Isolation and cellular properties of mesenchymal cells derived from the decidua of human term placenta

Differentiation 82 (2011) 77–88

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Isolation and cellular properties of mesenchymal cells derived from the decidua of human term placenta Daisuke Kanematsu a, Tomoko Shofuda b, Atsuyo Yamamoto b, Chiaki Ban d, Takafumi Ueda e, Mami Yamasaki c,f, Yonehiro Kanemura a,f,n a

Department of Regenerative Medicine, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, 2-1-14 Hoenzaka, Chuo-ku, Osaka 540-0006, Japan Department of Stem Cell Research, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, 2-1-14 Hoenzaka, Chuo-ku, Osaka 540-0006, Japan c Department of Molecular Medicine, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, 2-1-14 Hoenzaka, Chuo-ku, Osaka 540-0006, Japan d Department of Obstetrics and Gynecology, Osaka National Hospital, National Hospital Organization, Osaka 540-0006, Japan e Department of Orthopaedic Surgery, Osaka National Hospital, National Hospital Organization, Osaka 540-0006, Japan f Department of Neurosurgery, Osaka National Hospital, National Hospital Organization, Osaka 540-0006, Japan b

a r t i c l e i n f o

abstract

Article history: Received 25 August 2010 Received in revised form 6 May 2011 Accepted 26 May 2011

The clinical promise of cell-based therapies is generally recognized, and has driven an intense search for good cell sources. In this study, we isolated plastic-adherent cells from human term decidua vera, called decidua-derived-mesenchymal cells (DMCs), and compared their properties with those of bone marrow-derived-mesenchymal stem cells (BM-MSCs). The DMCs strongly expressed the mesenchymal cell marker vimentin, but not cytokeratin 19 or HLA-G, and had a high proliferative potential. That is, they exhibited a typical fibroblast-like morphology for over 30 population doublings. Cells phenotypically identical to the DMCs were identified in the decidua vera, and genotyping confirmed that the DMCs were derived from the maternal components of the fetal adnexa. Flow cytometry analysis showed that the expression pattern of CD antigens on the DMCs was almost identical to that on BMMSCs, but some DMCs expressed the CD45 antigen, and over 50% of them also expressed anti-fibroblast antigen. In vitro, the DMCs showed good differentiation into chondrocytes and moderate differentiation into adipocytes, but scant evidence of osteogenesis, compared with the BM-MSCs. Gene expression analysis showed that, compared with BM-MSCs, the DMCs expressed higher levels of TWIST2 and RUNX2 (which are associated with early mesenchymal development and/or proliferative capacity), several matrix metalloproteinases (MMP1, 3, 10, and 12), and cytokines (BMP2 and TGFB2), and lower levels of MSX2, interleukin 26, and HGF. Although DMCs did not show the full multipotency of BMMSCs, their higher proliferative ability indicates that their cultivation would require less maintenance. Furthermore, the use of DMCs avoids the ethical concerns associated with the use of embryonic tissues, because they are derived from the maternal portion of the placenta, which is otherwise discarded. Thus, the unique properties of DMCs give them several advantages for clinical use, making them an interesting and attractive alternative to MSCs for regenerative medicine. & 2011 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.

Keywords: Decidua-derived mesenchymal cell Mesenchymal stem cell Placenta Maternal cell Regenerative medicine

1. Introduction Mesenchymal stem cells (MSCs), a heterogeneous subset of stromal stem cells, show great promise as a cell source for clinical applications. MSCs proliferate as plastic-adherent cells with a

Abbreviations: DMC, decidua-derived mesenchymal cell; MSC, mesenchymal stem cell; AEC, amniotic epithelial cell; AMC, amniotic mesenchymal cell; STR, short tandem repeat; UCB, umbilical cord blood n Corresponding author at: Department of Regenerative Medicine, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, 2-1-14 Hoenzaka, Chuo-ku, Osaka 540-0006, Japan. Tel.: þ81 6 6942 1331; fax: þ 81 6 6946 3530. E-mail address: [email protected] (Y. Kanemura).

fibroblast-like morphology and can differentiate into cells of the mesodermal linage, including osteoblasts, chondrocytes, and adipocytes (Caplan, 1991; Pittenger et al., 1999; Dominici et al., 2006; Ohgushi and Caplan, 1999; Uccelli et al., 2008). They are typically isolated from the non-hematopoietic mononuclear cells of the bone marrow (bone marrow derived-MSCs: BM-MSCs), but they can also be isolated from various human fetal and adult tissues (Parolini et al., 2008; Koide et al., 2007; Ilancheran et al., 2009; Marcus and Woodbury, 2008; Caplan, 1991; Pittenger et al., 1999; Dominici et al., 2006; Uccelli et al., 2008). The fetal adnexa include the placenta, fetal membranes, and umbilical cord, and it contains extra-embryonic cells (Parolini et al., 2008; Ilancheran et al., 2009; Marcus and Woodbury, 2008). These are ephemeral organs that connect the developing fetal

0301-4681/$ - see front matter & 2011 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved. Join the International Society for Differentiation (www.isdifferentiation.org) doi:10.1016/j.diff.2011.05.010

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tissues to the uterine wall, encase the amniotic fluid in which the fetus is suspended during pregnancy, supply the fetus with maternal nutrients, allow fetal waste to be disposed of via the maternal kidneys, and contribute to fetal–maternal immune tolerance (Parolini et al., 2008; Ilancheran et al., 2009; Marcus and Woodbury, 2008). Many recent studies have shown that fibroblast-like adherent cells isolated from various components of the fetal adnexa, including the placenta, are phenotypically similar to MSCs, which suggests that the fetal adnexa represents a new source of human MSCs (Fukuchi et al., 2004; Igura et al., 2004; Zhang et al., 2004; In’tAnker et al., 2004; Wulf et al., 2004; Bailo et al., 2004; Yen et al., 2005; Li et al., 2005; Portmann-Lanz et al., 2006; Miao et al., 2006; Battula et al., 2007; Alviano et al., 2007; Soncini et al., 2007; Ilancheran et al., 2007; Barlow et al., 2008). However, the detailed biological properties of the MSCs derived from the human fetal adnexa remain to be investigated. We recently isolated the adherent cells from human term decidua vera, called decidua-derived mesenchymal cells (DMCs), and reported some of their properties (Nagase et al., 2009; Kanemura, 2010). In the present study, we examined their cellular properties in detail, and compared them with those of adult BMMSCs. The DMCs exhibited a typical fibroblast-like morphology and strongly expressed a mesenchymal cell marker, vimentin, in their cytoplasm and many antigens that are known to be expressed on MSCs, on their cell surface. They showed a high proliferation ability, differentiated well into chondrocytes and moderately into adipocytes, but hardly at all into osteoblasts, in vitro. These unique properties of DMCs should give them several advantages for clinical use, and indicate that they might be an attractive alternative to allogeneic MSCs for use in regenerative medicine.

