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TECH NOTE

Expression of CYP Enzymes in Cellartis Enhanced hiPS-HEP Cells

Overview

Hepatocytes derived from human pluripotent stem cells have the potential to serve as a predictive in vitro model system for toxicity testing and drug discovery studies. However, this application depends on the ability of the cells to accurately recapitulate hepatic function. Until recently, the functionality of stem cell-derived hepatocytes has been insufficient for applications requiring high expression of multiple drug metabolizing enzymes.

Using a novel 2D differentiation protocol, we have derived hepatocytes from human induced pluripotent stem (iPS) cells that have substantially improved functionality and display several adult hepatic characteristics. Here, we present data on the expression of drug metabolizing enzymes in Cellartis Enhanced hiPS-HEP cells.

Results

CYP activity and expression

Cytochrome P450 (CYP) enzyme activity and expression was measured in Cellartis Enhanced hiPS-HEP cells using several methods. Activity was analyzed using liquid chromatography/mass spectrometry (LC/MS) (Figure 1). The hiPS-HEP cells showed similar CYP1A, CYP3A, and CYP2C9 activity as cryoplateable human primary hepatocytes (cryo hphep). In contrast, the CYP2B6, CYP2D6, and CYP2C19 activities were lower in the hiPS-HEP cells than in the cryo hphep. CYP activities in hiPS-HEP cells were stable between days 6 and 11 after thawing, providing a much larger window for use than cryo hphep. Moreover, hiPS-HEP cells exhibited lower variability than cryo hphep, indicating better batch-to-batch consistency and reproducibility.

Figure 1. CYP activities in Cellartis Enhanced hiPS-HEP and cryoplateable human primary hepatocytes (cryo hphep). Cyropreserved Cellartis Enhanced hiPS-HEP cells were thawed and cultured for 6 or 11 days before analysis (n=5). The cryo hphep cells were cultured for 20 hours before analysis (n=4 different donors). Results are presented as the mean activity ± SEM.

CYP1A, CYP3A4, CYP2C, CYP2C19, and P450 reductase protein expression was also analyzed by Western blot (Figure 2). There was substantial expression of these factors in Cellartis Enhanced hiPS-HEP cells, at levels comparable to that in human liver samples.

Figure 2. Western blot analysis of CYP protein in Cellartis Enhanced hiPS-HEP cells and human liver extracts. Two different batches of hiPS-HEP were analyzed 7 days after thawing. Western blotting was performed at the Karolinska Institute (Stockholm, Sweden).

Substantial expression of CYP1A, CYP2C9, and CYP3A in Cellartis Enhanced hiPS-HEP cells could also be detected by immunocytochemical staining (Figure 3). Interestingly, distinct subgroups of hiPS-HEP cells were immuno-positive, an observation that is reminiscent of metabolic zonation in the liver lobe.

Figure 3. Immunocytochemical staining for CYP1A, CYP2C9, and CYP3A in Cellartis Enhanced hiPS-HEP cells (7 days after thawing). All immunostainings are merged with a nuclear counterstain (DAPI).

The adult enzymes CYP3A4 and CYP3A5 and the fetal enzyme CYP3A7 were analyzed by RT-PCR (Figure 4). The adult enzymes were expressed at levels comparable to cryoplatable primary hepatocytes, whereas the fetal enzyme was expressed at low levels in hiPS-HEP cells. Similar to the LC/MS data, hiPS-HEP cells exhibited lower variability than cryo hphep.

Figure 4. qRT-PCR analysis of CYP3A4, CYP3A5, and CYP3A7 mRNA expression in Cellartis Enhanced hiPS-HEP cells and cryoplateable human primary hepatocytes (cryo hphep). Cyropreserved hiPS-HEP cells were thawed and cultured for 7 or 11 days before analysis (n=3). The cryo hphep cells were analyzed directly after thawing (n=5 different donors). CYP3A4 and CYP3A5 were expressed at similar levels in both cell types; CYP3A7 was expressed at lower levels in the hiPS-HEP cells (*: % of hphep). In addition, less variation in expression level was seen for the hiPS-HEP cells compared to the cyro hphep cells.

Batch-to-batch variation

Batch-to-batch variability was assessed by both CYP enzyme activity (Figure 5) and cell morphology (Figure 6). The results indicate low batch-to-batch variation for Cellartis Enhanced hiPS-HEP cells both in terms of consistent expression of key CYP enzymes and homogenous cellular morphology.

Figure 5. CYP activity in five batches of Cellartis Enhanced hiPS-HEP cells. Cyropreserved hiPS-HEP cells were thawed and cultured for 6 or 11 days before analysis (n=5 batches). For each batch, the analysis was performed in triplicate. Data is presented as mean ± SEM. Consistent expression of all CYP enzymes tested was observed between different batches.

