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Higher expression of cytochrome P450 3A4 in human mesenchymal and adipose-derived stem cells than in dermal fibroblasts: With emphasis on the correlation with basal pregnane X receptor expression
Process Biochemistry 49 (2014) 1983–1989
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Higher expression of cytochrome P450 3A4 in human mesenchymal and adipose-derived stem cells than in dermal fibroblasts: With emphasis on the correlation with basal pregnane X receptor expression Tsai-Shin Chiang a,1 , Kai-Chiang Yang b,1 , Yao-Ming Wu c , Hong-Shiee Lai c , Ching-Chuan Jiang d , Ling-Ling Chiou e , Kuang-Lun Lee f , Guan-Tarn Huang f,∗ , Hsuan-Shu Lee a,f,g,∗∗ a
Institute of Biotechnology, National Taiwan University, Taipei, Taiwan School of Dental Technology, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan d Department of Orthopedics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan e Liver Disease Prevention and Treatment Research Foundation, Taipei, Taiwan f Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan g Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan b c
a r t i c l e
i n f o
Article history: Received 14 March 2014 Received in revised form 14 August 2014 Accepted 15 August 2014 Available online 23 August 2014 Keywords: Cytochrome P450 3A4 Pregnane X receptor Human dermal fibroblasts Adipose-derived stem cells Bone marrow-derived mesenchymal stem cells
a b s t r a c t Among the cytochrome P 450 (CYP) enzymes, CYP3A4 is most abundantly existed and metabolizes most of the drugs. We have shown human dermal fibroblasts (HDFs) express low levels of CYPs, but could be engineered to express much higher levels to be used as drug metabolism prediction. To seek better cell source for engineering, the basal mRNA expression and enzyme activities of CYP3A4 in HDFs (n = 5), bone marrow-derived mesenchymal stem cells (BMMSCs, n = 7), and adipose-derived stem cells (ADSCs, n = 3) were compared. The basal CYP3A4 expression and activity in BMMSCs and ADSCs were significantly higher than in HDFs. However, the coefficient of variation of CYP3A4 expression was very high in each cell type. Comparing the expression levels of 4 regulators glucocorticoid receptor, pregnane X receptor (PXR), hepatocyte nuclear factor 4␣, and hydrocarbon receptor nuclear translocator in the total 15 cell samples, we found the only basal PXR expression was significantly correlated with CYP3A4 expression. The gender and age of patients were not significant determinants in the expressions of PXR and CYP3A4. In conclusion, BMMSCs and ADSCs expressed higher levels of CYP3A4 than HDFs and the expression of CYP3A4 might be mainly determined by basal PXR expression. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Cytochrome P450 (CYP) enzymes are the major phase I drug metabolizing enzymes in the human body and are responsible for the oxidative metabolism of most endogenous compounds and exogenous xenobiotics, including clinically used drugs [1].
∗ Corresponding author at: No. 1, Section 1, Jen Ai Road, Taipei 10051, Taiwan. Tel.: +886 2 2312 3456x65015; fax: +886 2 2381 9723. ∗∗ Corresponding author at: 4F, No. 81, Chang-Xing Street, Taipei, Taiwan. Tel.: +886 2 2312 3456x65044; fax: +886 2 3366 6001. E-mail addresses: [email protected] (G.-T. Huang), [email protected] (H.-S. Lee). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.procbio.2014.08.006 1359-5113/© 2014 Elsevier Ltd. All rights reserved.
Among the CYPs, CYP3A4 has the greatest impact because it is the most abundant CYP enzyme in the human liver (30% of total hepatic CYP proteins), and it metabolizes more than 50% of the currently used drugs [2–4]. Hepatocytes are the major sources of CYP3A4. The other tissues expressing CYP3A4 include the intestine, kidney, adrenal gland, and brain [5]. Expression of these vital drug metabolism enzymes is coordinately regulated by several transcription factors and nuclear receptors that play roles in liver development and regeneration [2,6,7]. The important roles of pregnane X receptor (PXR), hepatocyte nuclear factor-4␣ (HNF-4˛), glucocorticoid receptor (GR), and aryl hydrocarbon receptor nuclear translocator (ARNT) have been reported in controlling the expression of several CYPs [8–11].
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The expressions of CYPs in human fibroblastic cells have seldom been investigated. The expression of CYP1B1 has been demonstrated in human mammary stromal fibroblasts [12]. Previously, we found that human dermal fibroblasts (HDFs) expressed low levels of CYP mRNAs and enzyme activities. By delivery of key regulators, HDFs can be engineered to express sufficiently high levels of CYP3A4 enzyme to metabolize a calcium channel blocker, nifedipine [13]. This strategy may become a rational alternative over an in vitro assay using human hepatocytes that are difficult to obtain. Moreover, HDFs are easily obtainable and are also easily expanded into large quantities to meet the requirement for establishing an in vitro human cell-based platform to predict drug metabolism. In addition to HDFs, bone marrow-derived mesenchymal stem cells (BMMSCs) and adipose-derived stromal cells (ADSCs) are fibroblastic cells that are also easily obtained and expanded in culture. Both cell types have received considerable attention for their possible application in regenerative medicine [14,15]. An interesting question is whether, and at what levels, these cell types express CYP3A4. This knowledge may help researchers to choose optimal cells for establishing the in vitro drug assays. Also, identifying the key regulators for the constitutive expression of CYPs in these cells may lead to further optimization of their CYPs expression. In this present study, HDFs, BMMSCs, and ADSCs were harvested from independent patients under informed consent. The correlations between basal and constitutive expression levels, and variability of CYP3A4 among these cell types were demonstrated. The relationships between CYP3A4 and the gender or age of the patients were also analyzed. The purpose of this study was to demonstrate that the mRNA and enzyme activity levels were significantly higher in BMMSCs and ADSCs than in HDFs, though at much lower levels compared to the commonly used hepatoma cell line Hep G2. Expression of PXR is the major determinant for the basal CYP3A4 expression in three human fibroblastic cells. 2. Materials and methods 2.1. Cell culture Retrieval and usage of human tissues for cell isolation have been approved by the Research Ethics Committee at National Taiwan University Hospital. HDFs were primarily cultured from human foreskin tissues obtained by circumcision (201201007RID) [13]. Bone marrow samples obtained from patients receiving total hip replacements were used to derive BMMSCs (201306035RIND). ADSCs were isolated from omentum resected from patients receiving abdominal surgery (201103081RC). The participants have provided their written informed consents and the ethics committees have approved this consent procedure. Human hepatoma Hep G2 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Hep G2, HDFs, and ADSCs were cultured in high glucose Dulbecco’s Modified Eagle’s medium (HGDMEM, GIBCO-BRL, Scotland, UK) supplemented with 10% fetal