2. Materials and methods 2.1. Human tissues and cells These studies were carried out in accordance with the principles of the Helsinki Declaration, and approval to use human fetal adnexa, umbilical cord blood cells (UCBCs), and adult bone marrow cells were obtained from the ethical committee of Osaka National Hospital. The donors’ blood was serologically tested for HBs, HCV, HIV, and syphilis. 2.2. Isolation of DMCs, amniotic epithelial cells (AECs), and amniotic mesenchymal cells (AMCs) Fetal membranes (amnions and chorions) with decidua vera (about 25 cm2) (Fig. 1) were collected, with written informed consent, from the vaginally delivered full-term fetal adnexa of normal healthy mothers at the Osaka National Hospital and stored in DMEM/F-12 (1:1) at 4 1C before use for various periods of time. The DMCs were prepared as follows. After extensive washes with phosphate-buffered saline (PBS), the amnion (Fig. 1, Am) was gently separated from the area above the chorion (Fig. 1, closed arrow). Next, the decidua vera (Fig.1, De) was scraped from the chorionic membrane (Fig. 1, Ch) (Fig. 1, open arrow). This procedure enabled a sample of decidual tissue to be obtained without chorionic contamination. The decidual tissue (Fig. 1, De) was dissected into small pieces and enzymatically dissociated in PBS containing collagenase (1 mg/ml; Invitrogen Corp., Carlsbad, CA), dispase (1 mg/ml; Invitrogen), and DNase I (final 0.01%, Invitrogen) for 1 h at 37 1C with gentle shaking. The suspensions were filtered through a 40-mm nylon mesh (Cell Strainer, BD Biosciences, San Jose, CA), and single-cell suspensions were propagated in 100-mm-diameter cell-culture dishes using DMEM/F-12 (1:1)-based culture medium supplemented with

Fig. 1. Structure of human term fetal membranes with the decidua vera. Human term fetal membranes with the decidua vera were stained by hematoxylin and eosin. The chorionic tissue is indicated between the closed arrow and open arrow. Am; amnion, Ch; chorionic tissue, De; decidua vera, *: fetal side, Scale bar¼100 mm.

10% fetal bovine serum (FBS), 15 mM HEPES, and antibiotic– antimycotic (Invitrogen) at 37 1C in 5% CO2. AECs were prepared as follows. The amnion (Fig. 1, Am) was dissected into 1 cm  1 cm pieces in PBS containing DNase I (final 0.03%) and enzymatically dissociated in trypsin (final 0.25%, Invitrogen) for 30 min at 37 1C with gentle shaking. The first digestion supernatant was discarded to remove debris, then the remaining amnion was repeatedly dissociated by trypsin, 5–7 times. All the digestion supernatants were collected and filtered through a 40-mm nylon mesh. The cells were collected by centrifugation, and single-cell suspensions were cultured in 100-mm-diameter cell-culture dishes using the same culture medium as for DMCs, at 37 1C in 5% CO2. AMCs were prepared as follows. The residual amniotic tissue used in the AEC isolation was further dissociated enzymatically in PBS containing collagenase Type-I (1 mg/ml; Invitrogen), dispase (1 mg/ml), and DNase I (final 0.01%) for 1 h at 37 1C with gentle shaking. The suspensions were filtered through a 40-mm nylon mesh, and single-cell suspensions were propagated in 100-mmdiameter cell-culture dishes using the same culture medium as for DMCs, at 37 1C in 5% CO2. 2.3. Isolation of BM-MSCs Adult bone marrow cells were extracted, after obtaining written informed consent, from the pelvis of an adult female patient undergoing orthopedic surgery, and mononuclear cells were collected in a BD Vacutainer CPT (REF 362753, BD Biosciences, San Jose, CA), according to the manufacturer’s instructions. The cells were suspended in DMEM low-glucose containing 10% FBS and antibiotic–antimycotic, and the BM-MSCs (BMMSC1) were propagated in 75T cell culture flasks at 37 1C in 5% CO2 (Marcus and Woodbury, 2008). Additional BM-MSCs (BMMSC2) were obtained from LONZA (Walkersville, MD) and were propagated by the same way. 2.4. Growth kinetics To assess the growth characteristics of the isolated cells, the culture medium was replaced 2 times every week, and the cells were passaged using trypsin–EDTA (final 0.05%, Invitrogen) once every week. Single-cell suspensions (2  104 cells/ml) were propagated in 100-mm-diameter cell-culture dishes. All the cells (AECs, AMCs, DMCs, and BM-MSC1) were cultured until they reached a poor proliferation rate, and the expanded number of cells in every passage was counted and plotted. 2.5. Colony-forming unit-fibroblast (CFU-F) assay The CFU-F assay was performed as described previously (Ito et al., 2010), with some modifications. The DMCs and BM-MSCs

