Alkaline phosphatase staining kit

Cells were seeded on 6-well plates at a concentration of 70 cells/cm². After 3 days, cells were washed in PBS and medium was replaced with 10-fold decreasing concentrations of LIF (from 100 to 0 units/ml). After 6 days, cells were fixed and stained with Alkaline Phosphatase Staining Kit (Sigma). Colonies were counted under a Zeiss Axiovert 40C microscope and scored into three distinct categories: stained (pluripotent), mixed and unstained (differentiated).

Methylsulfonylmethane (MSM), Ascorbic acid, β-glycerophosphate, Calcein Blue, MTT assay kit, Alkaline phosphatase staining kit, Cystamine, and GAPDH antibody were purchased from Sigma (St. Louis, MO). The following antibodies were bought from the company indicated in parenthesis: Collagen alpha 1 (Col 1; Novus Biological; Littleton, CO); Osteopontin (OPN; Abcam, Cambridge, UK), Transglutaminase (TG2; Abcam, Cambridge, UK), Runt-related transcription factor 2 (RUNX2) and HRP conjugated (mouse or rabbit) secondary antibodies (Santa Cruz Biotechnology, Dallas, TX); Osterix (Millipore, MA, USA). Alizarin Red S (ARS) 2% staining solution was from LifeNet® Cell Technology (CM-0058; Fredrick, MD). Fluorochrome-conjugated secondary antibody Alexa Fluor 488 (#4412) and Alexa Fluor 555 (#8953) (Cell Signaling technology®, Danvers, USA) Super Signal^™ West Pico Chemiluminescent substrate was bought from Thermo Fisher Scientific (Waltham, Massachusetts). Demineralized and mineralized bone particles were from LifeNet Health^®(Virginia Beach, Virginia).

Mouse ESCs were cultured without feeders on 0.1% gelatin‐coated plastics in Glasgow Minimal Essential Medium with 10% serum and Leukemia Inhibitory Factor (LIF) 19 and differentiation was performed according to our previous protocol 20. The cell lines used were derivatives of E14Tg2aIV (46C 21, OCRG9 22) or Oct4GiP 23. GlcNAcstatin C (GNS) was obtained from GlycoBioChem (Dundee, UK; www.glycobiochem.com) and used at 1 µM unless otherwise specified. For flow cytometry, cells were dissociated using Accutase (Millipore, Billerica, MA; www.emdmillipore.com) and resuspended in PBS/1% bovine serum albumin (BSA) containing 1 µg/ml propidium iodide (PI; Sigma, St. Louis, MO; www.sigmaaldrich.com). Cellular debris and PI‐positive cells were excluded from analysis. For clonal analysis, 600/100 cells were plated per 10 cm dish/well of six‐well plate and cultured for 6–8 days, then fixed, and stained using an Alkaline phosphatase staining kit (Sigma).

The RAW 264.7 cell line was purchased from American Type Culture Collection (ATCC® TIB-71™). C-PC was bought from Sigma-Aldrich (St. Louis, MO), dissolved in sterile water and stored at 4 °C. MTT assay kit, Alkaline phosphatase staining kit, and GAPDH antibody were also purchased from Sigma-Aldrich (St. Louis, MO). The following antibodies were bought from the company indicated in parenthesis: NFATc1 (SC-16657; Santa Cruz Biotechnology; Santa Cruz, CA), TRAP and CTSK (ab191406, ab19027; Abcam; Cambridge, United Kingdome), β3 Integrin, Caspase-3, caspase-9, c-Fos, and IκB-α (4702, 9662, 9504 S, 4384 S, 4814 S; Cell Signaling Technology; Danvers, MA), and HRP conjugated (mouse or rabbit) secondary antibodies (Santa Cruz Biotechnology; Santa Cruz, CA). Protein estimation reagents, molecular weight standards for proteins, and PAGE reagents were bought from Bio-Rad. Alizarin red solution was bought from Life-line Cell Technology (CM-0058; Fredrick, MD). Super Signal™ West Pico Chemiluminescent substrate was bought from Thermo Fisher Scientific (Waltham, Massachusetts). Rhodamine phalloidin and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

We verified the differentiation potential of MSCs to osteocytes and adipocytes. Differentiation to adipocytes was induced by culturing MSCs in DMEM supplemented with 10% FBS, 1 mM dexamethasone (Sigma, USA), 200 mM indomethacin (Sigma, USA), 1.7 mM insulin (Sigma, USA), 500 mM isobutyl-methylxanthine (Merck, Germany), 0.05 U/ml penicillin, and 0.05 mg/ml streptomycin for 2 weeks. We have used oil red-O staining (Sigma, USA) to identify the presence of adipocytes. oil red-O staining visualizes intracellular lipid accumulation. For staining, the cells were fixed in 10% formaldehyde (Merck, Germany) for 1 hour, after which they were washed with 60% isopropanol, (Merck, Germany) and stained with oil red-O solution in 60% isopropanol for 10 minutes. Next, cells were washed with distilled water and de-stained in 100% isopropanol for 15 minutes.
Differentiation of MSCs to osteocytes was induced Stem Cell Derived Endothelial Cells for Neovascularization in α-MEM (Gibco, USA) that contained 10% FBS, 0.1 mM dexamethasone, 10 mM β-glycerophosphate (Sigma, USA), and 50 mM ascorbate-phosphate (Sigma, USA) for two weeks. A specific histochemical stain for alkaline phosphatase (ALP) with an alkaline phosphatase staining kit (Sigma Chemical Co., USA) was used to identify the osteocytes.