bovine serum (FBS). BMMSCs were cultured in low glucose-DMEM (GIBCO-BRL) supplemented with 20% FBS. All of the cells were cultured at 37 ◦ C under an atmosphere containing 5% CO2 . In total, 5 HDF, 7 BMMSC, and 3 ADSC samples from respective patients were included in this study. 2.2. Characterization of HDFs, BMMSCs, and ADSCs by flow cytometry HDFs between 7 and 10 passages, and BMMSCs and ADSCs between 3 and 6 passages were used. Flow cytometric analyses were performed using antibodies against CD11b, CD34 (Santa Cruz, Santa Cruz, CA), CD29, CD31, CD44, CD45, CD73, CD90, and CD146 (BD Pharmingen, CA). Cells were incubated with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)conjugated monoclonal antibodies for 30 min at 4 ◦ C in the dark. Flow cytometry was performed using FACSCalibur (BD, Franklin Lakes, NJ), and the results were analyzed by FCS Express software (Version 4.0; Denovo Software, Los Angeles, CA). Negative controls using FITC-conjugated (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) or PE-conjugated (eBiosecience, San Diego, CA) isotype immunoglobulins were included in the experiments. 2.3. In vitro adipogenic and osteogenic differentiation Cells were seeded at a density of 1 × 104 cells/cm2 in six-well plates. Adipogenic and osteogenic differentiation were undertaken using AdvanceSTEM Adipogenic Differentiation Kit and AdvanceSTEM Osteogenic Differentiation Kit (Thermo Fisher Scientific Inc., Waltham, MA), respectively. The medium was changed every 2–3 days over a period of 21 days. 2.4. Evaluation of adipogenic and osteogenic differentiation Chemical staining and detection of specific gene expression were used to evaluate cell differentiation. For chemical staining, the differentiated cells were fixed in 10% neutral formalin and washed twice with phosphate-buffered saline. Adipogenic differentiation was determined by staining cells using oil Red-O (Sigma–Aldrich, St. Louis, MO) to show intracellular fat droplets, and osteogenic differentiation was evaluated by alizarin-red-S staining (Sigma–Aldrich) to show calcium phosphate deposition. For determination of the expression of specific differentiated genes, total RNAs of the cells were extracted using REzolTM C&T Reagent (Protech Technologies Inc., Taipei, Taiwan). Reverse transcription was performed by SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) with a total volume of 20 L, and the products were used for revere transcription-polymerase chain reaction (RT-PCR). The expression of lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor ␥ (PPAR ) mRNAs was used to indicate adipogenic differentiation, and the expression of runt-related transcription factor 2 (RUNX 2) mRNA was used to indicate osteogenic
Table 1 Primers for RT-PCR. Gene LPL PPAR Runx2 GAPDH
Sequence Sense Antisense Sense Antisense Sense Antisense Sense Antisense
Tm (◦ C)
Length (bp.)
60
278
60
605
55
163
60
496
5 -ATGGAGAGCAAAGCCCTGCTC-3 5 -TACAGGGCGGCCACAAGTTTT-3 5 -GCTGTTATGGGTGAAACTCTG-3 5 -ATAAGGTGGAGATGCAGGCTC-3 5 -CAGCGTCAACACCATCATTC-3 5 -CAGACCAGCAGCACTCCATA-3 5 -CAAGATCATCAGCAATGCC-3 5 -CTTGACAAAGTGGTCGTTGA-3
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Table 2 Characterization of HDF-1, BMMSC-1, and ADSC-1 by flow cytometry.
HDF-1 BMMSC-1 ADSC-1 a
CD11b
CD29
3.2 ± 0.2a 0.9 ± 0.2 0.4 ± 0.6
98.5 ± 0.9 97.5 ± 1.9 94.5 ± 5.3
CD31 0.3 ± 0.1 0.2 ± 0.01 0.4 ± 0.1
CD34
CD44
CD45
CD73
CD90
CD146
0.2 ± 0.1 0.2 ± 0.02 0.5 ± 0.2
98.3 ± 1.4 90.4 ± 6.3 65.7 ± 7.1
0.1 ± 0.01 0.05 ± 0.03 0.3 ± 0.1
99.1 ± 0.6 98.1 ± 0.4 98.1 ± 0.6
0.1 ± 0.03 98.6 ± 0.3 94.0 ± 1.9
14.1 ± 0.9 44.2 ± 4.9 11.2 ± 10.8
Positive rate (%) in mean ± SEM.
differentiation. The sense and antisense primers for the PCR are listed in Table 1.
protocol was denaturing at 95 ◦ C for 30 s, annealing at 60 ◦ C for 30 s, and extending at 72 ◦ C for 30 s for up to 40 cycles. 2.6. Statistics
2.5. Quantitative-polymerase chain reaction (Q-PCR) Q-PCR was performed on StepOne (Applied Biosystems, Foster City, CA, USA) using TaqMan® Fast Universal PCR Master Mix and specific primers/probes (Applied Biosystems) to measure the mRNA expression levels of CYP3A4 (Hs00604506 m1), HNF-4˛ (Hs00230853 m1), PXR (Hs01114267 m1), GR (Hs00353740 m1), ARNT (Hs01121918 m1), and house-keeping gene glyceraldehyde3-phosphate dehydrogenase (GAPDH, Hs02758991 g1). The PCR
Positive rates (%) in flow cytometry data were expressed as mean ± SEM of 3 independent measurements. The medians of CYP3A4 expression levels were compared between gender using Wilcoxon rank-sum test and among 3 kinds of cell using Kruskal–Wallis test. Spearman’s rank correlation was used to determine the correlation between CYP3A4 expression with the expression of PXR, HNF-4˛, GR, and ARNT, and also between the expression of CYP3A4 and PXR with patients’ age. Differences were
Fig. 1. Adipogenic and osteogenic differentiation potential of representative HDF, BMMSC, and ADSC. The adipogenic and osteogenic differentiation potential of HDF-1, BMMSC-1, and ADSC-1 were evaluated by chemical staining (A and B) and RT-PCR (C). HDF-1, BMMSC-1, and ADSC-1 were randomly selected and used as respective representatives from each group. (A) Adipogenic differentiation was evaluated by oil-red-O staining, and the fat droplets stained red in the differentiated adipocytes are indicated by white arrows. (B) Osteogenic differentiation was evaluated by alizarin-red-S staining, and the extracellular deposition of calcium phosphate is indicated by yellow arrows. Scale bars = 100 m. (C) RT-PCR was used to detect the expression of respective differentiation marker genes. RT-NC = reverse transcription-negative control using water as template; D0 = undifferentiated cells; D21 = cells 21 days after differentiation induction; LPL = lipoprotein lipase; PPAR = peroxisome proliferator-activated receptor ␥; RUNX 2 = runt-related transcription factor 2; GADPH = glyceraldehyde-3-phosphate dehydrogenase.