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(second passage) were seeded into 60-mm-diameter culture dishes at a density of 1000 or 500 cells/dish, and cultured for 9 days. The cells were fixed with methanol and stained with a 5% Giemsa solution. The number of colonies with a diameter greater than 1 mm was counted. 2.6. Flow cytometry (FCM) analysis Single-cell suspensions were prepared using enzymatic dissociation with trypsin–EDTA. The cells were incubated for 30 min at 4 1C with primary antibodies (Abs) (Supplemental Table 1). After several washes, the cells were incubated with AlexaFlours488-conjugated goat anti-mouse IgG or anti-mouse IgM Abs (Molecular Probes, Eugene, OR) at 4 1C for 30 min. The samples were suspended in DMEM/F-12 (1:1) medium containing propidium iodide (PI) (1 mg/ml, Sigma-Aldrich, Inc., St. Louis, MO) to label the dead cells, and filtered through 30-mm nylon mesh. Stained samples were analyzed by a FACSCalibur cytometer (BD Biosciences). 2.7. Genotyping of short tandem repeat (STR) polymorphisms Genomic DNA was extracted from DMCs by DNAzol Reagent (Invitrogen). To extract genomic DNA from amnion tissue, a DNeasy Blood & Tissue Kit (Qiagen Inc., Valencia, CA) was used; to extract it from UCBCs, a MagneSils ONE, Fixed Yield Blood Genomic System (Promega, Madison, WI) was used. STR loci were analyzed with the Powerplex 16 system (Promega) using a 310 Genetic analyzer and GeneMapper software (Applied Biosystems, Foster, CA), following the manufacturer’s instructions. 2.8. In vitro differentiation To induce chondrogenic differentiation, a pellet culture system was used (Mackay et al., 1998; Lee et al., 2003). Briefly, 2.5  105 cells were pelleted at the bottom of a 15-ml polypropylene tube, then cultured in DMEM-HG supplemented with dexamethasone (Dex) (0.1 mM; Wako Pure Chemical Industries, Ltd., Osaka, Japan), ascorbic acid 2-phosphate (170 mM; Wako), sodium pyruvate (1 mM; Invitrogen), proline (350 mM; Wako), recombinant human transforming growth factor-b3 (TGF-b3) (10 ng/ml; R&D Systems, Minneapolis, MN), and ITSþPremix [insulin (6.25 mg/ml), transferrin (6.25 mg/ml), selenious acid (6.25 ng/ml), linoleic acid (5.35 mg/ml), and bovine serum albumin (1.25 mg/ml); BD Biosciences]. The medium was replaced every 3–4 days for 28 days. To induce adipogenic differentiation, cells were cultured at a density of 5  104 cells/ml in DMEM high-glucose (DMEM-HG) supplemented with Dex (1 mM), 3-isobutyl-1-methylxanthine (IBMX) (500 mM; Wako), indomethacin (200 mM; Wako), recombinant human insulin (20 mg/ml; Sigma-Aldrich), and 10% FBS (Zhang et al., 2004). The medium was replaced every 3–4 days for 28 days. To evaluate the cytoplasmic neutral lipid content, the cells were fixed in PBS containing 4% paraformaldehyde (PFA) for 30 min at room temperature (RT), and stained with Oil red O (Sigma-Aldrich) for 15 min and counterstained with Mayer Hematoxylin (Wako). To induce osteogenic differentiation, cells were cultured at a density of 5  104 cells/ml in DMEM-HG supplemented with Dex (0.2 mM), b-glycerol phosphate (10 mM; Sigma-Aldrich), ascorbic acid 2-phosphate (100 mM), and 10% FBS (Lee et al., 2003). The medium was replaced every 3–4 days for 28 days. To evaluate the mineralized matrix, the cells were fixed in PBS containing 4% PFA for 30 min at RT, and stained with 0.1% Alizarin red S (SigmaAldrich) solution in water for 10 min.

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2.9. lmmunocytochemical and immunohistochemical staining Placental tissues and chondrogenic cell pellets were fixed in PBS containing 4% PFA at 4 1C, embedded in OCT compound (Sakura Finetek, Torrance, CA), frozen at  80 1C, and cut into 14-mm-thick sections on a cryostat (Leica, Nussloch, Germany). Cultured cells (DMCs, AECs, AMCs, and BM-MSCs) were fixed in PBS containing 4% PFA for 30 min at RT. Hematoxylin and eosin staining (HE staining) was performed using routine procedures. To detect the F-actin cytoskeleton, cells were incubated with AlexaFluors-568 phalloidin (Molecular Probes) in PBS for 20 min at RT (phalloidin staining). Samples were counterstained with TOPRO-3s iodide (1 mM, Molecular Probes) to detect nuclei. For immunocytochemical or immunohistochemical staining, the fixed cells or tissue samples were blocked with 10% normal rabbit serum for 1 h at RT, then incubated overnight at 4 1C with the following Abs: anti-human vimentin Ab (goat polyclonal, Chemicon, Temecra, CA), anti-human cytokeratin 19 (CK19) Ab (mouse monoclonal, SantaCruz, Santa Cruz, CA), and anti-human leukocyte antigen-G (HLA-G) Ab (mouse monoclonal, EXBIO, Vestec, Czech Republic). After several washes, the samples were incubated with AlexaFlours-488-conjugated rabbit anti-goat IgG Ab (Molecular Probes), AlexaFlours-568-conjugated goat antimouse IgG Ab (Molecular Probes), and TO-PRO-3s iodide (1 mM, Molecular Probes) for 1 h at RT. To evaluate chondrogenic differentiation, chondrogenic cell pellet sections were incubated in methanol/0.3% H2O2 for 30 min, blocked with 10% normal rabbit serum for 1 h at RT, and incubated with anti-collagen type-II Ab (goat polyclonal, SantaCruz) overnight at 4 1C. The sections were then incubated with a biotinylated anti-goat IgG Ab (Vector, Burlingame, CA) for 1 h at RT. Signals were detected by the avidin–biotin complex (ABC) method (VECTASTAIN Elite ABC Standard Kit, Vector) and metalenhanced DAB Kit (Pierce, Rockford, IL). The samples were examined using a confocal laser scanning microscope (LSM510, Carl Zeiss, Hallbergmoos, Germany) or a light microscope (IX70, Olympus, Tokyo, Japan). All stainings were performed with matched IgG isotype controls. 2.10. Microarray analysis The microarray study was carried out using the Human Genome U133 Plus 2.0 gene expression array (Affymetrix Inc., Santa Clara, CA). One hundred nanograms of total RNA was used to synthesize aRNA using the 30 IVT Express Kit, according to the manufacturer’s instructions (Affymetrix). After aRNA purification, 15 mg of aRNA was fragmented and hybridized with a preequilibrated GeneChip array at 45 1C for 16 h. The GeneChip array was then washed, stained, and scanned according to the manufacturer’s instructions. The gene expression data were extracted using Affymetrix Expression Console software, and the data sets were analyzed using GeneSpring GX software (Agilent Technologies, Inc., Santa Clara, CA) to identify genes with expression levels that fluctuated more than 2-fold between DMCs and BM-MSCs. 2.11. Quantitative reverse transcription-PCR (qRT-PCR) Total RNA was isolated using the RNeasy Mini kit (Qiagen, Valencia, CA). Complementary DNA was synthesized using the SuperScriptTM First-Strand Synthesis System for RT-PCR with random hexamers (Invitrogen). PCR was performed using the PowerSYBRs Green PCR Master Mix (Applied Biosystems) on an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems) with gene-specific primers (Supplemental Table 2), as previously described (Mori et al., 2006). The comparative cycle time (Ct) method was used to quantify the gene expression levels,

