Identification of mesenchymal progenitor cells in normal and osteoarthritic human articular cartilage

Authors


Abstract

Objective

To determine the presence of mesenchymal progenitor cells (MPCs) in human articular cartilage.

Methods

Primary cell cultures established from normal and osteoarthritic (OA) human knee articular cartilage were analyzed for the expression of CD105 and CD166, cell surface markers whose coexpression defines mesenchymal stem cells (MSCs) in bone marrow and perichondrium. The potential of cartilage cells to differentiate to adipogenic, osteogenic, and chondrogenic lineages was analyzed after immunomagnetic selection for CD105+/CD166+ cells and was compared with bone marrow–derived MSCs (BM-MSCs).

Results

Up to 95% of isolated cartilage cells were CD105+ and ∼5% were CD166+. The mean ± SEM percentage of CD105+/CD166+ cells in normal cartilage was 3.49 ± 1.93%. Primary cell cultures from OA cartilage contained significantly increased numbers of CD105+/CD166+ cells. Confocal microscopy confirmed the coexpression of both markers in the majority of BM-MSCs and a subpopulation of cartilage cells. Differentiation to adipocytes occurred in cartilage-derived cell cultures, as indicated by characteristic cell morphology and oil red O staining of lipid vacuoles. Osteogenesis was observed in isolated CD105+/CD166+ cells as well as in primary chondrocytes cultured in the presence of osteogenic supplements. Purified cartilage-derived CD105+/CD166+ cells did not express markers of differentiated chondrocytes. However, the cells were capable of chondrocytic differentiation and formed cartilage tissue in micromass pellet cultures.

Conclusion

These findings indicate that multipotential MPCs are present in adult human articular cartilage and that their frequency is increased in OA cartilage. This observation has implications for understanding the intrinsic repair capacity of articular cartilage and raises the possibility that these progenitor cells might be involved in the pathogenesis of arthritis.

Chondrocytes are thought to represent the only cell type in articular cartilage, although distinct chondrocyte phenotypes are recognized in the superficial, mid, deep, and calcified zones of the tissue (1, 2). Chondrocytes are a differentiated cell type that is derived from mesenchymal stem cells (MSCs), pluripotential progenitor cells whose progeny also include tendon cells, myocytes, bone marrow stromal cells, adipocytes, and osteoblasts (3, 4). Techniques for isolation and expansion of MSCs from bone marrow of different species have been established (5, 6).

Human MSCs can be characterized by the expression of certain cell surface antigens (7). Antibody to the cell surface antigen STRO-1 defines a subset of bone marrow cells that support hematopoiesis (8) and can differentiate into multiple mesenchymal lineages, including chondrocytes (9). Antibody SH-2 raised against human marrow-derived MSCs recognizes CD105 or endoglin, the transforming growth factor β (TGFβ) receptor type III (10). Antibody SB-10 reacts with CD166 or activated leukocyte cell adhesion molecule (ALCAM) (11). CD105 and CD166 are also expressed on many other cell types. The coexpression of CD105 and CD166 has been proposed to define a population of MSCs (12). Progenitor cells isolated from the bone marrow on the basis of CD105 expression are capable of chondrocytic differentiation (13). A subpopulation of cells in the perichondrium expresses CD166, and these cells have differentiation potential of MSCs (14).

Bone marrow is the most extensively characterized source of MSCs, and substantial heterogeneity in the relative abundance of MSCs has been demonstrated for rabbits (15) and among various inbred strains of mice (16). MSCs are normally present in the circulation of humans (17, 18). Umbilical cord blood contains cells that express MSC-related antigens and display a fibroblast-like morphology (19). Neonatal rat heart (20) and adult human skeletal muscle contain progenitor cells with MSC characteristics (21–23). Mesenchymal progenitor cells (MPCs) are also present in healing bone defect tissue (24) and in periodontal ligaments (25). The presence of progenitor cells with chondrogenic potential was demonstrated in rabbit (26) and human (27) synovium. Synovial fluid effusions from patients with osteoarthritis (OA) or rheumatoid arthritis also contain immature mesenchymal cells as characterized by the expression of bone morphogenetic protein receptors, CD44, type I collagen, and vimentin (28).

