Identification of Meflin as a Potential Marker for Mesenchymal Stromal Cells

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) in culture are derived from BM stromal cells or skeletal stem cells. Whereas MSCs have been exploited in clinical medicine, the identification of MSC-specific markers has been limited. Here, we report that a cell surface and secreted protein, Meflin, is expressed in cultured MSCs, fibroblasts and pericytes, but not other types of cells including epithelial, endothelial and smooth muscle cells. In vivo, Meflin is expressed by immature osteoblasts and chondroblasts. In addition, Meflin is found on stromal cells distributed throughout the BM, and on pericytes and perivascular cells in multiple organs. Meflin maintains the undifferentiated state of cultured MSCs and is downregulated upon their differentiation, consistent with the observation that Meflin-deficient mice exhibit increased number of osteoblasts and accelerated bone development. In the bone and BM, Meflin is more highly expressed in primitive stromal cells that express platelet-derived growth factor receptor α and Sca-1 than the Sca-1-negative adipo-osteogenic progenitors, which create a niche for hematopoiesis. Those results are consistent with a decrease in the number of clonogenic colony-forming unit-fibroblasts within the BM of Meflin-deficient mice. These preliminary data suggest that Meflin is a potential marker for cultured MSCs and their source cells in vivo.


(B)
Representative flow cytometric profile of non-hematopoietic CD45 -Ter119cells stained with PDGFRα and CD31 that were isolated from hind limb muscles of 8-to 10-week-old mice with collagenase treatment.
(C) Relative mRNA expression levels of Meflin were assessed by qPCR for each population from muscle-derived CD45 -Ter119cells. An asterisk indicates a statistically significant difference (P < 0.05).   (B) Recombinant Meflin (rMeflin) and control bovine serum albumin (BSA) were added to fibroblasts (NHDFs) starved in serum-free medium to achieve the final concentration of 1 g mL. After one h, the cells were stimulated by recombinant rat PDGF-BB ( ng mL), followed by Western blot analysis with the indicated antibodies. No apparent effect of rMeflin on PDGF signaling was observed when the activation of Akt or ERK was used as readout. We also used human mammary basal epithelial cells (MBE, ZenBio) and human pericytes from placenta (hPC-PL, PromoCell).

RNA interference
Target sequences for shRNA-mediated knockdown of human (NM_005545.

Expression of Meflin by the retroviral expression system
The cDNA encoding Meflin was inserted into the pRetroQ retroviral expression vector (Clonetech), followed by production of recombinant retrovirus and infection to C3H10T1/2 cells as described previously 10 .

Western blot analysis and immunoprecipitation
For Western blot analysis, cells were lysed with SDS sample buffer (10 mM Tris-HCl, 2% SDS, 2 mM EDTA, 0.02% bromophenol blue, 6% glycerol, pH 6.8), reduced with 80 mM dithiothreitol for 2 h at 50ºC and separated by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to nitrocellulose membranes, blocked in 4% milk in phosphate-buffered saline (PBS) containing 0.05% Tween 20, incubated with primary antibodies and detected by horseradish peroxidase-conjugated secondary antibodies (Dako). In Western blot analysis for the detection of Meflin, the primary antibodies were diluted with Can-Get-Signal Solution 1 (Toyobo, Osaka, Japan) to enhance antibody-antigen binding. For immunoprecipitation analysis, 293FT or COS7 cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 120 mM NaCl, 1 mM EDTA, and 1% Triton X-100 supplemented with Complete Protease Inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche, Indianapolis, Indiana). Lysates were cleared by centrifugation at 12,000 x g for ten min, followed by immunoprecipitation using the indicated antibodies and protein A/G beads (Sigma).

Isolation of GPI-anchored proteins and cell surface and nuclear proteins
Triton X-114 phase separation was performed according to protocols that were previously described with some modifications [11][12][13] . Dermal fibroblasts were suspended and lysed in an

Production and purification of recombinant Meflin
Recombinant mouse Meflin was produced by the silkworm expression system (ProCube, Sysmex, Kobe Japan) as described previously [14][15][16][17] . Mouse Meflin tagged with a secretion signal sequence (SP) and a Flag tag at the N-terminus (SP-Flag-Meflin) was subcloned into baculoviral expression vector pM01 (Sysmex) harboring the polyhedrin promoter, which was then cotransfected into BmN (B. mori) cells with baculovirus genomic DNA to produce recombinant virus. After propagation of the recombinant baculovirus in BmN cells, the virus was infected into silkworm larvae of the fifth instar early stage. Five days after inoculation, the hemolymph of the larvae containing recombinant Meflin was collected and centrifuged at 100,000 x g for 1 h. The supernatants (20 mL) were subjected to affinity purification using anti-Flag agarose followed by elution with the Flag peptide.

Mouse genotyping
Genomic DNAs extracted from mouse tails were used for PCR genotyping. Sequences of the primers are as follow: PCR1 forward, 5'-GCTGCATTTGAGCTGAGCCTCTGG-3'; PCR1 reverse,

Analysis of mouse skeleton
Skeletal preparations were made following the method described previously 18 . Fetuses were skinned, eviscerated, and dehydrated in 90% ethanol for 4 days and kept in acetone for another 2 days, followed by incubation in staining solution (1 vol. of 0.3% Alcian blue in 70% ethanol, 1 vol. of 0.1% alizarin red S in 95% ethanol, 1 volume of acetic acid, and 17 volumes of 70% ethanol) at 37°C for 4 days. The samples were rinsed in water and kept in 1% potassium hydroxide overnight at room temperature, followed by incubation in 1% potassium hydroxide/20% glycerol overnight at room temperature. The samples were then transferred into 50%, 80%, and 100% glycerol, and then photographed with a stereomicroscope (Olympus SZX7).

Bone histo-morphometric analysis
The tibiae of 10-week-old wild-type (n = 4) and Meflin-deficient (n = 5) male mice were subjected to histo-morphometric analysis at Kureha Special Laboratory (Tokyo, Japan). Briefly, bones were fixed with 70% ethanol, embedded (without decalcification) in carboxymethylcellulose, and with Giemsa solution, followed by counting fibroblastic colonies in each dish. Colonies containing more than 50 cells were counted after Giemsa coloration.

Flow cytometry and sorting
Isolation of mouse MSCs was performed as described previously 19 . BM suspensions isolated from the femurs and tibiae of C57BL/6 mice (8 -10 weeks) were hemolyzed with Lysing Buffer (BD Biosciences) to remove the red blood cells, followed by enrichment for CD45 Debris and dead cells were excluded by forward and side scatter.
For the isolation of PDGFRα + cells from skeletal muscle, we followed the protocol previously described by Uezumi et al 20 .
Hind limb muscles of C57BL/6 wild-type 8-to 10-week-old mice were excised, followed by digestion of the trimmed muscles with 0.2% type collagenase (Worthington) for 60 min at 37ºC. The digested muscles were filtered through a 70 µm cell strainer.
Erythrocytes were eliminated by adding Lysing Buffer (BD Biosciences), followed by enrichment for CD45 -TER119 cells using the EasySep Mouse Mesenchymal Progenitor Enrichment Kit. The cells were suspended in buffer consisting of PBS with 2% FBS, followed by the analysis on a FACSCanto flow cytometer and sorting on a FACSAria II.

Data analysis
Data are presented as the means ± S.D. Statistical significance was evaluated with Student's t test.