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following the manufacturer’s instructions. Glyceraldehyde-3phosphate dehydrogenase (GAPDH) or hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as the internal control gene for normalization.

71.6%, Table 1). Twelve attempts (14.8%) failed, apparently due to poor tissue condition, and 11 (13.6%) failed due to bacterial and/ or fungal contamination (Table 1). We did not observe a clear correlation between the storage time and the success of DMC isolation (R2 ¼0.0911, Table 1).

2.12. Statistical analysis Correlation between the storage time and the success of DMC isolation were analyzed by Pearson’s test. Results of the CFU-F assay are shown as a boxplot of three independent experiments performed for each cell line and analyzed by one-way Factorial ANOVA and Dunnett’s test, to evaluate the differences between DMCs and BM-MSCs. Significant differences in gene expression levels obtained by qRT-PCR were determined by unpaired Student’s t-tests.

3. Results 3.1. Relationship between the placental tissue storage time and the success of DMC isolation In this study, we attempted a total of 81 DMC isolations from 81 independent placental tissue samples stored for various amounts of time, and obtained 58 DMC lines (overall success rate

Table 1 Isolation of DMCs after various storage times. Storage time (hours)a

Trials

o24 24 o to 48 48 o to 72 72 o to 96 96 o to 120 120 o to 168

32 21 10 8 6 4

25 12 7 7 3 4

Total

81

58 (71.6)

Successes (%)

Failures Bacterial and/or fungal contamination (%)

(78.1) (57.1) (70) (87.5) (50) (100)

4 3 0 1 3 0

(12.5) (14.3) (0) (12.5) (50) (0)

11 (13.6)

Without contamination (%)

3 6 3 0 0 0

(9.4) (28.6) (30) (0) (0) (0)

12 (14.8)

a Duration of placental tissue storage at 4 1C before being used for DMC isolation.

Fig. 2. In vitro phenotypes of the DMCs, AECs, and AMCs. Phase-contrast images and Phalloidin (red)-staining in images of AECs (first passage, 15 days in culture), AMCs (third passage, 25 days in culture), and DMCs (third passage, 26 days in culture). Nuclei were counterstained with TO-PRO-3 (blue). Scale bar ¼200 mm (upper) and 20 mm (lower).

Fig. 3. Immunocytochemical analysis of DMCs, AECs, and AMCs. Immunocytochemical analysis of AECs (first passage, 15 days in culture), AMCs (third passage, 25 days in culture), and DMCs (third passage, 26 days in culture). Nuclei were counterstained with TO-PRO-3 (blue). Negative control is also shown. Scale bar¼ 20 mm.

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3.2. Isolation and in vitro phenotype analysis of the DMCs First, we isolated three different kinds of adherent cells from fetal membranes and decidua vera tissue (Fig. 1) and examined their cellular phenotypes in vitro. The cells dissociated from the amnion

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using repeated trypsin treatment displayed a rounded cobblestone appearance characteristic of epithelial cells, and filamentous actin was concentrated at their circumference, suggesting that these isolated cells had a preferential affinity for making lateral cell–cell contacts (Fig. 2). Immunohistochemical analysis showed that these

Fig. 4. Growth properties of the DMCs. (A) The growth rate of DMCs (n¼ 18; blue lines) is shown in comparison with that of AECs (n ¼7; green lines), AMCs (n¼6; red lines), and one line of BM-MSCs (BM-MSC1; orange line). (B) The number of CFU-F of BM-MSCs (second passage) and DMCs (second passage). n¼ 3, P, ANOVA; *, Po 0.01 (Dunnett’s test). Distribution is indicated by each box, and represents the first (25th percentile), second (50th percentile), and third (75th percentile) quartiles, with whiskers (the 10th and 90th percentiles). (C) Representative images of colonies derived from BM-MSCs and DMCs. Scale bar ¼5 mm (center), 200 mm (right).

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cells expressed both CK19 and vimentin, but not HLA-G (Fig. 3). From these properties, we defined these cells as AECs. The cells derived from the residual amnion, without AECs, showed morphology typical of fibroblast-like cells, and phalloidin staining showed the F-actin cytoskeleton extending throughout the entire cytoplasm, in a strong fibrillar pattern (Fig. 2). These cells expressed vimentin strongly, but not CK19 or HLA-G (Fig. 3). We concluded that these cells were of the mesenchymal linage, and defined them as AMCs. The cells derived from the decidua vera also showed a fibroblast-like cell morphology and a phalloidin staining pattern similar to that of the AMCs (Fig. 2). These cells also expressed vimentin but not CK19 or HLA-G (vimentin þ / CK19  /HLA-G  cells) (Fig. 3), suggesting they too were of the mesenchymal linage, and we defined them as DMCs.