The presence of MPCs in articular cartilage has not been reported. This report is the first to describe the presence of MPCs in articular cartilage from human adults. Their presence has implications for cartilage repair and pathologic cartilage remodeling as seen in arthritis.

MATERIALS AND METHODS

Cartilage procurement and characterization.

Cartilage from the femoral condyles and tibial plateaus of the knee joints was obtained from healthy organ donors or at autopsy from donors with no known history of joint disease. Tissue was also obtained at the time of total joint replacement surgery from patients with OA. All samples were graded according to a modified Mankin scale (29).

Isolation and culture of cells from cartilage.

Cartilage slices were cut into 2–3-mm3 pieces, washed with McCoy's 5A modified medium (Life Technologies, Rockville, MD), treated with trypsin (10% [volume/volume]) for 15 minutes in a 37°C shaking water bath, and washed 3 times with Hanks' balanced salt solution (HBSS). The tissues were transferred to McCoy's medium, supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 1 mM sodium pyruvate (Life Technologies), 100 units/ml penicillin, 100 μg/ml streptomycin, 25 mM HEPES buffered saline (HBS), 2 mg/ml clostridial collagenase type IV (Sigma, St. Louis, MO), and 0.15 mg/ml hyaluronidase (Sigma), and digested overnight on a gyratory shaker. The cells were washed 3 times with HBSS and cultured at high cell density in McCoy's medium supplemented as above. Cells were used in primary (passage 0) culture or passaged in monolayer culture, as indicated for each set of experiments.

Isolation and characterization of bone marrow–derived MSCs (BM-MSCs).

MSCs from the iliac crest bone marrow of healthy adult volunteers were isolated and cultured as described by Haynesworth et al and Pittenger et al (30, 31), with minor modifications. Briefly, human heparinized bone marrow aspirates were diluted with an equal volume of HBSS and centrifuged at 900g for 10 minutes at room temperature. The mononuclear cell pellet was resuspended in HBSS and 5 × 107 cells/ml were layered over a 1.073 gm/ml Percoll solution (Pharmacia, Piscataway, NJ) and centrifuged at 1,100g for 30 minutes at room temperature. Cells at the interface were collected and washed 3 times in HBSS. After washing, cells were resuspended in McCoy's medium supplemented with 10% characterized FBS, penicillin, streptomycin, and sodium pyruvate, counted, and cultured at a density of 2 × 105 cells/cm2. The experiments described here utilized MSCs that were grown in FBS lot number AFE5185, which was found to promote MSC expansion (31). Medium was replaced at 24 hours and 72 hours and every 3–4 days thereafter. MSCs were subcultured after 10–14 days by treatment with trypsin–EDTA (0.05% and 0.53 mM, respectively) for 5 minutes, subsequently washed in medium supplemented with FBS, and seeded into fresh flasks.

Flow cytometry.

After harvesting the cells and preparing a single cell suspension (105 cells per 50 μl phosphate buffered saline [PBS] supplemented with 1% bovine serum albumin [BSA; Sigma] and 0.1% NaN3), the cells were stained at 4°C for 45 minutes with R-phycoerythrin–conjugated anti-CD105 (monoclonal antibody SN6; Ancell, Bayport, MN), fluorescein isothiocyanate (FITC)–conjugated anti-CD166 (Ancell), anti–HLA class I (positive control), or mouse isotype (negative control). The cells were then washed 3 times in wash buffer (PBS, 1% BSA, 0.1% NaN3) and cell pellets were resuspended in 4% PBS–paraformaldehyde. The cells were subjected to fluorescence-activated cell sorting using a FACScan and CellQuest software (Becton Dickinson, San Jose, CA).

Confocal microscopy.