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3. Results 3.1. Characterization of HDFs, BMMSCs, and ADSCs by flow cytometry and induction of differentiation
Fig. 2. Boxplots of CYP3A4 expression levels in ADSCs, BMMSCs, and HDFs. The medians in ADSCs and BMMSCs were significantly higher than in HDFs. *P < 0.05.
considered statistically significant at P values of less than 0.05. For all statistics, data from triplicate or three independent experiments were used. The statistical analyses were performed by SAS 9.2 (Cary, NC, USA).
HDF-1, BMMSC-1, and ADSC-1 cells were used as respective representatives for each cell type to characterize their surface marker profiles by flow cytometric analysis and to determine their differentiation capacities into osteogenic and adipogenic lineages. Table 2 shows the positive rates of each cell for each surface marker. Each of the 3 cell types were consistently negative for the hematopoietic cell markers CD11b, CD31, CD34, and CD45 and were positive for some MSC markers, including CD29, CD44, and CD73. A striking difference was that the positive rate of CD90 in HDF-1 cells was quite low (0.1%) in contrast to the high positive rates in BMMSC-1 (98.6%) and ADSC-1 (94.0%) cells. The positive rates of CD146 were higher in BMMSC-1 (44.2%) cells and lower in ADSC-1 (11.2%) and HDF-1 (14.1%) cells. After induction of differentiation, accumulation of intracellular fat droplets (adipogenic differentiation) and extracellular calcium phosphate deposition (osteogenic differentiation) could be clearly identified in BMMSC-1 and ADSC-1, but not in HDF-1 cells (Fig. 1a and b). RT-PCR further confirmed the results. Adipocyte marker genes LDL and PPAR and osteocyte marker gene RUNX 2 expression was induced after adipogenic and osteogenic differentiation,
Fig. 3. Correlation between basal expression level of CYP3A4 with 4 key transcriptional regulators in the 15 cell samples. Basal CYP3A4 expression and basal PXR, HNF 4˛, GR, and ARNT expression in the 15 cell samples were analyzed to determine the correlation strength. Basal CYP3A4 expression was shown only significantly correlated with basal PXR expression linearly (r = 0.65, P < 0.005).
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respectively, in BMMSC-1 and ADSC-1 cells (Fig. 1c) [16,17]. HDF1 also weakly expressed these adipogenic and osteogenic makers at D0, which may be caused by fibroblast heterogeneity [18,19]. These mRNA expressions were down-regulated under adipogenic and osteogenic induction. These data are keeping in line with the findings of Bosch et al. [20]. 3.2. Basal expression levels of CYP3A4 in the 3 types of cell We compared the basal expression levels of CYP3A4 in Hep G2 and the 3 types of cell using the level of HDF-1 as the reference. The median level was 0.58 in HDFs, 3.00 in BMMSCs, and 6.41 in ADSCs. Fig. 2 shows significantly higher levels in ADSCs and BMMSCs than in HDFs by Kruskal–Wallis test. Compared to the level of Hep G2, the average expression level in HDFs, BMMSCs, and ADSCs was 2.0%, 15.5%, and 17.8%, respectively. However, the coefficient of variation of basal CYP3A4 expression within each group was very high (128% in HDFs, 87% in BMMSCs, and 74% in ADSCs). 3.3. Correlation of basal CYP3A4 expression level with the basal levels of 4 important hepatic factors It has been shown that the expression of most CYP genes is regulated by various liver-enriched transcription factors and nuclear receptors at transcriptional level [2,6,7,21]. Among the regulators, we examined the correlation between the respective basal gene expression levels of PXR, ARNT, GR, and HNF-4˛ with the basal expression levels of CYP3A4 in the 15 cell samples. Fig. 3 shows that the CYP3A4 level had only significant linear correlation with PXR level (r = 0.65, P < 0.005). 3.4. Correlation of basal CYP3A4 and PXR expression level with patients’ age The basal expression levels of CYP3A4 and PXR were then analyzed for correlation with age of the patients from whom the individual cells were derived. Fig. 4 shows no significant correlation of CYP3A4 (r = 0.36, P > 0.3) and PXR (r = 0.13, P > 0.7) with patients’ age in the 15 cells. 3.5. The role of gender on the basal expression levels of CYP3A4 and PXR Basal CYP3A4 and PXR expression levels were compared between male (n = 7) and female (n = 8) patients from whom the cells were derived. Fig. 5 shows no significant difference of the median expression levels of CYP3A4 (P > 0.45) and PXR (P > 0.09) between genders. 4. Discussion CYP expression in HDFs, BMMSCs, and ADSCs has been rarely investigated. In this study, we demonstrated that three types of human fibroblastic cells, i.e. HDFs, BMMSCs, and ADSCs, did constitutively express CYP3A4 mRNA, though at much lower levels compared to the commonly used hepatoma cell line Hep G2. The mRNA levels were significantly higher in BMMSCs and ADSCs than in HDFs (Fig. 2). The roles of CYP3A4 expression in these fibroblastic cells and the significance of higher expression levels in the stem cells BMMSCs and ADSCs than in non-stem cells HDFs remain unknown. A panel of antibodies against cell surface markers was chosen to characterize these cells. Cell surface marker profiles were very similar among HDFs, BMMSCs, and ADSCs (Table 2). All of the cells were negative for the hematopoietic cell markers CD11b, CD31, CD34, and CD45, and positive for certain MSC markers,
Fig. 4. Correlation between basal expression level of PXR and CYP3A4 with patients’ age in the 15 cell samples. No significant correlation was found.
