In a recent study of ours, we described the establishment process and culture method for all MSC lines derived from the bone marrow of GFP-transgenic mice: TGF-β-responsive SG-2, BMP-responsive SG-3 and TGF-β/BMP-non-responsive SG-5 cells (35). These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, GE Healthcare Life Sciences, Logan, UT, USA, Logan, UT, USA) at 37°C under hypoxic conditions (5% O₂, 5% CO₂ and 90% N₂).

Whole genome expression was analyzed for the bone-marrow derived SG-2, SG-3 and SG-5 MSC lines. Total RNA was extracted using ISOGEN reagent (Nippon Gene Co., Ltd., Tokyo, Japan). Filgen, Inc. (Nagoya, Japan) performed the DNA microarray analyses, including reverse transcription labeling, microarray hybridization, scanning and raw data analyses. For hybridization, 3 GeneChip Mouse Gene 2.0 ST arrays (Affymetrix, Santa Clara, CA, USA) were used. These analyses were conducted by the Research Institute of Bio-System Informatics (Tohoku Chemical Co., Ltd., Morioka, Japan).

Almost confluent SG-2, SG-3 and SG-5 cells (1.0×10⁵) were suspended in ice-cold phosphate-buffered saline (PBS) containing 0.5% FBS and 2 mM EDTA. The cells were incubated with phycoerythrin (PE)-conjugated anti-mouse VEGFR3 (CD310) antibody (1:10, clone AFL4, #130-102-216; Miltenyi Biotec, Bergisch Gladbach, Germany) for 1 h at 4°C in the dark. Acquisition was performed with an EPICS XL ADC system (Beckman Coulter, Inc., Brea, CA, USA).

The SG-2, SG-3 and SG-5 cells were serum-starved overnight and stimulated with 10 ng/ml VEGF-C (R&D Systems, Inc., Minneapolis, MN, USA) for 1 h at 37°C under hypoxic conditions. The cells were washed twice with ice-cold PBS and then lysed in RIPA buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich). Protein content was measured using BCA reagent (Pierce/Thermo Fisher Scientific, Waltham, MA, USA). Samples containing equal amounts of protein were separated using 12.5% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). After blocking with 5% non-fat dry milk in T-TBS (50 mM Tris-HCl, pH 7.2, 150 mM NaCl and 0.1% Tween-20), the membrane was incubated with primary anti-Akt (#9272), anti-phospho-Akt (Ser473) [phosphorylated (p-)Akt; #9271], anti-p44/42 mitogen-activated protein kinase (MAPK; ERK1/2; #9102), anti-phospho-p44/42 MAPK (Thr202/Tyr204) (p-ERK1/2; #9101), anti-p38 MAPK (p38; #9212), anti-phospho-p38 MAPK (T180/Y182) (p-p38; #9211), anti-stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) (JNK; #9252) and anti-phospho-SAPK/JNK (Thr183/Tyr185) (p-JNK; #9251) antibodies (all from Cell Signaling Technology, Danvers, MA, USA), and anti-β-actin (clone C4; Santa Cruz Biotechnology, Dallas, TX, USA) antibody as a loading control for normalization. The blots were incubated with an alkaline phosphatase-conjugated secondary antibody and developed using the BCIP/NBT membrane phosphatase substrate system (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA).

Cell proliferation was analyzed using a colorimetric assay for cleavage of the tetrazolium salt WST-1 (Roche Diagnostics, Basel, Switzerland) by mitochondrial dehydrogenases in viable cells. The measured absorbance of the dye directly correlates with the number of metabolically active cells in the culture. The cells were cultured in 96-well plates (Nunc; Thermo Fisher Scientific) in growth medium with/without 10 ng/ml VEGF-C under hypoxic conditions. After 5 days, the cells were incubated for a further 1 h at 37°C with 100 µl medium containing 10 µl WST-1 reagent. The samples were shaken for 1 min, and absorbance was measured at 450 nm using an MPR-A4i microplate reader (Tosoh Corp., Tokyo, Japan).

The migration assay was performed as reported previously using Transwell cell culture inserts (BD Biosciences, Franklin Lakes, NJ, USA) that were 6.5 mm in diameter with 8-µm pore filters (11). The cells (5.0×10⁴) were suspended in 350 µl serum-free DMEM containing 0.1% BSA (Sigma-Aldrich) and seeded into the upper well; 600 µl normal growth medium with/without 10 ng/ml VEGF-C was placed in the lower well of the Transwell plate. Following incubation for 15 h under hypoxic conditions, the cells that had not migrated from the upper side of the filter were scraped off with a cotton swab, and the filters were stained with the three-step stain set (Diff-Quik; Sysmex, Kobe, Japan). The number of cells that had migrated to the lower side of the filter was counted under a light microscope in 5 high-power fields (×400 magnification; Olympus IX70; Olympus Corp., Tokyo, Japan). The experiment was performed in triplicate.