Cells were grown to subconfluence on coverslips (22 × 22 mm) in 10-cm petri dishes covered with McCoy's medium supplemented with 10% FBS for 2–3 days, then fixed with 4% paraformaldehyde for 10 minutes or methanol for 4 minutes at room temperature. Adherent cells were treated with blocking reagents (5% FBS and 2% BSA) for 30 minutes and then stained for 30–45 minutes in a humidified chamber at room temperature with R-phycoerythrin–conjugated antibody to human CD105 (1:50 dilution), FITC-conjugated antibody to human CD166 (1:50 dilution), or control mouse IgG conjugated with FITC or phycoerythrin. The cells were then washed twice in PBS, and Slowfade light antifade reagent (Molecular Probes, Eugene, OR) was applied to the coverslips to prevent photobleaching. Fluorescently labeled cells were visualized and photographed using a laser scanning confocal microscope (MRC1024; Bio-Rad, Richmond, CA).

Immunomagnetic cell separation.

The monoclonal or polyclonal antibodies to the cell surface antigens (mouse anti-CD105 was generously provided by Dr. Elizabeth A. Wagner and Gregory Vercellotti, Fred Hutchinson Cancer Research Center, Seattle, WA, through the Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA; anti-CD166 from Ancell) were preincubated with the appropriate magnetic secondary antibody, and the magnetic antibody complex was then incubated with the cell suspension. The procedure was used for both negative and positive cell selection, as recommended by the manufacturer (MagnaBind Separation; Pierce, Rockford, IL), with some modifications. After placing 107 cells in a tube in 10 ml medium containing 5% FBS and antibiotics, MagnaBind goat anti-mouse IgG beads (50 particles per cell) were washed and resuspended in sterile medium with antibiotics. The specific monoclonal antibodies were added to the resuspended goat anti-mouse IgG and incubated at 4°C for 20 minutes. The goat anti-mouse IgG/monoclonal antibody complexes were washed 3 times, resuspended in 1 ml sterile medium, and added to the cells (total particle-to-cell ratio 50:1). The cell–particle mixture was resuspended by gentle agitation and incubated at 4°C for 20 minutes with agitation every 5 minutes to promote attachment.

After magnetic separation for 10 minutes, the pellets (CD105+/CD166+ cells) and the supernatants (negative selection) were collected and centrifuged. The cell–bead mixture was resuspended in a chymopapain-containing solution (Sigma) at a concentration of 10 units/106 cells and incubated at 4°C for 10–20 minutes to break the antigen–antibody interaction. The cells were then cultured for 3–5 days to allow the remaining magnetic beads to separate before a second enrichment for the CD166+ cells was performed. Cells obtained with this procedure were >90% CD105+/CD166+, as shown by flow cytometry.

Immunohistochemistry and cytochemistry.

Human BM-MSCs and immunomagnetically selected CD105+/CD166+ cells from normal cartilage and OA lesions were cultured as micromass pellets. The pellets were fixed with freshly prepared 4% paraformaldehyde overnight and embedded in paraffin. Tissue blocks were cut into 4–5-μm sections that were placed on slides and dried overnight at 37°C. Deparaffinization was carried out in xylene (2 times, 3 minutes each), and sections were hydrated in graded series of ethanol, after which they were washed in PBS. Detection of proteoglycan was performed using Alcian blue staining. The following primary monoclonal antibodies were used: anti–type I collagen (Chemicon, Temecula, CA), anti–type II collagen (Chemicon), anti–type III collagen (Chemicon), antiaggrecan (Serotec, Raleigh, NC), anti–cartilage-derived morphogenetic protein 1 (anti–CDMP-1) (Santa Cruz Biotechnology, Santa Cruz, CA), and mouse isotype control.

CD105+/CD166+ cells were cultured in monolayer for up to 17 passages. BM-MSCs and primary chondrocytes were included as controls in the cytochemistry experiments. Cells on chamber slides were fixed for 10 minutes with buffered 4% paraformaldehyde, washed 3 times with PBS, and blocked with 10% BSA for 30 minutes before adding the primary antibodies. Immunocytochemistry was performed using the alkaline phosphatase–anti–alkaline phosphatase method (32).

Adipogenesis.