including CD29, CD44, and CD73 [22,23]. However, HDFs were negative for another MSC marker CD90 that was positive in both BMMSCs and ADSCs. Another striking difference was the positive rates of CD146, which were lower in HDFs and ADSCs (14.1% and 11.2%, respectively) than in BMMSCs (44.2%). This is consistent with a previous report showing higher rate of CD146 in MSCs than in fibroblasts [24]. Regarding the differentiation potential, both BMMSCs and ADSCs, but not HDFs, were shown capable of differentiating into both adipogenic and osteogenic lineages (Fig. 1). Among the 15 cell samples, the basal expression levels of CYP3A4 were significantly higher in BMMSCs and ADSCs than in HDFs (Fig. 2). However, the coefficients of variation were very high within each group. These results suggested that the constitutive cellular conditions of the 15 individual cells might play an important role in basal CYP3A4 expression. Therefore, we tried to figure out the roles of age and gender in the expression level of CYP3A4. Age and gender have been shown to be determinants for the expression of CYP3A4 in the liver [25,26]. The fetal liver does not express CYP3A4, but expression in the liver increases to approximately 40% of adult level at four months of age and to 72% at 12 months of age [27]. Liver CYP3A4 mRNA expression was shown to be 1.91-fold higher in women compared with men [26]. In contrast, our data demonstrated that age and gender were not determinants in the basal expression level of CYP3A4 (Figs. 4 and 5), suggesting there must be some other factors controlling the levels in these fibroblastic cells.
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in these 15 fibroblastic cells were unknown. Genetic polymorphisms may be important determinants [37,38]. For example, a single nucleotide polymorphism in intron 6 of CYP3A4 strongly affects the hepatic expression of CYP3A4 [39]. In addition, some polymorphisms in the promoter or intron of PXR may significantly affect CYP3A4 expression [40,41]. Other reports showed that the expression of PXR is tissue-specific and co-regulated by other vital nuclear receptors, such as HNF 4␣ [42] and GR [11], and its expression is also epigenetically regulated by DNA methylation [43] and by miRNA [44]. Signaling pathways activated by xenobiotics [45,46] or drugs [35,47–50] may also regulate PXR expression. It is known that age, gender, daily dose, metabolism profile, drug interactions, alcohol, and other factors can also result in the expression variability of CYPs and other drug metabolism enzymes [51]. However, there was no significant role of age or gender in the basal expression levels of PXR and CYP3A4 (Figs. 4 and 5) based on our findings. We are cautions that the sample size in this study is still limited and further studies with more samples are warranted to clarify the relationships between patients’ profiles and the CYPs activities.
5. Conclusions Expression of CYP3A4 was higher in the stem cells BMMSCs and ADSCs than in the non-stem cells HDFs and was highly variable within each cell type. CYP3A4 expression was shown to be more highly correlated with the expression of PXR than HNF 4˛, GR, and ARNT. No significant role was observed between patients’ age or gender and their expression of PXR or CYP3A4 within the enrolled 15 patients. Fig. 5. The influence of patient’s gender on basal expression level of PXR and CYP3A4 in the 15 cell samples. No difference was found between genders (P > 0.05).
Conflicts of interest Previous studies have concluded the regulation of the expression of most CYP genes depends on several transcription factors and nuclear receptors at the transcriptional level [2,6,7,19,28]. Recent studies have shown that HNF 4␣ and other factors are important regulators for controlling the expression of several CYP genes in human hepatocytes [8,28], and GR has a synergistic effect with PXR in regulating CYP3A4 and CYP2C expressions [18,29]. In addition, CYP1 families are regulated by a heterodimer composed of the aryl hydrocarbon receptor (AhR) and its nuclear translocator ARNT [10,30,31]. Most importantly, PXR, an orphan nuclear receptor, is the predominant regulator of the CYP3A subfamily in response to the exposure of various xenobiotics [9,32,33]. PXR is broadly expressed in many types of human tissues, including the liver, kidney, intestine, and colon [34,35]. Consistently, CYP3A4 is highly expressed in these tissues [36]. These findings collectively suggested that PXR is a key regulator of CYP3A4 expression. Among the basal expression of PXR, GR, HNF 4˛, and ARNT, we found basal CYP3A4 was best correlated with basal PXR expression in the 15 cell samples (Fig. 3). Though higher in stem cells than non-stem cells, the basal CYP3A4 expression level was highly variable among the cells in each group, indicating the variation of CYP3A4 expression was not only tissue-dependent but also individual-dependent. Similar trends were also found in basal PXR expression (data not shown), further suggesting the key role of basal PXR in basal CYP3A4 expression. The 15 cell samples in this study were derived from 15 individual patients; thus we could not compare the basal PXR levels in different cells from a single patient. The underlying mechanisms for the high inter-individual variability of basal PXR and CYP3A4 expression
The authors declare that they have no conflicts of interest.
Contributions Tsai-Shin Chiang participated in research design, carrying out of the experiments, data analysis, and writing of the manuscript. KaiChiang Yang and Kuang-Lun Lee participated in data analysis, and writing of the manuscript. Yao-Ming Wu, Hong-Shiee Lai, ChingChuan Jiangd, and Ling-Ling Chiou participated in carrying out the experiments. Feng-Huei Lin participated in data analysis. GuanTarn Huang and Hsuan-Shu Lee participated in research design, writing of the manuscript, and grant application.
Acknowledgments This work was supported by grants from the Ministry of Science and Technology of Taiwan (MOST 101-3114-B-002-003 and MOST 102-2313-B-002-014) and Liver Disease Prevention and Treatment Research Foundation, Taipei, Taiwan. The flow cytometry was performed at the Joint Center for Instruments and Researches, College of Bioresources and Agriculture, National Taiwan University, Taiwan. The authors acknowledge statistical experiment assistance provided by the Taiwan Clinical Trial Bioinformatics and Statistical Center, Training Center, and Pharmacogenomics Laboratory (which is founded by National Research Program for Biopharmaceuticals (NRPB) at the Ministry of Science and Technology of Taiwan; MOST 103-2325-B-002-033).
