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TISSUE-SPECIFIC STEM CELLS In Vitro Differentiation of Human Placenta-Derived Multipotent Cells into Hepatocyte-Like Cells CHIH-CHENG CHIEN,a,b BETTY LINJU YEN,c FA-KUNG LEE,e TSUNG-HSUAN LAI,a,e YAO-CHANG CHEN,c,d SHU-HUI CHAN,a HSING-I HUANGa,b a

Cathay Medical Research Institute, eDepartment of Obstetrics and Gynecology, Cathay General Hospital, Taipei, Taiwan; bDepartment of Medicine, Fu-Jen Catholic University, Taipei, Taiwan; cStem Cell Research Center, National Health Research Institutes, Taipei, Taiwan; dDepartment of Forensic Medicine, National Taiwan University, Taipei, Taiwan Key Words. Placenta • Differentiation • Hepatocyte • Regenerative medicine

ABSTRACT Multipotent cells isolated from human term placenta (placenta-derived multipotent cells [PDMCs]) have been known to be able to differentiate into mesodermal lineage cells, including adipocytes and osteoclasts. The low infection rate and young age of placenta compared with other tissue origins of adult stem cells make theses cells attractive target for cell-based therapy. However, the differentiation potential of PDMCs toward hepatic cells has not been evaluated yet. In this study, we cultivated PDMCs with hepatic differentiation medium to evaluate the ability of these cells in differentiating toward hepatic cells. After treatment, the morphologies of differentiated PDMCs changed to polygonal epithelial cell-like. The differentiated cells not only show the hepatocyte-like morphol-

ogies but also express hepatocyte-specific markers, including albumin and cytokeratin 18. The bioactivity assays revealed that these hepatocyte-like cells could uptake lipoprotein and store glycogen. Furthermore, the addition of rifampicin increased the gene expression of CYP3A4, which is similar with the activities of human liver cells. According to our previous results, PDMCs were capable of differentiating into mesodermal and ectodermal lineage cells. Our results indicate that PDMCs can differentiate into three germ layer cells, which is similar to embryonic stem cells. In conclusion, placenta might be an easily accessible source for progenitor cells that are capable of differentiating toward hepatocyte-like cells in vitro. STEM CELLS 2006;24:1759 –1768

INTRODUCTION

hepatocyte-like cells [5– 8]. These findings, important for liver regeneration, indicated that some adult stem cells are also capable of differentiating into hepatic lineage cells. The differentiated cells expressed liver-specific markers and had hepatocyte-specific bioactivities, including urea production, albumin secretion, and glycogen storage [7, 8]. Moreover, the transplantation of bone marrow cells rescued fumarylacetate-hydrolase-deficient mice and restored their liver functions [9]. The administration of cord blood stem cells enhanced liver regeneration and reduced mortality rates in a murine model of toxic liver injury [10]. These results indicated that stem cells might have a clinically applicable therapeutic potential in liver diseases. Recently, we and others isolated placenta-derived multipotent cells (PDMCs) from human term placenta and showed that these cells have multilineage differentiating ability [11, 12]. PDMCs are fibroblast-like cells that attach on plastic surfaces. These cells could be expanded for more than 20 population

In mammals, repair or regeneration of the liver has long been thought possible. Mature hepatocytes may divide and be responsible for hepatic cell replacement [1]. However, under certain circumstances, especially when the ability of differentiated hepatocytes to divide is impaired, hepatocyte progenitors are still required [2]. Endogenous progenitor cells have been observed when liver has undergone extensive injury or hepatocyte proliferation is blocked chemically [3]. These cells, named oval cells, are about 10 ␮m in size and have a high nuclear/cytoplasmic ratio. They play important roles in liver regeneration via their ability to differentiate into parenchymal hepatocytes and bile ductular cells [4]. Oval cells are induced to differentiate and expand, whereas liver cells undergo severe damage. However, their mechanisms of action are still not fully understood. Stem cells derived from nonliver sources, such as bone marrow and umbilical cord blood, were recently shown to differentiate into

Correspondence: Hsing-I Huang, Ph.D., Cathay Medical Research Institute, Cathay General Hospital, No. 32, Ln 160, Jian-Cheng Road, 22141, Hsi-Chih, Taipei, Taiwan, R.O.C. Telephone: 886-2-26907965, ext. 2312; Fax: 886-2-26907963; e-mail: [email protected] Received October 20, 2005; accepted for publication March 15, 2006. ©AlphaMed Press 1066-5099/2006/ $20.00/0 doi: 10.1634/stemcells.2005-0521

