In Vitro Differentiation of Human Placenta-Derived Multipotent Cells into Hepato - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:04 |只看该作者 |倒序浏览 |打印

作者：Chih-Cheng Chiena,b, Betty Linju Yenc, Fa-Kung Leee, Tsung-Hsuan Laia,e, Yao-Chang Chenc,d, Shu-Hui Chana, Hsing-I Huanga,b


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      【摘要】/ o& h; n" E) }0 T
   Multipotent cells isolated from human term placenta (placenta-derived multipotent cells ) 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 morphologies 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.
      【关键词】 Placenta Differentiation Hepatocyte Regenerative medicine/ E/ d& h( {, [& O
   INTRODUCTION

In mammals, repair or regeneration of the liver has long been thought possible. Mature hepatocytes may divide and be responsible for hepatic cell replacement . Oval cells are induced to differentiate and expand, whereas liver cells undergo severe damage. However, their mechanisms of action are still not fully understood.
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Stem cells derived from nonliver sources, such as bone marrow and umbilical cord blood, were recently shown to differentiate into hepatocyte-like cells . These results indicated that stem cells might have a clinically applicable therapeutic potential in liver diseases.; }1 m* B3 t, j. U; \4 V6 P
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Recently, we and others isolated placenta-derived multipotent cells (PDMCs) from human term placenta and showed that these cells have multilineage differentiating ability . 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.4 {+ ?% z& @: d
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MATERIALS AND METHODS

Isolation and Culture of Human PDMCs$ M2 Z  n  @' J1 o* X7 E% t
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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¡ãC 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 1x penicillin/streptomycin (Gibco-Invitrogen). The cells were then incubated at 37¡ãC in an incubator with 5% CO2.1 [5 O$ g. v+ S) b! u( Z

Flow Cytometry# T2 ?! T! X4 R) b8 ]3 _4 X; w
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PDMCs were cultured and examined for the immunophenotype. Cells (2 x 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¡ãC. 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.
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Hepatogenic Differentiation
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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 (Sigma-Aldrich) supplemented with 1x Insulin-Transferrin-Selenium (ITS), 4.7 µg/ml linoleic acid, 1 mg/ml bovine serum albumin, 10¨C8 M dexamethasone (Decadron; Merck & Co., Whitehouse Station, NY, http://www.merck.com), 10¨C4 M ascorbic acid (all from Sigma-Aldrich), 10 ng/ml epidermal growth factor (Gibco-Invitrogen), 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.
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Total RNA Isolation of Reverse Transcriptase-Polymerase Chain Reaction- Q$ q+ H  B! d# }% @# Q2 E
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Total RNA was isolated from PDMCs using Purescript (Gentra, Minneapolis, http://www.gentra.com/), and 2 µg 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 glyceraldehyde-3-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, Göttingen, Germany, http://www.biometra.de/) at 94¡ãC for 30 seconds, 55¡ãC for 30 seconds, and 72¡ãC 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./ f- u. @; e0 K/ `( ^
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Immunocytochemistry

Cultured cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 5 minutes at room temperature and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 20 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¡ãC. 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 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¡ãC and stained with 4',6-diamino-2-phenylindole (KPL, Gaithersburg, MD) to identify cell nuclei. The cells were visualized and photographed by fluorescence microscopy (Olympus BX51; Tokyo).
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Western Blot  q! |1 m+ @& a: x: u! Z7 w
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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 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¡ãC overnight. Horseradish peroxidase-conjugated 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.- v1 v9 m! G  K9 v3 u
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Uptake of Low-Density Lipoprotein
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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.& j4 I+ _; N- J1 R; u4 A( ]+ X
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Periodic Acid-Schiff Staining, x- s  \* Z( _' N

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.5 b* B$ ]6 W4 V6 {& J1 N
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Rifampicin Treatment of Differentiated Cells& H+ ~7 A) Y" @3 K& k
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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 . Untreated and differentiated PDMCs, along with HepG2, were plated on a six-well culture plate at a concentration of 2 x 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 transcription-polymerase chain reaction (RT-PCR) analysis for detection of CYP3A4 mRNA levels.

RESULTS/ R& l$ _( Y4 y. c) |4 @' x$ `* M
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Characterization of Human Placenta-Derived Multipotent Progenitors
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We isolated PDMCs from human placenta according to the protocol described previously (sample number = 20) . 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.5 z  B  u( \2 S3 _0 ~

Figure 1. Characterization of PDMCs. (A): Flow cytometric analysis for freshly isolated placental cells and expanded PDMCs. Freshly isolated placental cells were CD14( /¨C), CD34(¨C), CD90(¨C), and CD105(¨C). The isolated PDMCs were CD14(¨C), CD34(¨C), 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 peroxidase-conjugated secondary antibody and counterstained by hematoxylin (magnification x200). (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 x300). Abbreviations: FL, fluorescence; PDMC, placenta-derived multipotent cell.1 D+ v* l2 J5 q  h/ |' q
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Hepatogenic Differentiation

The multilineage differentiation ability of PDMCs was previously demonstrated , 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.% w: d1 |, D8 r# K8 N7 x) q6 ]
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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 fibronectin-coated dishes. Undifferentiated PDMCs assumed the fibroblast morphology on untreated (A), fibronectin-coated (B), and poly-L-lysine-coated (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 x200). (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.

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¨CC) (magnification x200). (D¨CF): After 14 days of differentiation induction, cells of different groups were fixed and stained with anti-CK-18 antibody (magnification x200). (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.

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 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¨C5D). 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¨C5H). 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 .# W  d8 P2 [2 x2 z0 L

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 x200). (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.. i7 V) `/ ]: J" J

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¨CD) and anti-CK 18 (E¨CH) 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 x100). Abbreviation: D, day.

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 . 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.
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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 x200). 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 x100).

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 . After treatment with growth-factor-containing differentiation medium for 7 days, hepatocyte-like cells derived from PDMCs, but not undifferentiated PDMCs, expressed CYP3A4 mRNA. Like HepG2 cells, differentiated PDMCs showed increased expression of CYP3A4 transcripts after rifampicin treatment (Fig. 7).

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.2 L, y: v: w' X. d9 N% J. G

DISCUSSION1 G3 v" B7 h) ^. T+ q6 A

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 .8 x8 C1 b& S+ x/ p' B
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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 . 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.$ Q  E7 Z9 P1 Q# h6 T+ N, i. w

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 . Thus, the expression of these markers by these progenitor cells might indicate their potential to differentiate into hepatic lineage cells.
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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 cell-like 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 , 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 markers for hepatocyte differentiation . 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 . 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.
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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 . 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., D* Z3 I. c% I& I  C- j/ E- T0 P
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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.# |* Z  ~% T: v" y* }

DISCLOSURES
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS. w4 g( Z1 I: L2 S
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This work was supported by grant MR9405 from Cathay General Hospital.
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