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Higher expression of cytochrome P450 3A4 in human mesenchymal and adipose-derived stem cells than in dermal fibroblasts: With emphasis on the correlation with basal pregnane X receptor expression Tsai-Shin Chiang a,1 , Kai-Chiang Yang b,1 , Yao-Ming Wu c , Hong-Shiee Lai c , Ching-Chuan Jiang d , Ling-Ling Chiou e , Kuang-Lun Lee f , Guan-Tarn Huang f,∗ , Hsuan-Shu Lee a,f,g,∗∗ a
Institute of Biotechnology, National Taiwan University, Taipei, Taiwan School of Dental Technology, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan d Department of Orthopedics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan e Liver Disease Prevention and Treatment Research Foundation, Taipei, Taiwan f Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan g Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan b c
a r t i c l e
i n f o
Article history: Received 14 March 2014 Received in revised form 14 August 2014 Accepted 15 August 2014 Available online 23 August 2014 Keywords: Cytochrome P450 3A4 Pregnane X receptor Human dermal fibroblasts Adipose-derived stem cells Bone marrow-derived mesenchymal stem cells
a b s t r a c t Among the cytochrome P 450 (CYP) enzymes, CYP3A4 is most abundantly existed and metabolizes most of the drugs. We have shown human dermal fibroblasts (HDFs) express low levels of CYPs, but could be engineered to express much higher levels to be used as drug metabolism prediction. To seek better cell source for engineering, the basal mRNA expression and enzyme activities of CYP3A4 in HDFs (n = 5), bone marrow-derived mesenchymal stem cells (BMMSCs, n = 7), and adipose-derived stem cells (ADSCs, n = 3) were compared. The basal CYP3A4 expression and activity in BMMSCs and ADSCs were significantly higher than in HDFs. However, the coefficient of variation of CYP3A4 expression was very high in each cell type. Comparing the expression levels of 4 regulators glucocorticoid receptor, pregnane X receptor (PXR), hepatocyte nuclear factor 4␣, and hydrocarbon receptor nuclear translocator in the total 15 cell samples, we found the only basal PXR expression was significantly correlated with CYP3A4 expression. The gender and age of patients were not significant determinants in the expressions of PXR and CYP3A4. In conclusion, BMMSCs and ADSCs expressed higher levels of CYP3A4 than HDFs and the expression of CYP3A4 might be mainly determined by basal PXR expression. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Cytochrome P450 (CYP) enzymes are the major phase I drug metabolizing enzymes in the human body and are responsible for the oxidative metabolism of most endogenous compounds and exogenous xenobiotics, including clinically used drugs [1].
∗ Corresponding author at: No. 1, Section 1, Jen Ai Road, Taipei 10051, Taiwan. Tel.: +886 2 2312 3456x65015; fax: +886 2 2381 9723. ∗∗ Corresponding author at: 4F, No. 81, Chang-Xing Street, Taipei, Taiwan. Tel.: +886 2 2312 3456x65044; fax: +886 2 3366 6001. E-mail addresses: [email protected] (G.-T. Huang), [email protected] (H.-S. Lee). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.procbio.2014.08.006 1359-5113/© 2014 Elsevier Ltd. All rights reserved.
Among the CYPs, CYP3A4 has the greatest impact because it is the most abundant CYP enzyme in the human liver (30% of total hepatic CYP proteins), and it metabolizes more than 50% of the currently used drugs [2–4]. Hepatocytes are the major sources of CYP3A4. The other tissues expressing CYP3A4 include the intestine, kidney, adrenal gland, and brain [5]. Expression of these vital drug metabolism enzymes is coordinately regulated by several transcription factors and nuclear receptors that play roles in liver development and regeneration [2,6,7]. The important roles of pregnane X receptor (PXR), hepatocyte nuclear factor-4␣ (HNF-4˛), glucocorticoid receptor (GR), and aryl hydrocarbon receptor nuclear translocator (ARNT) have been reported in controlling the expression of several CYPs [8–11].
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The expressions of CYPs in human fibroblastic cells have seldom been investigated. The expression of CYP1B1 has been demonstrated in human mammary stromal fibroblasts [12]. Previously, we found that human dermal fibroblasts (HDFs) expressed low levels of CYP mRNAs and enzyme activities. By delivery of key regulators, HDFs can be engineered to express sufficiently high levels of CYP3A4 enzyme to metabolize a calcium channel blocker, nifedipine [13]. This strategy may become a rational alternative over an in vitro assay using human hepatocytes that are difficult to obtain. Moreover, HDFs are easily obtainable and are also easily expanded into large quantities to meet the requirement for establishing an in vitro human cell-based platform to predict drug metabolism. In addition to HDFs, bone marrow-derived mesenchymal stem cells (BMMSCs) and adipose-derived stromal cells (ADSCs) are fibroblastic cells that are also easily obtained and expanded in culture. Both cell types have received considerable attention for their possible application in regenerative medicine [14,15]. An interesting question is whether, and at what levels, these cell types express CYP3A4. This knowledge may help researchers to choose optimal cells for establishing the in vitro drug assays. Also, identifying the key regulators for the constitutive expression of CYPs in these cells may lead to further optimization of their CYPs expression. In this present study, HDFs, BMMSCs, and ADSCs were harvested from independent patients under informed consent. The correlations between basal and constitutive expression levels, and variability of CYP3A4 among these cell types were demonstrated. The relationships between CYP3A4 and the gender or age of the patients were also analyzed. The purpose of this study was to demonstrate that the mRNA and enzyme activity levels were significantly higher in BMMSCs and ADSCs than in HDFs, though at much lower levels compared to the commonly used hepatoma cell line Hep G2. Expression of PXR is the major determinant for the basal CYP3A4 expression in three human fibroblastic cells. 2. Materials and methods 2.1. Cell culture Retrieval and usage of human tissues for cell isolation have been approved by the Research Ethics Committee at National Taiwan University Hospital. HDFs were primarily cultured from human foreskin tissues obtained by circumcision (201201007RID) [13]. Bone marrow samples obtained from patients receiving total hip replacements were used to derive BMMSCs (201306035RIND). ADSCs were isolated from omentum resected from patients receiving abdominal surgery (201103081RC). The participants have provided their written informed consents and the ethics committees have approved this consent procedure. Human hepatoma Hep G2 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Hep G2, HDFs, and ADSCs were cultured in high glucose Dulbecco’s Modified Eagle’s medium (HGDMEM, GIBCO-BRL, Scotland, UK) supplemented with 10% fetal