Figure 6. Cell morphology of different batches of Cellartis Enhanced hiPS-HEP cells. Cyropreserved hiPS-HEP cells were thawed and cultured for 6 days before analysis (n=5 batches). Consistent morphology is observed between different batches by microscopy.

Conclusions

Cellartis Enhanced hiPS-HEP cells have substantial expression of CYP enzymes as detected by activity assay and expression (Western blot and immunocytochemistry). These cells have several features characteristic of adult hepatic cells, including substantial expression of the adult enzymes CYP2C9, CYP2C19, and CYP3A4, and low expression of fetal genes (e.g., CYP3A7). Our analysis also indicates that hiPS-HEP cells have high batch-to-batch consistency with regard to CYP activity levels and homogeneity (i.e., >90% pure hepatocytes). Taken together these results suggest that Cellartis Enhanced hiPS-HEP cells can provide an inexhaustible source of functional human hepatocytes for applications that demand hepatic functionality, high reproducibility, and continuous supply of material with the same genetic background.

Methods

Cell culture

Cryopreserved Cellartis Enhanced hiPS-HEP cells were thawed and plated in 96-well plates according to the recommended protocol. Cells were maintained for 6 to 11 days, with a media change every other day, before analysis.

CYP activity assay

The CYP activities of Cellartis Enhanced hiPS-HEP cells were analyzed at days 6 and 11 after thawing. LC/MS was used to measure the formation of specific metabolites, Acetaminophen (CYP1A), 4-OH-Diclofenac (CYP2C9), 4-OH-Mephenytoin (CYP2C19), OH-Bufuralol (CYP2D6), and 1-OH-Midazolam (CYP3A). LC/MS analysis was performed at Pharmacelsus GmbH. Briefly, the cells were carefully washed twice with pre-warmed William medium E (+ 0.1% PEST). Then, the activity assay was started by adding 110 µl/cm2 culture area of pre-warmed William medium E containing 0.1% PEST, 25 mM HEPES, 2 mM L-Glutamine, and the probe substrate cocktail (Table I). After 2 hours at 37°C, 100 µl of the supernatant was collected and kept at -80°C until LC/MS analysis. The metabolite concentrations measured by LC/MS were normalized to the amount of protein per well (determined using the Pierce BCA Protein Assay Kit) and the assay duration (120 min).

Table I. Probe Substrate Cocktail
CYP Substrate Assay concentration
1A Phenacetin 10 µM
2B6 Bupropion 10 µM
2C19 Mephenytoin 50 µM
2C9 Deiclofenac 10 µM
2D6 Bufuralol 10 µM
3A Midazolam 5 µM

Western blot

Cellartis Enhanced hiPS-HEP cells were thawed and plated in T75 flasks and maintained in culture for 8 days before being harvested for Western blot analysis. Fifteen micrograms of protein were used for analysis. Pooled human liver microsomes were used as a control. The following antibodies were used for detection of CYP450 enzymes and P450 reductase: anti-human CYP3A4 (1:1000, PAP 011, Cypex), anti-human CYP2C C-terminal peptide (1:1000, gift from Dr. R. Edwards), anti-CYP2C19 (1:1000, HPA015066, Sigma Aldrich), and anti-cytochrome P450 reductase (1:1000, ab13513, Abcam).

Immunocytochemistry

Cellartis Enhanced hiPS-HEP cells were fixed 7 days after plating and stained with the following primary antibodies: rabbit anti-CYP1A2 (1:100, BML-CR3130, Enzo), rabbit anti-CYP2C9 (1:2500, PAP091, CYPEX), and rabbit anti-CYP3A4 (1:200, PAP011, CYPEX). A donkey anti-rabbit Alexa Fluor 5948 IgG (1:1000, A21207, Invitrogen) secondary antibody was used for detection.

qRT-PCR

Total RNA from Cellartis Enhanced hiPS-HEP cells was extracted using the MagMAX-96 Total
RNA Isolation Kit (Life Technologies) according to the manufacturer’s instructions. cDNA was synthesized and qRT-PCR amplification reactions were performed using a ABI 7500 Real-Time PCR System (Life Technologies). Gene expression was analyzed using TaqMan Gene Expression Assays (Life Technologies) according to the manufacturer’s recommendations. Each sample was analyzed in duplicate. The following assays were used (Life Technologies): CYP3A4 (Hs00604506_m1), CYP3A5 (Hs00241417_m1), and CYP3A7 (Hs00426361_m1).

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