Human msc phenotyping kit

MNC were seeded at 4 × 10⁵/cm² in MSC medium and incubated at 37°C/5% CO₂. After 3 days, non-adherent haematopoietic cells were replaced with fresh medium, with cultures maintained by weekly demi-depletion until 70% confluency was reached. Cells were passaged with trypsin (0.05% trypsin/0.02% EDTA) and reseeded at 4 × 10³/cm² (passage one; P1) and for all further passages. Cells were cryopreserved (MSC medium containing 25% FBS and 10% DMSO) in liquid nitrogen vapour-phase at the end of P1, resuscitated and experimental work performed at P4. Purity of MSC cultures was confirmed by immunophenotype and differentiation capacity; at the same passage as experimentation (P4) and according to international guidelines (45 (link)). Confirmatory immunophenotyping was achieved on all samples using the Human MSC Phenotyping Kit (Miltenyi Biotec, Woking, UK) according to manufacturer’s instructions, staining positively for CD73-APC, CD90-FITC and CD105-PE, and negatively for CD14-PerCP, CD20-PerCP, CD34-PerCP and CD45-PerCP (data not shown). MSC from three independent cultures (011B, 016, 025) were also induced to undergo tri-lineage differentiation using StemPro® Differentiation Kits (Thermo Fisher Scientific) for adipogenesis, chondrogenesis and osteogenesis, according to manufacturer’s instructions (data not shown).

Human bone marrow MSCs (from seven donors, 18–25 years of age) were isolated from human bone marrow mononuclear cells (Lonza Biosciences, Berkshire, U.K.) by adherence for more than 24 hours to tissue culture plastic and were expanded in monolayer culture in mesenchymal stem cell growth medium (Lonza Biosciences) supplemented with 5 ng/ml fibroblast growth factor‐2 (R&D Systems, Abingdon, U.K.). Cultures were maintained in a humid atmosphere of 5% CO₂/95% air at 37°C. Once cells reached confluence, they were passaged (P1) using Trypsin/EDTA at a split ratio of 1:3. Experiments were performed using cells between P2 and P7, and all experiments were repeated with cells from three to four donors. The phenotypes of all donors of MSCs were tested by flow cytometry on a FACSCanto II system (Becton Dickinson, Oxford, U.K.) using a human MSC Phenotyping Kit (Miltenyi Biotec, Bisley, U.K.) with positive staining for CD73, CD90, and CD105 and negative staining for CD14, CD20, CD34, and CD45 (three of which can be found in the supplementary material of reference [20]). Cells were also demonstrated to be capable of differentiation into osteoblastic and adipogenic lineages (Supporting Information Fig. S1 and Supporting Information Materials and Methods).

Before application, immunophenotypic analysis of BM-MSCs was performed with a human MSC phenotyping kit (Miltenyi Biotec, Bergisch Gladbach, Germany). A minimum of 2 × 10⁵ cells were resuspended in PBS (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) with 2% FBS (FBS, Thermo Fisher Scientific, Inc., Waltham, MA, USA), centrifuged at 300× g for 10 min, stained with a cocktail of antibodies CD 45 FITC, CD 90 FITC, CD 105 PE, CD 73 APC for 10 min in dark, washed with PBS containing 2% FBS and centrifuged at 300× g for 10 min. Cells were analyzed with a Becton Dickinson FACSCalibur using CellQuestPro software (Becton Dickinson, San Jose, CA, USA).

The identification and phenotyping of cultured MSCs was based on the defined International Society for Cellular Therapy (ISCT) standards and the use of human MSC Phenotyping Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) [20 (link)]. The expression of CD90, CD105, CD14, CD20, CD34 and CD45 was assessed by BD FACSCanto™ II Flow cytometer (Becton Dickinson, USA) equipped with the FacsDiva program, and the data were then analyzed by FCS Express program.

hPDCs cultured in monolayer or aggregates were characterized for expression of stemness markers (CD73, CD90, and CD105) by flow cytometry using human MSC Phenotyping kit (Lot# 130-095-198, Miltenyi Biotec, NL). hPDCs were dispersed using TripLE (Life Technologies), suspended in a flow cytometry staining buffer solution (eBioscience Inc.,USA, Lot#E00015-1639), and stained in accordance to manufacturer’s instructions. In brief, 100 μl of cell suspension (up to 1 × 10⁶) was mixed with 10 μl of MSC Phenotyping Cocktail and incubated for 10 minutes without light at 4 °C. Subsequently, hPDCs were washed and analyzed using BD FACS CantoTM using the cell analyzer (BD Biosciences, San Jose, CA) and FlowJo V10 software.

MSCs in primary tissue or culture were quantified as previously described [5 (link), 13 (link)]. Briefly, cells were labeled with a MSC Phenotyping Cocktail (anti-CD14 PerCP, anti-CD20-PerCP, anti-CD34-PerCP, anti-CD45-PerCP, anti-CD73-APC, anti-CD90-FITC, and anti-CD105-PE) or Isotype Control Cocktail (Mouse IgG1-FITC, Mouse IgG1-PE, Mouse IGG1-APC, Mouse IgG1-PerCP, and Mouse IgG2a-PerCP) included in the human MSC Phenotyping kit (Miltenyi) in a volume of 100 μl per 1×10⁶ cells for 10m at 4°C according to the manufacturer's instructions. Anti-HLA-DR-PerCP (Miltenyi) was also added to the MSC Phenotyping Cocktail. Analysis was performed using a BD FACSCalibur flow cytometer. All compensation controls were performed using anti-EpCAM antibodies directly conjugated to FITC, PE, APC, or Biotin followed by anti-Biotin-PerCP on aliquots of the same cell suspension to ensure proper gating and instrument settings.