bovine serum (FBS). BMMSCs were cultured in low glucose-DMEM (GIBCO-BRL) supplemented with 20% FBS. All of the cells were cultured at 37 ◦ C under an atmosphere containing 5% CO2 . In total, 5 HDF, 7 BMMSC, and 3 ADSC samples from respective patients were included in this study. 2.2. Characterization of HDFs, BMMSCs, and ADSCs by flow cytometry HDFs between 7 and 10 passages, and BMMSCs and ADSCs between 3 and 6 passages were used. Flow cytometric analyses were performed using antibodies against CD11b, CD34 (Santa Cruz, Santa Cruz, CA), CD29, CD31, CD44, CD45, CD73, CD90, and CD146 (BD Pharmingen, CA). Cells were incubated with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)conjugated monoclonal antibodies for 30 min at 4 ◦ C in the dark. Flow cytometry was performed using FACSCalibur (BD, Franklin Lakes, NJ), and the results were analyzed by FCS Express software (Version 4.0; Denovo Software, Los Angeles, CA). Negative controls using FITC-conjugated (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) or PE-conjugated (eBiosecience, San Diego, CA) isotype immunoglobulins were included in the experiments. 2.3. In vitro adipogenic and osteogenic differentiation Cells were seeded at a density of 1 × 104 cells/cm2 in six-well plates. Adipogenic and osteogenic differentiation were undertaken using AdvanceSTEM Adipogenic Differentiation Kit and AdvanceSTEM Osteogenic Differentiation Kit (Thermo Fisher Scientific Inc., Waltham, MA), respectively. The medium was changed every 2–3 days over a period of 21 days. 2.4. Evaluation of adipogenic and osteogenic differentiation Chemical staining and detection of specific gene expression were used to evaluate cell differentiation. For chemical staining, the differentiated cells were fixed in 10% neutral formalin and washed twice with phosphate-buffered saline. Adipogenic differentiation was determined by staining cells using oil Red-O (Sigma–Aldrich, St. Louis, MO) to show intracellular fat droplets, and osteogenic differentiation was evaluated by alizarin-red-S staining (Sigma–Aldrich) to show calcium phosphate deposition. For determination of the expression of specific differentiated genes, total RNAs of the cells were extracted using REzolTM C&T Reagent (Protech Technologies Inc., Taipei, Taiwan). Reverse transcription was performed by SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) with a total volume of 20 L, and the products were used for revere transcription-polymerase chain reaction (RT-PCR). The expression of lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor ␥ (PPAR ) mRNAs was used to indicate adipogenic differentiation, and the expression of runt-related transcription factor 2 (RUNX 2) mRNA was used to indicate osteogenic
Table 1 Primers for RT-PCR. Gene LPL PPAR Runx2 GAPDH
Sequence Sense Antisense Sense Antisense Sense Antisense Sense Antisense
Tm (◦ C)
Length (bp.)
60
278
60
605
55
163
60
496
5 -ATGGAGAGCAAAGCCCTGCTC-3 5 -TACAGGGCGGCCACAAGTTTT-3 5 -GCTGTTATGGGTGAAACTCTG-3 5 -ATAAGGTGGAGATGCAGGCTC-3 5 -CAGCGTCAACACCATCATTC-3 5 -CAGACCAGCAGCACTCCATA-3 5 -CAAGATCATCAGCAATGCC-3 5 -CTTGACAAAGTGGTCGTTGA-3
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Table 2 Characterization of HDF-1, BMMSC-1, and ADSC-1 by flow cytometry.
HDF-1 BMMSC-1 ADSC-1 a
CD11b
CD29
3.2 ± 0.2a 0.9 ± 0.2 0.4 ± 0.6
98.5 ± 0.9 97.5 ± 1.9 94.5 ± 5.3
CD31 0.3 ± 0.1 0.2 ± 0.01 0.4 ± 0.1
CD34
CD44
CD45
CD73
CD90
CD146
0.2 ± 0.1 0.2 ± 0.02 0.5 ± 0.2
98.3 ± 1.4 90.4 ± 6.3 65.7 ± 7.1
0.1 ± 0.01 0.05 ± 0.03 0.3 ± 0.1
99.1 ± 0.6 98.1 ± 0.4 98.1 ± 0.6
0.1 ± 0.03 98.6 ± 0.3 94.0 ± 1.9
14.1 ± 0.9 44.2 ± 4.9 11.2 ± 10.8
Positive rate (%) in mean ± SEM.
differentiation. The sense and antisense primers for the PCR are listed in Table 1.
protocol was denaturing at 95 ◦ C for 30 s, annealing at 60 ◦ C for 30 s, and extending at 72 ◦ C for 30 s for up to 40 cycles. 2.6. Statistics
2.5. Quantitative-polymerase chain reaction (Q-PCR) Q-PCR was performed on StepOne (Applied Biosystems, Foster City, CA, USA) using TaqMan® Fast Universal PCR Master Mix and specific primers/probes (Applied Biosystems) to measure the mRNA expression levels of CYP3A4 (Hs00604506 m1), HNF-4˛ (Hs00230853 m1), PXR (Hs01114267 m1), GR (Hs00353740 m1), ARNT (Hs01121918 m1), and house-keeping gene glyceraldehyde3-phosphate dehydrogenase (GAPDH, Hs02758991 g1). The PCR
Positive rates (%) in flow cytometry data were expressed as mean ± SEM of 3 independent measurements. The medians of CYP3A4 expression levels were compared between gender using Wilcoxon rank-sum test and among 3 kinds of cell using Kruskal–Wallis test. Spearman’s rank correlation was used to determine the correlation between CYP3A4 expression with the expression of PXR, HNF-4˛, GR, and ARNT, and also between the expression of CYP3A4 and PXR with patients’ age. Differences were
Fig. 1. Adipogenic and osteogenic differentiation potential of representative HDF, BMMSC, and ADSC. The adipogenic and osteogenic differentiation potential of HDF-1, BMMSC-1, and ADSC-1 were evaluated by chemical staining (A and B) and RT-PCR (C). HDF-1, BMMSC-1, and ADSC-1 were randomly selected and used as respective representatives from each group. (A) Adipogenic differentiation was evaluated by oil-red-O staining, and the fat droplets stained red in the differentiated adipocytes are indicated by white arrows. (B) Osteogenic differentiation was evaluated by alizarin-red-S staining, and the extracellular deposition of calcium phosphate is indicated by yellow arrows. Scale bars = 100 m. (C) RT-PCR was used to detect the expression of respective differentiation marker genes. RT-NC = reverse transcription-negative control using water as template; D0 = undifferentiated cells; D21 = cells 21 days after differentiation induction; LPL = lipoprotein lipase; PPAR = peroxisome proliferator-activated receptor ␥; RUNX 2 = runt-related transcription factor 2; GADPH = glyceraldehyde-3-phosphate dehydrogenase.