3.3. Growth properties of the DMCs We compared the growth of the DMCs with that of the AECs, AMCs, and BM-MSCs. The AECs grew very poorly in 10% FBSsupplemented medium and reached a poor proliferation rate within several passages (Fig. 4A, green lines). Although several AMC (Fig. 4A, red lines) and adult BM-MSC (Fig. 4A, orange line) lines showed exponential growth for various periods of time, they also reached poor proliferation within 70 days in vitro (DIV). Other AMC lines showed worse growth, quickly reaching a poor proliferation rate, like the AECs (Fig. 4A, red lines). In contrast, all of the DMC lines showed reproducibly good growth, growing exponentially for almost 90 DIV, and gradually reaching poor proliferation after 120 DIV (Fig. 4A, blue lines). We estimated that the DMCs underwent 34.4 74.5 (mean 7S.D.) population doublings before reaching the poor proliferation rate. To evaluate the colony-forming ability of the DMCs, we performed CFU-F assays for two BM-MSC lines and three lines of DMCs (Fig. 4B and C). At the second passage, there were significantly more DMC52 than BM-MSC1 colonies at both high (1000 cells/dish) and low (500 cells/dish) cell densities (po0.01), while the other DMC lines and the BM-MSC2 showed similar colony numbers as the BM-MSC1 (Fig. 4B). All of the DMC colonies were compact formations at high cell densities, while those of BM-MSCs were more loose and sparse (Fig. 4C). These findings indicated that the cell growth properties were different between BM-MSCs and DMCs.

3.4. Cell-surface antigens of the DMCs FCM analysis was performed using exponentially growing DMCs (58.3 74.36 DIV, 6.1 71.73 passages; mean7S.D). Most of the DMCs (over 90%) highly expressed CD13, CD29, CD44, CD73, CD90, and CD166 antigens and HLA-ABC, and over 60% of them also expressed CD105 antigen (Fig. 5). In contrast, few cells (o3%) expressed CD14, CD19, or CD34 antigen or HLA-DR (Fig. 5). In addition, some cells expressed SSEA-4 or CD45 antigen, and over 50% of them also expressed anti-fibroblast antigen, but only a very minor population expressed CD271/low-affinity nerve growth factor receptor (LNGFR) (Fig. 5). 3.5. In situ identification and genotyping of the DMCs To examine the origin of the DMCs in situ, we examined placental tissue histologically by HE staining and immunohistochemistry. As shown in Fig. 1 (HE staining) the placental tissue consisted of three layers, which were – from the fetal to the maternal side – the amnion (Am), chorion (Ch), and decidua (De). We identified the chorion by its position in the central portion of the fetal membrane, its high cellularity, and by the cells’ strong expression of HLA-G, vimentin, and CK19 (Fig. 6A and B arrows). In the decidua vera, which lines the uterine wall, we found vimentinpositive cells that did not express either HLA-G or CK19 (Fig. 6A and B arrowheads). Based on their properties, we reasoned that these vimentin þ /CK19  /HLA-G  cells were DMCs in situ. To confirm that the DMCs were of pure maternal origin, we extracted the genomic DNA from six DMC lines and examined their genotypes for 15 STR loci plus Amelogenin, to identify the sex chromosome (Table 2). All the examined samples had homozygous or heterozygous alleles of the 16 loci, and none of the six STR genotypes derived from DMCs completely matched the genotypes from the amnion or UCB, which are derived from the fetus (Table 2). These findings indicated that each DMC line was derived from a single donor, and their origin was not fetal but maternal. 3.6. In vitro multilineage differentiation potential of the DMCs The in vitro differentiation analysis was performed using exponentially growing DMCs (57.472.34 DIV, 6 70.63 passages; mean7S.D.).

Fig. 5. Expression profiles of the DMC cell-surface antigens. Expression profiles of the cell-surface antigens of the DMCs (58.3 7 4.36 DIV, 6.1 71.73 passages; mean 7S.D) were examined using FCM, and representative results are shown in the figure. Closed histograms in gray represent the distribution of cells stained by the respective antibodies, and open histograms are the negative controls without staining. The bar shows the percentage of gated, positive cells. The rate of each marker is shown as the mean 7 S.D. (n¼ 8).

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specifically, collagen, type II, alpha 1(COL2A1), and collagen, type X, alpha 1 (COL10A1) (Fig. 7A). The expression of aggrecan (ACAN) was also significantly up-regulated in four of the six DMC lines (Fig. 7A). The two BM-MSCs lines also significantly expressed SOX9, ACAN, COL2A1, and COL10A1 after 4 weeks of differentiation (Fig. 7A). In addition, immunohistochemical analysis confirmed the induction of COL2A1 protein in all the cell pellets by 4 weeks of differentiation, to a level similar to that seen in BMMSCs (Fig. 7A). 3.6.2. Adipogenic differentiation capacity Although the expression of peroxisome proliferator–activated receptor gamma (PPARG), a master regulator of adipogenesis, was significantly up-regulated in five of the six DMC lines, to the same level as in two BM-MSC lines (Fig. 7B), the adipocyte differentiation-related genes adiponectin (ADIPOQ) and leptin (LEP) increased significantly in only three and two of the six DMC lines, respectively, after 4 weeks of differentiation. At the phenotypic level, a certain amount of lipoidal substance was revealed by oil red O staining in all six DMC lines after differentiation (Fig. 7B). However, the amount was modest compared with that in the BMMSCs (Fig. 7B). 3.6.3. Osteogenic differentiation capacity The expression of runt-related transcription factor 2 (RUNX2), a master regulator of osteogenesis, was significantly up-regulated in DMC line 39, but was not significantly changed in the others (Fig. 7C). The levels of osteoblast differentiation-related genes, alkaline phosphatase (ALPL), secreted protein, acidic, cysteinerich (SPARC; osteonectin) were also significantly up-regulated in four (ALPL), and five (SPARC) of the six DMC lines to various extents, but they were generally lower than in the BM-MSCs (Fig. 7C), and no up-regulation of secreted phosphoprotein 1 (SPP1) was observed in any of the six DMC lines (Fig. 7C). Alizarin red S staining clearly showed the precipitation of calcium in the two BM-MSC lines after differentiation, indicating mineralization of the cell surface during osteogenic differentiation; however, no such staining was observed on the induced DMCs, which looked the same as uninduced cells (Fig. 7C). 3.7. Gene expression profile comparison between DMCs and BM-MSCs

Fig. 6. In situ phenotypes of the DMCs in placental tissue. (A) Immunocytochemical analysis of full-term fetal membranes with the decidua vera, stained with anti-vimentin (green) and anti-CK19 (red) antibodies, or with anti-vimentin (green), and anti-HLA-G (red) antibodies. Nuclei are stained with TO-PRO-3 (blue). Negative control is also shown. *: fetal side, Scale bar ¼100 mm. (B) Highmagnification images of the areas in white boxes in (A). Arrows indicate nonDMC chorionic cells, and arrowheads show DMCs. Scale bar ¼ 10 mm.