STEM CELLS 2006;24:1759 –1768 www.StemCells.com

In Vitro Hepatic Differentiation of PDMCs

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doublings and could be induced to differentiate into cells of various mesenchymal tissues, including adipocytes, osteoblasts, and chondrocytes [11, 12]. Under appropriate culture conditions, these cells can also differentiate toward neural lineage cells (B.L. Yen, C.-C. Chien, Y.-C. Chen, J.T. Chen, J.S. Huang, F.-K. Lee, and H.-I. Huang, manuscript submitted for publication). In comparison with stem cells isolated from other adult tissues, PDMCs are young, and there are no ethical problems associated with their study and clinical application [13, 14]. Furthermore, the collection of placenta samples does not harm mother or infant. The ability of PDMCs to differentiate and the ease with which they can be handled make them a novel source for cell-based therapeutic strategies. However, the ability of placenta-derived multipotent progenitor cells to differentiate toward hepatic lineage cells has not yet been demonstrated. In this study, we have conducted in vitro investigations to determine whether placenta progenitor cells can be induced toward the hepatic lineage. To characterize differentiated PDMCs, we have analyzed their gene expression of liver cell markers, such as albumin, ␣-fetoprotein (AFP), and tyrosine aminotransferase (TAT). In addition, we have used immunostaining to assess the expression of albumin, cytokeratin 18 (CK-18), and AFP at the protein level. Functional assays indicate that the differentiated PDMCs acquire liver cell-specific bioactivities, including lipoprotein uptake and glycogen storage. In addition to hepatic lineage cells, the PDMCs have been shown to differentiate into mesodermal mesenchymal and ectodermal neural lineage cells. Hence, our results suggest that placenta may be an alternative source of multipotent progenitor cells with multigermline potential and may therefore serve as a novel source of cell-based therapeutic strategies.

MATERIALS

AND

METHODS

Isolation and Culture of Human PDMCs Human placenta for this study was collected after delivery in Cathay General Hospital after informed consent. To isolate PDMCs, the placental tissue was dissected into small pieces by sterilized scissors and forceps. The chopped tissues were then washed with an equal volume of phosphate-buffered saline (PBS) and digested with trypsin-EDTA (Gibco-Invitrogen, Grand Island, NY, http://www.invitrogen.com) at 37°C for 10 minutes. An equal volume of Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-Invitrogen) containing 10% fetal bovine serum (HyClone, Logan, UT, http://www.hyclone.com) was added and centrifuged at 1,200g for 10 minutes to get the cell pellet. The cells were then resuspended in Dulbecco’s modified Eagle’s Medium with 1.0 g/l glucose (DMEM-LG) (Gibco-Invitrogen) supplemented with 10% fetal bovine serum and 1⫻ penicillin/streptomycin (Gibco-Invitrogen). The cells were then incubated at 37°C in an incubator with 5% CO2.

Flow Cytometry PDMCs were cultured and examined for the immunophenotype. Cells (2 ⫻ 105 cells) were trypsinized and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD14 (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com) and anti-CD90 (BD Biosciences, San Diego, http://www. bdbiosciences.com) for 60 minutes at 4°C. Then the cells were washed three times. For unconjugated anti-CD34 (BD Biosciences), and anti-CD105 (R&D Systems Inc., Minneapolis, http:// www.rndsystems.com), FITC-conjugated rat anti-mouse IgG

antibody (Ab) (BD Biosciences) was used as secondary antibody. Flow cytometry with FACSCalibur (BD Biosciences) was performed.

Hepatogenic Differentiation To induce the hepatogenic differentiation, PDMCs after five passages were plated on 5-mm culture dishes untreated or coated with fibronectin or poly-L-lysine (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) in the expansion medium. After 24 hours, medium was removed, cells were washed twice with PBS, and cells were cultured in differentiation medium consisting of 60% DMEM-LG and 40% MCDB201 (SigmaAldrich) supplemented with 1⫻ Insulin-Transferrin-Selenium (ITS), 4.7 ␮g/ml linoleic acid, 1 mg/ml bovine serum albumin, 10⫺8 M dexamethasone (Decadron; Merck & Co., Whitehouse Station, NY, http://www.merck.com), 10⫺4 M ascorbic acid (all from Sigma-Aldrich), 10 ng/ml epidermal growth factor (GibcoInvitrogen), and 10 ng/ml platelet-derived growth factor-BB (R&D Systems). After 16 hours of incubation, the medium was removed, and cells were washed three times. Then, the cells were treated with differentiation medium or differentiation medium supplemented with 20 ng/ml hepatocyte growth factor (HGF) (Peprotech, Rocky Hill, NJ, http://www. peprotech.com) and 10 ng/ml fibroblast growth factor-4 (FGF-4) (Sigma-Aldrich). Two milliliters of medium was added to each culture dish, and that medium was changed once a week.

