Specimens from patients with human meningioma were freshly obtained from the operating room with the approval of Institutional Review Boards of our institutes. Informed consent was provided according to the Declaration of Helsinki. Neuropathologists diagnosed these surgical specimens according to World health Organization (WHO) classification (30). Candidate MS-MSLCs were isolated from meningioma specimens within 60 min of meningioma removal using mechanical dissociation methods proven effective for MSC isolation from bone marrow (31,32), normal brain (19) and gliomas (12,28). Briefly, surgical specimens were minced and dissociated with a scalpel in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F-12; Mediatech, Manassas, VA, USA) and then passed through a series of cell strainers with a 100-μm nylon mesh (BD Falcon, Franklin Lakes, NJ, USA). Cell suspensions were washed twice in minimal essential medium-α (MEMα; Mediatech, Herndon, VA, USA) and single-cell suspensions were placed in a 10-cm² cell culture dish at a density of 2×10⁶ cell/cm². These cells were cultured in complete MSC medium consisting of MEMα, 10% fetal bovine serum (FBS; Lonza, Basel, Switzerland), 2 mM L-glutamine (Mediatech) and antibiotic-antimycotic solution (100X, Gibco, Invitrogen Korea, Seoul, Korea). After 24 h, non-adherent cells were removed by washing twice with phosphate-buffered saline (PBS; Mediatech) and the adherent cells were cultured until they reached confluence. The cells were then trypsinized (0.25% trypsin with 0.1% EDTA) and sub-cultured at a density of 5,000 cells/cm². The cells were cultured continuously through 3–4 passages, consistent with their role as progenitor/stem cells. Cell cultures were observed with an IX71 inverted phase-contrast microscope (Olympus, Tokyo, Japan) to determine their morphology. Images of cells were obtained at each passage using a DP70 Digital Microscope Camera (Olympus) equipped with DP Controller software (Olympus).

To investigate the surface antigen expression profile, candidate MS-MSLCs were first counted and washed in PBS (Mediatech) by centrifugation, after which pellets were resuspended in fluorescent-activated cell sorting (FACS) buffer (PBS with 10% FBS) at a concentration of 5×10⁵ cells/100 μl. These single-cell suspensions were incubated at 4°C for 30 min with phycoerythrin-, fluorescein isothiocyanate (FITC)-, Alexa Fluor 647-, or allophycocyanin-conjugated antibodies against CD105 (0.25 μg/100 μl; eBioscience, San Diego, CA, USA), CD45 (5 μg/100 μl; BD Pharmingen, San Diego, CA, USA), CD73 (5 μg/100 μl; BD Pharmingen), CD90 (0.25 μg/100 μl; eBioscience), CD31 (0.5 μg/100 μl; eBioscience) and nerve/glial antigen 2 (NG2, 2.5 μg/100 μl; R&D Systems, Minneapolis, MN, USA). All antibody solutions were prepared in FACS buffer. For the detection of NG2 proteoglycan, a FITC-conjugated secondary NG2 antibody (Millipore, Billerica, MA, USA) was used following primary antibody incubation (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). FACS analysis was performed using a FACS Vantage SE (BD Biosciences) flow cytometry system equipped with FlowJo software (Tree Star, Inc., Ashland, OR, USA) and 30,000 events were recorded for each sample. Because we merely sought to show the presence of MSLCs among heterogeneous cells instead of isolating a uniform population of MSCs (33), heterogeneous cell populations in which FACS showed that >10% of cells expressed surface antigen were considered positive and those with <5% by FACS were considered negative (12).