Cells were cultured in McCoy's 5A modified medium with 10% FBS, 1 mM sodium pyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin, 25 mM HBS, and L-glutamine, and grown to ∼80% confluence. Cells were washed with serum-free medium and transferred to adipogenesis medium with 500 μM methylisobutylxanthine, 1 μM dexamethasone, 60 μM indomethacin, and 5 μg/ml insulin (all from Sigma) for 4 weeks, as previously described (33, 34), with minor modifications. All reagents used for adipogenesis supplements were dissolved in DMSO (Sigma). In control experiments, cells were cultured with the final concentration of DMSO as above for the same period of time. The cells containing lipid vacuoles were visualized using phase-contrast microscopy prior to oil red O staining.

After 4 weeks in culture, culture plates were fixed in 10 mM sodium periodate, 2% paraformaldehyde, 75 mML-lysine dihydrochloride, and 37.5 mM sodium phosphate (dibasic), pH 7.4, for 15 minutes at room temperature and then air dried and stained in a filtered solution of 0.3% oil red O in 60% isopropanol for 15 minutes, as previously described (35). The number of cells containing stained lipid vacuoles was determined by microscopic examination.

Chondrogenesis.

The chondrogenesis assay was performed on BM-MSCs and on magnetic bead–selected CD105+/CD166+ cells isolated from normal (N-38+/+) and OA (OAL-36+/+) cartilage. The micromass pellet culture method for chondrogenesis was performed as described by Johnstone et al (36). BM-MSCs (from 5 donors) in passages 10–12 and CD105+/CD166+ enriched adherent cells (2 donors, N-38+/+ and OAL-36+/+ lines) were detached using trypsin–EDTA, and 2.5 × 105 cells were pelleted at 500g in 15 ml polypropylene conical tubes and cultured using defined medium (low-glucose Dulbecco's modified Eagle's medium supplemented with insulin, transferrin, selenious acid, BSA, and linoleic acid) (Becton Dickinson, Franklin Lakes, NJ), sodium pyruvate (1 mM), ascorbate 2-phosphate (37.5 μg/ml), dexamethasone (10−8M), and recombinant human TGFβ1 or TGFβ3 (10 ng/ml). The pelleted cells form a spherical aggregate that does not adhere to the walls of the tube. Aggregate cultures were incubated at 37°C, 5% CO2, and medium was changed every 2–3 days. Aggregates were harvested after 1–4 weeks for Alcian blue staining and immunohistochemistry using antibodies to type II collagen, CDMP-1, and aggrecan.

Osteogenesis.

Cells were plated at 3,000 cells/cm2 in 6-well plates in control medium (DMEM, 10% FBS). The following day, the medium was replaced with DMEM, 10% FBS for control cultures, and osteogenic supplements (100 nM dexamethasone, 10 mM β-glycerophosphate, and 0.05 mML-ascorbic acid 2-phosphate) were added. These conditions have been established as optimal for osteogenic differentiation of BM-MSCs (5). After 12–16 days, the cultures were assayed for calcium content with a commercially available kit (Sigma). The assay utilizes o-cresolphtalein as a chromogenic substrate that forms a purple-colored complex with calcium in alkaline medium. Intensity of the color in test and standard samples was detected on a spectrophotometer at 575 nm.

Statistical analysis.

Statistical analysis was performed using Student's t-test or Spearman's rank correlation. Results were expressed as the mean ± SEM.

RESULTS

CD105+/CD166+-expressing MPCs in normal and OA human cartilage.

The coexpression of CD105 and CD166 has been suggested to define BM-MSCs (12). CD166 is also expressed on the progenitor cell population in the perichondrium (14). We determined the number of cartilage cells expressing these markers by flow cytometry (Figure 1). In primary cell cultures from normal cartilage, CD166 was expressed on a subpopulation of cells (<10%), whereas CD105 was expressed on the majority of cartilage cells (>80%) (Figure 1A). Although most cells isolated from normal cartilage are thought to represent differentiated chondrocytes, the expression of CD166 appears to define a subset of not fully differentiated chondrocytes or potential chondroprogenitors. The mean ± SEM percentage of CD105+/CD166+ cells in normal cartilage was 3.49 ± 1.93% (n = 13). There was no apparent correlation between the frequency of these cells and donor sex or age in this group of relatively young donors with completely normal cartilage (age range 17–39 years; n = 13) (Figure 2C). As expected, CD105 and CD166 were detected on the majority of BM-MSCs (Figure 1C).

