Human Placenta-Derived CD146-Positive Mesenchymal Stromal Cells Display a Distinct Osteogenic Differentiation Potential

Abstract

Mesenchymal stromal cells (MSCs) are multipotent cells that can be differentiated in vitro into a variety of cell types, including adipocytes or osteoblasts. Our recent studies indicated that a high expression of CD146 on MSCs from bone marrow correlates with their robust osteogenic differentiation potential. We therefore investigated if expression of CD146 on MSCs from the placenta correlates with a similar osteogenic differentiation potential. The MSCs were isolated specifically from the endometrial and fetal parts of human term placenta and expanded in separate cultures and compared with MSCs from bone marrow as controls. The expression of cell surface antigens was investigated by flow cytometry. Differentiation of MSCs was documented by cytochemistry and analysis of typical lineage marker genes. CD146-positive MSCs were separated from CD146-negative cells by magnet-assisted cell sorts (MACS). We report that the expression of CD146 is associated with a higher osteogenic differentiation potential in human placenta-derived MSCs (pMSCs) and the CD146^pos pMSCs generated a mineralized extracellular matrix, whereas the CD146^neg pMSCs failed to do so. In contrast, adipogenic and chondrogenic differentiation of pMSCs was not different in CD146^pos compared with CD146^neg pMSCs. Upon enrichment of pMSCs by MACS, the CD146^neg and CD146^pos populations maintained their expression levels for this antigen for several passages in vitro. We conclude that CD146^pos pMSCs either respond to osteogenic stimuli more vividly or, alternatively, CD146^pos pMSCs present a pMSC subset that is predetermined to differentiate into osteoblasts.

Introduction

Human mesenchymal stromal cells (MSCs) are multipotent cells found in different tissues of the adult body, and MSCs from the bone marrow seem to be the reference stem cells in this field [1,2]. They express a variety of cell surface antigens, including CD29, CD73, CD90, CD140b, CD105, CD146, and CD166, but lack expression of antigens characteristic for hematopoietic or endothelial cells, such as CD11b, CD14, CD34, CD31, CD45, or CD133 [3 –8]. Widely used protocols for preparation of MSCs from the bone marrow (bmMSCs) start with a crude mix of cells and debris in suspension. The mononuclear cells are enriched by gradient centrifugation, followed by expansion in suitable media [3,9]. Despite intensive research, a unique MSC-defining epitope is not known [3,10 –13]. However, the bright expression of nerve growth factor receptor (CD271), frizzled-9 (CD349), and tissue nonspecific alkaline phosphatase (TNAP) determined early differentiation stages of bmMSCs [14,15]. In addition to these antigens, expression of the stage-specific embryonic antigen 4 (SSEA-4), CD146, fibroblast activation protein-α (FAP), and sushi domain containing 2 (SUSD2) were suggested as marker antigens for bmMSCs [2,4,13,16 –18].

Some reagents, including monoclonal antibodies (mAb) W3D5 and W5C5 to SUSD2 [18], W8B2 (=MSCA-1 antigen) to TNAP [19], or to the molecules detectable by mAb clones W5C4, W12D1, or 58B1, generated distinct histogram patterns, indicating that bmMSCs ex vivo present as a complex blend of different cells [18,20,21]. These bmMSC subsets were further separated ex vivo by FACS, and the CD271⁺CD56⁺ subset had a more prominent chondrogenic potential compared with the CD271⁺CD56⁻ fraction, whereas this CD271⁺CD56⁻ population yielded more adipogenic cells, and the osteogenic potential of bmMSCs was high and not different in these fractions [15]. The CD271⁺ fraction of periosteum-derived progenitor cells contained the osteoblast precursor cells, which deposited a mineralized matrix, whereas the CD271⁻ subset failed to do so [22]. For pMSCs, functionally different subsets of cells were not defined or separated to a comparable extent.

Compared with bmMSCs, bulk MSCs or MSC subsets isolated from sources other than the bone marrow may differ in their regenerative potential [8]. We recently provided evidence that bmMSCs express CD146, alkaline phosphatase, and Runt-related transcription factor 2 (Runx2) significantly higher than pMSCs and this correlated with a better osteogenic differentiation capacity of bmMSCs [23,24]. CD146, also referred to as melanoma adhesion molecule (MCAM), cell surface glycoprotein MUC18, or S-endo1 antigen, is a cell adhesion molecule expressed on endothelial cells [25], B and T lymphocytes [26], smooth muscle cells [27,28], pericytes [29], and its expression has been described on MSCs from different sources [4,7,30 –34]. It binds to laminin α4 and thereby contributes to the interaction of cells to the basal lamina of vessels [35] and to the evasation of cells [36].

CD146 has been considered a key marker for differentiation-competent MSCs [4,33], but it has not been described as an antigen that can facilitate enrichment or depletion of MSCs based on their osteogenic differentiation capacity. We therefore extended our recent studies [23,24] and investigated if expression of CD146 on pMSCs also correlated with a higher osteogenic potential. In this study, we report that the CD146⁺ pMSCs generated osteoblasts in vitro upon osteogenic induction, whereas CD146⁻ pMSCs failed to do so. This finding may be important for tissue engineering of soft tissue to reduce unwanted pro-osteogenic cells.

Materials and Methods

Preparation of MSCs

MSCs were isolated from normal, healthy human term placenta (from n>10 donors) as described recently [23,24]. The endometrial maternal part of the placenta was separated from the fetal part to enrich for maternal (pmMSC) and fetal (pfMSC) subsets of placenta MSCs (pMSCs). The tissue was minced and digested by proteolysis for 1h at 37°C (12 U/mL collagenase type XI, SIGMA-Aldrich, Taufkirchen, Germany; 2.4 U/mL Dispase II; Roche, Mannheim, Germany). Proteolysis was stopped by addition of FBS (final concentration 1% vol/vol), and debris was removed by filtration of the cell extract. Live mononuclear cells were enriched by gradient centrifugation (Ficoll, ρ=1.078 g/mL; GE Healthcare, Freiburg, Germany, 20 min 400 g, 20°C), washed twice with PBS, and incubated for 24 h (37°C, 5% CO₂, humidified cell incubator) in complete MSC expansion medium (Lonza, Basle, Switzerland) or in a GMP-compliant medium containing 5% pooled human serum and 5% human platelet lysate (both from the Red Cross and Blood Bank at UKT, Tübingen), HEPES, heparin, l-glutamine, and antibiotics as described [37]. After 24 h, nonadherent cells were removed and the adherent cells were expanded in GMP-compliant DMEM for 1 week. The adherent cells were harvested using trypsin-EDTA (Sigma-Aldrich, St. Louis, MO), counted, and first passage cells were inoculated at a starting density of 2,000 cells/cm², and expanded in GMP medium. Unless otherwise noted, cells in the second passage were used for the experiments.