Total RNA Isolation of Reverse TranscriptasePolymerase Chain Reaction Total RNA was isolated from PDMCs using Purescript (Gentra, Minneapolis, http://www.gentra.com/), and 2 ␮g of total RNA was used for reverse transcription using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, http://www.invitrogen. com). The cDNA was amplified using Taq Platinum (Invitrogen). The primers used were designed according to the corresponding human genes. The following oligonucleotide primers were used: albumin (sense, 5⬘-TTGGAAAAATCCCACTGCAT-3⬘; antisense, 5⬘-CTCCAAGCTCAAAAAGC-3⬘), AFP (sense, 5⬘-AGCTTGGTGGATGAAAC-3⬘; antisense, 5⬘-TCCAACAGGCCTGAGAAATC-3⬘), TAT (sense, 5⬘-TGAGCAGTCTGTCCACTGCCT; antisense, 5⬘-ATGTGAATGAGGAGGATCTGAG), CYP3A4 (sense, 5⬘-CCTTACACATACACACCCTTTGGAAGT; antisense, 5⬘-AGCTCAATGCATGTACAGAATCCCCGGTTA-3⬘) and human glyceraldehyde3-phosphate dehydrogenase (GAPDH) primers (sense, 5⬘-TGAAGGTCGGAGTCAACGGATTTGGT-3⬘; antisense, 5⬘-CATGTGGGCCATGAGGTCCACCAC-3⬘; Clontech, Palo Alto, CA, http://www.clontech.com) were used as internal control for polymerase chain reactions (PCRs). Amplification reactions were performed on Biometra T1 thermal cycler (Whatman Biometra, Go¨ttingen, Germany, http://www.biometra.de/) at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 60 seconds for 30 cycles. The PCR products were then separated by electrophoresis on 1.5% agarose gels. The sequence of each PCR product was confirmed using automatic sequencing.

Immunocytochemistry Cultured cells were fixed with 4% paraformaldehyde (SigmaAldrich) in PBS for 5 minutes at room temperature and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 20

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Figure 1. Characterization of PDMCs. (A): Flow cytometric analysis for freshly isolated placental cells and expanded PDMCs. Freshly isolated placental cells were CD14(⫹/⫺), CD34(⫺), CD90(⫺), and CD105(⫺). The isolated PDMCs were CD14(⫺), CD34(⫺), CD90(⫹), and CD105(⫹). Cells were labeled with specific monoclonal antibodies for indicated molecules (filled histograms) or isotype controls (open histograms). These experiments were repeated at least three times, and similar results were observed. (B): Expression of ␣-fetoprotein (AFP) of isolated PDMCs. Undifferentiated PDMCs after passage 5 were fixed and stained with anti-AFP antibody (Ab) (1:400) and then reacted with horseradish peroxidaseconjugated secondary antibody and counterstained by hematoxylin (magnification ⫻200). (C): Immunofluorescence staining of c-Met on PDMCs. Undifferentiated PDMCs after passage 5 were fixed and stained with anti-c-Met Ab (1:50) and then reacted with fluorescein isothiocyanate-conjugated anti-rabbit secondary antibody and counterstained by 4⬘,6-diamino-2-phenylindole (magnification ⫻300). Abbreviations: FL, fluorescence; PDMC, placenta-derived multipotent cell.

minutes. Cells were then incubated with blocking solution consisting of PBS, 10% horse serum (Chemicon International, Temecula, CA, http://www.chemicon.com) at room temperature for 30 minutes. For immunohistochemistry, endogenous peroxide activity was quenched with 0.3% hydrogen peroxide (Sigma-Aldrich) solution. Slides were then incubated sequentially with primary antibody overnight at 4°C. Primary antibodies against human hepatocyte (1:100) and AFP (1:400) (DakoCytomation) were applied, followed with biotinylated anti-mouse antibody and biotinylated anti-rabbit antibody, respectively. After being washed three times, slides were incubated with avidin-biotin conjugate of horseradish peroxidase (Vector Laboratories, Burlingame, CA, http://www. vectorlabs.com) in PBS for 30 minutes at room temperature. To reveal the resulting peroxidase activity, the slides were treated with diaminobenzidine tetrahydrochloride solution (Vector Laboratories). The slides were counterstained with hematoxylin and mounted on glass slides with mounting medium (Vector Laboratories). For immunofluorescence staining, primary antibodies against HGF receptor (c-Met) (1:50) (Santa Cruz Biotechnology www.StemCells.com

Inc., Santa Cruz, CA, http://www.scbt.com), albumin (1:100), and CK-18 (1:100) (DakoCytomation) were used. After being incubated with primary antibody, cells were incubated with fluorescein-labeled anti-rabbit, Cy3-conjugated anti-rabbit, or fluorescein-labeled anti-mouse (Chemicon) secondary antibodies for 1 hour at 37°C and stained with 4⬘,6-diamino-2phenylindole (KPL, Gaithersburg, MD) to identify cell nuclei. The cells were visualized and photographed by fluorescence microscopy (Olympus BX51; Tokyo).