3.6.1. Chondrogenic differentiation capacity qRT-PCR analysis showed that the expression of SRY-box 9 (SOX9), a master regulator of chondrogenesis, was significantly up-regulated in all six DMC lines after 4 weeks of differentiation, as were other chondrocyte differentiation-related genes,

To examine the differences between DMCs and BM-MSCs further, we analyzed four additional genes: TWIST1, TWIST2/ DERMO1, telomerase reverse transcriptase (TERT), and msh homeobox 2 (MSX2) with RUNX2, which were reported to be associated with early mesenchymal development and/or proliferative capacity, in the cells’ undifferentiated condition (Fig. 8A) (Psaltis et al., 2010; Isenmann et al., 2007, 2009; Matsubara et al., 2008). The DMCs expressed significantly higher TWIST2 (Po0.01) and RUNX2 (Po0.05) and lower MSX2 (Po0.01) levels than BMMSCs (Fig. 8A). Neither the DMCs nor the BM-MSCs expressed TERT, and there was no significant difference in TWIST1 expression between the DMCs and BM-MSCs (Fig. 8A). For a more detailed analysis, we obtained global geneexpression profiles of DMCs and BM-MSCs using microarray analysis (Fig. 8B). The two cell types showed similarity with a Pearson correlation coefficient of 0.949 (Fig. 8B), and we obtained a list of probe sets with more than 2-fold differential expression (Supplemental Tables 3 and 4). To confirm the microarray results, the expression levels of 19 selected genes were also examined by qRT-PCR analysis. DMCs expressed significantly higher levels of matrix metalloproteinases (MMP1, 3, 10, 11, and 12), tumor necrosis factor ligand superfamily 4 (TNFSF4),

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Table 2 Short tandem repeat (STR) genotyping of the DMCs. Locus

Lot 32

D3S1358 TH01 D21S11 D18S51 Penta_E D5S818 D13S317 D7S820 D16S539 CSF1PO Penta_D vWA D8S1179 TPOX FGA AMEL

34

39

48

49

52

Amnion

DMC

Amnion

DMC

UCB

DMC

UCB

DMC

Amnion

DMC

UCB

DMC

15, 16 7, 8 29, 30 14 16, 17 10, 11 8, 10 10 9, 12 10, 12 11, 12 17, 18 12 8, 11 23, 25 X

15, 17 6, 8 29, 31 14 16, 20 10 8, 12 8, 10 12, 14 10, 11 11, 12 14, 17 10, 12 9, 11 24, 25 X

15, 17 6, 7 30 16, 19 15, 20 12, 13 8, 11 11, 12 13 12 9, 10 14, 17 15, 16 8 24.2, 25 X

15 6, 9 29, 30 16, 21 13, 20 9, 12 11, 13 12 12, 13 12 9, 10 14, 18 13, 15 8 21, 24.2 X

15, 17 7, 8 29 14 5, 20 13 8, 9 11, 12 12 11, 12 9, 12 18 13, 14 9, 11 23 X, Y

16, 17 6, 7 29 12, 14 5, 20 10, 13 8, 9 11, 12 12, 13 12 9, 12 17, 18 11, 13 8, 11 23 X

15, 16 7, 9 30, 31 16, 19 5, 19 11, 12 8, 12 8, 12 10 10, 11 9, 10 14 11, 12 8, 9 23, 25 X, Y

15, 16 9 29, 31 13, 19 19, 21 9, 11 12 11, 12 10 11, 12 9, 11 14, 18 12, 13 8, 9 23, 25 X

17 7 29, 30 13, 17 11, 15 9, 11 11, 12 11 9, 12 11, 13 12 16, 18 12, 16 8 21, 23 X, Y

15, 17 7 29, 30 16, 17 10, 11 9, 13 8, 12 11 10, 12 12, 13 11, 12 18 10, 16 8 21, 23 X

15 7, 9 30, 32 13, 14 10, 11 10, 13 8, 9 9, 10 9 12 9, 13 14, 19 10, 11 8 23 X

15 6, 7 31, 32 13 10, 11 10, 12 9, 11 10, 11 9, 10 12, 14 9, 13 18, 19 11, 14 8 23 X

Fifteen polymorphic STR DNA loci plus Amelogenin (AMEL) for the sex choromosome were analyzed. DMC, decidua-derived mesenchymal cells; UCB, umbilical cord blood cells.

wingless-type MMTV integration site family member 2 (WNT2), bone morphogenetic protein 2 (BMP2), Platelet-derived growth factor subunit B (PDGFB), transforming growth factor beta 2 (TFGB2), aldehyde dehydrogenase 1 family A2 (ALDH1A2), and neuropilin-2 (NRP2) than BM-MSCs (Fig. 8C). On the other hand, DMCs expressed significantly lower levels of filaggrin (FLG), cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1), vascular cell adhesion molecule 1 (VCAM1), pentraxin3 (PTX3), interleukin 26 (IL26), fibroblast growth factor receptor 2 (FGFR2), hepatocyte growth factor (HGF), and neuregulin 1 than BM-MSCs (Fig. 8C).