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3. Results 3.1. Characterization of HDFs, BMMSCs, and ADSCs by flow cytometry and induction of differentiation
Fig. 2. Boxplots of CYP3A4 expression levels in ADSCs, BMMSCs, and HDFs. The medians in ADSCs and BMMSCs were significantly higher than in HDFs. *P < 0.05.
considered statistically significant at P values of less than 0.05. For all statistics, data from triplicate or three independent experiments were used. The statistical analyses were performed by SAS 9.2 (Cary, NC, USA).
HDF-1, BMMSC-1, and ADSC-1 cells were used as respective representatives for each cell type to characterize their surface marker profiles by flow cytometric analysis and to determine their differentiation capacities into osteogenic and adipogenic lineages. Table 2 shows the positive rates of each cell for each surface marker. Each of the 3 cell types were consistently negative for the hematopoietic cell markers CD11b, CD31, CD34, and CD45 and were positive for some MSC markers, including CD29, CD44, and CD73. A striking difference was that the positive rate of CD90 in HDF-1 cells was quite low (0.1%) in contrast to the high positive rates in BMMSC-1 (98.6%) and ADSC-1 (94.0%) cells. The positive rates of CD146 were higher in BMMSC-1 (44.2%) cells and lower in ADSC-1 (11.2%) and HDF-1 (14.1%) cells. After induction of differentiation, accumulation of intracellular fat droplets (adipogenic differentiation) and extracellular calcium phosphate deposition (osteogenic differentiation) could be clearly identified in BMMSC-1 and ADSC-1, but not in HDF-1 cells (Fig. 1a and b). RT-PCR further confirmed the results. Adipocyte marker genes LDL and PPAR and osteocyte marker gene RUNX 2 expression was induced after adipogenic and osteogenic differentiation,
Fig. 3. Correlation between basal expression level of CYP3A4 with 4 key transcriptional regulators in the 15 cell samples. Basal CYP3A4 expression and basal PXR, HNF 4˛, GR, and ARNT expression in the 15 cell samples were analyzed to determine the correlation strength. Basal CYP3A4 expression was shown only significantly correlated with basal PXR expression linearly (r = 0.65, P < 0.005).
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respectively, in BMMSC-1 and ADSC-1 cells (Fig. 1c) [16,17]. HDF1 also weakly expressed these adipogenic and osteogenic makers at D0, which may be caused by fibroblast heterogeneity [18,19]. These mRNA expressions were down-regulated under adipogenic and osteogenic induction. These data are keeping in line with the findings of Bosch et al. [20]. 3.2. Basal expression levels of CYP3A4 in the 3 types of cell We compared the basal expression levels of CYP3A4 in Hep G2 and the 3 types of cell using the level of HDF-1 as the reference. The median level was 0.58 in HDFs, 3.00 in BMMSCs, and 6.41 in ADSCs. Fig. 2 shows significantly higher levels in ADSCs and BMMSCs than in HDFs by Kruskal–Wallis test. Compared to the level of Hep G2, the average expression level in HDFs, BMMSCs, and ADSCs was 2.0%, 15.5%, and 17.8%, respectively. However, the coefficient of variation of basal CYP3A4 expression within each group was very high (128% in HDFs, 87% in BMMSCs, and 74% in ADSCs). 3.3. Correlation of basal CYP3A4 expression level with the basal levels of 4 important hepatic factors It has been shown that the expression of most CYP genes is regulated by various liver-enriched transcription factors and nuclear receptors at transcriptional level [2,6,7,21]. Among the regulators, we examined the correlation between the respective basal gene expression levels of PXR, ARNT, GR, and HNF-4˛ with the basal expression levels of CYP3A4 in the 15 cell samples. Fig. 3 shows that the CYP3A4 level had only significant linear correlation with PXR level (r = 0.65, P < 0.005). 3.4. Correlation of basal CYP3A4 and PXR expression level with patients’ age The basal expression levels of CYP3A4 and PXR were then analyzed for correlation with age of the patients from whom the individual cells were derived. Fig. 4 shows no significant correlation of CYP3A4 (r = 0.36, P > 0.3) and PXR (r = 0.13, P > 0.7) with patients’ age in the 15 cells. 3.5. The role of gender on the basal expression levels of CYP3A4 and PXR Basal CYP3A4 and PXR expression levels were compared between male (n = 7) and female (n = 8) patients from whom the cells were derived. Fig. 5 shows no significant difference of the median expression levels of CYP3A4 (P > 0.45) and PXR (P > 0.09) between genders. 4. Discussion CYP expression in HDFs, BMMSCs, and ADSCs has been rarely investigated. In this study, we demonstrated that three types of human fibroblastic cells, i.e. HDFs, BMMSCs, and ADSCs, did constitutively express CYP3A4 mRNA, though at much lower levels compared to the commonly used hepatoma cell line Hep G2. The mRNA levels were significantly higher in BMMSCs and ADSCs than in HDFs (Fig. 2). The roles of CYP3A4 expression in these fibroblastic cells and the significance of higher expression levels in the stem cells BMMSCs and ADSCs than in non-stem cells HDFs remain unknown. A panel of antibodies against cell surface markers was chosen to characterize these cells. Cell surface marker profiles were very similar among HDFs, BMMSCs, and ADSCs (Table 2). All of the cells were negative for the hematopoietic cell markers CD11b, CD31, CD34, and CD45, and positive for certain MSC markers,
Fig. 4. Correlation between basal expression level of PXR and CYP3A4 with patients’ age in the 15 cell samples. No significant correlation was found.