Bone marrow-derived MSCs (bmMSCs, from n>10 donors) were prepared as described recently [38] and expanded as illustrated above. All tissue samples were obtained from volunteers after informed consent. The study was approved by the Ethics Committee of the University of Tübingen.

Flow cytometry and cell sorts

Expression of cell surface antigens on MSCs was explored by flow cytometry as described recently [6]. The cells were detached by mild proteolysis (Accutase; Sigma-Aldrich) and counted with the aid of a hematocytometer, and 2×10⁵ MSCs were incubated with diluted preimmune IgG (Gamunex (50 μL, 4°C, 20 min; Grifols, Barcelona, Spain) to reduce unspecific binding of antibodies [6], washed, and resuspended in 10 μL PFEA buffer (PBS containing 2% FBS, 2 mM EDTA, and 0.01% sodium azide). Then, the following mAbs were added to the MSCs according to the supplier's recommendations: anti-CD14-FITC (clone MΦP9; BD Bioscience, Franklin Lakes, NJ); anti-CD34-PE (clone 4H11; Biolegend, San Diego, CA); anti-CD45-APC (clone 2D1; R&D Systems, Minneapolis, MN); anti-CD73-PE (clone: AD2; BD Biosciences); anti-CD90-PE (clone: Thy-1A1; R&D Systems); anti-CD105-FITC (clone: SN6; AdD Serotec, Martinsried, FRG), anti-CD146 (clone: 128018; R&D Systems), or anti-SUSD2 (clone W5D5, generous gift of Dr. Bühring) [18]. After incubation for 30 min on ice, the cells were washed twice with cold PFEA buffer, and binding of unlabeled primary antibodies to the MSCs was visualized by counterstaining of the cells with fluorochrome-labeled goat anti-mouse IgG (30 min on ice, 1:100; Jackson ImmunoResearch, Newmarket, Suffolk, GB). Staining of cells with this secondary antibody alone served as the control.

The samples were analyzed by flow cytometry (LSRII; BD Biosciences). Before each experiment, mouse or hamster/rat κ-chain Comp Beads (BD Biosciences) were reacted with the corresponding fluorochrome-labeled antibodies and incubated for 20 min at room temperature in the dark. Negative Comp Beads were used as an unstained negative control (BD Biosciences). After washing with PFEA, the beads were resuspended in 200 μL PFEA to allow automatic compensation with the BD FACSDiva acquisition software. The MSCs were then measured accordingly. All data were processed and analyzed using FACSDiva and FlowJo 7.2.2 (Treestar, Inc., Ashland, OR) following recently updated guidelines [39]. Flow cytometry data were computed as geometrical means of fluorescence intensity (MFI).

Separation of MSCs by magnetic-activated cell sort

The MSCs were detached by Accutase as described above and washed in MACS^® running buffer (0.5% BSA, 2 mM EDTA in PBS; Miltenyi Biotech, Bergisch Gladbach, FRG), and 10⁷ MSCs were blocked by incubation of the cells in Gamunex (50 μL, 4°C, 20 min; Grifols). The MSCs were rinsed in MACS running buffer (Miltenyi Biotech) and reacted with the PE-labeled anti-CD146 mAB (clone: 128018; 10 μL mAB per 10⁷ MSCs; R&D Systems). After washing, MSCs were reacted with 20 μL anti-PE magnetic beads, rinsed again, resuspended in MACS running buffer, and separated in an autoMACS separator (Miltenyi Biotech) as described by the supplier utilizing the programs, possel and possel s, for enrichment of the CD146^pos MSCs. After magnetic separation, the MSCs were analyzed by flow cytometry for enrichment of CD146^pos cells. Depending on the experimental setup, MSCs were then expanded in GMP-compliant medium in separate cultures (CD146^high, CD146^dim, CD146^low) for later analyses or were incubated in differentiation medium to investigate their trilineage differentiation potential.

Determination of telomere lengths

The lengths of telomeres of pmMSCs and pfMSCs were determined by hybridization of FITC-labeled DNA oligonucleotides to the repetitive telomere motive [.TTAGGG_(n).] with the aid of the Telomere PNA Kit following the instructions of the supplier (Dako, Glostrup, Denmark). As a reference, telomeres of T lymphoma 1301 were included. The nuclei of all cells were counterstained by propidium iodide. The mean of fluorescence intensity of FITC-labeled telomeres of MSCs was determined relative to the telomere length of T lymphoma 1301 (ie, 30 kb) by flow cytometry as described above.

Differentiation of MSCs in vitro and cytochemical staining

The differentiation of MSCs after expansion in the GMP-compliant medium was investigated as described recently [1]. Adipogenic differentiation of MSCs was induced by an adipogenic differentiation medium supplemented with 10 μg/mL insulin, 100 μM indomethacin, 0.5 mM isobutyl xanthine, and 1 μM dexamethasone. Osteogenic differentiation was achieved by an osteogenic differentiation medium containing 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and 50 μM ascorbic acid, and chondrogenic differentiation was performed in micromass pellet cultures in a serum-free chondrogenic differentiation medium containing 50 mM ascorbic acid, 0.1 M L-proline, 1 mM dexamethasone, and 10 ng/mL TGF-β3. Unless stated otherwise, the MSCs were incubated in the respective differentiation media for 4 weeks. The differentiation media were refreshed twice a week, and progress of differentiation was monitored as described [40,41]; adipocytes were stained utilizing the Oil Red O method, mineralization by osteoblasts was detected by von Kossa staining, and generation of proteoglycans by chondrocytes was documented in sections of the micromass cultures by Alcian Blue dye.

Immunocytochemistry and immunohistochemistry

To detect expression of CD146 on human MSCs, immunocytochemistry (ICC) was performed. The MSCs were seeded in eight-well PCA chamber slides (Sarstedt, Nümbrecht, Germany) at a starting density of 15,000 MCSs/well and inoculated for 18 h to reach the optimal density. Then, the cells were washed twice with cold PBS, fixed (cold ethanol, 15 min), and washed twice with PBS again. Then, cells were incubated for 1 h at ambient temperature with blocking solution (1% BSA in PBS), followed by incubation in antibody staining solution to detect CD146 (anti-CD146-PE, mouse mAB IgG1, clone 128018, 1:100 in PBS/BSA; R&D Systems) or Thy-1 (anti-CD90-FITC, mouse mAb IgG2a, clone Thy1A1, 1:100 in PBS/BSA; Dianova, Hamburg, Germany), for 4 h at ambient temperature in a humidified chamber in the dark.

