Adipose tissue harvesting, isolation of ASCs, and their culture

The Institutional Animal Care and Use Committee of Osaka Medical and Pharmaceutical University approved all of the following research protocols (approval ID: 21050-A), including the surgical procedures and animal care, and all methods were performed in accordance with the relevant guidelines and regulations. Mice were maintained under a specifically controlled pathogen-free environment (temperature, 25°C; humidity, 50–70%; light/dark cycle, 12 h/12 h) with free access to food and water. Female 8-week-old Balb/c mice (SHIMIZU Laboratory Supplies, Kyoto, Japan) were sacrificed by cervical dislocation under isoflurane anaesthesia. Adipose tissue was harvested from the inguinal areas and used in all experiments as subcutaneous adipose tissue. Mouse ASCs (mASCs) were isolated from each adipose tissue sample (as previously described) with minor modifications [32]. Briefly, adipose tissue was placed in a collection tube, washed in phosphate-buffered saline (PBS) (pH 7.4) and cut into 0.5–1.0 mm small fragments using fine scissors. The tissues were then transferred into 15-mL tubes. Type I collagenase (1.0 mg/mL in 1% BSA/HBSS(+)) was then added to the tube at an identical volume. The mixture was immediately agitated using a water bass shaker (150 rpm) at 37 °C for 30 min. The digested tissue was filtered through a 40-μm cell strainer and centrifuged at 200×g for 5 min. The supernatant was aspirated and the cell pellet was resuspended in erythrocyte lysis buffer (168 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA-4Na; 10 mL) at 4 °C for 10 min. After erythrocyte lysis, 5 mL of medium was added, and the tube was centrifuged at 200×g for 5 min. The cell pellet was resuspended in medium and filtered through sterilised 100- and 40-μm cell strainers (Corning Inc., NY, USA). The mASCs were cultured and used in the third passage.

Cell proliferation assay

The mASCs were seeded into collagen-coated wells of a 96-well plate (Corning Inc.) at a density of 5000 cells/100 μL per well. They were cultured in DMEM/F-12 containing 10% FBS and 1.0% Pen-Strep for 24 h at 37 °C in a 5% CO2/95% air atmosphere. The medium was changed to a medium supplemented with LMWH at 0, 10, and 100 μg/mL, respectively, and cultured for 24 h at 37 °C. The medium was subsequently exchanged with 100 μL of fresh medium containing WST-8 of Cell Count Reagent SF (10 μL) and incubated for 30 min. The absorbance of the medium was measured at 450 nm wavelength. Cell viability was expressed as a percentage compared with cells cultured in DMEM/F-12 containing 10% FBS and 1.0% Pen-Strep. This experiment was repeated five times for each sample.

Cell migration assay

The migration activity of mASCs was evaluated using a modified Boyden chamber method. mASCs (50,000 cells per well) were seeded into the upper chambers of 24-well culture plates, and the lower chambers were filled with DMEM/F-12 medium containing 2% FBS and 1.0% Pen-Strep supplemented with LMWH at 0, 1, 10, and 100 μg/mL, respectively, followed by incubation for 6 h at 37 °C. The migrated cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) and counted in three randomly selected high-power fields (HPFs: ×200, 0.15 mm2 per HPF) per chamber under a fluorescence microscope, and the resulting numbers were averaged. The experiments were independently performed five times for each sample.

HGF immunoassay

The HGF content of the supernatants was evaluated using an HGF ELISA Quantikine Kit (R&D Systems Inc., MN, USA) in accordance with the protocol of the manufacturer. The data were normalised to the relative cell numbers obtained from the cell proliferation assay. The experiments were carried out six times independently for each sample and in duplicate.

Quantitative real-time reverse transcription-PCR (RT-qPCR) using mASCs

Total RNA was extracted from mASCs cultured in DMEM/F-12 containing 2% FBS and 1.0% Pen-Strep supplemented with LMWH at 0, 1, 10, or 100 μg/mL. After RNA extraction with an RNeasy mini kit (Qiagen Ltd., Manchester, UK), cDNA was synthesised using an ExScript RT kit (Takara, Shiga, Japan) and amplification was performed in a Sequence Detection System 7000 (Applied Biosystems) according to the manufacturer’s instructions. The primer sequences for stromal derived factor-1 (SDF-1), C-X-C chemokine receptor (CXCR) type 4 (CXCR-4), CXCR type 7 (CXCR-7), and glyceraldehyde 6-phosphate dehydrogenase (GAPDH) are summarised in Table 1. Each target gene expression was normalised to the housekeeping gene expression. The experiments were repeated in triplicate, and results were averaged.

Table 1 Primers used in the RT-qPCR analyses in vitroAnimals and experimental groups

For inducing skin fibrosis, the back skins of 8-week-old female BALB/c mice were shaved, and for 21 days, the mice were daily subcutaneously injected with 100 μg/100 μL bleomycin (BLM) (Nippon Kayaku, Tokyo, Japan) in sterile saline [33] (Additional file 1). The mASCs activated with LMWH (hep-mASCs) were cultured in DMEM/F-12 containing 10% FBS and 1.0% Pen-Strep supplemented with LMWH (10 and 100 μg/mL). In this study, to assess the effect of hep-mASCs, mice were assigned to the following groups, with n = 9 in each group: BLM-induced SSc (BLM-alone), BLM-induced SSc administered mASCs (BLM-mASC), BLM-induced SSc administered mASCs activated with 10 μg/mL LMWH (BLM-hep10-mASC), and BLM-induced SSc administered mASCs activated with 100 μg/mL LMWH (BLM-hep100-mASC). The mASCs cultured with or without LMWH were intravenously injected into the tail vein with 100 μL of PBS. In the BLM-alone group, 100 μL of PBS was intravenously injected into the tail vein. The number of administered cells was 2.5 × 104 cells. All three treatments were performed on day 7 following BLM administration. The mice were euthanised and their skins were harvested at 21 days after administering BLM.