Western Blot PDMCs were differentiated toward hepatic lineage for 21 days. Cell lysate was prepared. The concentration of protein lysate was determined using the bicinchoninic acid method (BioWhittaker Molecular Applications, Rockland, ME, http://www. bmaproducts.com). The protein samples was fractionated on SDS-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene membrane (GE Healthcare Biosciences Ltd., Taipei, Taiwan, http://www1.amershambiosciences.com). The

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membrane was then blocked by 10% skim milk (Sigma-Aldrich) in PBS. After being washed three times, the membrane was incubated with antibodies against human AFP (1:1000) (DakoCytomation) and GAPDH (1:500) (Ambion, Austin, TX, http:// www.ambion.com) at 4°C overnight. Horseradish peroxidaseconjugated anti-rabbit/mouse antibodies (Vector Laboratories) were incubated with membrane at room temperature for 1 hour. After washing, the bands were detected by enhanced chemiluminescence reagents (Amersham Biosciences). Expression of GAPDH was used as internal control.

Uptake of Low-Density Lipoprotein The uptake of lipoprotein was detected with the Dil-Ac-LDL staining kit (Biomedical Technologies, Stoughton, MA, http://www. btiinc.com). The assay was performed according to then manufacturer’s instructions.

Periodic Acid-Schiff Staining The medium was taken out from culture plates and cells were washed with PBS three times. Then, the cells were fixed using 4% paraformaldehyde for 30 minutes. After being oxidized in 10 g/l periodic acid for 10 minutes and washed three times with dH2O, cells were treated with Schiff’s reagent (Sigma-Aldrich) for 15 minutes. Afterwards, cells were rinsed in dH2O for 10 minutes and counterstained with hematoxyline. The staining results were observed under an invert microscope.

Rifampicin Treatment of Differentiated Cells HepG2 is a human hepatoma cell line obtained from American Type Culture Collection (Rockville, MD, http://www.atcc.com) and used as positive control in this experiment [15]. Untreated and differentiated PDMCs, along with HepG2, were plated on a six-well culture plate at a concentration of 2 ⫻ 105 cells per ml. Rifampicin (Sigma-Aldrich) was first dissolved in dimethyl sulfoxide (DMSO) first and then added to the medium at a final concentration of 50 mM. After 48 hours, total RNA was isolated from different cell groups and applied in reverse transcriptionpolymerase chain reaction (RT-PCR) analysis for detection of CYP3A4 mRNA levels.

Figure 2. Effects of poly-L-lysine-coated tissue culture plates on the morphological change and expression of albumin transcripts of placenta-derived multipotent cells (PDMCs) treated with differentiation medium. Cells were cultured in differentiation medium or expansion medium with the use of untreated, poly-L-lysine-coated, or fibronectincoated dishes. Undifferentiated PDMCs assumed the fibroblast morphology on untreated (A), fibronectin-coated (B), and poly-L-lysinecoated (C) culture dishes. After 4 weeks of hepatogenic initiation, a portion of cells grown on fibronectin-coated plates changed into round cells (E), and some of the cells cultured on poly-L-lysine-coated plates turned into polygonal cells (F). However, the treatment of differentiation medium on cells grown on untreated culture vessels showed no effect on changes of cell morphology (D) (magnification ⫻200). (G): Cells from different treatments were harvested at day 28, and total RNA was isolated for reverse transcription-polymerase chain reaction analysis. The mRNA expression levels of albumin and GAPDH were examined. Abbreviations: D, differentiation medium; E, expansion medium; Fn, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PLL, poly-L-lysine; Un, untreated.

RESULTS Hepatogenic Differentiation Characterization of Human Placenta-Derived Multipotent Progenitors We isolated PDMCs from human placenta according to the protocol described previously (sample number ⫽ 20) [11]. To define the immunophenotypes of the isolated cells, we assayed their surface protein expression by flow cytometry and immunocytochemistry. These cells expressed CD29, CD44, CD90 (Thy 1), CD105, SH3, and SH4, but not CD34, CD45, CD14, CD117 (c-kit), HLA-DR, or STRO-1, as demonstrated previously [11]. The antigenic properties of PDMCs differed from those of freshly dissociated placenta cells, which lacked CD90 and CD105 (Fig. 1A). CD90 is a marker of hepatic progenitor cells [16]. To determine whether PDMCs express other endodermal markers, we used immunostaining for c-Met and AFP. PDMCs expanded for five passages were fixed and interacted with Abs against human c-Met and AFP. As can be seen in Figure 1B and 1C, PDMCs were weakly positive for AFP and c-Met.