To detect CD146⁺ cells in the placenta, in situ immunohistochemistry (IHC) was performed. The tissue samples were prepared, soaked in Tissue Tec (Sakura Finetek, Alphen aan den Rijn, The Netherlands), shock-frozen in liquid nitrogen, and stored in airtight tubes for later use at −70°C. Cryosections were prepared (7 mm, −15°C) and the samples were mounted on SuperFrost slides (Menzel, Braunschweig, Germany), fixed with cold acetone (−20°C), air-dried, and stained. The samples were washed twice with PBS, preincubated with 1% BSA in PBS, and then stained with PE-labeled anti-CD146 mAb (clone 128018, 1:100; R&D Systems) and FITC-labeled anti-NG2 mAb (mouse mAB IgG1, clone LHM2, 1: 200 in 1% BSA/PBS; Santa Cruz Biotechnology, Dallas, TX). After incubation (16 h, 4°C, humidified chamber), unbound primary antibodies were rinsed away and the samples were incubated for 30 min in BSA/PBS enriched with DAPI (1:500) to visualize the nuclei. Staining with PE-conjugated goat anti-mouse IgG (1:100 in 1%BSA/PBS; Jackson ImmunoResearch) alone served as the control. The slides were covered, sealed with clear nail polish, evaluated, and recorded microscopically (Zeiss, LSM, Oberkochen, Germany) utilizing the AxioVision and Zen software (Zeiss).

Statistics

To obtain the mean values of results from individual experiments, all data were processed by spreadsheet software (Excel^®, Microsoft). To evaluate differences between experimental groups, a two-sided t-test was performed, and probability values (P) below 0.05 were considered to indicate statistical significance.

Results

Characterization of human term placenta-derived MSCs

The MSCs were isolated from human term placenta as described recently [23,24] and characterized [3,5]. MSCs from the maternal and fetal parts expressed CD73, CD90, and CD105, but not CD14, CD34, or CD45. There was no difference in expression of these MSC marker antigens between placenta-derived maternal MSCs (pmMSC, Fig. 1A–F) versus placenta-derived fetal MSCs (pfMSC, Fig. 1A′–F′). However, the proliferation rates of pfMSCs were higher (20%, P<0.03, n=5 each), telomere lengths were longer (1.29-fold, P<0.008, n=4 each), and expression of the SUSD2 protein was elevated (P<0.04, n=9, not shown) in pfMSCs compared with the corresponding pmMSCs, corroborating that two distinct pMSC populations were prepared from human term placenta.

FIG. 1.

Expression of cell surface antigens on human placenta-derived mesenchymal stromal cells (pMSCs). MSCs were detached and incubated with mAB to CD73 (A, A′), CD90 (B, B′), CD105 (C, C′), CD14 (D, D′), CD34 (E, E′), and CD45 (F, F′) and analyzed by flow cytometry to explore if the pmMSCs (A–F) and pfMSCs (A′–F′) met the quality criteria defined by the International Society for Cellular Therapy (ISCT) consensus conference [3]. The results document that both populations express the mesenchymal antigens, but not hematopoietic or endothelial antigens. Numbers added to the histograms illustrate the percentage of cell staining in the given gated area (horizontal bars). The dotted histograms present the MSCs incubated with secondary antibodies only, the solid black histograms document the MSCs reacted with the respective primary antibodies.

To investigate the differentiation potential of pmMSCs and pfMSCs, adipogenic, osteogenic, and chondrogenic differentiation were induced in vitro. Adipocytic differentiation of pmMSCs (Fig. 2B) and pfMSCs (Fig. 2B′) was visualized by Oil Red O staining, osteogenic differentiation was detected by von Kossa staining (Fig. 2C, C′), and chondrogenesis by Alcian Blue staining of micromasses (Fig. 2D, D′). Differentiation of bmMSCs served as controls (Fig. 2B°–D°). Overall, there was no obvious difference in adipogenic, osteogenic, or chondrogenic differentiation between pmMSCs and pfMSCs. However, the osteogenic differentiation potential of pmMSCs and pfMSCs (Fig. 2C, C′) was rather low compared with bmMSCs (Fig. 2C°), confirming our recent results [23,24].

FIG. 2.

Exploring the trilineage differentiation capacity of human pMSCs. MSCs were harvested and incubated in six-well plates (osteogenesis, adipogenesis) or round-bottom 96-well plates to induce differentiation by addition of the respective differentiation media for 4 weeks. MSCs incubated in expansion medium served as control cells (A, A′, A°). Adipogenesis was detected by Oil Red O staining (B, B′, B°), osteogenesis by von Kossa staining (C, C′, C°), and chondrogenesis by Alcian Blue stain (D, D′, D°). Trilineage differentiation capacity was recorded in bulk pmMSCs (A–D) and pfMSCs (A′–D′), but the osteogenic potential in bmMSCs (A°–D°) was more prominent than in pmMSCs and pfMSCs, corroborating our recent study [23]. Color images available online at www.liebertpub.com/scd

Expression of CD146 on placenta-derived MSCs

Next, we investigated the expression of CD146 on pMSCs over time. Early passage pmMSCs and pfMSCs were separated by magnetic sort (MACS) to generate CD146^high, CD146^dim, and CD146^low fractions, and the efficacy of the separation was confirmed by flow cytometry immediately after cell sorting (Fig. 3). The pmMSCs and pfMSCs, which were not retained in the MACS columns by magnetic anti-CD146 mAb, were confirmed not to display a robust CD146 signal by flow cytometry (Fig. 3A, A′). The CD146^dim and CD146^high fractions of pmMSCs (Fig. 3B, C) and pfMSCs (Fig. 3B′, C′) yielded higher fluorescence intensities, indicating that the MACS technology enriched CD146^pos and CD146^low subsets from suitable pMSC populations with sufficient efficacy.

FIG. 3.