Figure 1.

Fluorescence-activated cell sorting (FACS) of CD105 and CD166 expression on primary cartilage cells and bone marrow–derived mesenchymal stem cells (BM-MSCs). Normal and osteoarthritic (OA) cartilage was digested with collagenase and cells were allowed to adhere to the culture flasks. These primary cell cultures were then harvested, stained with antibodies to CD105 and CD166, and subjected to FACS. Human BM-MSCs (passages 2–5) were used as a reference. Representative examples are shown for A, normal cartilage, B, OA cartilage, and C, BM-MSCs. SSC = side scatter; FSC = forward scatter; FITC = fluorescein isothiocyanate; PE = phycoerythrin; NC = negative control; T = test, CD105, CD166; PC = positive control.

Figure 2.

Frequencies of CD105+/CD166+ cells in normal and osteoarthritic (OA) cartilage. Primary cultures of normal and OA cartilage cells were analyzed by flow cytometry for CD105 and CD166 expression. A, The normal donor group (n = 13). B, OA tissues (n = 10). The macroscopically normal-appearing (OAN) and fibrillated or lesional (OAL) areas were separated. C, Statistical analysis of the normal and OA groups. Values are the mean ± SEM.

Cartilage from OA joints was harvested separately from macroscopically normal-appearing areas and fibrillated OA lesions. Cells were isolated and analyzed in primary culture for CD105 and CD166 expression (Figure 1B). The results showed that normal-appearing and degraded OA cartilage did not differ in the number of single-positive or double-positive cells (Figure 2B). There was no apparent correlation between the frequency of CD105+/CD166+ cells and OA grade or donor age (data not shown). However, the percentage of CD105+/CD166+ cells was significantly higher in cartilage obtained from OA joints as compared with cartilage from normal joints (Figure 2C).

Analysis by confocal microscopy confirmed the coexpression of CD105 and CD166 at the single-cell level in a similar distribution on BM-MSCs and cartilage cells (Figure 3). The expression of CD105 and CD166 messenger RNA (mRNA) in normal human articular cartilage was also demonstrated (results not shown). The STRO-1 antigen that is expressed on BM-MSCs (8) was not detected on chondrocytes (results not shown).

Figure 3.

Confocal microscopy of CD105 and CD166 on mesenchymal stem cells (MSCs) and cartilage-derived progenitor cells. Bone marrow–derived MSCs in passages 2–5 (top) and normal cartilage–derived progenitor cells in passages 3–5 (bottom) were stained with R-phycoerythrin (R-PE)–conjugated anti-CD105 and fluorescein isothiocyanate (FITC)–conjugated anti-CD166 and analyzed by confocal microscopy. Controls using mouse IgG conjugated with FITC and R-PE were negative (results not shown).

Collectively, these results indicate that normal cartilage contains CD105+/CD166+ cells that have the phenotype of MPCs. The frequency of these cells is increased in OA cartilage.

Adipogenic potential of cartilage-derived progenitor cells.

To analyze the multilineage potential of the progenitor cells from cartilage, cells were transferred to culture medium known to promote adipogenesis in BM-MSCs. The typical appearance of adipocytes in 3-week cultures of BM-MSCs is shown in Figures 4A and B. Cells from normal cartilage that were maintained in adipogenesis medium for 3–4 weeks showed colonies of cells that contained lipid vacuoles (Figure 4C) and were oil red O positive (Figure 4D). To determine whether the development of adipocytes originated from CD105+/CD166+ cells, this cell population was isolated by immunomagnetic selection and maintained in adipogenesis medium. CD105+/CD166+ cells differentiated to adipocytes (Figures 4E and F).

Figure 4.