The multilineage differentiation ability of PDMCs was previously demonstrated [11]. To determine the optimal conditions for PDMCs to differentiate into hepatocyte-like cells, we tested different cytokines and growth factors known to induce hepatocyte differentiation for their effect on these cells. We also assessed how coating culture plates with different proteins affected PDMCs. Cells cultured in expansion medium retained their fibroblast-like morphology (Fig. 2A–2C). However, when PDMCs were cultured in differentiation medium on poly-Llysine-coated plates, their morphology changed to polygonal (Fig. 2F). When the cells were cultured on uncoated or fibronectin-coated plates, their morphological changes were less obvious (Fig. 2D, 2E). We isolated total RNA from these cells 28 days after treatment and analyzed the expression of albumin transcripts by RT-PCR (Fig. 2G). PDMCs cultured in differentiation medium on poly-L-lysine-coated culture vessels had the highest level of albumin mRNA expression. We then assessed the effect of HGF and FGF-4 on the differentiation of PDMCs into


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Figure 3. Effects of hepatocyte growth factor (HGF)/fibroblast growth factor-4 (FGF-4) on differentiation of PDMCs. Morphology of human PDMCs cultured in differentiation medium with or without growth factors for 12 days (A–C) (magnification ⫻200). (D–F): After 14 days of differentiation induction, cells of different groups were fixed and stained with anti-CK-18 antibody (magnification ⫻200). (A, D): PDMCs cultured in expansion medium. (B, E): Differentiation medium alone. (C, F): Differentiation medium with HGF (20 ng/ml) and FGF-4 (10 ng/ml). (G): Reverse transcription-polymerase chain reaction analysis was also performed; the expression levels of TAT are shown. GAPDH was used as internal control. Abbreviations: D, differentiation medium; D/H⫹F, differentiation medium supplemented with hepatocyte growth factor and fibroblast growth factor-4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TAT, tyrosine aminotransferase; Un, untreated.

hepatocyte-like cells. In comparison with cells cultured in differentiation medium alone (Fig. 3B), the addition of 20 ng/ml HGF and 10 ng/ml HGF did not significantly affect the changes in cell morphology (Fig. 3C). Cells treated with HGF and FGF-4 appeared smaller, and some cells of polygonal morphology formed colonies (Fig. 3C). Seo et al. reported that DMSO might enhance changes in cell morphology [17]. However, treatment with 0.1% DMSO had no apparent effect on the differentiation of PDMCs (data not shown). Upon immunofluorescence staining for CK-18, only PDMCs treated in differentiation medium containing HGF and FGF-4 showed positive (Fig. 3F). When we assayed the mRNA expression levels of TAT, the late differential marker of hepatic development [18], by RT-PCR, only PDMCs cultured in growth-factor-containing medium expressed TAT transcripts (Fig. 3G). In subsequent differentiation experiments, the PDMCs were cultured on poly-L-lysine-coated plates in differentiation medium containing HGF and FGF-4.

Characterization of Differentiated PDMCs The morphological changes in differentiated PDMCs could be observed as early as day 4 in culture. After 7 days of treatment, only some of the cells retained their fibroblast-like shape (Fig. 4B). On day 14, some cells had aggregated in clusters (Fig. 4C). Total RNA isolated from undifferentiated and treated cells at different time points was PCR-amplified for albumin and AFP (Fig. 4D). The levels of albumin mRNA increased in treated cells in a time-dependent manner, whereas the levels of AFP mRNA decreased significantly. Western blot analysis showed that the protein levels of AFP decreased in differentiated PDMCs (Fig. 4E). For further characterization, the immunocytochemical staining against CK18 and albumin, the markers for hepatocyte differentiation, was applied on the differentiated www.StemCells.com

hepatocyte-like cells. Undifferentiated PDMCs did not express albumin or CK-18 (Fig. 5A, 5E). Albumin could be detected in treated cells on days 7, 21, and 28 of culture in differentiation medium, and the intensity of the fluorescent staining increased in a time-dependent manner (Fig. 5B–5D). In contrast, CK-18 could be seen on differentiated cells on days 7 and 14, but it was almost undetectable on day 21 (Fig. 5F–5H). Immunocytochemical analysis with an anti-human hepatocyte antibody strongly stained differentiated cells (Fig. 5J), whereas untreated PDMCs were negative for this antigen (Fig. 5I). This antibody has been used to label human hepatocytes [19].