Separation of pMSC subsets by a monoclonal antibody to CD146. Human pmMSCs (A–C) or pfMSCs (A′–C′) were incubated with anti-CD146 and with a paramagnetic detection reagent. The cells were separated by MACS to yield three subsets: the run-through (unbound) fraction defining the CD146^low MSCs (A, A′), the CD146^dim fraction eluted after mild rinsing of the column (B, B′), and the CD146^high cells bound to the column (C, C′). After magnetic sorting, an aliquot of the cells was analyzed by flow cytometry to explore the success of separation (solid black histograms). MSCs incubated with secondary antibodies only served as controls (dotted black histograms). Cells of the 2×3 individual subsets were expanded in separate cultures for 4 more weeks, and expression of CD146 was investigated again (solid gray histograms). In both, pmMSCs (A–C) and pfMSCs (A′–C′), the expression of CD146 remained basically stable over 4 weeks and showed a similar histogram to those recorded immediately after cell sorting. To avoid confusion, the gating areas and dotted gray histograms of the staining control after further expansion are not included. Figure 3 is representative for three consecutive experiments.

As expression of CD146 was possibly changing in time during continued culture of MSCs in vitro, the CD146^high, CD146^dim, and CD146^low fractions of pmMSCs and pfMSCs were expanded over four more passages in individual cultures to investigate if the expression of CD146 changed significantly over time. The results indicated that CD146^low MSCs did not acquire a high expression of this cell surface antigen, whereas the CD146^dim or CD146^high pMSCs maintained expression of CD146 (Fig. 3).

Differentiation potential of CD146^high and CD146^low placenta-derived MSCs

To investigate if the expression of CD146 on pMSCs correlated with their osteogenic differentiation potential, pmMSCs and pfMSCs were separated by MACS to generate CD146^high and CD146^low subsets (Fig. 3) and expanded in individual cultures. Spontaneous osteogenesis was not observed in the CD146^high subsets of pmMSCs (Fig. 4A, B) and pfMSCs (Fig. 4A′, B′), but the CD146^high subsets of pmMSCs (Fig. 4C, D) and pfMSCs (Fig. 4C′, D′) underwent moderate osteogenic differentiation in the osteogenic differentiation medium and mineralization was observed. However, CD146^high pfMSCs and pmMSCs did not deposit the significant amounts of mineralized extracellular matrix after induction of osteogenesis to yield the intensities and patterns of von Kossa staining that were observed with bmMSCs (Fig. 2C°). Similar to CD146^high pMSC subsets, spontaneous osteogenesis was not recorded in the CD146^low subsets of pmMSCs (Fig. 4E, F) and pfMSCs (Fig. 4E′, F′). In contrast to the CD146^high pMSCs, mineralization following incubation in osteogenic differentiation medium was also not observed in the CD146^low subsets of pmMSCs (Fig. 4G, H) and pfMSCs Fig. 4G′, H′).

FIG. 4.

Exploring the osteogenic differentiaton in CD146^high and CD146^low subsets of pMSCs. The pmMSCs (A - H) and the pfMSCs (A′–H′) were separated to yield CD146^high (A-D′) and CD146^low (E-H′) subsets of MSCs. The individual cells were incubated in expansion medium and stained by von Kossa reagents to detect spontaneous ossification. Spontaneous mineralization in CD146^high pmMSCs (A) or pfMSCs (A′) was not observed, nor observed by microscopy in pmMSCs (B) and pfMSCs (B′). Upon incubation in osteogenic differentiation medium for 4 weeks, the CD146^high pmMSCs (C) and pfMSCs (C′) displayed a moderate von Kossa stain, and black silver grains were visualized by microscopy in pmMSCs (D) and pfMSCs (D′). Comparably, no sign of spontaneous osteogenesis was found in CD146^low pmMSCs (E, F) and CD146^low pfMSCs (E′, F′) after incubation in expansion medium. In contrast to the CD146^high pmMSCs and pfMSCs (C–D′), mineralization was not detected after 4 weeks of osteogenic differentiation in the CD146^low pmMSC (G, H) and CD146^low pfMSC (G′, H′) subsets. Figure 4 is representative for three individual experiments.

To test if the general differentiation capacity of the CD146^high versus the CD146^low subsets of pMSCs was distinct, we next explored their adipogenic and chondrogenic differentiation capacities as well. Significant differences in adipogenesis (Fig. 5A–C) and chondrogenesis (Fig. 5D–F) were not seen in bulk pMSCs (Fig. A, D) compared with both the CD146^high (Fig. B, E) and CD146^low (Fig. D, F) subsets of pMSCs. We conclude that the expression of CD146 on pMSCs correlates specifically with an elevated osteogenic differentiation potential.

FIG. 5.

Exploring the adipogenic and chondrogenic differentiaton potential in CD146^high and CD146^low pMSCs. pMSCs were expanded in vitro and either separated by MACS to generate CD146^high and CD146^low subsets or maintained as unseparated bulk cultures. Adipogenic or chondrogenic differentiation was induced by addition of the specific differentiation media for 4 weeks, and success of differentiation of unseparated bulk pMSCs (A, D), CD146^high (B, E), and CD146^low (C, F) pMSCs was investigated utilizing the Oil Red O (A–C) or Alcian Blue (D–E) staining methods, respectively. Color images available online at www.liebertpub.com/scd

Detection of CD146 on human MSCs in vitro and its localization in situ

The expression of CD146 on characterized human pMSCs in vitro was visualized by ICC on cells in the second passage of in vitro culture (Fig. 6). However, ICC is a qualitative method and signal intensities obtained by ICC do not allow an exact quantification of protein expression. Expression of CD146 in placenta tissue was also explored by IHC, and cells surrounding small vessels were stained with NG2, an antibody reactive with pericytes, MSCs, and related mesenchymal cells [4,28,31]. Expression of CD146 was confirmed in placenta tissue in close proximity of NG2 signals, suggesting that perivascular cells expressed CD146 in situ (Fig. 6).

FIG. 6.

Detection of CD146 on MSCs in vitro and its localization in placenta tissue. Human pMSCs were expanded and seeded in chamber slides (top), stained with FITC-labeled mAb to CD90 (A) or PE-labeled mAB to CD146 (B), and counterstained with DAPI to visualize the nuclei. Sections of human placenta (bottom) were reacted with PE-labeled mAb to CD146 and FITC-labeled mAb to NG2 (C). Nuclei were counterstained with DAPI. Expression of CD146 was seen around larger lacunae of placenta and smaller vessels (marked by *). Expression of the NG2 antigen was observed in the tissue and more prominently around small vessels. Samples stained with FITC-labeled secondary antibody and DAPI only served as controls (D). Size bar=100 μm. Color images available online at www.liebertpub.com/scd

