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Human PD-MSCs were isolated from the chorionic plate of the placenta with approval by the Institutional Review Board of CHA General Hospital, Seoul, Republic of Korea (IRB 08-17). PD-MSCs were obtained from the human placentas of the consented women after uncomplicated elective Caesarean delivery by separating via blunt dissection from the placental body. Then the samples were characterized by examining their morphology and expression of stem cell markers, along with FACS analysis of the surface markers. The details of the method and results can be found in our previous studies [23 (link), 24 (link)]. PD-MSCs were cultured in alpha-minimum essential medium (α-MEM, HyClone Laboratories Inc., South Logan, UT, USA) with 10% fetal bovine serum (FBS, GIBCO-BRL, Langley, OK, USA), 1% penicillin/streptomycin (GIBCO-BRL), 25 ng/mL FGF-4 (Peprotech, Rocky Hill, NJ, USA), and 1 μg/mL heparin (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a humidified 5% CO₂/95% air atmosphere, and stained with PKH67 by using a PKH67 Fluorescent Cell Linker Kit (Sigma-Aldrich).

Osteoclasts were generated from murine bone marrow cells as previously described27 (link). Bone marrow cells obtained via flushing the long bones of 6-week-old mice were cultured in α-MEM (HyClone Laboratories) containing 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin (Life Technologies, Carlsbad, CA, USA) in the presence of M-CSF (30 ng/mL) for 3 days. After non-adherent cells were removed, adherent BMMs were plated onto 96-well plates (1 × 10⁴ cells/well) and further cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL). Cultured cells were fixed and stained for TRAP. TRAP-positive multinuclear cells (>3 nuclei/cell) [TRAP⁺ MNCs] were counted as osteoclasts. Cells were observed using a Leica DMIRB microscope equipped with an N Plan 10 × 0.25 numerical aperture objective lens (Leica Microsystems, Wetzler, Germany). Images were captured using ProgRes® Capture Pro software (Jenoptik, Jena, Germany).

For osteoblast differentiation assays, the primary mesenchymal stem cells (MSCs) were isolated from the bilateral femur and tibia of naïve, saline control or CPD-treated (100mg/kg/day) male C57BL/6 mice. Bone marrow cavity was flushed with α-MEM (Hyclone laboratories, Utah, USA) containing 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin. MSCs were cultured with osteoblast differentiation introduction medium (α-MEM complete medium containing 10mmol/L β-Glycerophosphate (Sigma), 50mg/L L-ascorbic acid (Sigma), 10^-8mol/L Dexamethasone (Sigma)) for 7ds with media refreshed twice weekly. Then cells were fixed for 20min with 10% Neutral Buffered Formalin followed by washing twice with PBS. ALP staining reagent 1-Step NBT-BCIP (Pierce, Rockford, IL, USA) were added to wells of 24-well plate for 0.5-1h at 37°C and the positive staining was shown by Lyons blue. MSCs from naïve mice were cultured with CPD-Con, CPD-B, CPD-D, and CPD-H for 3 days before cultured in the osteoblast differentiation introduction medium for ALP staining or Alizarin red staining. To examine the capacity of mineralized nodule formation, prolonged osteoblast differentiation induction was performed until 21ds with media refreshed twice weekly. The cells were fixed and washed as the ALP staining, then were stained with 1% alizarin red at 37°C for 30 min, washed for observation.

Primary osteoblast precursors from mouse calvaria were isolated as described previously [45 (link)]. Briefly, calvaria were isolated from 4-day-old mice and digested with 0.25% trypsin and 0.2% collagenase at 37 °C for 30 min. Released cells were plated in a 100-mm dish, grown in α-MEM (Hyclone Laboratories Inc.) supplemented with 10% FBS (Hyclone Laboratories Inc.), and incubated in a 37 °C incubator with 5% CO₂. After 3 days, adherent cells were used as osteoblast precursors. The osteoblast precursors were used for osteogenic differentiation with L15 EPS.

hDPSCs were seeded at 15,000 cells/cm² onto 12-well culture dishes in osteogenic differentiation media consisting of α-MEM (Hyclone Laboratories Inc.) supplemented with 10% FBS (Hyclone Laboratories Inc.), 100 nM dexamethasone (Sigma-Aldrich), 10 mM β-glycerophosphate (Sigma-Aldrich) and 0.05 mM ascorbic acid 2-phosphate (Sigma-Aldrich) [36 (link)]. The media was changed every 2 to 3 days and osteogenic differentiation was conducted for 28 days.

For the fatty acid oxidation assay, cells were incubated in α-MEM (HyClone Laboratories, Inc., Logan, UT, USA) containing 0.1 mM palmitate (9,10-[³H]palmitate, 5 μci/mL) (PerkinElmer, Waltham, MA, USA) and 1% bovine serum albumin for 24 h. Following incubation, the medium was harvested and precipitated with 10% trichloroacetic acid (Sigma-Aldrich/Millipore), vortexed, and incubated for 20 min at room temperature, then centrifuged at 16000 × g for 10 min at 4 °C. The supernatants were transferred to a new tube and incubated in a scintillation vial containing 0.5 mL water at 60 °C for 12 h. After incubation, the vial was removed and radioactivity (³H₂O in water) was quantified using a liquid scintillation counter (LKB Instruments, Victoria, Australia).