Mol Cell Toxicol (2013) 9:185-193 DOI 10.

1007/s13273-013-0023-2

ORIGINAL PAPER

The effect of ginsenosides on hepatogenic differentiation using placenta-derived stem cells as an in vitro screening system
Hyun-Jung Lee1, So Young Eun2, Seung-Gwan Lee2, Boo-Yong Lee3 & Gi Jin Kim1
Received: 5 March 2013 / Accepted: 26 April 2013 The Korean Society of Toxicogenomics and Toxicoproteomics and Springer 2013

lated from human full-term placentas are able to grow through self-renewal and differentiate into multiple lineages. PDSCs have been reported to have a potential for hepatogenic differentiation and therapeutic effects in an animal model of liver injury. However, the frequency of the hepatogenic differentiation of PDSCs remains low. Saponins are a class of chemical compounds called glycosides and have various activities, including anti-inflammatory actions, anti-tumor effects, and anti-allergy and hepatoprotective activity. Because little is known about the effect of various ginsenosides on PDSC differentiation, we evaluated whether ginsenosides could induce the hepatogenic differentiation of PDSCs. PDSCs cultured under several different ginsenosides were induced to differentiate into hepatogenic lineages and were then analyzed. We found that the morphologies changed from a spindle shape to a characteristic polygonal hepatocyte-like morphology after hepatogenic differentiation induction. In addition, the differentiated cells expressed several hepatocyte-specific markers and increased their indocyanine green (ICG) uptake, although different efficacies for hepatogenic differentiation were observed according to the different ginsenoside conditions. Taken together, these data suggest that ginsenosides may contribute to enhanced hepatogenic differentiation of PDSCs and that PDSCs may be used as a source for in vitro drug screening.
1

Abstract Placenta-derived stem cells (PDSCs) iso-

Keywords Placenta-derived stem cells, Ginsenosides, Hepatogenic differentiation, In vitro screening Mesenchymal stem cells (MSCs), which are present in several tissues and organs, are currently being investigated as potential therapeutic agents for various degenerative diseases1-3 because they can propagate in large quantities, differentiate into various tissue cell types, and lack the ethical problems associated with other stem cells4. However, bone marrow-derived mesenchymal stem cells (BM-MSCs), which are used as representative MSCs, have limitations (e.g., lower yields, age-dependency of the donor, and obtainment through invasive procedures) for study and use in clinical approaches to regenerative medicine. Due to these reasons, many scientists have reported new alternatives to embryonic stem cells or BM-MSCs as sources for MSCs5,6. Among the adult stem cells, placenta-derived stem cells (PDSCs) have several advantages, including fewer ethical problems because the PDSCs are isolated from a full-term placenta, which is discarded after delivery of the baby. Moreover, PDSCs have a higher proliferative potential that is associated with a short population doubling time. PDSCs also contain other types of stem cells, including MSCs, which have the ability to differentiate into various types of cells (i.e., adipocytes, chondrocytes, osteocytes, and neuronal cells) under the appropriate differentiation induction conditions7. In particular, PDSCs have the ability to differentiate into hepatocyte-like cells in vitro8-10. Recently, we reported the potential of chorionic plate-derived MSCs (CP-MSCs) from the fetal placenta membrane to differentiate into functional hepatocyte-like cells, and we evaluated the therapeutic potential of CP-MSCs by measuring their effects on the structural and functional

Department of Biomedical Science, CHA University, Seoul 135-081, Korea 2 Department of Biomedical Science, College of Health Science, Korea University, Seoul, Korea 3 Department of Food Science and Biotechnology, CHA University, Seongnam 463-836, Korea Correspondence and requests for materials should be addressed to G. J. Kim ( gjkim@cha.ac.kr)

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regeneration of the liver in CCl4-injured rats11. However, it remains difficult to increase the frequency of MSC hepatogenic differentiation in an in vitro culture system due to the lower frequency of these cells. Therefore, a study for new compounds or factors that can enhance the differentiation of CP-MSCs into hepatocytes is required. Ginseng, the root and rhizomes of different Panax species (Araliaceae), is one of the most commonly used traditional herbal medicines in East Asia12. Ginsenosides, which are the active components of ginseng, belong to a group of steroidal saponins and are distributed in many parts of the ginseng plant, including the root, leaf, and berry13. Moreover, an advantage of ginseng is that it does not produce harmful pharmacological side effects. Ginsenosides, which are glycosides containing the aglycones protopanaxadiol (PD) or protopanaxatriol (PT), are the major effective components of ginseng and have been shown to have a wide variety of biological activities, including immunomodulatory effects and antioxidant, anti-inflammatory and antitumor activities14-17. Interestingly, ginsenosides have been shown to improve liver function18-20; indeed, ginseng has been used to treat hepatitis and liver fibrosis and cancer, and ginsenosides have been known to possess anti-tumor and liver-protective effects21. However, it is difficult to isolate and evaluate the effects of a single ginsenoside compound because ginsenosides appear to affect multiple pathways, and their effects