Biological Activities of Differentiated Hepatocyte-Like Cells To determine whether the hepatocyte-like cells derived from PDMCs had biological functions of hepatocytes, we performed three different assays. Low-density lipoprotein (LDL) is a lipoprotein that carries cholesterol. LDL uptake is observed in hepatocytes [20]. We assessed LDL uptake by incubating the differentiated PDMCs with Dil-Ac-LDL. After 14 days in differentiation medium, we observed LDL uptake by the hepatocyte-like cells, but not by untreated PDMCs (Fig. 6A, 6B). Because human hepatocytes can make and store glycogen, we analyzed the levels of stored glycogen by periodic acid-Schiff (PAS) staining of differentiated PDMCs at day 14 (Fig. 6C, 6D). The positive staining was observed on differentiated cells. We also tested whether hepatocyte-like cells derived from PDMC increase their levels of CYP3A4 mRNA in response to rifampicin. Indeed, rifampicin treatment upregulates CYP3A4 transcription in cultured human hepatocytes [21]. After treatment with growth-factor-containing differentiation medium for 7 days, hepatocyte-like cells derived from PDMCs, but not

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Figure 4. Characterization of hepatocyte-like cells derived from PDMCs. The PDMCs were cultured with hepatocyte growth factor (HGF)/ fibroblast growth factor-4 (FGF-4) supplemented differentiation medium on poly-L-lysine-coated culture dishes for 14 days. Phase contrast view on day 0 (A), day 7 (B), and day 14 (C) (magnification ⫻200). (D): Total RNA was isolated from cells treated at different time points. Reverse transcription-polymerase chain reaction analysis of albumin, AFP, and GAPDH was performed. (E): Western blot analysis was applied to detect the AFP protein expression levels. Protein was extracted from cells cultured in differentiation medium containing HGF and FGF-4 at days 0, 7, 14, and 21. GAPDH was applied as internal control. Abbreviations: AFP, ␣-fetoprotein; D, day; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

undifferentiated PDMCs, expressed CYP3A4 mRNA. Like HepG2 cells, differentiated PDMCs showed increased expression of CYP3A4 transcripts after rifampicin treatment (Fig. 7).

DISCUSSION Stem cells have great potential for clinical applications in regenerative medicine and tissue/organ replacement. Mesenchymal stem cells (MSCs) are a promising source. First, they can be easily isolated and expanded in quantities relevant to clinical application. Second, because autologous MSCs can be used, there is no risk of rejection. Third, MSCs can be cryopreserved for long periods without losing their stem cell properties. Fourth, MSCs have a broader differentiation potential than previously anticipated and can differentiate into all mesodermal lineage cells, including osteocytes, adipocytes, and chondrocytes [22–24]. MSCs can also differentiate into neural lineage cells, such as neuronal and glial cells [25, 26]. Thus, MSCs are multipotent adult stem cells. Adult bone marrow is now the main source for MSCs. MSCs derived from bone marrow (BMMSCs) can self-renew and have multilineage differentiation potential [27]. After systemic injection, BM-MSCs can incorporate into numerous organs and differentiate along tissuespecific lineages [28 –31]. However, disadvantages, which include the risk of infection associated with cell aspiration and the decreased cell content in elderly patients, limit the application of BM-MSCs in clinical stem-cell-based therapy [32–35]. Thus, the search for an alternative source of MSCs is important in the development of stem-cell-based therapy. Recent work has shown that MSCs are also present in other adult tissues [36]. Recently, we showed that progenitor cells could be isolated from human term placenta. These cells can be expanded beyond 20 cell doublings and can generate both mesodermal and neural lineage cells. PDMCs express CD90, SH105, SH3, and SH4; expression of these proteins is characteristic of MSCs [11].