4. Discussion 4.1. The DMCs are mesenchymal cells of purely maternal origin In this study, we successfully isolated DMCs from the decidua vera. The DMCs exhibited a typical fibroblast-like morphology and had an F-actin cytoskeleton that extended throughout the entire cytoplasm (Fig. 2). The DMCs strongly expressed a mesenchymal cell marker, vimentin, but not CK19 (an epithelial cell marker) or HLA-G, which belongs to the non-classical class Ib HLAs and is predominantly expressed on fetal-derived cells, like the chorionic trophoblasts (Fig. 3). In situ, these vimentin þ /CK19  /HLA-G  cells were identified in the side of the fetal adnexa adjacent to the uterus (Fig. 6), and genotyping analysis clearly showed that each DMC line was purely derived from a single donor that was not the fetus (Table 2). The expression pattern of CD antigens on the DMCs was almost identical to that on BM-MSCs (Fig. 5) (Dominici et al., 2006; Miao et al., 2006; Barlow et al., 2008), and in vitro assays showed that the DMCs could differentiate into chondrocytes and adipocytes, although their expression of osteoblastic markers was very limited (Fig. 7). These findings revealed that the DMCs, which are mesenchymal lineage cells of purely maternal origin, have MSC-like properties in placental tissue, although they have less multipotency than BM-MSCs (Parolini et al., 2008; Dominici et al., 2006; Barlow et al., 2008). The fetal membranes and decidua vera are major components of the fetal adnexa. They comprise three tissue layers from two different origins; that is, the amnion and chorion are from the

fetus, and the decidua is from the mother (Parolini et al., 2008; Ilancheran et al., 2009; Marcus and Woodbury, 2008). Many previous papers have reported the isolation of MSCs from fetal adnexal tissues. Some groups isolated MSCs from specific parts of the tissues, including the amnion (In’tAnker et al., 2004; Bailo et al., 2004; Portmann-Lanz et al., 2006; Alviano et al., 2007; Ilancheran et al., 2007), chorion (Bailo et al., 2004; Portmann-Lanz et al., 2006), chorionic villi (Igura et al., 2004; Zhang et al., 2006), villous stroma of the para-umbilical area (Wulf et al., 2004), and the internal portion of the placental lobules (Fukuchi et al., 2004). Others have isolated MSCs from mixed adnexal tissues, consisting of two or three layers (Zhang et al., 2004; Yen et al., 2005; Li et al., 2005; Miao et al., 2006; Battula et al., 2007; Soncini et al., 2007; Barlow et al., 2008). Although a few reports clearly showed that the isolated MSCs are of maternal origin (In’tAnker et al., 2004; Wulf et al., 2004; Barlow et al., 2008), many others did not investigate this matter thoroughly, and concluded that the MSCs isolated from placental tissues were of fetal origin. Others report that the genotype of placental MSCs examined by minisatellite polymorphism is altered after several passages (Soncini et al., 2007) and that proliferative capacity differs between cells of fetal versus maternal origin (In’tAnker et al., 2004). In a very preliminary phase of the present study, we used whole placental tissues or samples containing a mixture of chorion and decidua to isolate MSCs and simply designated the isolated cells as being of fetal origin, as in many previous reports. However, interestingly, even when these cells were isolated from whole tissues and genotyped after several passages, they turned out to be purely maternal in origin cells. To confirm and extend this finding, we modified the cell separation protocols as described in this paper. Although it was possible that chorionic tissues were present at the boundary of the amnion or decidua when the AMCs or DMCs were isolated, no HLA-G þ cells were observed in the DMC population (Fig. 3), and genotyping analysis clearly showed that the DMCs were of purely maternal origin. We think these findings indicate that the DMCs had a good proliferative ability compared with chorionic cells. Furthermore, our present results suggested that the DMCs had a better potential for in vitro expansion than the AMCs or AECs from the amnion, or the BM-MSCs, consistent with previous reports (In’tAnker et al., 2004; Barlow et al., 2008).

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Fig. 7. In vitro multipotency of the DMCs. The multipotency of the DMCs was examined using six different lines of DMCs (DMC 32, 34, 39, 48, 49, and 52) that had been cultured for 57.4 7 2.34 DIV, 6 70.63 passages at the beginning of the in vitro differentiation assay. The BM-MSC1 and BM-MSC2 had been cultured for 63 DIV and 7 passages, 40 DIV and 5 passages, respectively, at the beginning of the in vitro differentiation assay: (A) chondrogenic differentiation, (B) adipogenic differentiation, and (C) osteogenic differentiation. Graphs show the fold change in gene expression level determined by qRT-PCR (mean 7 S.D., n¼3), S9: SOX9, AC: ACAN, C2: COL2A1, C10: COL10A1, PG: PPARG, AD: ADIPOQ, LE: LEP, R2: RUNX2, AP: ALPL, SC: APARC, S1: SPP1. *: P o 0.01. Photographs show the results of collagen type-II immunostaining, with negative control and not-differentiated cells (Chondrogenesis, Scale bar ¼200 mm), Oil red O staining (differentiated and not differentiated) (Adipogenesis, Scale bar ¼50 mm), Alizarin red S staining (differentiated and not differentiated) (Osteogenesis, Scale bar ¼ 50 mm), respectively. After osteogenic differentiation, the DMC line 39 cells were too fragile to survive the Alizarin red S staining procedure.

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Fig. 8. Comparison of the transcriptomes of DMCs versus BM-MSCs. (A) Differences between DMCs and BM-MSCs in the expression level of genes associated with early mesenchymal development and/or proliferative capacity were confirmed by qRT-PCR analysis (mean7 S.D., n ¼3). Fold change values were calculated with reference to the transcript quantity in the BM-MSC sample. *; P o0.05, **; P o 0.01. (B) Scatter plot showing the comparison of the global gene expression between DMCs and BM-MSCs. P: Pearson Correlation coefficient value. The parallel lines represent 2- and 10-fold differences between samples. (C) Nineteen genes identified as having significantly different expression levels between DMCs and BM-MSCs were confirmed by qRT-PCR analysis (mean7 S.D., n¼3). All results showed statistically significant differences (Po 0.01).