Adipogenic differentiation in cartilage-derived progenitor cells. CD105+/CD166+ cells were isolated from primary cell cultures of normal and OA cartilage by immunomagnetic selection and transferred to adipogenic culture conditions. Cultures were maintained for 3–4 weeks and analyzed by phase-contrast microscopy and oil red O staining.A andB, Human BM-MSCs after 3 weeks in adipogenesis medium (A, phase contrast; B, oil red O). (Results represent 6 different cell preparations.)C andD, Normal human cartilage cells after 3–4 weeks in adipogenesis medium (C, phase contrast; D, oil red O). (Results represent 10 different donors.)E andF, CD105+/CD166+ cells selected with immunomagnetic beads from normal human cartilage cells after 3–4 weeks in adipogenesis medium (E, phase contrast; F, oil red O). G andH, Human BM-MSCs after 3–4 weeks in medium containing DMSO (G, phase contrast; H, oil red O). Normal and OA human cartilage cells and CD105+/CD166+ cells from normal and OA cartilage did not differentiate to adipocytes after culturing in DMSO-containing medium for 3–5 weeks (results not shown). See Figure 1 for definitions.

The lipid vacuoles that developed in cartilage progenitor cells during adipogenic differentiation were similar in size and staining intensity to those found in BM-MSCs. Adipogenic differentiation of MSCs in vitro did not occur in all cells at the same time, but it began in clusters that increased in frequency with time. In addition, culturing BM-MSCs or cartilage-derived CD105+/CD166+ cells for >5–6 weeks under these same adipogenic conditions resulted in loss of lipid vacuoles and eventually cell death.

Control cultures that were performed in the presence of 0.1% DMSO, the diluent for the adipogenic factors in the same medium, did not develop cells with adipocyte morphology or oil red O staining (Figures 4G and H). The percentage of CD105+/CD166+ progenitor cells from cartilage was higher than the percentage of cells that differentiated to adipocytes (Figure 5). This discrepancy could be explained by the possibility that some of the cartilage progenitor cells may be committed to another lineage or may be undergoing chondrogenesis. Alternatively, adipogenesis may be inhibited by factors secreted from preadipocytes (37, 38) or chondrocytes (39–41).

Figure 5.

Comparison of the percentage of progenitor cells in cartilage obtained by flow cytometric analysis and by adipogenic differentiation. These findings illustrate the discrepancy of the results obtained by the characterization of the cells on the basis of cell surface markers CD105 and CD166 and by the functional capacity of these cells to differentiate to adipocytes.

Osteogenesis from cartilage-derived progenitor cells.

To further document multilineage potential of cartilage-derived progenitor cells, selected CD105+/ CD166+ cells, unfractionated primary chondrocytes, and BM-MSCs were cultured for 18 days in the presence or absence of osteogenic factors. Deposition of calcium as a measure of osteogenesis was detected in all cultures performed in the presence but not in the absence of osteogenic supplements (Figure 6). As expected, levels were highest in BM-MSCs, but significant levels were observed in purified CD105+/CD166+ cells (Figure 6). Osteogenesis was also observed in unfractionated chondrocytes, and this was reduced after depletion of CD166+ cells.

Figure 6.

Osteogenesis from cartilage-derived progenitor cells. Human bone marrow–derived mesenchymal stem cells (hMSCs) and CD105+/CD166+ cells selected by immunomagnetic beads, unfractionated chondrocytes, and chondrocytes depleted of CD166+ cells were cultured for 18 days in monolayers in the absence or presence of osteogenic supplements. Values are the mean and SEM levels of total calcium content in the cultures.

Chondrogenic potential of cartilage-derived progenitor cells.

To analyze whether CD105+/CD166+ cells from cartilage are able to differentiate to chondrocytes and form cartilage, we used the micromass pellet culture technique, which was described previously, to initiate chondrogenesis in human BM-MSCs (6, 42). CD105+/CD166+ cells from cartilage did not express markers of differentiated chondrocytes, such as type II collagen and CDMP-1 (27), as demonstrated by immunocytochemistry (Figure 7). CD105+/CD166+ cells from cartilage, as well as BM-MSCs, expressed type I collagen and aggrecan (Figure 7). These results were confirmed in mRNA analysis by reverse transcriptase–polymerase chain reaction (results not shown). Upon culture in micromass pellets for 1 week, the majority of progenitor cells from cartilage differentiated to chondrocytes and formed cartilage tissue, which contained type II collagen and CDMP-1 (Figure 8).