Discussion

CD146 was originally described to be primarily expressed at the intercellular junction of endothelial cells and plays a role in cell–cell and cell–matrix interaction and in angiogenesis [42]. Recent evidence indicates that CD146 is a multifaceted molecule actively involved in miscellaneous processes, such as development, signaling transduction, cell migration, and MSC differentiation [43]. Recently, CD146⁺ periodontal ligament stem cells (PDLCs) showed a greater proliferative and osteogenic potential than CD146⁻ PDLCs [44]. Our previous studies provided evidence on differences in expression of CD146 and correlated this with an osteogenic potential [23]. This study complements these previous reports very well in several aspects. In this study, we confirm that expression of CD146 correlates with the osteogenic differentiation capacity not only in human bmMSCs as shown previously [2,4] but also in MSCs from human term placenta. This includes the MSCs derived from both the maternal and fetal sections of the placenta and is also in line with our recent studies [23,24]. For bmMSCs, CD146 has been considered to be an indicator of stemness since its expression correlated with the trilineage differentiation capacity of bmMSC clones [33]. However, for pMSCs, we did not observe significant differences in the adipogenic or chondrogenic differentiation of CD146^pos compared with CD146^neg pMSCs. In addition, in our recent experiments, all human bmMSCs expressed CD146 at a bright signal intensity [23,24]. We still attempted to separate human bmMSCs in CD146^pos or CD146^neg subsets by FACS, but functionally different subsets of bmMSCs were not obtained (data not shown), supporting recent results [28]. Therefore, the default setting for differentiation of bmMSCs tends to go toward the osteogenic lineage [2], and in about a third of samples investigated, spontaneous ossification of bmMSCs was observed upon injection of the cells in muscle tissue [45]. The ostensive conflict of results in these details—correlation of CD146 with osteogenesis, but to a lesser extent adipogenesis and chondrogenesis of MSCs—may be explained by a recent finding, which provided evidence that knockdown of CD146 in MSCs significantly reduced the expression of genes associated with osteogenesis and adipogenesis, but overexpression of CD146 enhanced osteogenesis without facilitating adipogenesis [46]. In this sense, CD146 seems not to describe a distinct lineage of MSCs, but its intensity of expression appears to be an indicator of its osteogenic differentiation potential.

In the present study, expression of CD146 was detected on human MSCs in situ, ex vivo, and after primary culture. Expression of CD146 was previously shown to be elevated on bmMSCs [4] and also expressed on pMSCs [23], but was not detected on fibroblasts [4]. Therefore, this antigen may allow the discrimination of differentiation-competent MSCs from contaminating fibroblasts. Expression of CD146 on bmMSCs and pMSCs does vary among samples from different donors and also depends to some degree on cell culture conditions [23,47]. Enrichment of CD146^high bmMSCs yielded cells with a significantly elevated production of proteoglycans compared with the CD146^low subset, whereas osteogenesis and adipogenesis remained unchanged [47]. In our hands, separation of CD146^high pMSCs from CD146^low pMSCs facilitated an enrichment of osteogenic, but not adipogenic or chondrogenic, subsets of pMSCs. Recently, others reported that CD146⁺ endometrial stem cells gave rise not only to mesenchymal cells such osteoblast and adipocytes but may also have an even wider differentiation capacity [48]. This suggests that CD146 plays a role in defining functional subsets of MSCs at least upon expansion in vitro. At the same time, it also suggests that MSCs from different niches or tissues differ to some degree in their expression of receptors and the processing of such receptor signals.

Technical differences, such as the composition of the expansion media in the experimental conditions, may also explain the dissimilarities in results obtained. Adult stem cells typically remain in a quiescent stage and do not necessarily proliferate [49 –52]. Cloning of MSCs from primary culture may therefore yield clones of cells different from bulk cultures as overgrowth of mitotically active cells over slow growing cells is avoided. Therefore, by cloning MSCs ex vivo or early on in vitro, different subtypes within the MSC population may be resolved [28,33]. In addition, in many investigations, media containing bovine serum were employed for expansion of bmMSCs [28,32,33], and it is known that the composition of the expansion media influences the phenotype, mitotic activity, and differentiation potential of such cells considerably [37,41,53 –55]. We expanded the MSCs in a fully GMP-compliant medium complemented with human serum and platelet lysate [37]. We therefore may yield primarily trilineage-competent CD146^pos bmMSCs and at the same time may lose the less differentiation-competent CD146^low bmMSCs or other CD146⁻ mesenchymal cells that possibly contaminated the bulk MSC cultures [4].

Distinct subsets of bmMSCs have been described in ex vivo analyses [12,15,18 –20,56] as well as after expansion in vitro [33,53,57] and by in vivo functional analyses [2]. Expression of CD271, alkaline phosphatase (TNAP/MSCA-1), and CD56 on human bmMSCs ex vivo correlated with higher clonogenic and chondrogenic capacities, whereas the CD271⁺,TNAP⁺,CD56⁻ population preferably gave rise to adipocytes and yielded chondrocytes with a rather low efficacy [15]. Expression of CD146 on MSCs correlated with self-renewal, regeneration of bone in vivo, and with their potential to organize a stem cell niche for hematopoiesis [4,46]. These reports provide evidence that cell surface antigens can determine physiological differences between distinct subsets of cells.

Specific differences between bulk MSCs isolated from different sources such as bone marrow versus adipose tissue, placenta, and others have been described as well [1,5,13,16,30,32,58 –62], and phenotypic changes during continued in vitro culture were noticed early on [63,64]. Therefore, the correlation of a distinct cell surface marker with a given MSC population may be related to the source of the cell as well, not to mention interindividual differences between human donors or between cells obtained from different species.

In contrast to bmMSCs, which presented as CD146^high cells in our hands, MSCs from human term placenta resulted in a larger population of CD146^dim or CD146^low cells as detected in in vitro cultures [6]. The isolation of MSCs from solid tissues requires several steps of dissection and mincing, followed by enzymatic degradation, filtration, and purification. It is very likely that fibroblast-like cells, smooth muscle cells, or pericytes may be prepared when MSCs are isolated from such sources. By microscopy and flow cytometry, contaminating mesenchymal cells cannot be discriminated from true MSCs when simple criteria are administered to define the stem cells [3]. Sophisticated analyses exploring the expression of several cell surface antigens or lineage markers may help to better define truly differentiation-competent MSCs [31,32]. The CD146^low cells investigated in this study seem to be MSC-like cells with a two-lineage differentiation potential as the CD146^low pMSCs generated chondrocytes and adipocytes, but not osteoblasts in vitro. Accordingly, the CD146^high cells represent the MSCs with a trilineage differentiation capacity. Again, this is in line with the results reported from bmMSCs and PDLCs recently [4,33,44].