Taken together, these findings indicate that, although cells isolated as MSCs from placental tissues may represent a heterogeneous population of fetal and maternal origin when first cultured, because the DMCs have the highest proliferative potential among the placental-derived mesenchymal cells, and because passaging selects for the fastest growing population, their population may become homogeneous with time in culture. The fetal and maternal contributions of placenta-derived MSCs must therefore be very carefully determined, using highly sensitive and reproducible methods, like genotyping. 4.2. The DMCs have MSC-like properties but differ from BM-MSCs The DMCs had several unique cellular properties. Although MSCs are usually defined as having tri-lineal multipotency, e.g. giving rise to osteogenic, adipogenic, and chondrogenic cells, several reports have indicated that this multipotency varies with the cell source. We found that the DMCs differentiated well into chondrocytes (Fig. 7A); similarly, MSCs derived from chorionic villi have a good chondrogenic capability (Zhang et al., 2006). The DMCs differentiated moderately into adipocytes (Fig. 7B), and MSCs derived from placenta or UCBs have low or no adipogenic capacity, in contrast to BM-MSCs or adipose tissue-derived MSCs (Barlow et al., 2008; Kern et al., 2006). The strong propensity to

differentiate into the chondrogenic lineage and modest ability to differentiate into the adipogenic lineage might be characteristic of MSCs derived from extra-embryonic tissues. It was also reported that the cells derived from human placental decidua basalis (PDBMSCs) have a multipotent differentiation potential (Huang et al., 2009). We think that the DMCs might resemble PDB-MSCs, since their cell shape and oil-drop formation upon adipogenic differentiation are very similar. In contrast, unlike BM-MSCs, the DMCs showed few osteogenic characteristics (Fig. 7C). Previous work showed that AECs had an altered phenotype in culture and a reduced osteogenic potential (Stadler et al., 2008). In our differentiation assays, we used DMCs in their exponential phase of growth, after about 57 DIV. This long culture time might have affected the differentiation potential of the DMCs, or their poor propensity osteogenic capability might be a differentiation property specific to DMCs. Overall, the differentiation capacity of the DMCs had MSC-like characteristics, but differed from that of BM-MSCs. The frequency of CFU-F in unfractionated bone marrow mononuclear cells was reported to be 12.672.2 per 105 cells plated (Psaltis et al., 2010). Although it is difficult to compare our results directly with these previously reported findings, because of the different culture protocols, our present findings indicate that the colony-forming ability of DMCs is at least equal to, and probably

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better than that of BM-MSCs (Fig. 4B and C). This growth property of DMCs may result in their high reproduction rate and exponential growth. The expression pattern of cell-surface antigens on the DMCs closely resembled that of BM-MSCs. However, one interesting exception was that a minor population of DMCs expressed CD45 antigen, which is well known as the leukocyte common antigen, i.e., it is present on all leukocytes. Although MSCs are thought not to express the CD45 antigen, several recent papers suggest that some non-hematopoietic and multilineage stem cells express it (Rogers et al., 2007; Kaiser et al., 2007). In the decidual tissues, decidual stromal cells (DSCs) have been identified (Oliver et al., 1999). It was shown that DSCs express vimentin strongly, like the DMCs, as well as CD45 antigen (Oliver et al., 1999). These findings suggest that the DMCs are in fact a heterogeneous population that contains a subpopulation of CD45-positive stem cells, like the DSCs. Since CD45-positive cells show enhanced chondrogenic marker gene expression in MSCs (Ahmed et al., 2006), the CD45-positive subpopulation of DMCs may contribute to their good chondrogenic differentiation. Moreover, the DMCs also expressed anti-fibroblast antigen, which is a surface membrane molecule expressed on human dermal fibroblasts (Singer et al., 1989). Several recent publications showed that human dermal-skin-derived fibroblasts have a multilineage differentiation potential like that of the BM-MSCs (Sudo et al., 2007; Lorenz et al., 2008). The expression of antifibroblast antigen on the DMCs might indicate some correlation or biological similarity between the DMCs and multipotent stem cells that exist in the skin. These points should be addressed in future studies. Our gene expression analyses also indicated differences between DMCs and BM-MSCs. Of the genes associated with early mesenchymal development and/or proliferative capacity (Psaltis et al., 2010; Isenmann et al., 2007, 2009; Matsubara et al., 2008), DMCs expressed higher TWIST2 and RUNX2 levels and a lower MSX2 level than BM-MSCs (Fig. 8A). Furthermore, microarray analysis showed that DMCs expressed higher levels of several MMPs (MMP1, 3, 10, and 12) and cytokines (BMP2 and TGFB2) and lower levels of IL26 and HGF than BM-MSCs (Fig. 8C, Supplemental Tables 3 and 4). These findings may represent important molecular properties of DMCs in additional to their endogenous cytokine production profiles. Further studies are needed to clarify and better understand these findings. 4.3. DMCs may be useful as allogeneic MSCs in regenerative medicine Although DMCs are not superior to BM-MSCs in multipotency, their greater proliferative ability means that their cultivation might require less maintenance, and their derivation from the maternal portion of the human fetal adnexal tissues, which are otherwise discarded, would resolve many ethical concerns associated with the use of embryonic stem cells. The high success rate of DMC isolation from tissues stored more than 24 h (Table 1) indicates that it might be feasible to develop a system for collecting or banking fetal adnexal tissue from multiple or even remote hospitals. Although the bacterial and/or fungal contamination rate was not negligible in the present study (Table 1), the contamination was probably introduced during vaginal delivery. The use of cesarean-delivered samples, or the institution of other quality controls, should reduce the pathogenic contamination and improve the DMCs. DMCs are already considered an attractive source of allogeneic MSCs as well as a potential cell source for generating humaninduced pluripotent stem (hiPS) cells and establishing hiPS cell banking systems (Kanemura, 2010). Furthermore, the pericellular

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matrix generated by the DMCs (PCM-DM) supports the growth and pluripotency of human embryonic stem cells and hiPS cells (Nagase et al., 2009; Kanemura, 2010). The PCM-DM should contribute to the establishment of xeno-free and feeder-free culture systems for hiPS cells. These unique properties of the DMCs give them several advantages for clinical use and make them an attractive alternative to allogeneic MSCs for regenerative medicine.

Acknowledgments We thank Ms. Chika Teramoto at Osaka National Hospital for her support in collecting the human fetal adnexal tissues and UCBs, and we thank all the staff members of our laboratories. This study was supported by the Project for the Realization of Regenerative Medicine, from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and the Cooperative Link of Unique Science and Technology for Economy Revitalization (CLUSTER) project from MEXT, Japan.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.diff.2011.05.010.

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