are complex. Furthermore, a correlation between ginsenosides and the hepatogenic differentiation of stem cells has not been reported. In the present study, we investigated whether ginsenosides have beneficial effects on the cultivation of CP-MSCs and on the enhancement of the hepatogenic differentiation potential of CP-MSCs into hepatocytes. In addition, we compared the different ginsenosides (i.e., Rb1, Rd, Rh2, F2, compound K, Rh1, and Re) to determine whether they play a positive role in stem cell differentiation.
The structure of ginsenosides

Ginsenosides have a 4-ring, steroid-like structure with sugar moieties attached; thus far, more than 40 different ginsenosides have been identified and isolated from the root of P. ginseng22,23. Each ginsenoside has at least 2 (carbon-3 and -20) or 3 (carbon-3, -6 and -20) hydroxyl groups that are free or bound to monomeric, dimeric, or trimeric sugars. Ginsenosides also exist as stereoisomers depending on the position of the hydroxyl group on carbon-20. Based on their chemical structures, ginsenosides are generally divided into 2 groups: PD and PT. Whereas the sugar moieties in the PD group attach to the 3-position of dammarane-type triterpine (e.g., Rb, Rd, Rh2, F2 and compound K), the sugar moieties in the PT group attach to the 6-position of dammaranetype triterpine (e.g., Rh1 and Re) (Figure 1)24.

Figure 1. Chemical structure of gensenosides, which are glycosides containing the aglycones protopanaxadiol (PD) or protopanaxatriol (PT).

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Figure 2. Comparison of cell viability in CP-MSCs according to gensenosides treatment for dose courses as determined by MTT assays. (A) The cell viability of CP-MSCs in protopanaxadiol types of gensenosides treatment after 24 hours. (B) The cell viability of CP-MSCs in protopanaxatriol types of gensenosides treatment after 24 hours.

Cell viability of the CP-MSCs depends on the concentration of ginsenosides

To analyze the ginsenoside cytotoxicity in the CPMSC culture, we determined the cell viabilities of CPMSCs under different concentrations of ginsenosides and different time periods. In the MTT assay, CP-MSC cytotoxicity was found at high ginsenoside concentrations, regardless of the saponin type (PD or PT) and time period (30 g/mL). As shown in Figure 2, the concentration range of the ginsenosides showing no cytotoxicity in the CP-MSCs was 0.003 to 3 g/mL (Figure 2). For the PT saponins, the viability of Rh2 at the 3 g/mL concentration was similar to the control; however, the viability of compound K at 30 g/ mL was lower than the other compounds (Figure 2A). In addition, the viability of Rh1 was shown to be higher than Re at several concentrations (Figure 2B). These findings suggest that there are differences in cytotoxicity depending on the concentration and the ginsenoside type, although all ginsenosides have cytotoxicity at high doses.
Proliferation activity of the PDSCs depends on the concentration of ginsenosides

sure the cell growth, we counted the cell number every day for a week. As shown in Figure 3, the growth of the CP-MSCs under the low-dose condition of the Rd and Rh2 PD saponins was shown to be greater than that under the high dose of these components compared with the control. However, the growth of the CPMSCs in F2 and compound K was lower than in the control, regardless of the dose, and there was no difference in the cell growth in response to Rb1 (Figure 3A). The growth of the CP-MSCs incubated with the Rh1 and Re (PT saponins) was greater at the higher dose, compared with the control (Figure 3B). These findings suggest a difference between the PD and PT saponins and a difference between the types of ginsenosides that induce CP-MSC proliferation.
The effect of ginsenosides on inducing the hepatogenic differentiation of CP-MSCs