including CD29, CD44, and CD73 [22,23]. However, HDFs were negative for another MSC marker CD90 that was positive in both BMMSCs and ADSCs. Another striking difference was the positive rates of CD146, which were lower in HDFs and ADSCs (14.1% and 11.2%, respectively) than in BMMSCs (44.2%). This is consistent with a previous report showing higher rate of CD146 in MSCs than in fibroblasts [24]. Regarding the differentiation potential, both BMMSCs and ADSCs, but not HDFs, were shown capable of differentiating into both adipogenic and osteogenic lineages (Fig. 1). Among the 15 cell samples, the basal expression levels of CYP3A4 were significantly higher in BMMSCs and ADSCs than in HDFs (Fig. 2). However, the coefficients of variation were very high within each group. These results suggested that the constitutive cellular conditions of the 15 individual cells might play an important role in basal CYP3A4 expression. Therefore, we tried to figure out the roles of age and gender in the expression level of CYP3A4. Age and gender have been shown to be determinants for the expression of CYP3A4 in the liver [25,26]. The fetal liver does not express CYP3A4, but expression in the liver increases to approximately 40% of adult level at four months of age and to 72% at 12 months of age [27]. Liver CYP3A4 mRNA expression was shown to be 1.91-fold higher in women compared with men [26]. In contrast, our data demonstrated that age and gender were not determinants in the basal expression level of CYP3A4 (Figs. 4 and 5), suggesting there must be some other factors controlling the levels in these fibroblastic cells.
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in these 15 fibroblastic cells were unknown. Genetic polymorphisms may be important determinants [37,38]. For example, a single nucleotide polymorphism in intron 6 of CYP3A4 strongly affects the hepatic expression of CYP3A4 [39]. In addition, some polymorphisms in the promoter or intron of PXR may significantly affect CYP3A4 expression [40,41]. Other reports showed that the expression of PXR is tissue-specific and co-regulated by other vital nuclear receptors, such as HNF 4␣ [42] and GR [11], and its expression is also epigenetically regulated by DNA methylation [43] and by miRNA [44]. Signaling pathways activated by xenobiotics [45,46] or drugs [35,47–50] may also regulate PXR expression. It is known that age, gender, daily dose, metabolism profile, drug interactions, alcohol, and other factors can also result in the expression variability of CYPs and other drug metabolism enzymes [51]. However, there was no significant role of age or gender in the basal expression levels of PXR and CYP3A4 (Figs. 4 and 5) based on our findings. We are cautions that the sample size in this study is still limited and further studies with more samples are warranted to clarify the relationships between patients’ profiles and the CYPs activities.
5. Conclusions Expression of CYP3A4 was higher in the stem cells BMMSCs and ADSCs than in the non-stem cells HDFs and was highly variable within each cell type. CYP3A4 expression was shown to be more highly correlated with the expression of PXR than HNF 4˛, GR, and ARNT. No significant role was observed between patients’ age or gender and their expression of PXR or CYP3A4 within the enrolled 15 patients. Fig. 5. The influence of patient’s gender on basal expression level of PXR and CYP3A4 in the 15 cell samples. No difference was found between genders (P > 0.05).
Conflicts of interest Previous studies have concluded the regulation of the expression of most CYP genes depends on several transcription factors and nuclear receptors at the transcriptional level [2,6,7,19,28]. Recent studies have shown that HNF 4␣ and other factors are important regulators for controlling the expression of several CYP genes in human hepatocytes [8,28], and GR has a synergistic effect with PXR in regulating CYP3A4 and CYP2C expressions [18,29]. In addition, CYP1 families are regulated by a heterodimer composed of the aryl hydrocarbon receptor (AhR) and its nuclear translocator ARNT [10,30,31]. Most importantly, PXR, an orphan nuclear receptor, is the predominant regulator of the CYP3A subfamily in response to the exposure of various xenobiotics [9,32,33]. PXR is broadly expressed in many types of human tissues, including the liver, kidney, intestine, and colon [34,35]. Consistently, CYP3A4 is highly expressed in these tissues [36]. These findings collectively suggested that PXR is a key regulator of CYP3A4 expression. Among the basal expression of PXR, GR, HNF 4˛, and ARNT, we found basal CYP3A4 was best correlated with basal PXR expression in the 15 cell samples (Fig. 3). Though higher in stem cells than non-stem cells, the basal CYP3A4 expression level was highly variable among the cells in each group, indicating the variation of CYP3A4 expression was not only tissue-dependent but also individual-dependent. Similar trends were also found in basal PXR expression (data not shown), further suggesting the key role of basal PXR in basal CYP3A4 expression. The 15 cell samples in this study were derived from 15 individual patients; thus we could not compare the basal PXR levels in different cells from a single patient. The underlying mechanisms for the high inter-individual variability of basal PXR and CYP3A4 expression
The authors declare that they have no conflicts of interest.
Contributions Tsai-Shin Chiang participated in research design, carrying out of the experiments, data analysis, and writing of the manuscript. KaiChiang Yang and Kuang-Lun Lee participated in data analysis, and writing of the manuscript. Yao-Ming Wu, Hong-Shiee Lai, ChingChuan Jiangd, and Ling-Ling Chiou participated in carrying out the experiments. Feng-Huei Lin participated in data analysis. GuanTarn Huang and Hsuan-Shu Lee participated in research design, writing of the manuscript, and grant application.
Acknowledgments This work was supported by grants from the Ministry of Science and Technology of Taiwan (MOST 101-3114-B-002-003 and MOST 102-2313-B-002-014) and Liver Disease Prevention and Treatment Research Foundation, Taipei, Taiwan. The flow cytometry was performed at the Joint Center for Instruments and Researches, College of Bioresources and Agriculture, National Taiwan University, Taiwan. The authors acknowledge statistical experiment assistance provided by the Taiwan Clinical Trial Bioinformatics and Statistical Center, Training Center, and Pharmacogenomics Laboratory (which is founded by National Research Program for Biopharmaceuticals (NRPB) at the Ministry of Science and Technology of Taiwan; MOST 103-2325-B-002-033).
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