Figure 7.

Representative immunocytochemical analysis of monolayer cultured chondroprogenitor cells obtained from normal (N-38+/+) and osteoarthritic (OAL-36+/+) cartilage. Cells were fixed and stained using antibodies against aggrecan, type I collagen, type II collagen, cartilage-derived morphogenetic protein 1 (CDMP-1), and isotype control antibodies (NC).

Figure 8.

Chondrogenic differentiation of cartilage-derived progenitor cells in comparison with bone marrow–derived mesenchymal stem cells (hMSC). Immunoselected CD105+/CD166+-expressing cells obtained from normal cartilage (N-38+/+), osteoarthritic cartilage lesions (OAL-36+/+) in passage 10, and hMSC were cultured as micromass pellets for 1 week. The pellets were fixed and stained with Alcian blue or with antibodies against type II collagen, aggrecan, and cartilage-derived morphogenetic protein 1 (CDMP-1), with isotype control (mouse IgG) as a negative control (NC).

DISCUSSION

Stem cells are self-renewing multipotential progenitors with a broad developmental potential in a given tissue (43). Stem cells have been isolated from fetal tissues and bone marrow (44). The presence of stem cells in mature organs has recently been recognized and has led to revisions of previous concepts on tissue homeostasis and regeneration (45–48). These discoveries also opened avenues for potential new therapeutic approaches by enhancing the capacity of these cells for tissue repair and renewal after trauma, disease, or aging. The presence of stem cells in mature organs might also contribute to abnormal organ remodeling in pathologic conditions, in which these cells are activated but not properly regulated to develop into differentiated cell types.

The presence of stem cells in adult articular cartilage has not been evaluated. The current study used a combination of phenotypic and functional analyses to address this issue. These results provide the first evidence for the presence of MPCs in adult human normal and OA cartilage.

The phenotypic characterization was centered on CD105 and CD166. Monoclonal antibody SB-10 had originally been shown to react with BM-MSCs, but it also binds to cells in the periosteum, the developing brain, the esophagus, and the lungs (49). SB-10 immunoreactivity is lost upon MSC differentiation. The SB-10 antigen was shown to be identical to ALCAM or CD166 (11), a CD6 ligand that is also expressed on thymic epithelial cells, activated T cells, B lymphocytes, and monocytes (50). A subpopulation of cells in the perichondrium expresses CD166, and these cells have the differentiation potential of MSCs (14). We show here that CD166 is expressed on a subpopulation of human cartilage cells (<10%).

Antibody SH-2, which was also raised against human MSCs, is now known to recognize CD105, the TGFβ receptor endoglin (10). CD105 is also expressed on endothelial cells, syncytiotrophoblasts, macrophages, and connective tissue stromal cells and has been suggested to play a role in TGFβ signaling during chondrogenic differentiation. Cells that are selected from adherent bone marrow cell cultures on the basis of CD105 expression are capable of chondrocytic differentiation (13).

BM-MSCs express both CD105 and CD166, and the coexpression of these receptors is a criterion to phenotypically define this cell type (12). The majority of cells in primary cultures established from normal cartilage are CD105+, a subset of ∼10% are CD166+, and cells that express both CD105 and CD166 were detected at an average frequency of ∼3.2%.

Demonstration of multilineage potential is essential for the identification of stem cells. In this study, differentiation to adipocytes and osteoblasts was selected because these mesenchymal cell types are not present in cartilage and also do not spontaneously develop during chondrocyte monolayer culture. The results from these experiments show that primary monolayer cultures from cartilage contain cells that are capable of undergoing adipogenesis when cultured in the appropriate medium. CD105+/CD166+ cells were purified by immunomagnetic selection and were shown to undergo adipogenesis and osteogenesis, further supporting the notion that this subset represents MPCs in cartilage. Cartilage cell preparations that were depleted of CD166-expressing cells were not capable of differentiating to adipocytes (results not shown).