Based on the ginsenoside cytotoxicity screening results for the various concentrations and types, we selected two concentrations of ginsenosides demonstrating low cytotoxicity and analyzed the proliferation of CP-MSCs after exposure to these compounds. We used 3 ng/mL and 30 ng/mL of the PD ginsenoside saponins (with the exception of Rh2), otherwise, 300 ng/mL and 3 g /mL of the PT ginsenoside saponins were used. The morphologies of the CP-MSCs cultured in medium containing the two concentrations of ginsenosides for a week were similar to those of the control. To mea-

Because we confirmed a positive effect of ginsenosides on the cultivation of CP-MSCs, we investigated whether ginsenosides could enhance the hepatogenic differentiation potential of CP-MSCs. The CP-MSCs were differentiated into hepatocytes using a hepatic induction medium containing ginsenosides. After hepatic differentiation, the morphology of the CP-MSCs changed from a spindle shape to a polygonal hepatocytelike cell shape, which was similar to that of the control hepatic-differentiated CP-MSCs (Figure 4). Moreover, the morphological changes were increased in the differentiated CP-MSCs cultured with the PD saponins Rh2 and F2 and the PT saponin Rh1. To compare the expression of hepatocyte-specific markers, we isolated total RNA from the cell pellet after hepatic differentiation and performed an RT-PCR analysis. As shown in Figure 5, the analysis demonstrated that the expression of hepatogenic marker genes

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Figure 3. Comparison of proliferation activity of CP-MSCs depend on concentration of gensenosides. (A) The proliferation of CP-MSCs in protopanaxadiol types of gensenosides treatment for 7 days. (B) The proliferation of CP-MSCs in protopanaxatriol types of gensenosides treatment for 7 days.

Figure 4. Morphological changes of CP-MSCs after inducing hepatogenic differentiation by gensenosides treatment. The morphology of CP-MSCs changed to a round form, similar to that of hepatocytes following hepatogenic differentiation. Original Magnification: 200.

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Figure 5. Expression of hepatocyte-specific genes in CP-MSCs depend on hepatogenic differentiation with and without gensenosides treatment. Expression of hepatogenic-specific genes (HNF1, HNF-4a, CXCR4, Albumin, and TAT) in CP-MSCs during hepatogenic differentiation with protopanaxadiol types and protopanaxatriol types of gensenosides treatment by RT-PCR analysis. -actin was used as the internal standard (A). The graphs were represented the fold change of relative value of RT-PCR products inducing hepatogenic differentiation with high concentration of each ginsenoside (B). * is the value versus undifferentiated cells without saponins and is the value versus hepatic differentiated cells without saponins.

in the CP-MSCs cultured with ginsenosides was higher than in the control (without ginsenosides) and that the higher dose resulted in a higher gene expression (Figure 5). In particular, the expression of the hepatocyte-specific transcription factor, hepatocyte nuclear factor (HNF)-1a, was increased in the CP-MSCs during hepatogenic differentiation, compared with the control, even in cases when full hepatogenic differentiation was not induced. In addition, the expression of HNF-4a and albumin in the differentiated CP-MSCs cultured with the higher ginsenoside dose was increased when compared to that of the lower ginsenoside dose. Furthermore, the expression level of TAT, a functional hepatocyte enzyme, was higher in the differentiated CP-MSCs cultured with ginsenosides, compared with the control. The expression of hepatocyte-specific genes in the differentiated cells treated with Rh2 and F2 was greater than that induced by the other PD saponins. In the case of the PT saponins, Rh1 treatment

significantly induced an increase in albumin and TAT expression (Figure 5A). Therefore, these results support the idea that ginsenosides play a role in enhancing the expression of hepatocyte-specific genes during the induction of CP-MSC hepatogenic differentiation. Especially, Rh1, Rh2, and F2 significantly induce the expression of hepatocyte-specific markers such as HNF-4a, Albumin, and TAT than other saponins at comparison of fold change of relative value of RT-PCR (P0.05, Figure 5B). However, there are no statistical differences the expressions for HNF-1a and CXCR4 when they are inducing hepatogenic differentiation according to types of saponins and different concentration of sapoinins treatment.
ICG uptake assay after the hepatic differentiation of CP-MSCs

To analyze the function of the hepatocyte-like cells,

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Figure 6. ICG uptake assay of CP-MSCs depend on hepatogenic differentiation with and without gensenosides treatment. Right upper images shows ICG uptake in undifferentiated CP-MSCs without gensenosides treatment. Original Magnification: 200.

we used an ICG uptake assay to test the CP-MSCs cultured with or without ginsenosides. In general, the uptake of ICG by the CP-MSCs was rare prior to differentiation and in the control; however, the CP-MSCs took up large amounts of ICG following hepatogenic differentiation with the ginsenosides. The ICG uptake in the differentiated CP-MSCs cultured with Rd, Rh2, and F2 was greater than that observed with the other PD saponins. However, there was no difference in the ICG uptake between the PT saponins Rh1 and Re. These results suggest that culture medium containing ginsenosides could promote CP-MSC hepatogenic differentiation and that functional hepatocyte-like cells are derived from CP-MSCs through different PD and PT saponins.