A technical aspect not discussed yet also merits critical examination. The preparation of subsets of human MSCs by FACS or MACS clearly differs in the yield of cells obtained by separation, in efficacy and purity of the subsets generated, and the viability of the cells acquired. For this study, the MACS technique was preferred as the yield and viability of pMSCs were higher, but, of course, at the cost of the purity of the cells gained. This decision was mainly made due to the fact that many cells were needed for the subsequent expansion and differentiation experiments. In particular, chondrogenesis required many cells for the micromass cell cultures. Therefore, in this study, the term separation does not mean an absolute distinction between CD146^pos and CD146^neg cells, but it rather reflects a relative enrichment of one over the other population. In contrast to many other lineage markers, including CD73, CD90, and CD105, expression of CD146 on human pMSCs generated in many samples a somewhat wide histogram [23], indicating that different cells expressed this antigen to a quite variable extent. A stringent sorting by FACS might dissect functional subsets, but this requires prolonged expansion to generate the number of cells needed. Still, to the best of our knowledge, we show for the first time that CD146 can be used to functionally separate pMSCs into an osteogenic subset and a nonosteogenic subset. The expression of CD146 on pMSCs seems to be stable at least over a few passages as seen in this study. This finding grants new aspects for tissue engineering or clinical purposes: if osteogenic cells have to be depleted (eg, in soft tissue engineering/regenerative medicine), the pro-osteogenic cells can be reduced by MACS or FACS using a CD146 mAb without compromising too much on proadipogenic or chondrogenic cells. Whether this applies also for differentiation of pMSCs in other cells, such as smooth muscle cells, for instance, remains to be explored. In animal studies, spontaneous heterotopic ossification was observed upon injection of bulk MSCs [4,45]. Unwanted osteogenesis can possibly be ameliorated by functional enrichment of MSC subsets. However, selection of defined MSC subsets may require extended expansion of the cells in vitro. However, the differentiation potential, and here especially the chondrogenesis, may be significantly compromised in MSCs at later stages of expansion in vitro [63]. This has to be taken into account when planning MSC-based therapies.

Summary and Conclusion

Bone marrow-derived MSCs have been enriched ex vivo to obtain more adipogenic or chondrogenic subsets by mAB to CD271, TNAP1, and CD56 [15]. However, to the best of our knowledge, enrichment or depletion of osteogenic cells from bulk pMSCs by a monoclonal antibody has not been reported. We report on a functional difference in CD146^pos compared with CD146^neg human pMSCs regarding their osteogenic differentiation potential and show that CD146^pos pMSCs generated osteoblasts in vitro upon osteogenic induction, whereas CD146^neg pMSCs failed to do so. Furthermore, the CD146^low pMSCs did not gain expression of the cell surface antigen, nor did CD146^pos pMSCs lose its expression after prolonged expansion in vitro. We therefore conclude that CD146^pos and CD146^neg pMSCs represent two subsets of cells that share the chondrogenic and adipogenic differentiation capacities and can be functionally separated to enrich or deplete osteogenic pMSCs. Such separations of MSCs into functionally distinct subsets can be advantageous when osteogenesis and mineralization cause an adverse problem in tissue engineering or in regenerative medicine studies aiming at restoration of soft tissue.

Footnotes

Acknowledgments

The authors thank their colleagues in the hospitals for taking the extra time during and after surgery to collect, wrap, and ship biopsy samples, which were key elements for our studies, and Chaim Goziga for help in preparation of the artwork. This project was supported by grants to WKA from the BMBF (ReGiNA TP1) and the DFG (KFO273) and, in part, by institutional funding.

Author Disclosure Statement

In the name of all coauthors, we declare that there are no actual or potential conflicts of interest to be disclosed for any of the authors of this investigation and manuscript.

References

Pittenger

, Mackay

, Beck

, Jaiswal

, Douglas

, Mosca

, Moorman

, Simonetti

, Craig

and Marshak

. (1999). Multilineage potential of adult human mesenchymal stem cells. Science, 284:143–147.

Bianco

, Cao

, Frenette

, Mao

, Robey

, Simmons

and Wang

. (2013). The meaning, the sense and the significance: translating the science of mesenchymal stem cells into medicine. Nat Med, 19:35–42.

Dominici

, Le Blanc

, Mueller

, Slaper-Cortenbach

, Marini

, Krause

, Deans

, Keating

, Prockop

and Horwitz

. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8:315–317.

Sacchetti

, Funari

, Michienzi

, Di Cesare

, Piersanti

, Saggio

, Tagliafico

, Ferrari

, Robey

, Riminucci

and Bianco

. (2007). Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell, 131:324–336.

Parolini

, Alviano

, Bagnara

, Bilic

, Buhring

, Evangelista

, Hennerbichler

, Liu

, Magatti

, et al. (2008). Concise review: isolation and characterization of cells from human term placenta: outcome of the first international Workshop on placenta derived stem cells. Stem Cells, 26:300–311.

Pilz

, Braun

, Ulrich

, Felka

, Warstat

, Ruh

, Schewe

, Abele

, Larbi

and Aicher

. (2011). Human mesenchymal stromal cells express CD14 cross-reactive epitopes. Cytometry A, 79:635–645.

Tormin

, Li

, Brune

, Walsh

, Schüz

, Ehinger

, Ditzel

, Kassem

and Scheding

. (2011). CD146 expression on primary nonhematopoietic bone marrow stem cells is correlated with in situ localization. Blood, 117:5067–5077.

Aicher

, Buhring

, Hart

, Rolauffs

, Badke

and Klein

. (2011). Regeneration of cartilage and bone by defined subsets of mesenchymal stromal cells—potential and pitfalls. Adv Drug Deliv Rev, 63:342–351.

Friedenstein

, Chailakhyan

and Gerasimov

, V. (1987). Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet, 20:263–272.

10.

Chamberlain

, Fox

, Ashton

and Middleton

. (2007). Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells, 25:2739–2749.

11.

Kolf

, Cho

and Tuan

. (2007). Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation. Arthritis Res Ther, 9:204.

12.

Buhring

, Treml

, Cerabona

, de Zwart

, Kanz

and Sobiesiak

. (2009). Phenotypic characterization of distinct human bone marrow-derived MSC subsets. Ann N Y Acad Sci, 1176:124–134.

13.

Sivasubramaniyan

, Lehnen

, Ghazanfari

, Sobiesiak

, Harichandan

, Mortha

, Petkova

, Grimm

, Cerabona

, et al. (2012). Phenotypic and functional heterogeneity of human bone marrow- and amnion-derived MSC subsets. Ann N Y Acad Sci, 1266:94–106.

14.