To determine the mesenchymal differentiation potential of candidate MS-MSLCs, we used a proven trilineage differentiation test identical to that described previously (12,31,32). Briefly, we tested the capacity of candidate MS-MSLCs to differentiate along adipogenic, osteogenic and chondrogenic lineages. For adipogenic differentiation, MS-MSLCs were seeded in a 6-well plate at a density of 4×10⁴ cells/cm² in complete MSC medium. At confluence, cell differentiation was induced with adipogenic differentiation medium from the adipogenic differentiation BulletKit (Lonza Walkersville, Walkersville, MD, USA). These cells were fed with fresh medium every 3–4 days for 3 weeks. In control experiments, cells were incubated for the same period of time in complete MSC medium. On day 21, the cells were washed in PBS (Mediatech) and fixed in 10% formalin (Fisher Scientific, Fair Lawn, NJ, USA) for 1 h at room temperature. After fixation, the cells were rinsed with deionized water several times, after which of 60% isopropanol (Pharmco-AAPER, Brookfield, CT, USA) was added and cells were allowed to sit for 5 min. Oil red O solution (Sigma) was then added to each well. After 5 min, the cells were rinsed with deionized water and briefly counter-stained with hematoxylin (Sigma). For osteogenic differentiation, candidate MS-MSLCs were plated at a density of 3×10⁴ cells/cm² in a 6-well plate. The next day, the medium was replaced with osteogenic differentiation medium from the osteogenic differentiation BulletKit (Lonza Walkersville). These cells were fed with fresh medium every 3–4 days for 3 weeks. In control experiments, cells were incubated for the same period of time in complete MSC medium. On day 21, cell cultures were washed twice with PBS (Mediatech) and fixed in 70% ice-cold ethanol (Pharmco-AAPER) for 1 h, followed by washing with deionized water. The cells were stained with 40 mM Alizarin Red (pH 4.2; Sigma) for 10 min at room temperature with rotation, followed by washing with deionized water five times. For chondrogenic differentiation, candidate MS-MSLCs were trypsinized and washed in serum-containing medium. Aliquots of 2.5×10⁵ cells suspended in 0.5 ml of medium were placed in 15-ml conical polypropylene tubes (SPL, Pocheon, Gyeonggi, Korea). The cells were then gently centrifuged for 5 min at 150 x g and left at the bottom of the tubes, which were placed in an incubator with caps loosened to permit gas exchange. The cells formed small pellets that were cultured for 3 weeks in chondrogenic differentiation medium from the chondrogenic differentiation BulletKit (Lonza Walkersville) supplemented with 20 μg/ml of transforming growth factor (TGF)-β3 (Ontogeny Research Products, Cambridge, MA, USA). Every 3–4 days, the cells were fed with fresh medium. In control experiments, the cells were incubated for the same period of time in complete MSC medium. These pellets were fixed in 10% formalin for 1 h at room temperature, then embedded in paraffin sections and stained with toluidine blue (Sigma) for proteoglycans and glycosaminoglycans.

Four-to-eight-week-old male athymic nude mice (Central Laboratory Animal Inc., Seoul, Korea) were used to assess the tumorigenicity of candidate MS-MSLCs. Mice were housed in micro-isolator cages under sterile conditions and observed for ≥1 week before study initiation to ensure proper health. Lighting, temperature and humidity were controlled centrally. All experimental procedures were approved by our Institutional Animal Care and Use Committee. The body weights of mice were checked daily. If body weight decreased by >15% compared with the original body weight, mice were euthanized as proscribed by the approved protocol. The brain was dissected and placed in formalin for pathological studies.

Mice were anesthetized with a solution of Zoletil (30 mg/kg; Virbac Korea, Seoul, Korea) and xylazine (10 mg/kg; Bayer Korea, Seoul, Korea) delivered intraperitoneally. Candidate MS-MSLCs were implanted into the right frontal lobe of nude mice using a guide-screw system within the skull, as described previously (34). Mice received 5×10⁵ candidate MS-MSLCs via a Hamilton syringe (Dongwoo Science Co., Seoul, Korea) inserted to a depth of 4.5 mm. Each sample of candidate MSLCs was injected into three mice simultaneously using a multiple microinfusion syringe pump (Harvard Apparatus, Holliston, MA, USA) at a speed of 0.5 μl/min, as previously described (11,12,19,34,35). At least 180–200 days after injection, mouse brains were carefully removed, sectioned, stained with hematoxylin and eosin (H&E) and examined for tumors.

The possible location of MS-MSLCs in human meningioma specimens was investigated using double immunofluorescence labeling. Meningioma specimens were immediately removed and post-fixed in 4% paraformaldehyde at 4°C overnight. After dehydration with 30% sucrose in PBS, meningioma specimens were frozen with OCT compound (Sakura Finetek USA. Inc., Torrance, CA, USA) at −80°C. Frozen sections were processed for immunofluorescence labeling using goat anti-human CD105 (1:100; R&D Systems), rabbit anti-human CD31 (1:50, an endothelial cell marker; Abcam, MA, USA) and rabbit anti-human NG2 antibodies (1:100, a pericyte marker; Millipore, Danvers, MA, USA). Alexa Fluor 488- and Alexa Fluor 555-conjugated goat anti-rabbit IgG antibodies (1:2,000; Invitrogen, CA, USA) were used as secondary antibodies. Samples were mounted in DAPI (4′,6-diamidino-2-phenylindole)-containing Vectashield mounting medium (H-1200; Sunil Technopia, Seongnam, Korea) to stain nuclei and were examined under a fluorescence inverted microscope (IX71; Olympus) equipped with DP Controller software (Olympus).

Data are expressed as means ± standard deviations. Survival curves for MS-MSLC-implanted mice were obtained using the Kaplan-Meier method. SPSS version 18.0KO software (SPSS Korea, Seoul, Korea) was used for calculations.

