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 h 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]. Collagen deposition was quantified on trichrome stained sections as the ratio of blue-stained area to total stained area using digital analyzer software ImageJ (http://rsb.info.nih.gov).
Protein extracts and measurement of soluble collagen
Tissue samples were homogenized in 2 ml of buffer solution and centrifuged at 3,000 rpm for 20 min, after which supernatants were harvested. Total protein concentrations in supernatant were determined using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Total soluble collagen was quantified using the Sircol Soluble Collagen Assay (Bio-color, Belfast, Ireland) as previously described [19].
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 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. The quantity of mRNA was calculated using the 2-ΔΔCt method, and β-actin was used to normalize total RNA quantities.
Bronchoalveolar lavage (BAL)
BAL was performed in situ four times with Hanks’ balanced salt solution (35 mL/kg; pH 7.2–7.4, WelGENE Inc.). Cell pellets were resuspended in 1 ml of HBSS, and then counted using a hemocytometer.
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, cells were stained with PE-conjugated anti-CD11b and APC-Cy7-conjugated anti-CD4.
Measurements of IgE by ELISA
Concentrations of IgE in the supernatants of homogenized tissues were measured by ELISA (Biolegend).
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.