Discussion
Drug screening using stem cells has been a focus in stem cell research due to several limitations of traditional in vitro assays using primary cells or cell lines25-27. However, standard differentiation technology to induce target tissue-specific cells derived from stem cells had been not established thus far. Therefore, the optimization and validation of differentiation was required to establish reproducible in vitro toxicity screening methods using stem cells. Many chemicals and cytokines (e.g., oncostatin M and HGF) were used to induce the hepatogenic differentiation of MSCs to enhance the efficiency of hepatogenic differentiation28. In addition, the hepatogenic differentiation of stem cells could be used to induce hepatogenesis-related transcription fac-

tors, including HNF-1, HNF-4 and C/EBP29,30. However, the efficiency of the hepatogenic differentiation of MSCs remains approximately 30% in a total cell population, and the conventional protocol has complex steps and is labor intensive. Therefore, simple and effective procedures for hepatogenic differentiation are required. There have been approximately 30 types of ginsenosides identified thus far because saponins are capable of changing their structures during the process of isolation and extraction, and they all possess a steroidal aglycone core consisting of 27 C-atoms or a triterpenoidal aglycone core of 30 C-atoms31. The diversity of monosaccharides and the multiple modes linking them result in a large array of saponins31. The composition of the monosaccharides may vary substantially even in saponins from one natural source32. However, it is still unknown which ginsenosides have beneficial effects on the growth and differentiation of stem cells although they have various positive effects. In this report, we demonstrated the possibility of using ginsenosides as supplemental agents for the cultivation and the induced hepatogenic differentiation of CP-MSCs. MSCs have the capacity for self-renewal and differentiation into various lineages of mesenchymal tissues. These characteristics may reveal MSCs as possible therapeutic agents for degenerative diseases with regard to tissue engineering and cell-based therapies. Although BM-MSCs represent the main source of MSC availability, the use of BM-MSCs is not always acceptable because the cell number and the capacity for proliferation and differentiation significantly decrease with the age of the donor. Therefore, new alterna-

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tive sources for stem cells should be established to obtain cells with a higher proliferative potency and capacity for differentiation and a lower risk of contamination. The placenta is likely a feasible source of MSCs for the following reasons: the placenta, which is normally discarded, can be easily obtained and contains a great deal of MSCs formed from the extraembryonic mesoderm; furthermore, it lacks the ethical problems associated with other stem cells. In addition, the placenta has immunomodulation properties and multipotential for differentiation33,34. We previously reported that CPMSCs isolated from full-term placentas have the potential for differentiation into hepatic and mesodermal lineages, including osteogenic, chondrogenic, and adipogenic cells, and that naive CP-MSCs expressing ATP-binding cassette transporters G2 (ABCG2) can be used as the source of an in vitro screening system for hepatotoxicants35. This study is the first to report that the cell morphology of CP-MSCs cultivated in the presence of saponins was maintained without changes and that the growth rate increased with specific saponin concentrations. In addition, saponins enhance the CP-MSC hepatogenic differentiation potential when used as supplements. Interestingly, there are differences in the potential for the hepatogenic differentiation of CP-MSCs that depend on the differences between the PD and PT saponins and a difference in the type of ginsenosides. Notably, the potential for the hepatogenic differentiation of CP-MSCs was more enhanced when they were cultured with Rh1, Rh2, and F2 than other saponins. However, the function of saponins and the mechanism for inducing hepatogenic differentiation in stem cells should be evaluated further. In conclusion, the ginsenosides Rh1, Rh2, and F2 contribute to a greater hepatogenic differentiation and proliferation of CP-MSCs than other saponins. In addition to, serving as alternative MSC sources, CP-MSCs may be used in in vitro systems for drug screening.

a concentration of 5104 cells/well, cultured with medium containing various concentrations of saponins for 7 days, and then counted. The ginsenosides (Rb1, Rd, Rh2, F2, compound K, Rh1, and Re) were purchased from BTGin Co., Ltd. (Okcheon, Chungbuk, South Korea) and dissolved in dimethyl sulfoxide (DMSO). The chemical structures of the ginsenosides are shown in Figure 1.
MTT assay