The CD105+/CD166+ cells from cartilage do not express markers of differentiated chondrocytes, such as type II collagen or CDMP-1 (27). However, upon culture in micromass pellets in the presence of chondrogenic supplements, the cells expressed chondrocyte markers and formed cartilage-like tissue.

The combined results from the cell surface marker expression, adipocyte, and chondrocyte differentiation studies suggest that normal adult human cartilage contains CD105+/CD166+ cells, which are capable of differentiation along multiple lineages, thus defining them as MPCs. The percentage of CD105+/CD166+ progenitor cells from cartilage was higher than the percentage of cells that differentiated to adipocytes. This may indicate that some of these cells are committed to or undergoing chondrocytic differentiation and are no longer able to differentiate to adipocytes. It is also possible that adipogenesis is inhibited by factors secreted from preadipocytes (37, 38) or chondrocytes (39–41).

The normal cartilage donors tested ranged in age from 17 to 39. They were selected to represent skeletally mature individuals who are completely devoid of aging-associated or OA changes in cartilage. The frequency of CD105+/CD166+ cells did not show an aging-associated variation in this group of young, healthy individuals. It remains to be determined whether the frequency of progenitor cells is altered in normal cartilage from older individuals. An aging-associated decline in the frequency of MSCs has been reported for human (51, 52) and murine (53) bone marrow. Bone marrow from OA patients contains MSCs with reduced chondrogenic and adipogenic potential (54).

In the present study, we also analyzed OA cartilage. OA represents one of the most common aging-associated musculoskeletal diseases. Decompensation of cartilage repair capacity and aging-associated changes in chondrocyte function have been suggested as potential pathogenic factors (55). The results from the analysis of OA cartilage revealed a significant increase in the percentage of CD105+/CD166+ cells as compared with normal cartilage from the younger donor group. Comparison of normal-appearing cartilage from OA joints with fibrillated OA lesions showed that both types of tissue contained increased numbers of CD105+/ CD166+ cells.

The increased frequency of progenitor cells in OA cartilage could result either from proliferation of resident progenitor cells or from the recruitment from other sites. Synovial membrane has recently been shown to contain chondroprogenitors (26, 28), and similar cells have been detected in synovial fluid (28). The cells in the synovial membrane may be similar to synovial nurse-like cells that migrate from the bone marrow through specialized vascular channels to the joint space (56–59).

The demonstration of MPCs in normal cartilage has implications for cartilage repair and pathologic cartilage remodeling, as seen in arthritis. It is possible that the apparent failure of repair in mature cartilage (60) is due to an inappropriate programming of the MPCs in cartilage, and the therapeutic administration of growth and differentiation factors may improve the outcome of the cartilage response to injury. In arthritic joints, the milieu of inflammatory mediators may lead to an abnormal differentiation of the progenitor cells, and this could be involved in the aberrant calcification of cartilage matrix and the formation of fibrocartilage that is seen (61). Cytokines, such as interleukin-1 or tumor necrosis factor, which are present in arthritic joints, have already been shown to interfere with chondrocytic differentiation of MSCs (62). This can occur via the inhibition of transcription factors, such as sex-determining region Y–type high mobility group box proteins, which promote cartilage-specific gene expression, or via the expression of inhibitors of chondrogenesis, such as nuclear factor of activated T cells (63).

In conclusion, the present study demonstrates that normal adult human articular cartilage contains MPCs. This opens new possibilities to achieve intrinsic cartilage repair and adds a novel aspect to the pathogenesis of cartilage degradation in arthritis.

Acknowledgements

We thank Dr. Joe Trotter, director of the FACS Core Laboratory, the Scripps Research Institute (TSRI), for help in performing flow cytometry and for valuable discussions, and Dr. Malcolm Wood for help in performing confocal microscopy. We appreciate Dr. N. J. Zvaifler's comments on the manuscript.

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