Hbm msc

We quantified adipogenic differentiation response by measuring protein content of PPARγ (Cell Signaling Technology, Inc., Danvers, MA, USA) and FABP4 (Cell Signaling). We also measured intracellular SIRT1 protein content (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Beta(β)-actin was measured for total protein normalization (Cell Signaling). Primary antibodies for these proteins were selected based on their successful application by the manufacturer to either immunohistochemistry or in-cell ELISA platforms. Goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Goat≠Rabbit; Abcam, Cambridge, MA, USA) was also selected based on this criterion.
Antibody optimization for the ICE was performed on purchased human bone marrow-derived mesenchymal stem cells (hBM-MSC; Lonza). hBM-MSCs were grown in 96-well plates for 21 days according to the adipogenic differentiation protocol above (without treatment conditions). Antibody dilutions were optimized in-house, listed in S2 Table. The slope profiles for each antibody dilution were analyzed. Dilutions presenting with multiple stable slope values over 5 minute intervals within the 30-minute read time were chosen as optimal dilution factors for the respective antibody.

Human bone marrow derived-mesenchymal stem cells (hBM-MSC) were purchased from Lonza (Walkersville, MD) and maintained in MSC basal media supplemented with L-glutamine, gentamicin sulfate, amphotericin and mesenchymal cell growth supplement (Lonza; Walkersville, MD). After the third passage, the cells were cultured in vesicle-depleted media. Vesicle depletion from media was performed by tangential flow filtration (TFF) using a sterile 500 kDa molecular weight cut off MidiKros filter lined with a modified polyethersulfone membrane (Repligen; Waltham, MA). The permeate, containing the vesicle-depleted MSC media, was collected and passed through a 0.22 μm filter before storing at 4°C. Human hepatocytes (HH; Sciencell, United Kingdom) and PLC cells (ATCC; Manassas, VA) were cultured in untreated plates. KMBC (provided by Dr. Gregory Gores, Mayo Clinic), HepG2 and Hep3B (ATCC; Manassas, VA) cells were cultured in collagen-coated plates. The aforementioned cells were maintained in Dulbecco’s modified eagle media (DMEM) high glucose media supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). HL-60 promyeloblasts (ATCC; Manassas, VA) were cultured with Iscove’s modified Dulbecco’s medium supplemented with 20% FBS in T75 flasks. RAW264.7 murine macrophages (ATCC; Manassas, VA) were cultured with DMEM high glucose media supplemented with 10% FBS.

Human bone marrow-derived MSCs (hBM-MSCs) were purchased from Lonza (Lonza, Walkersville, MD, USA), and periodontal ligament-derived MSCs (hPDL-MSCs) were prepared as previously described [49 (link)]. Briefly, periodontal ligament (PDL) tissue samples separated from the middle third of a patient’s wisdom tooth were digested with collagenase (Collagenase NB 6 GMP Grade from Clostridium histolyticum; Serva, Heidelberg, Germany)/dispase (Godo Shusei Co., Tokyo, Japan), and single-cell suspensions were passed through a 70-μm cell strainer (Falcon, Franklin Lakes, N.J., USA). The collected cells were incubated in a culture plate (Falcon T-25 flask, Primaria; BD Biosciences, San Jose, CA, USA) in complete medium: α-MEM (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Waltham, MA, USA), 2 mM l-glutamine (Sigma-Aldrich, St. Louis, MO, USA), and 82.1 μg/ml l-ascorbic acid phosphate magnesium salt n-hydrate (Wako Junyaku, Tokyo, Japan) with the antibiotics gentamicin (40 μg/ml, GENTCIN; Schering-Plough, Osaka, Japan) and amphotericin B (0.25 μg/m, FUNGIZONE; Bristol-Myers Squibb, Tokyo, Japan). After three passages for expansion in T-225 flasks, the cells were preserved in freezing media (STEM-CELLBANKER GMP grade; Nihon Zenyaku Kogyo, Fukushima, Japan) and stored in liquid nitrogen.

Ethical approval with written informed patient consent for the use of human umbilical cords for this study was given by the Institutional Domain Specific Review Board (DSRB), Ministry of Health, Singapore. Commercial human bone marrow mesenchymal stem cells (hBMMSCs) were purchased from Lonza (Basel, Switzerland) and cord blood CD34+ cells from Stem Cell Technologies (Vancouver, Canada). The ethical approval for their use was given by the National University of Singapore, Institutional Review Board (NUS-IRB). Approval for all animal procedures was given by the National University of Singapore Institutional Animal Care and Use Committee (IACUC).

The frozen hBMMSCs (Lonza, Basel, Switzerland) were thawed and cultured in medium consisting of DMEM-high glucose (Invitrogen) supplemented with 10% FBS (GE Healthcare Life Sciences), 1% antibiotic-antimycotic mixture, and 2 mM l-glutamine (Invitrogen).