Human BM-MSCs were obtained from healthy persons who voluntarily donated their bone marrow stem cells. Approximately 10-20 mL of BM was aspirated from the posterior iliac crests of humans under local anesthesia. BM mononuclear cells were isolated through density-gradient centrifugation (Histopaque-1077, Sigma-Aldrich, St. Louis, MO, USA). Mononuclear cells (2-3×10⁵ cells/cm²) were plated in a 75-cm² flask (Falcon, Franklin Lakes, NJ, USA) with Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco) and then cultured at 37℃ in a 5% CO₂ atmosphere. When the cultures approached 80% confluence, the cells were harvested by treatment with a trypsin/EDTA solution (Gibco) and replated at a density of 4-5×10³ cells/cm² in 175-cm² flasks. The human procedures and protocols were approved by the Institutional Review Board (IRB) at Yonsei University Wonju Severance Hospital (CR109021), and conducted according to the principles of the Declaration of Helsinki. All participants provided written informed consent prior to participation in the study.

The immunophenotypes of the BM-MSCs (CD14, CD34, CD45, CD73, and CD105) were analyzed prior to experimental use, and their differentiation potentials were determined (osteogenesis and adipogenesis; Fig. 1). For the immunophenotype analysis, BM-MSCs were stained with antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE): CD14-FITC, CD34-FITC, CD45-FITC, CD73-PE, and CD105-PE (BD Biosciences, San Jose, CA, USA). Briefly, 5×10⁵ cells were resuspended in 0.2 mL of phosphate-buffered saline (PBS) and incubated with FITC- or PE-conjugated antibodies for 20 min at room temperature. FITC- or PE-conjugated mouse IgGs were used as the control isotype at the same concentration as the specific primary antibodies. The fluorescence intensity of the cells was evaluated through flow cytometry (Epics XL; Beckman Coulter, Miami, FL, USA). Osteogenic differentiation was determined by first plating the cells at 2×10⁴ cells/cm² in six-well plates and then incubating them in the following osteogenic medium for 2-3 weeks: DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% FBS, 10 mM β-glycerophosphate, 10^-7 M dexamethasone, and 0.2 mM ascorbic acid (Sigma-Aldrich).20 The osteogenic differentiation was quantified from the release of p-nitrophenol from p-nitrophenyl phosphate by the enzyme alkaline phosphatase.21 For adipogenic differentiation, BM-MSCs were plated at 2×10⁴ cells/cm² in six-well plates and cultured for 1 week; then, differentiation was induced with adipogenic medium (10% FBS, 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 10 µg/mL insulin, and 100 µM indomethacin in high-glucose DMEM) for an additional 3 weeks. The differentiated cells were fixed in 4% paraformaldehyde for 10 min and stained with fresh Oil Red-O solution (Sigma-Aldrich) to display lipid droplets (Fig. 1B). The criteria regarding the use of MSCs included viability greater than 80%; the absence of microbial contamination (bacteria, fungus, virus, or mycoplasma) when tested 3-4 days before experimentation; CD73 and CD105 expression in more than 90% of the cells; and the absence of CD14, CD34, and CD45 in less than 3% of the cells, as assessed by flow cytometry.

We used immortalized human HSCs that were established by retroviral expression of human telomerase reverse transcriptase (hTERT) for the majority of in vitro experiments. Characterization by immunofluorescence for α-SMA revealed a purity of >96%. HSCs were plated in a 75 cm² flask (Falcon) with DMEM (Gibco) containing 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco), and cultured at 37℃ in a 5% CO₂ atmosphere.

For co-culture system of HSCs and BM-MSCs, cells were seeded at a 1:1 ratio in each well of a 12 or 96 well plate (Costar, Corning Inc., Corning, NY, USA). An indirect co-culture system of HSCs and BM-MSCs was assembled using Transwell membranes (12 mm diameter, 0.4 µm pore size, Costar, Corning Inc., Corning, NY, USA). HSCs were placed in the lower chamber with BM-MSCs placed on the membrane insert. Indirect co-culture system were maintained in DMEM for 48 hrs.

An indirect co-culture system of activated HSCs with BM-MSCs were maintained in DMEM containing 10% FBS at a density of 1:1 for 48 hrs. Supernatants were collected after 48 hrs of indirect co-culture system. Quantification of human TGF-β1, interleukin (IL)-6, IL-10, and hepatocyte growth factor (HGF) from culture supernatant was determined using enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer's instructions. The absorbance of each well was measured at 490 nm wavelength using a microplate reader ELX 800 (Bio-Tek Instruments, Inc., Winooski, VT, USA).

Cell viability assays performed using a CellTiter 96® AQueous One Solution Reagent ELISA kit (Promega, Madison, WI, USA). The CellTiter 96 Aqueous One Solution contains MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] compound and an electron coupling reagent phenazine ethosulfate (PES). The MTS compound is bioreduced by mitochondrial enzymes of cells into a colored formazan product that is soluble in cell culture medium. Briefly, for direct co-culture system of HSCs and BM-MSCs, cells were seeded at a 1:1 ratio in each well of a 96 well plate (Corning). Co-cultures were maintained in DMEM for 4 days. Assays are performed by adding a small amount of the CellTiter 96® AQueous One Solution Reagent direct to culture wells, incubating for 2 hrs and then recording the absorbance at 490 nm wave-length using a microplate reader ELX 800 (Bio-Tek Instruments).

