Experimental allo-HSCT and MSCs
Female B10.D2 (H-2d) and BALB/c (H-2d) mice (8 to 12 weeks old) were purchased from Shizuoka Institute for Laboratory Animals (Japan SLC, Shizuoka, Japan). Briefly, recipient (BALB/c) mice were lethally irradiated with 650 cGy using a Gammacel137Cs source. Approximately 6 hours later, they were injected i.v. via the tail vein with donor (B10.D2) T cell-depleted bone marrow (5 x 106 cells/mouse) and spleen cells (3 x 106 cells/mouse) (referred to as Scl-GVHD mice). A control group of BALB/c recipient mice received either B10.D2 donor BM without T cells (non-GVHD controls) or BALB/c BM with T cells (syngeneic controls). The primary human BM and AD MSCs were obtained from a stem cell bank. BM-MSCs (Scl-GVHD + hBM-MSCs) or AD-MSCs (Scl-GVHD + hAD-MSCs) were administered after allo-HSCT at a dose of 3 x 105 cells/mouse.
GVHD skin score and histopathological analysis
The clinical skin GVHD score was modified as previously described (7). The minimum score was 0, and the maximum score was 8. Formalin-fixed, paraffin-embedded tissue sections were subjected to hematoxylin-eosin (H&E)-staining for microscopic examination and Masson’s trichrome staining for fibrosis. Slides were scored by a pathologist (blinded to experimental groups). Dermal thickening from the bottom of the epidermis to the fat was evaluated for each animal as previously described (18). The Masson’s trichrome stained section was standardized for the photographic area (1200 x 1200 pixels) allowing a direct comparison between images. The collagen area was traced and measured using the Image J software (http://rsb.info.nih.gov) and the percentage of collagen per standardized field of view (100x magnification) was calculated.
Protein extracts and measurement of soluble collagen and IgE
Tissue samples were immediately frozen in liquid nitrogen were disrupted using a Polytron homogenizer (Pellet pestles cordless motor, Sigma) and centrifuged at 3,000 rpm for 20min. Proteins were purified from the supernatant and the concentration was assessed using the Bradford method (Bio-Rad, Hercules, CA). Total soluble collagen was quantified using the Sircol Soluble Collagen Assay (Bio-color, Belfast, Ireland) as previously described (19). Concentrations of IgE were determined with a Mouse IgE ELISA MAX Standard kit (Biolegend, 432401).
Immunohistochemical (IHC) staining
Tissue sections (4 ㎛) were mounted on super frost glass sliders and deparaffinized in xylene and a graded series of ethanol, followed by antigen retrieval. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Nonspecific binding sites were saturated by exposure to 10% normal goat serum diluted in PBS for 60 min. Slides were then incubated overnight at 4℃ with primary antibodies against mouse MMP1 (1:250 dilution, Abcam), PTEN (1:100 dilution, Abcam), pSmad3 (1:200 dilution, Invitrogen), CD3 (1:400 dilution, Santa Cruz), CD68 (1:250 dilution, Abcam) or EPX (1:200 dilution, Abcam), then washed with PBS for 10 min. Biotinylated goat anti-rabbit IgG and rabbit anti-goat IgG (Vector Laboratories, Burlingame, CA) secondary antibodies were applied to tissue sections, and the slides were incubated at room temperature for 30 min. After the sections were washed and incubated for 30 minutes with peroxidase-conjugated streptavidin (Dako, Glostrup, Denmark) at room temperature, 3,3’-diaminobenzidine was added to visualize antigens. Sections were counterstained with Mayer’s hematoxylin, dehydrated, cleared, and mounted. Negative control tissue samples were prepared in the same manner as described above, except that the primary antibody was omitted or replaced with an isotype control antibody (R&D Systems, Minneapolis, MN).
IHC stains were evaluated for the presence of positively staining cells in the dermis as previously described (20). In brief, the following semiquantitative scale, based on the percentage of positive cells, was used: 0 (no staining), 1 (<25% staining), 2 (25–50% staining), 3 (50–75% staining), or 4 (75–100% staining). Stained cells were counted under a high-power microscopic field (400 X original magnification) on a light microscope.
Fluorescent detection for in vivo tracking of MSCs
Primary hMSCs were labeled with PKH-26 according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, Mo, USA) and injected into recipient mice. To evaluate MSC recruitment, tissues were immediately embedded in the OCT (CellPath) embedding matrix, placed on dry ice, and stored at -80℃. Tissues were then sectioned on a cryostat (4 ㎛), fixed with acetone, and, after washing, stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Slides were examined using a confocal microscope (LSM700; Carl Zeiss).
Quantitative real-time (qRT)-PCR analysis
Total RNA was isolated from skin and lung homogenates with Trizol (Invitrogen, Carlsbad, USA) in accordance with the manufacturer’s instructions. One microgram of total RNA was reverse transcribed into cDNA. Quantitative assessment of target mRNA levels was performed by quantitative RT-PCR using a CFX96TM real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Results were normalized to b-actin expression and expressed as fold change compared with non GVHD control mice, where specified, using the 2–DDCt method. The sequences of forward and reverse primers are shown in Table 1.
Bronchoalveolar lavage (BAL)
BAL was performed in situ four times with Hanks’ balanced salt solution (35 ml/kg; pH 7.2–7.4), and the recovered BAL fluid (BALF) was immediately cooled in ice. BALF returns were centrifuged (1500 rpm, 5 min at 4oC), and the pooled cell pellets from all BALF were combined and resuspended in 1 ml of Hanks’ balanced salt solution, and the number of BAL cells was counted with a hemacytometer. Aliquots (100 μl) of cell resuspension were cytocentrifuged, and the cytospin slides were stained with Diff-Quick for differential cell counting (markers of cellular inflammation and epithelial injury). Percentage and absolute numbers of each cell type was calculated in triplicate.
Cell isolation and flow cytometric analysis
Mononuclear cells were isolated from skin as previously described (18). Briefly, minced skin was digested for 30 min in complete medium supplemented with Liberase and DNase (both purchased from Roche), and leukocytes were isolated by density gradient centrifugation on Accuprep medium (Accurate Chemical, Oslo, Norway). To determine the surface phenotype, single-cell suspensions were stained with PE-conjugated anti-CD11b and APC-Cy7-conjugated anti-CD4 at 4 °C for 30 min. Samples were analyzed using an LSRII (BD Pharmingen, San Diego, CA).
Statistical analysis
All values are expressed as mean ± standard error of the mean (SEM). Statistical comparisons between groups were performed using a parametric independent sample t-test if there were >5 animals per group, or using the Mann-Whitney test if there were <5 animals per group.