In Vitro Hepatic Differentiation of PDMCs PDMCs are negative for CD34, CD45, and CD117 (c-kit) and therefore differ from hematopoietic stem cells isolated from bone marrow or umbilical cord blood [37, 37]. The properties of PDMCs are similar to those of BM-MSCs [39]. MSCs derived from adult bone marrow can differentiate into hepatic lineage cells [8]. However, the ability of PDMCs for hepatic differentiation has not yet been evaluated. Since PDMCs and BM-MSCs share several stem cell properties, we hypothesized that, despite their different origins, these progenitor cells may possess equivalent hepatic differentiation potential. In this study, we have attempted to differentiate placenta-derived cells toward a hepatic lineage. Undifferentiated PDMCs were positive for CD90 (Thy1), an antigen expressed on CD34(⫹) cells from human bone marrow, cord blood, and fetal liver. CD90 was recently identified as a new marker for hepatic oval cells [40]. However, these cells also express AFP and c-Met, which can be detected at the protein level. Both are markers of hepatic stem cells in early-stage human embryo [41, 42]. AFP is a major serum protein produced primarily by the visceral endoderm of the yolk sac, as well as by hepatoblasts and more differentiated fetal hepatic cells [43, 44]. It is one of the earliest markers for endodermal differentiation [45]. AFP is not expressed by all stem cells. Adult stem cells, including human umbilical cord blood-derived mesenchymal stem cells [46] and human bone marrow-derived MSCs [8], are absent for AFP expression. However, other progenitor cells, such as rat bone marrow-derived MSCs [47] and human peripheral blood monocyte-derived pluripotent stem cells [48] positively express AFP. The AFP-producing cells isolated from embryonic stem cells can differentiate into mature hepatocytes [49]. In addition, the enriched c-Met(⫹) cells from umbilical cord blood have been shown to be induced to hepatocyte-like cells with liver-specific functions [50]. Thus, the expression of these markers by these progenitor cells might indicate their potential to differentiate into hepatic lineage cells. To the best of our knowledge, this is the first report demonstrating that placenta-derived cells are capable of differentiating toward endodermal hepatic lineage cells. In this study, we treated the cells directly with hepatogenic medium. The morphology of the differentiated cells changed from fibroblast celllike to polygonal epitheloid cell-like as early as 4 days after treatment. The differentiated cells sometimes aggregated to form cell colonies. To optimize the differentiation conditions, different combinations of cytokines and various coatings of the culture plates were tested. The growth factors chosen, HGF and FGF-4, and the chemical DMSO were shown to play important roles in hepatocyte differentiation [51]. HGF, a growth factor produced by mesenchymal cells, plays a critical role in the development of liver cells [52], and the addition of HGF helps in driving stem cell differentiation toward hepatocyte-like cells [17, 53]. FGF-4 is a member of the fibroblast growth factor family. It can induce bone-marrow-derived progenitor cells to form immature hepatocyte-like cells, and it plays an important role in initial endoderm patterning and specification [54]. Compared with individual treatment, the combined use of HGF and FGF-4 has a synergistic effect on promoting hepatic cell differentiation [7]. Addition of HGF and FGF-4 to the culture led to increased numbers of differentiated cells. Furthermore, immunochemical analysis showed that PDMCs treated in differentiation medium containing HGF and FGF-4 express CK-18 after 14 days of differentiation, whereas PDMCs treated with differentiation


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Figure 5. Immunofluorescence and immunohistochemical staining of differentiated placenta-derived multipotent cells (PDMCs). PDMCs were treated with differentiation medium containing hepatocyte growth factor and fibroblast growth factor-4 for 4 weeks, and cells harvested different time points (as indicated) were fixed and stained with anti-albumin (A–D) and anti-CK 18 (E–H) monoclonal antibodies and then stained with Cy3-conjugated anti-rabbit and fluorescein isothiocyanate-conjugated anti-mouse antibodies (Abs) respectively. The results were observed using fluorescence microscope. The cells cultured in expansion medium were applied as controls (A, E). For immunohistochemical analysis, treated cells were fixed at day 14 and stained with anti-hepatocyte Ag Ab (J). Biotinylated anti-mouse antibody was applied as secondary antibody. The untreated PDMCs were negative for this antigen (I). Samples were counterstained with hematoxylin (magnification ⫻100). Abbreviation: D, day.

medium alone are negative for CK-18 at this time point. RT-PCR analysis indicated that the expression of TAT, a mature hepatocyte marker, increased in cells treated with differentiation medium and HGF/FGF-4. Our results suggest that HGF and FGF-4 could induce PDMCs to differentiate toward hepatic lineage cells, an effect also observed with BM-MSCs [55]. DMSO, a chemical that has been used for cryopreservation, could be applied to maintain the functions of adult hepatocytes in vitro [56]. Seo et al. showed that the addition of DMSO promotes the differentiation of adipose stromal cells into hepatocyte-like cells [17]. However, in our study, DMSO was ineffective in promoting the differentiation of PDMCs toward hepatocyte-like cells. In tests to evaluate the influence of different culture plate coatings on differentiation, we found that poly-L-lysine, a synthetic molecule used to enhance cell attachment to plastic surfaces and promote neurite outgrowth in neural cells [57], induces the differentiation of PDMCs into cells with hepatic morphologies and phenotypes better than fibronectin. However, the mechanisms by which poly-L-lysine exerts this effect are not fully understood. We conducted several assays to further characterize the differentiated PDMCs. The results of the RT-PCR analysis indicated that the expression of albumin was upregulated in the differentiated cells. In contrast to undifferentiated cells, the treated cells expressed albumin and CK-18, which are specific www.StemCells.com