The SG-2, SG-3 and SG-5 cells were stimulated with 10 ng/ml VEGF-C (R&D Systems) or 5.0 ng/ml TGF-β (Calbiochem, Merck Millipore). After 48 h, total RNA from each cell was isolated using ISOGEN reagent (Nippon Gene Co., Ltd.) according to the manufacturer's instructions. First-strand cDNA was synthesized from total RNA using the PrimeScript RT Reagent kit (Takara Bio, Otsu, Japan). RT-qPCR was performed on a Thermal Cycler Dice Real-Time system with SYBR Premix Ex Taq II (both from Takara Bio) and specific oligonucleotide primers (Table I) using a two-step cycle procedure (denaturation at 95°C for 5 sec, annealing and extension at 60°C for 30 sec) for 40 cycles. For each test run, cDNA derived from 50 ng total RNA as a template and 0.4 µM primer pair was used. mRNA expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh), and the relative amounts of each mRNA in each sample were calculated using the ΔΔCq method. The relative mRNA expression levels are expressed as fold increase or decrease relative to the control.

All experiments were repeated at least 3 times. Representative images or data are shown. The numerical data are presented as the means ± standard deviation (SD). Differences between averages and percentages between control and tests were statistically analyzed using paired two-tailed Student's t-tests. A P-value <0.05 was considered to indicate a statistically significant difference.

To identify the genes that modulate the regenerative ability of MSCs, we used DNA microarrays to characterize the specific gene expression profiles observed in the TGF-β-responsive SG-2 cells. We identified 105 genes that were ≥10-fold more strongly expressed in the SG-2 cells compared to the SG-3 and SG-5 cells (Table II). Among these genes, we focused on VEGFR3, the Flt4 gene product, since, as a cell surface antigen, it is useful for identifying and sorting MSCs from various tissues. The gene expression level of Flt4 in the SG-2 cells was more than 16.5- and 32.0-fold higher than that in the SG-3 and SG-5 cells, respectively. These results were further confirmed by flow cytometry (Fig. 1) and indicated that the SG-2 cells expressed higher levels of VEGFR3 on the cell surface than the SG-3 and SG-5 cells.

Subsequently, we examined the effects of VEGF-C, a specific ligand of VEGFR3, on the MSC lines (SG-2, SG-3 and SG-5). Indeed, VEGF-C significantly stimulated SG-2 cell proliferation (Fig. 2A) and cell migration (Fig. 2B), but had no effect on the SG-3 or SG-5 cells. These results strongly suggest that VEGF-C specifically promotes the proliferative activity and migratory ability of the SG-2 cells through VEGFR3.

To clarify the signaling pathways activated by VEGF-C in SG-2 cells, we evaluated the phosphorylation status of molecules in the PI3K/Akt- and MAPK-mediated pathways. ERK1/2 phosphorylation was markedly upregulated in the SG-2 cells upon VEGF-C stimulation, whereas that in the SG-3 and SG-5 cells was unaffected (Fig. 3). These results suggest that VEGF-C enhances the proliferative activity and migratory ability of the MSCs through the ERK1/2 pathway in Flt4-positive SG-2 cells.

The VEGF-C gene encodes a ligand for VEGFR3 that is expressed mainly in lymphatic endothelia (18). Furthermore, the VEGF-C/VEGFR3 pathway was the first critical pathway described for the development of the lymphatic vascular tree (36). As the SG-2 cells respond to both TGF-β and VEGF-C, we examined the effects of TGF-β or VEGF-C stimulation on their multi-differentiation potential. VEGF-C clearly and significantly increased the mRNA expression levels of the lymphatic endothelial cell markers, prospero homeobox 1 (Prox1) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) (p<0.01), in the SG-2 cells, whereas the levels were clearly and significantly decreased following stimulation of the cells with TGF-β (p<0.05; Fig. 4A and B). We previously reported that TGF-β promotes the osteogenic differentiation of bone marrow-derived MSCs (10). Therefore, in this study, we further investigated the mRNA expression of osteogenic differentiation markers in the SG-2 cells following TGF-β or VEGF-C stimulation. TGF-β clearly and significantly increased the expression of the early-stage osteogenic differentiation marker genes, Runt-related transcription factor 2 (Runx2) and alkaline phosphatase, liver/bone/kidney (Alpl) (p<0.01), in the SG-2 cells; by contrast, VEGF-C clearly and significantly decreased the expression of these early-stage osteogenic differentiation markers (p<0.05; Fig. 4C and D). Of note, TGF-β unexpectedly decreased the expression of the late-stage osteogenic differentiation markers, integrin-binding sialoprotein (Ibsp) and bone gamma-carboxyglutamate (Gla) protein (Bglap) (p<0.05; Fig. 4E and F). On the other hand, as expected, VEGF-C suppressed the expression of these late-stage differentiation markers (p<0.01; Fig. 4E and F). These results suggest that VEGF-C and TGF-β reciprocally regulate the commitment of MSCs to differentiate into lymphatic endothelial or osteoblastic phenotypes, respectively. On the other hand, both TGF-β and VEGF-C appear to suppress the final maturation of osteoblastic MSC differentiation during late-stage osteogenesis.