Saponins were diluted in DMSO, and the saponin cytotoxicity was assayed in 96-well culture plates. The CP-MSCs were seeded at a concentration of 2103 cells/well and treated with various saponin concentrations for 24 and 48 hrs. Next, the saponin cytotoxicity was estimated using the MTT assay according to the protocol of the manufacturer. Briefly, 10 L of MTT (5 mg/mL) was added to each well and incubated for 4 hrs. The media were discarded, and 50 L of DMSO was added; the plate then was gently mixed on a gyratory shaker at room temperature for 5 min. The optical density was measured at 562 nm. The results are expressed as the percentage of cell viability in comparison with the control cells (i.e., the cells without any saponin).
Hepatic differentiation

Materials & Methods

Cell culture and Materials

CP-MSCs were harvested from normal placentas after full-term delivery as described previously11. The isolated cells were cultured in medium containing DMEM /F12 supplemented with penicillin (100 U/mL)-streptomycin (100 g/mL), 25 ng/mL FGF4, 1 g/mL heparin, 50 g/mL gentamicin (Invitrogen) and 10% fetal bovine serum. The cells were passaged every 48-72 h at a 1 : 3 ratio. To analyze the effect of saponins on the cultivation of the CP-MSCs, the cells were seeded at

To direct the differentiation of the PDSCs into hepatocytes in vitro, we designed a series of inducing media (Table 1). The cells were cultured on 0.1% collagencoated dishes in medium containing 2% fetal bovine serum, 100 U/mL penicillin and 100 g/mL streptomycin. Approximately 2 days later, when the cells were 70-80% confluent, the culture medium was replaced with early inducing medium to allow for hepatic differentiation. The protocol was designed with different combinations of EGF, bFGF, HGF and BMP4 as indicated in Table 1. After the terminal step at day 7, the hepatic-induced cells were assayed for indocyanine green (ICG) uptake for hepatic differentiation identification. Next, the hepatic-induced cells were harvested for the analysis of hepatocyte-specific gene expression using RT-PCR. To assay the saponin effect, the early inducing medium, mid-term inducing medium and terminal inducing medium included various concentrations of saponins.
Hepatocyte-specific gene expression analysis by RT-PCR

Total RNA was extracted from the hepatic induced cells using the RNeasy plus mini kit and 1 g was reverse transcribed into cDNA with the superscript III

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first-strand synthesis system according to the protocol of the manufacturer. The first-strand cDNAs were amplified in a final volume of 25 L containing 0.5 U h-Taq DNA polymerase and 10 pmol of each mature hepatic gene primer. The PCR primers and the length of the amplified products are listed in Table 2. The PCR cycling conditions were as follows: initial denaturation at 95 C, 15 min; cycling at 95 C, 20 s, 55 C, 58 C, 59 C, 40 s, and 72 C, 1 min; and final extension at 72 C, 5 min. All of the PCRs were performed for 40 cycles. The amplified PCR products were analyzed by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. The cDNA samples were adjusted to yield equal -actin amplifications.
Indocyanine green (ICG) uptake assay

To analyze the ICG uptake, the differentiated cells were incubated in ICG solution (100 mg/mL in DMSO) at a final concentration of 0.5 mg/mL, incubated at 37 C for 1.5 h, rinsed three times with PBS and replenished with DMEM containing 10% FBS. ICG was completely eliminated from the cells after 72 hrs.
Statistical analysis

Results are presented as the meansSD. Statistical significance measured multiple comparisons were performed using the t-test with a significance level of P 0.05. * is the value versus undifferentiated cells without saponins and is the value versus hepatic differentiated cells without saponins. Abbreviations bFGF, basic fibroblast growth factor; BM-MSCs, bone marrow-derived mesenchymal stem cells; BMP4, bone morphogenetic protein 4; CP-MSCs, chorionic plate-derived MSCs; DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; FGF4, fibroblast growth factor 4; HGF, hepatocyte growth factor; HNF-1a, hepatocyte nuclear factor-1a; ICG, indocyanine green; MSCs, mesenchymal stem cells; MTT, 3-(4,5-dimethylthiazol2yl)-2,5,-diphenyl tetrazolium bromide; PDSCs, placentaderived stem cells; TAT, tyrosine amino transferase Acknowledgements This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MEST) (KRF-2008-313-E00247).

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