MS-MSLCs were obtained from a total of 20 meningioma specimens (10 WHO grade I and 10 WHO grade II) and grown under MSC culture conditions, as described previously (12,31,32). Candidate MS-MSLCs with general properties of human BM-MSCs and MSLCs, characterized by their spindle shape and ability to adhere to plastic, were selected from WHO grade II (Fig. 1A) and grade I (Fig. 1B) meningiomas. Five of the ten WHO grade II meningioma samples and 2 of the 10 WHO grade I meningioma samples passed step 1 and were selected for characterization (Table I). Although the proportion of spindle-shaped, adherent cells in each of these selected specimens was different, their morphology showed little difference between WHO grade I and II.

Flow cytometry analysis was used to assess surface antigen expression in spindle-shaped cells that adhered to plastic under MSC/MSLC culture conditions. Although there are no specific pathognomonic markers for human BM-MSCs, it is generally agreed that CD105, CD90 and CD73 are positive markers and CD45 is negative marker for most MSLCs (12,32,33). Using these criteria, we tested whether candidate cells are MS-MSLCs (Fig. 2). Because MSLCs from mice normal brains (19), glioma xenografts (11) and Korean glioma specimens (12) are found around vessels, CD31, a marker of endothelial cells and NG2, a marker of pericyte were additionally used to discriminate MS-MSLCs and vessel-related cells. MS-MSLCs were negative for CD31 and NG2 (Fig. 2), as expected for these non-endothelial, non-pericyte cells. Of the meningioma specimens that passed step 1, three of five WHO grade II and one of two WHO grade I specimens showed proper surface antigen expression (Table II).

MSCs/MSLCs exhibit trilineage - osteocyte, adipocyte and chondrocyte - differentiation capacity (11,12,19,33). To validate the mesenchymal trilineage differentiation potential of MS-MSLCs, we tested candidate cells that passed steps 1 and 2 for their ability to differentiate into osteocytes, adipocytes and chondrocytes when cultured in induction medium (Fig. 3A, C and E). Differentiation into only two of the three cell types meant failure to pass step 3. No trilineage differentiation was observed in control medium (Fig. 3B, D and F). Among the selected candidate MS-MSLCs, only WHO grade II meningioma cells satisfied the criterion of trilineage differentiation potential (Table III).

Unlike CSCs, MSCs/MSLCs are not tumorigenic in vivo. To satisfy this criterion, most mice intracranially implanted with candidate MS-MSLCs that passed steps 1, 2 and 3 should survive for more than 6 months. Tests of the three candidate MS-MSLCs from WHO grade II meningioma samples that passed steps 1, 2 and 3 showed that mice implanted with candidate MS-MSLCs from two samples (MS-MSLC0817 and MS-MSLC1025) survived for >6 months (Fig. 4), whereas those implanted with the third sample (MS-MSLC0802) died ∼4 months later (Table IV). Notably, however, mice in the group implanted with MS-MSLC0802 meningioma cells that failed to survive >4 months died from infection and not because of a tumor. Accordingly, two groups of MS-MSLCs (MS-MSLC0817 and MS-MSLC1025) isolated from meningioma specimens passed step 4, the final test for MS-MSLC selection, convincingly demonstrating no tumorigenicity or general toxicity (Table V).

The results of steps 1–4 corroborate the hypothesis that MSLCs exist in meningioma specimens, although the question of where MS-MSLCs are located remained. Previous studies of MSLCs in normal mouse brains (19), mouse glioma xenografts (11) and Korean glioma specimens (12) have suggested that these cells were located in a perivascular site. To verify that MS-MSLCs might also be located in a perivascular niche, we analyzed meningioma specimens for expression of the markers CD105, CD31 and NG2 by immunofluorescence. CD105, a surface marker present in most MSCs/MSLCs, was selected for establishing the presence of MS-MSLCs. To determine whether CD105-positive cells were near endothelial cells, we performed double-immunofluorescence labeling for the endothelial cell markers, CD31. Histological analyses suggested that some CD105-positive cells were closely associated with CD31-positive cells (Fig. 5A). To determine whether CD105-positive cells were associated with pericytes, we performed double-immunofluorescence labeling for CD105 and the pericyte marker NG2. Similar to the results obtained with CD105 and CD31 double-immunofluorescence labeling, some CD105-positive cells were intimately associated with NG2-positive cells (Fig. 5B). Accordingly, we infer that some CD105-positive candidate MS-MSLCs are located in the perivascular niche (Fig. 5, arrows). However, not all CD105-positive cells were found near vessels. These cells may be niche-independent cells (Fig. 5, arrowheads).