markers for hepatocyte differentiation [58]. Our results showed that the expression of CK-18 had declined on day 21. These data differ from those of hepatic differentiation from other mesenchymal stem cells. Recently, Sharma et al. reported the generation of CK-18-negative hepatocyte-like cells derived from human cord blood in injured mouse liver [59]. Thus, PDMCs might act in the process of hepatic differentiation similarly to stem cells isolated from human cord blood. Immunohistochemical staining with anti-human hepatocyte antibody showed that differentiated PDMCs express this antigen. Western blotting data confirmed that the levels of AFP, a marker for immature hepatocytes [58], decreased in the differentiated PDMCs. However, expression of hepatocyte-specific markers at mRNA and protein levels does not mean that differentiated hepatocyte-like cells have acquired functional hepatocyte activities. Therefore, we carried out functional assays to evaluate the bioactivities of PDMC-derived hepatocyte-like cells. The results showed that these differentiated cells could uptake Dil-Ac-LDL, contain glycogen, and trigger CYP3A4 gene expression in response to rifampicin treatment. Although uptake of LDL is seen in hepatocytes [20], other cells, including endothelial cells, also possess this ability [21]. Generation and storage of glycogen is one of the characteristics of liver cells. The differentiated PDMCs were stained by PAS, whereas undifferentiated PDMCs were not. In


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Figure 6. Low-density lipoprotein (LDL) uptake by placenta-derived multipotent cell (PDMC)-derived hepatocyte-like cells. Undifferentiated cells (A) and PDMCs cultured on poly-L-lysine-coated dishes with hepatocyte growth factor (HGF) and fibroblast growth factor-4 (FGF-4) for 14 days (B) were incubated with Dil-acil-LDL. The labeled cells were counterstained with 4⬘,6-diamino-2-phenylindole and photographed by fluorescence microscopy (magnification ⫻200). Periodic acid-Schiff (PAS) staining of undifferentiated and differentiated PDMCs. PDMCs were cultured on poly-L-lysine-coated dishes with HGF and FGF-4 for 14 days. PAS staining was described in Materials and Methods. (C): Undifferentiated PDMCs. (D): PDMC-derived hepatocyte-like cells (magnification ⫻100).

Figure 7. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CYP3A4 mRNA levels before and after RFP treatment in undifferentiated and differentiated PDMCs. Differentiated hepatocyte-like PDMCs and untreated cells were incubated with 50 mM RFP for 48 hours. Total RNA was isolated from cells, and the levels of CYP3A4 transcripts were evaluated by RT-PCR. The levels of GAPDH mRNA served as internal control. Abbreviations: D/H⫹F, differentiation medium supplemented with hepatocyte growth factor and fibroblast growth factor-4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RFP, rifampicin; Un, untreated.

metabolism [60]. Taken together, these results indicate that PDMCs derived from human term placenta treated in vitro not only express hepatocyte markers, but also acquire the functional activities of hepatocytes. As shown in previous studies, several cell types, including liver stem cells, embryonic stem cells, and adult stem cells, can be induced to differentiate into hepatocyte-like cells [2, 6, 17, 61]. Mesenchymal stem cells isolated from bone marrow were shown to differentiate toward liver cells in vitro and in vivo [5– 8]. These differentiated cells have hepatocyte phenotypes and hepatocyte-specific biological activities. An in vivo study has shown that administration of bone marrow-derived MSCs protects animals from experimental liver fibrosis [62]. Thus, transplantation of BM-MSCs might have clinically applicable therapeutic potential. However, because of the disadvantages in clinical application of BM-MSCs, including low stem cell content in elderly patients and the risk of infection by various pathogens, the search for alternative sources of mesenchymal stem cells is intensive. Recently, stem cells derived from several other origins were isolated, which could differentiate toward hepatocyte-like cells and had relevant biological activities, such as uptake of lipoprotein, urea production, and glycogen storage [6 – 8, 16]. However, cord blood-derived MSCs have a low isolation rate and the differentiation ability of adipose tissue-derived MSCs is limited. Hence, PDMCs could be a superior source of MSCs because of the high stem cell content in placenta tissue, the low infection risk associated with their use, and their differentiation ability, which is similar to that of BM-MSCs. Thus, our study has provided evidence that PDMCs can differentiate toward cells with hepatocyte characteristics. PDMCs may serve as cells for in vivo therapy of genetic or acquired disorders of the liver. Together with our previous studies showing that PDMCs have the ability to differentiate toward mesodermal and neural lineage cells, our present findings suggest that these progenitor cells have the ability to differentiate into cells of three different germ layers. The differentiation ability of these cells is therefore broader than we had previously thought. In addition, because PDMCs are progenitor cells that can be easily obtained with a low-risk of pathogen infection, they might contribute to the development of new therapeutic concepts for liver diseases.

ACKNOWLEDGMENTS This work was supported by grant MR9405 from Cathay General Hospital.

response to rifampicin, the mRNA expression of CYP3A4 increases in differentiated PDMCs. This effect has been observed in human liver cells and been used for in vitro analysis of drug

DISCLOSURES

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