Msc phenotyping kit

To determine MSC surface marker expression cells were detached by accutase treatment and stained with MSC phenotyping kit (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) according to manufacturer’s instructions. Stained cells (5 × 10⁵ cells per aliquot) were resuspended in 300 µL flow cytometry buffer and acquisition was carried out on a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Between 1–5 × 10⁴ gated events were recorded. Subsequent analysis was performed with Kaluza Flow Cytometry software (version 1.3, Beckman Coulter, Brea, CA, USA).

UC-MSCs were detached with StemPro^® Accutase^® Cell Dissociation Reagent (Gibco, ThermoFisher Scientific), washed in PBS and analyzed for CD73, CD105, CD90, CD45, CD34, CD14, CD11b, HLA-DR expression (Dominici et al., 2006 (link)) using the MSC-Phenotyping Kit (Milteny Biotec GmbH, Germany). Appropriate IgG isotype-matched antibodies and unstained cells were used as controls. Dead cells were excluded by adding 7-aminoactinomycin D (7-AAD; BD Bioscience) prior to analysis. After staining procedures, cells were acquired by FACSCanto II flow cytometer (BD Biosciences) and analyzed by FACSDiva software (BD Biosciences).

The surface marker expression of MSCs and iMSCs was analyzed using MSC Phenotyping Kit (Miltenyi). The cells were trypsinized, washed with PBS and stained with labeled antibodies as well as analyzed according to the manufacturer’s instructions. For the analysis of the stained cells, fluorescence-activated cell sorting (FACS) calibur (BD) flow cytometer was used, the program CellQuestPro for data acquisition, and the softwares Cyflogic (http://www.cyflogic.com) and Microsoft Excel for data analysis.

Bovine serum albumin (BSA, MW), fluorescein isothiocyanate isomer I (FITC), phosphate-buffered saline (PBS), calcium chloride, sodium carbonate, poly (allylamine hydrochloride) (PAH), poly (sodium 4-styrenesulfonate) (PSS), minimum Essential Medium Eagle Alpha Modification (α-MEM), ITS Liquid Media Supplement, fetal bovine serum (FBS), L-glutamine, and penicillin/streptomycin were purchased from Sigma–Aldrich (St. Louis, MO, United States). Ethylenediaminetetraacetic acid (EDTA) was purchased from Helicon, MSC Phenotyping Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), and trypan blue solution (Invitrogen, United Kingdom).

To confirm that MSC were multipotent mesenchymal stromal cells we analyzed their immunophenotype according to the published criteria [39 (link)]. Cells were stained with anti- CD73, CD90, CD105, CD14, CD20, CD34, CD45 antibodies and appropriate isotype control antibodies (MSC Phenotyping Kit, Miltenyi Biotec, Bergisch Gladbach, Germany) and analyzed using flow cytometry.
The potential of MSC for osteogenic and adipogenic differentiation was tested using standard techniques in vitro. Briefly, osteogenic differentiation was induced by plating 6 × 10⁴ MSC on a 24-well plate and incubated in an Advance Stem Cell Osteogenic Medium (HyClone) containing 10% Advance Stem Cell Supplement and 100 U/mL penicillin/streptomycin for 21 days. Differentiation efficiency was analyzed using Alizarin Red S staining for calcium accumulation. Adipogenic differentiation was induced by the incubation of MSC in Advance Stem Cell Adipogenic Medium (HyClone) containing 10% Advance Stem Cell Supplement and 100 U/mL penicillin/streptomycin for 18 days. Cells accumulated intracellular lipids were analyzed using Oil-Red-O staining.

Flow cytometry was performed using a FACSCalibur Cytometer (BD Biosciences); a phenotyping kit (MSC phenotyping kit, Miltenyi) was used to characterize the tMSCs. The following antibodies were used: anti-CD34PerCP, anti-CD45PerCP, anti-CD20PerCP, anti-CD14PerCP, anti-CD73APC, anti-CD9FITC, and anti-CD105PE (BD Pharmingen). Matched isotype controls were applied to determine background fluorescence levels.