Quantification of cell apoptosis was measured using a Cell Death Detection ELISA kit (Roche Applied Science, Indianapolis, IN, USA). Cell Death Detection ELISA kit was used to quantify histone-complexed DNA fragments (nucleosomes) in cytoplasm of the apoptotic cells after induction of apoptosis. Briefly, for direct co-culture system of HSCs and BM-MSCs, cells were seeded at a 1:1 ratio in each well of a 12 well plate (Corning). After direct co-culture system of HSCs and BM-MSCs for 4 days, cells were lysed with cell lysis buffer. Monoclonal antibodies against single-strand DNA and histones (H1, H2a, H2b, H3, and H4) specifically detected and bound mononucleosomes and oligonucleosomes derived from cells undergoing apoptosis. Biotinylated antihistone antibodies then fixed the antibody-nucleosome complexes to the streptavidin-coated microtiter plate. The anti-DNA antibodies were conjugated with a peroxidase that reacted with the substrate ABTS (2,2V-azino-di[3-ethylbenzthiazolinsulfonate]) to form a colored product. The remaining steps were carried out according to the instructions supplied by the manufacturer. The absorbance of each well was measured at 405 nm wavelength using a microplate reader ELX 800 (Bio-Tek Instruments).

Cells were cultured on 4-well chamber slide (BD Falcon, Bedford, MA, USA) and treated with or without TGF-β1 (R&D Systems, Abingdon, UK). After 24hrs of TGF-β1 (1 ng/mL) treatment, cells were washed three times with PBS and fixed with 4% paraformaldehyde in PBS for 15 min and followed by incubation with Protein Block for 15 min at room temperature. The cells were incubated with anti-α-SMA monoclonal antibody (Abcam, Cambridge, MA, USA) overnight at 4℃. The cells were washed with PBS and incubated with Alexa Fluor® 488 (Invitrogen, Carlsbad, CA, USA) secondary antibody for 1 hr at room temperature in dark room. After washing, the slides were mounted with Vecta-shield (Vector Labs, Burlingame, CA, USA) mounting media containing 4',6-diamino-2-phenylindole (DAPI) for detection of all nuclei. Fluorescence images were observed with a laser scanning confocal microscope (TCS SPE, Leica Microsystems GmbH, Wetzlar, Germany).

Values are expressed as means ± standard deviation. Analysis was conducted using nonparametric analysis with the Mann-Whitney U tests. All analyses were performed using SPSS software version 18.0 (SPSS Inc., Chicago, IL, USA). For all tests, P-values of <0.01 were considered statistically significant.

The immunophenotypes for CD14, CD34, CD45, CD73, and CD105 cells were determined and osteogenic or adipogenic differentiation was induced on experimental the day (Fig. 1). In cell populations, CD73 or CD105 (which are positive markers of BM-MSCs) were expressed in more than 99% of the cells. However, CD14, CD34, or CD45 (which are known to be negative markers of BM-MSCs) were expressed in less than 1% of the cells (Fig. 1A). BM-MSCs could be differentiated into osteocytes and adipocytes (Fig. 1B).

The expression of α-SMA, a specific marker for activated HSCs was observed by fluorescent immunocytochemistry. We observed that the activation of HSCs resulted in the expression of α-SMA, which more increased by TGF-β1 treatment (Fig. 2A and 2B). The expression of α-SMA in direct co-culture system of activated HSCs with BM-MSCs were significantly decreased compared to the HSCs (Fig. 2).

To determine whether BM-MSC paracrine factors could modulate activated HSCs via indirect co-culture system. Measurement of TGF-β1, HGF, IL-6, and IL-10 from supernatant of co-cultured medium were done by ELISA. An indirect co-culture system of activated HSCs with BM-MSCs decreased the production of TGF-β1 by 75% and IL-6 by 16%, respectively (P<0.01, Fig. 3). Whereas, co-culture system of activated HSCs with BM-MSCs increased the production of HGF by 3.0-fold and IL-10 by 3.2-fold, respectively (P<0.01, Fig. 3).

At a 1:1 co-culture ratio, to determine whether BM-MSCs have the capacity to inhibit activated HSCs viability, we quantified the CellTiter 96® AQueous One Solution Reagent ELISA kit. And to determine whether BM-MSCs also have the capacity to reduce activated HSCs numbers by inducing their apoptosis, we quantified the Cell Death Detection ELISA kit. HSCs viability was decreased by 34% and apoptosis was increased by 3.7-fold with direct co-culture system of activated HSCs with BM-MSCs, respectively (P<0.01). We confirmed that BM-MSCs induced apoptosis of HSCs and also significantly inhibited viability of HSCs (Fig. 4).