate 2000 cells per well, add various doses of a test chemical 48 h after inoculation, add a detection dye 10 h after treatment, and measure optical density 2 h after addition of the reagent for detection.

Efficient Generation of Cynomolgus Monkey Induced Pluripotent Stem Cell-Derived Intestinal Organoids with Pharmacokinetic Functions.

In preclinical studies, the cynomolgus monkey (CM) model is frequently used to predict the pharmacokinetics of drugs in the human small intestine, because of its evolutionary closeness to humans. Intestinal organoids that mimic the intestinal tissue have attracted attention in regenerative medicine and drug development. In this study, we generated intestinal organoids from CM induced pluripotent stem (CMiPS) cells and analyzed their pharmacokinetic functions. CMiPS cells were induced into the hindgut; then, the cells were seeded on microfabricated culture vessel plates to form spheroids. The resulting floating spheroids were differentiated into intestinal organoids in a medium containing small-molecule compounds. The mRNA expression of intestinal markers and pharmacokinetic-related genes was markedly increased in the presence of small-molecule compounds. The organoids possessed a polarized epithelium and contained various cells constituting small intestinal tissues. The intestinal organoids formed functional tight junctions and expressed drug transporter proteins. In addition, in the organoids generated, cytochrome P450 3A8 (CYP3A8) activity was inhibited by the specific inhibitor ketoconazole and was induced by rifampicin. Therefore, in the present work, we successfully generated intestinal organoids, with pharmacokinetic functions, from CMiPS cells using small-molecule compounds.

Evaluation of a repeated-dose liver micronucleus assay with 2,6-dinitrotoluene using young adult rats.

As part of a collaborative study by the Mammalian Mutagenicity Study Group of the Environmental Mutagen Society of Japan, we examined micronucleus induction in hepatocytes following oral administration of 2,6-dinitrotoluene (2,6-DNT) at 30, 40, and 50mg/kg/day for 14 days or at 20, 30, and 40mg/kg/day for 28 days to young adult male rats. This compound is known to be a rat liver carcinogen. The formation of micronucleated hepatocytes was confirmed to be dose-dependent with statistically significant increases observed in both treatments. In contrast, no statistically significant changes in the percentage of micronucleated immature erythrocytes were observed in any dose group in the bone marrow micronucleus assay. These results indicated that the repeated-dose liver micronucleus assay has the potential to detect genotoxic hepatocarcinogens and can be integrated into general toxicological studies.

Evaluation of human hepatocytes cultured by three-dimensional spheroid systems for drug metabolism.

We investigated the utility of three-dimensional (3D) spheroid cultures of human hepatocytes in discovering drug metabolites. Metabolites of acetaminophen, diclofenac, lamotrigine, midazolam, propranolol and salbutamol were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) to measure enzyme activities in this system cultured for 2 and 7 days. Sequential metabolic reactions by Phase I and then Phase II enzymes were found in diclofenac [CYP2C9 and UDP-glucuronyltransferases (UGTs)], midazolam (CYP3A4 and UGTs) and propranolol (CYP1A2/2D6 and UGTs). Moreover, lamotrigine and salbutamol were metabolized to lamotrigine-N-glucuronide and salbutamol 4-O-sulfate, respectively. These metabolites, which are human specific, could be observed in clinical studies, but not in conventional hepatic culture systems as in previous reports. Acetaminophen was metabolized to glucuronide and sulfate conjugates, and N-acetyl-p-benzo-quinoneimine (NAPQI) and its metabolites were not observed. In addition, mRNA of drug-metabolism enzymes [CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, UGT1A1, UGT2B7, sulfotransferase 1A1 (SULT1A1) and glutathione S-transferase pi 1 (GSTP1)], which were measured by qRT-PCR, were expressed in the human hepatocyte spheroids. In conclusion, these results suggest that human hepatocyte spheroids are useful in discovering drug metabolites.

Characterization of marmoset CYP2B6: cDNA cloning, protein expression and enzymatic functions.

The common marmoset is a promising species for evaluating the safety of drug candidates. To further understand the capacity for drug metabolism in marmosets, a cDNA encoding a CYP2B enzyme was cloned from the total RNA fraction of marmoset liver by 3'- and 5'-RACE methods. Nucleotide and deduced amino acid sequences showed 90.8 and 86.2% identity, respectively, with human CYP2B6. The marmoset CYP2B6 (marCYP2B6) protein was expressed in insect cells, and its enzymatic properties were compared with those of human (humCYP2B6) and cynomolgus monkey (cynCYP2B6) orthologs in liver and insect cell microsomes. Enzymatic functions were examined for the oxidation of 7-ethoxy-4-(trifluoromethyl)coumarin (7-ETC), bupropion (BUP) and efavirenz (EFV). The kinetic profiles for the oxidation of the three substrates by liver microsomal fractions were similar between humans and cynomolgus monkeys (biphasic for 7-ETC and monophasic for BUP and EFV), but that of marmosets was unique (monophasic for 7-ETC and biphasic for BUP and EFV). Recombinant enzymes, humCYP2B6 and cynCYP2B6, also yielded similar kinetic profiles for the oxidation of the three substrates, whereas marCYP2B6 showed activity only for 7-ETC hydroxylation. In silico docking simulations suggested that two amino acid residues, Val-114 and Leu-367, affect the activity of marCYP2B6. In fact, a marCYP2B6 mutant with substitutions V114I and L367V exhibited BUP hydroxylase activity that was 4-fold higher than that of humCYP2B6, while its EFV 8-hydroxylase activity was only 10% that of the human enzyme. These results indicate that the amino acids at positions 114 and 367 affect the enzymatic capacity of marmoset CYP2B6.