Battula

, Bareiss

, Treml

, Conrad

, Albert

, Hojak

, Abele

, Schewe

, Just

, Skutella

and Buhring

. (2007). Human placenta and bone marrow derived MSC cultured in serum-free, b-FGF-containing medium express cell surface frizzled-9 and SSEA-4 and give rise to multilineage differentiation. Differentiation, 75:279–291.

15.

Battula

, Treml

, Bareiss

, Gieseke

, Roelofs

, de Zwart

, Müller

, Schewe

, Skutella

, et al. (2009). Isolation of functionally distinct mesenchymal stem cell subsets using antibodies against CD56, CD271 and mesenchymal stem cell antigen-1 (MSCA-1). Hematologica, 94:19–30.

16.

Gang

, Bosnakovski

, Figueiredo

, Visser

and Perlingeiro

RCR

. (2007). SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood, 109:1743–1751.

17.

Bae

, Park

, Son

, Ju

, Paik

, Jeon

, Koh

, Kim

and Kim

. (2008). Fibroblast activation protein alpha identifies mesenchymal stromal cells from human bone marrow. Br J Haematol, 142:827–830.

18.

Sivasubramaniyan

, Harichandan

, Schumann

, Sobiesiak

, Lengerke

, Maurer

, Kalbacher

and Buhring

. (2013). Prospective isolation of mesenchymal stem cells from human bone marrow using novel antibodies directed against Sushi domain containing 2. Stem Cells Dev, 22:1944–1954.

19.

Sobiesiak

, Sivasubramaniyan

, Hermann

, Tan

, Orgel

, Treml

, Cerabona

, de Zwart

, Ochs

, et al. (2010). The mesenchymal stem cell antigen MSCA-1 is identical to tissue non-specific alkaline phosphatase. Stem Cells Dev, 19:669–677.

20.

Vogel

, Grünebach

, Messam

, Kanz

, Brugger

and Bühring

. (2003). Heterogeneity among human bone marrow-derived mesenchymal stem cells and neural progenitor cells. Haematologica, 88:126–133.

21.

Buhring

, Battula

, Treml

, Schewe

, Kanz

and Vogel

. (2007). Novel markers for the prospective isolation of human MSC. Ann N Y Acad Sci, 1106:262–271.

22.

Alexander

, Schäfer

, Munz

, Friedrich

, Klein

, Hoffmann

, Bühring

and Reinert

. (2009). NGFR: a new osteogenic differentiation marker in mineralizing periosteal cells. Tissue Eng, 15:715

23.

Pilz

, Ulrich

, Abruzzese

, Abele

, Schäfer

, Buhring

and Aicher

. (2011). Human term placenta-derived mesenchymal stromal cells are less prone to osteogenic differentiation than bone marrow-derived mesenchymal stromal cells. Stem Cells Dev, 20:635–646.

24.

Ulrich

, Rolauffs

, Abele

, Bonin

, Nieselt

, Hart

and Aicher

. (2013). Low osteogenic differentiation potential of placenta-derived mesenchymal stromal cells correlates with low expression of the transcription factors Runx2 and Twist2. Stem Cells Dev, 22:2859–2872.

25.

Anfosso

, Bardin

, Frances

, Vivier

, Camoin-Jau

, Sampol

and Dignat-George

. (1998). Activation of human endothelial cells via S-Endo-1 antigen (CD146) stimulates the tyrosine phosphorylation of focal adhesion kinase p125FAK. J Biol Chem, 273:26852–26856.

26.

Elshal

, Khan

, Raghavachari

, Takahashi

, Barb

, Bailey

, Munson

, Solomon

, Danner

and McCoy

Jr. (2007). A unique population of effector memory lymphocytes identified by CD146 having a distinct immunophenotypic and genomic profile. BMC Immunol, 8:29

27.

Yang

, Zhang

, Weakley

, Lin

, Yao

and Chen

. (2011). Transforming growth factor-beta increases the expression of vascular smooth muscle cell markers in human multi-lineage progenitor cells. Med Sci Monit, 17:BR55–BR61.

28.

Espagnolle

, Guilloton

, Deschaseaux

, Gadelorge

, Sensebe

and Bourin

. (2014). CD146 expression on mesenchymal stem cells is associated with their vascular smooth muscle commitment. J Cell Mol Med, 18:104–114.

29.

Crisan

, Corselli

, Chen

WCW

and Péault

. (2012). Perivascular cells for regenerative medicine. J Cell Mol Med, 16:2851–2860.

30.

Zannettino

ACW

, Paton

, Arthur

, Khor

, Itescu

, Gimble

and Gronthos

. (2008). Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo. J Cell Physiol, 214:413–421.

31.

Covas

, Panepucci

, Fontes

, Silva

Jr. , Orellana

, Freitas

, Neder

, Santos

, Peres

, Jamur

and Zago

. (2008). Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivascular cells and fibroblasts. Exp Hematol, 36:642–654.

32.

Crisan

, Yap

, Casteilla

, Chen

, Corselli

, Park

, Andriolo

, Sun

, Zheng

, et al. (2008). A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell, 3:301–313.

33.

Russell

, Phinney

, Lacey

, Barrilleaux

, Meyertholen

and O'Connor

. (2010). In vitro high-capacity assay to quantify the clonal heterogeneity in trilineage potential of mesenchymal stem cells reveals a complex hierarchy of lineage commitment. Stem Cells, 28:788–798.

34.

Masuda

, Anwar

, Buhring

, Rao

and Gargett

. (2012). A novel marker of human endometrial mesenchymal stem-like cells. Cell Transplant, 21:2201–2214.

35.

Flanagan

, Fitzgerald

, Baker

, Regnstrom

, Gardai

, Bard

, Mocci

, Seto

, You

, et al. (2012). Laminin-411 is a vascular ligand for MCAM and facilitates TH17 cell entry into the CNS. PLoS One, 7:e40443

36.

Bardin

, Blot-Chabaud

, Despoix

, Kebir

, Harhouri

, Arsanto

, Espinosa

, Perrin

, Robert

, et al. (2009). CD146 and its soluble form regulate monocyte transendothelial migration. Arterioscler Thromb Vasc Biol, 29:746–753.

37.

Felka

, Schäfer

, deZwart

and Aicher

. (2010). Animal serum- free differentiation of human mesenchymal stem cells. Cytotherapy, 12:143–153.

38.

Felka

, Schäfer

, Schewe

, Benz

and Aicher

. (2009). Hypoxia reduces the inhibitpry effect of IL-1beta on chondrogenic differentiation of FCS-free expanded MSC. Osteoarthritis Cartilage, 17:1368–1376.