Histological analysis

The back skins of each mouse were harvested and fixed for 6 h in 4% PFA/PBS followed by overnight incubation in 20% sucrose/PBS. The tissues were embedded in optimal cutting temperature compound (Sakura FineTek, Tokyo, Japan), cut into 5-μm sections, and stained with haematoxylin and eosin (H&E) or Masson’s trichrome stain. The thickness of the dermis was measured to evaluate skin sclerosis. The distance between the epidermal–dermal junction and the dermal–fat junction was measured at 10 randomly selected sites per section and was averaged.

Hydroxyproline assay

Hydroxyproline content was determined by measuring the skin tissue. The injected skin sites of each BLM-SSc mouse were punch-biopsied (6 mm) and stored at −70 °C. Each sample was treated with 0.5 mol/L acetic acid-containing pepsin (0.3 mg/10 mg) at 4 °C for 24 h. The hydroxyproline content of the skin tissue was measured using a SircolTM soluble collagen assay kit (Biocolor Ltd, Northern Ireland).

Fluorescent immunocytochemistry

The skin sections were washed with PBS, and the samples were blocked in an antibody dilution buffer of 2% BSA/PBS for 15 min at room temperature (RT). After the blocking solution was removed, primary antibodies/markers were added to the antibody dilution buffer at 37 °C for 2 h: anti-CD3 (1:200) (eBioscience, San Diego, CA, USA) for T lymphocytes; anti-F4/80 antibody (1:200) (eBioscience) for macrophages. After washing with PBS, cells were incubated for 30 min at RT with secondary antibodies prepared at 1:500 in antibody dilution buffer: Alexa 594 donkey anti-rat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). After the secondary antibodies were removed and the tissues had again been washed with PBS, nuclear counterstaining was performed by incubating with 4’,6-diamidino-2-phenylindole (DAPI) solution (1 μg/mL in PBS; Fujifilm Wako Japan K.K.) for 10 min at RT. A mounting medium (ImmunoBioScience, Mukilteo, WA) was added to the sample slides before they were covered with cover slips and sealed with nail varnish. They were then evaluated under a fluorescence microscope (BZx-700, Keyence, Osaka, Japan). The positively stained cells in each sample were counted in five different high-power fields (×200).

Quantitative real-time RT-PCR using skin in vivo

Total RNA was extracted from skin tissues harvested at 21 days after BLM administration. Following RNA extraction with an RNeasy fibrous tissue mini kit (Qiagen Ltd.), cDNA was synthesised and amplified. The primer sequences for interleukin-2 (IL-2), interferon-gamma (INF-γ), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-13 (IL-13), interleukin-17 (IL-17), interleukin-6 (IL-6), α-smooth muscle actin (α-SMA), transforming growth factor β1 (TGF-β1), collagen type 1 alpha 1 (COL1α1), tissue inhibitor of metalloproteinase 2 (TIMP-2), and GAPDH are summarised in Table 2. The primers for gremlin-1 (GREM-1) were purchased from Sino Biological, Beijing, China. The experiments were repeated in triplicate, and the results were averaged.

Table 2 Primers used in the RT-qPCR analysesWestern blotting using skin

The skin tissues were crushed into pieces using an automill machine (2 sets of 2 min at 1500 rpm) in RIPA buffer supplemented with protease inhibitor: PMSF (0.1 mM) and phosphatase inhibitor: sodium fluoride (5 mM).

The protein concentration was analysed using Qubit protein assay kit (Thermo Fisher Scientific, Cleveland, OH, USA).

Western blotting was performed using the Protein Simple WES System (Protein Simple, SanJose, CA, USA) following the protocols given by the manufacturer. Primary antibodies: anti-Smad2/3 (1:20) (Cell Signaling Technologies, Danvers, MA, USA), anti-Phospho-Smad2/3 (1:20) (Cell Signaling Technologies), and secondary antibodies: Anti-Rabbit Secondary HRP Antibody (Protein Simple) were used the western blot. Protein expression was normalized to total protein using the Total Protein Detection Module (Protein Simple). Simple Western data was analysed using Compass Software version 3.1 (Protein Simple).

Bioluminescence imaging for in vivo analysis of mASCs

The mASCs were incubated with indocyanine green (ICG) in PBS (25 μM) for 30 min at 37 °C followed by rinsing thrice with PBS. The in vivo migration ability and distribution of the hep-mASCs were investigated. The ICG-labelled mASCs—cultured with LMWH (0, 10, and 100 μg/mL)—were intravenously injected into nine mice after a week of BLM administration. Twenty-four hours after administering mASCs, the mice were sacrificed and their skin, thymus, spleen, lungs, heart, and kidneys were harvested. The cell accumulation in each tissue was evaluated as signal intensities using an IVIS Imaging System 200 Series (Calliper Life Science, Hopkinton, MA, USA), and the differences in average signal intensities depending on the LMWH concentration was examined.

Statistical analysis

All values are presented as mean ± standard error of mean (SEM). Comparisons between two groups were tested using the Mann-Whitney U test, and those among multiple groups were tested for significance via analysis of variance (ANOVA) followed by post hoc testing with a Tukey procedure. Statistical significance was set at P < 0.05. Statistical analyses were performed using commercially available software (GraphPad Prism, MDF Co. Ltd., Tokyo, Japan).