Abstract
Background aimsMesenchymal stromal cells (MSCs) are regenerative and immuno-privileged cells that are used for both tissue regeneration and treatment of severe inflammation-related disease. For quality control of manufactured MSC batches in regard to mature fat cell contamination, a quantitative method for measuring adipogenesis is needed.MethodsFour previously proposed methods were validated with the use of bone marrow (BM) MSCs during a 21-day in vitro assay. Oil red staining was scored semiquantitatively; peroxisome proliferator activated receptor-γ and fatty acid binding protein (FABP)4 transcripts were measured by quantitative real-time polymerase chain reaction; FABP4 protein accumulation was evaluated by flow cytometry; and Nile red/4′,6-diamidino-2-phenylindole (DAPI) ratios were measured in fluorescent microplate assay. Skin fibroblasts and MSCs from fat pad, cartilage and umbilical cord were used as controls.ResultsOil red staining indicated considerable heterogeneity between BM donors and individual cells within the same culture. FABP4 transcript levels increased 100- to 5000-fold by day 21, with large donor variability observed. Flow cytometry revealed increasing intra-culture heterogeneity over time; more granular cells accumulated more FABP4 protein and Nile red fluorescence compared with less granular cells. Nile red increase in day-21 MSCs was ∼5- and 4-fold, measured by flow cytometry or microplate assay, respectively. MSC proliferation/apoptosis was accounted through the use of Nile red/DAPI ratios; adipogenesis levels in day-21 BM MSCs increased ∼13-fold, with significant correlations with oil red scoring observed for MSC from other sources.ConclusionsFlow cytometry permits the study of MSC differentiation at the single-cell level and sorting more and less mature cells from mixed cell populations. The microplate assay with the use of the Nile red/DAPI ratio provides rapid quantitative measurements and could be used as a low-cost, high-throughput method to quality-control MSC batches from different tissue sources.
Highlights
The capacity of multipotential stromal cells (MSCs) to differentiate into a range of cell types including chondrocytes, tenocytes, adipocytes and osteoblasts is well established (1e3)
Semiquantitative scoring of adipogenesis of MSCs with the use of oil red staining The most common staining for adipogenically differentiated MSCs was initially used and quantified by means of a visual grading system [25] whereby the level of adipogenic progression in 500 cells in a central area of the well was ranked from 1e4 on the basis of the proportion of cytoplasm occupied by fat in each cell (Figure 1A)
MSCs from three bone marrow (BM) donors and negative control skin fibroblasts were grown in adipogenic medium for 21 days (Figure 1B), and scoring was performed on days 0, 3, 7, 14 and 21 after induction (Figure 1BeD)
Summary
The capacity of multipotential stromal cells (MSCs) to differentiate into a range of cell types including chondrocytes, tenocytes, adipocytes and osteoblasts is well established (1e3). MSCs represent only a small fraction of nucleated cells in the BM aspirates (between 0.001e0.01%), they can expanded with high efficiency (3e5), representing a rapidly expandable, easy to access source of multipotent cells to treat a wide variety of conditions through tissue engineering and cell therapy Another feature that makes MSCs desirable for use in regenerative medicine is the reported immunoprivileged nature of these cells and their ability to reduce inflammation and exhibit immunosuppressive effects (6e8). MSC proliferation/apoptosis was accounted through the use of Nile red/DAPI ratios; adipogenesis levels in day-21 BM MSCs increased w13-fold, with significant correlations with oil red scoring observed for MSC from other sources. The microplate assay with the use of the Nile red/DAPI ratio provides rapid quantitative measurements and could be used as a low-cost, high-throughput method to quality-control MSC batches from different tissue sources
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