title: Assay validation for the assessment of adipogenesis of multipotential stromal cells--a direct comparison of four different methods.
abstract: OBJECTIVE
  Mesenchymal 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.
  
  
  METHODS
  Four 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.
  
  
  RESULTS
  Oil 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.
  
  
  CONCLUSIONS
  Flow 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.
authors.affiliation: Institute of Molecular and Cellular Biology, Institute of Medical and Biological Engineering, Faculty of Biological Sciences, University of Leeds, Leeds, West Yorkshire, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Aldridge
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Kouroupis
authors.forename: Sarah
authors.initials: S
authors.lastname: Churchman
authors.forename: Anne
authors.initials: A
authors.lastname: English
authors.forename: Eileen
authors.initials: E
authors.lastname: Ingham
authors.forename: Elena
authors.initials: E
authors.lastname: Jones
mesh_terms: D050156:Adipogenesis / Q000235:genetics / Q000502:physiology*; D000293:Adolescent; D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D002648:Child; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008875:Middle Aged; D055815:Young Adult