The protocol used in this study was approved by the Gyeongsang National University Animal Use Committee. Feline bone marrow was harvested by bone marrow aspiration from a greater trochanter of the femur. Mononuclear cells were isolated from the collected samples by the Ficoll 400 (Sigma, USA) density gradient method. The cells were then rinsed twice with PBS, and grown in Dulbeco's Modified Eagle high glucose medium (DMEM, 4.5 g/l glucose; Gibco-BRL, USA) with 10% fetal bovine serum (FBS; Gibco-BRL, USA) and penicillin/streptomycin (50 IU/ml / 50 µg/ml; Sigma, USA). After 5 days, the resulting monolayer of cells, hereafter named bone marrow-derived MSCs, was trypsinized and aliquots were frozen and stored or cultivated further.

MSCs were from passage 4. The study was divided into three experimental groups: a MSC group, a PC12 cell group, and the combined MSC + PC12 cell group. The cells were seeded at 1.5 × 10⁵ cells of each cell type (3 × 10⁵ total in the combined group) onto 60 mm dishes and grown in DMEM supplemented with 10% FBS and penicillin/streptomycin (50 IU/ml / 50 µg/ml). Cells were incubated at 37℃ in a humidified atmosphere of 5% CO₂ for 3 days.

Ten microliters of CD9 (Serotec, USA), CD18, CD44, and CD45-like (VMRD, USA) cat-specific antibodies were added to the bottom of tubes, followed by 100 µl of 6 × 10⁶ cells/ml single cell suspensions of cultured MSCs in the 4th passage. The mixture was incubated for 30 min at 4℃ in the dark and washed. Five microliters fluorescent-tagged secondary antibodies were then added to the resuspended cells for 30 min at 4℃ in the dark. Positive cells were then detected on flow cytometers.

To identify MSCs from feline derived bone marrow, the primary chicken anti-vimentin antibody (1 : 5,000, polyclonal; Chemicon, USA) was employed; the secondary antibody was a FITC-conjugated antibody against chicken IgG (1 : 320; Sigma, USA). To identify TH-positive differentiation of feline MSCs in co-cultures experiment, MSCs were prelabeled using Dil-Ac-LDL (1 : 20; Biomedical Technologies, USA) and then co-cultured with PC12 for three days. Anti-TH (1 : 500, polyclonal; Chemicon, USA) was the primary antibody used to identify TH in all groups; a FITC-labeled antibody against rabbit IgG (1 : 80; Sigma, USA) was used as the secondary antibody. The cells were cultured in a four-well chamber slide and fixed with 4% paraformaldehyde for 10 min. Following fixation, the cells were treated with 0.3% Triton X-100 containing 10% normal goat serum for 30 min. Cells were then incubated with the primary antibody at room temperature for 2 h, followed by incubation with the FITC-conjugated secondary antibody at 37℃ for 30 min. To prevent non-specific staining, secondary antibodies were used at the same concentrations as the primary antibodies. The slides were examined with an microscope (Olympus, Japan) equipped with fluorescent illumination.

Western blot analysis was performed to determine whether the presence of MSCs affected TH protein expression in PC12 cells. The cells were lysed using a SDS-sample buffer. After ultracentrifugation (12,000 rpm/4℃/10 min), the supernatant was collected. Fifteen µg of protein was separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, USA). Immunoreactions were performed with antibodies against TH (1 : 500, polyclonal; Chemicon, USA) and GAPDH (1 : 300, monoclonal; Chemicon, USA). The membrane was washed and incubated with horseradish peroxidase conjugated anti-rabbit IgG (1 : 1,000; Santa Cruz Biotechnology, USA) or anti-mouse IgG (1 : 5,000; Chemicon, USA). Antigen protein was visualized using chemiluminescence with an ECL-detecting reagent (Amersham Pharmacia Biotech, USA) according to the protocol of the manufacturer.

Experiments were repeated three times. The band from the western blot was scanned and analyzed by densitometry using the Sigma Gel System (SPSS, USA). Density values are expressed as the mean ± SD. Statistical analysis was performed using one-way ANOVA analysis followed by a posthoc LSD test. The acceptance level for statistical significance was p<0.05.

The feline bone marrow-derived mononuclear fraction was isolated and then cultured. Attached cells were observed at 10-14 days after the initial plating. These cells comprised a morphologically heterogeneous population of cells that included spindle-shaped cells, large flat cells, and small round cells (Fig. 1A).

Upon a fourth passage of adherent cells, a homogeneous population of fibroblast-like cells progressively arose from the feline bone marrow-derived mononuclear cells (Fig. 1B). Identification of mesenchymal stem cells was performed with a primary anti-vimentin antibody (Fig. 1C).

The feline MSC-surface antigen profile was ascertained by staining with feline-specific monoclonal antibodies followed by flow cytometry (Fig. 2). Feline MSCs were strongly positive for CD9 and CD44, but negative for CD18 and CD45. They demonstrated typical characteristics of mesenchymal cells, including the capacity to transform into osteocytes and adipocytes.

In the MSCs alone, the cells showed an elongated fibroblast shape (Fig. 3A). In the PC12 cells alone, the cells showed modest growth and tended to grow in small clusters (Fig. 3B). In the co-cultured group, both cells appeared robust. Although the PC12 cells did not change morphology significantly, the MSCs possessed long, thin, complex processes (Fig. 3C).

Representative immunoblot bands for TH proteins are shown in Fig. 4. TH protein demonstrated a stronger signal at 60 kDa in the co-culture group than the PC12 cell group (p<0.001). In contrast, TH wasn't expressed in the MSC alone group.

MSCs (red) did not show immunostaining of TH protein (green) (Figs. 5A-C), indicating that the MSCs had not differentiated into TH positive cells under our experimental conditions.