39.

Herzenberg

, Tung

, Moore

, Herzenberg

and Parks

. (2006). Interpreting flow cytometry data: a guide for the perplexed. Nat Immunol, 7:681–685.

40.

Pittenger

, Mosca

and McIntosh

. (2000). Human mesenchymal stem cells: progenitor cells for cartilage, bone, fat and stroma. Curr Top Microbiol Immunol, 251:3–11.

41.

Warstat

, Meckbach

, Weis-Klemm

, Hack

, Klein

, de Zwart

and Aicher

. (2010). TGF-β enhances the integrin α2β1-mediated attachment of mesenchymal stem cells to type I collagen. Stem Cells Dev, 19:645–656.

42.

Johnson

. (1999). Cell adhesion molecules in the development and progression of malignant melanoma. Cancer Metastasis Rev, 18:345–357.

43.

Wang

and Yan

. (2013). CD146, a multi-functional molecule beyond adhesion. Cancer Lett, 330:150–162.

44.

Zhu

, Tan

, Qiu

, Li

, Huang

, Fu

and Liang

. (2013). Comparison of the properties of human CD146+ and CD146- periodontal ligament cells in response to stimulation with tumour necrosis factor alpha. Arch Oral Biol, 58:1791–1803.

45.

Hashemi

, Ghods

, Kolodgie

, Parcham-Azad

, Keane

, Hamamdzic

, Young

, Rippy

, Virmani

, Litt

and Wilensky

. (2008). A placebo controlled, dose-ranging, safety study of allogenic mesenchymal stem cells injected by endomyocardial delivery after an acute myocardial infarction. Eur Heart J, 29:251–259.

46.

Stopp

, Bornhauser

, Ugarte

, Wobus

, Kuhn

, Brenner

and Thieme

. (2013). Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation, differentiation, and maintenance of hematopoietic stem and progenitor cells. Haematologica, 98:505–513.

47.

Hagmann

, Frank

, Gotterbarm

, Dreher

, Eckstein

and Moradi

. (2014). Fluorescence activated enrichment of CD146+ cells during expansion of human bone-marrow derived mesenchymal stromal cells augments proliferation and GAG/DNA content in chondrogenic media. BMC Musculoskelet Disord, 15:322

48.

Fayazi

, Salehnia

and Ziaei

. (2014). Differentiation of human CD146-positive endometrial stem cells to adipogenic-, osteogenic-, neural progenitor-, and glial-like cells. In Vitro Cell Dev Biol Anim. [Epub ahead of print]; DOI: 10.1007/s11626-014-9842-2.

49.

Cheng

, Rodrigues

, Shen

, Yang

Y-g

, Dombkowski

, Sykes

and Scadden

. (2000). Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science, 287:1804–1808.

50.

Pietras

, Warr

and Passegué

. (2011). Cell cycle regulation in hematopoietic stem cells. J Cell Biol, 195:709–720.

51.

Fuchs

, Tumbar

and Guasch

. (2004). Socializing with the neighbors: stem cells and their niche. Cell, 116:769–778.

52.

Orford

and Scadden

. (2008). Deconstructing stem cell self-renewal: genetic insights into cell-cycle regulation. Nat Rev Genet, 9:115–128.

53.

Wagner

, Feldmann

Jr. , Seckinger

, Maurer

, Wein

, Blake

, Krause

, Kalenka

, Burgers

, et al. (2006). The heterogeneity of human mesenchymal stem cell preparations—evidence from simultaneous analysis of proteomes and transcriptomes. Exp Hematol, 34:536–548.

54.

Azouna

, Jenhani

, Regaya

, Berraeis

, Othman

, Ducrocq

and Domenech

. (2012). Phenotypical and functional characteristics of mesenchymal stem cells from bone marrow: comparison of culture using different media supplemented with human platelet lysate or fetal bovine serum. Stem Cell Res Ther, 3:6

55.

Hagmann

, Moradi

, Frank

, Dreher

, Kammerer

, Richter

and Gotterbarm

. (2013). Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells. BMC Musculoskeletal Disorders, 14:223

56.

Battula

, Treml

, Abele

and Buhring

. (2008). Prospective isolation and characterization of mesenchymal stem cells from human placenta using a frizzled-9-specific monoclonal antibody. Differentiation, 76:326–336.

57.

Ben Azouna

, Jenhani

, Regaya

, Berraeis

, Ben Othman

, Ducrocq

and Domenech

58.

De Bari

, Dell'Accio

, Tylzanowski

and Luyten

. (2001). Multipotent mesenchymal stem cells from adult synovial membrane. Arthritis Rheum, 44:1928–1942.

59.

Zuk

, Zhu

, Mizuno

, Huang

, Futrell

, Katz

, Benhaim

, Lorenz

and Hedrick

. (2001). Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng, 7:211–228.

60.

Yumi

, Hideaki

, Daisuke

, Imiko

, Toshio

and Kohichiro

. (2004). Human placenta-derived cells have mesenchymal stem/progenitor cell potential. Stem Cells, 22:649–658.

61.

Kern

, Eichler

, Stoeve

, Kluter

and Bieback

. (2006). Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells, 24:1294–1301.

62.

Kanematsu

, Shofuda

, Yamamoto

, Ban

, Ueda

, Yamasaki

and Kanemura

. (2011). Isolation and cellular properties of mesenchymal cells derived from the decidua of human term placenta. Differentiation, 82:77–88.

63.

Muraglia

, Cancedda

and Quarto

. (2000). Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci, 113:1161–1166.

64.

Mitchell

, McIntosh

, Zvonic

, Garrett

, Floyd

, Kloster

, Di Halvorsen

, Storms

, Goh

, et al. (2006). Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers. Stem Cells, 24:376–385.

Human Placenta-Derived CD146-Positive Mesenchymal Stromal Cells Display a Distinct Osteogenic Differentiation Potential

Abstract

Introduction

Materials and Methods

Preparation of MSCs

Flow cytometry and cell sorts

Separation of MSCs by magnetic-activated cell sort

Determination of telomere lengths

Differentiation of MSCs in vitro and cytochemical staining

Immunocytochemistry and immunohistochemistry

Statistics

Results

Characterization of human term placenta-derived MSCs

Expression of CD146 on placenta-derived MSCs

Differentiation potential of CD146high and CD146low placenta-derived MSCs

Detection of CD146 on human MSCs in vitro and its localization in situ

Discussion

Summary and Conclusion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Differentiation potential of CD146^high and CD146^low placenta-derived MSCs