title: Automated microscopy as a quantitative method to measure differences in adipogenic differentiation in preparations of human mesenchymal stromal cells.
abstract: OBJECTIVE
  Multipotent stromal cells, also called mesenchymal stromal cells (MSCs), are potentially valuable as a cellular therapy because of their differentiation and immunosuppressive properties. As the result of extensive heterogeneity of MSCs, quantitative approaches to measure differentiation capacity between donors and passages on a per-cell basis are needed.
  
  
  METHODS
  Human bone marrow-derived MSCs were expanded to passages P3, P5 and P7 from eight different donors and were analyzed for colony-forming unit capacity (CFU), cell size, surface marker expression and forward/side-scatter analysis by flow cytometry. Adipogenic differentiation potential was quantified with the use of automated microscopy. Percentage of adipogenesis was determined by quantifying nuclei and Nile red-positive adipocytes after automated image acquisition.
  
  
  RESULTS
  MSCs varied in expansion capacity and increased in average cell diameter with passage. CFU capacity decreased with passage and varied among cell lines within the same passage. The number of adipogenic precursors varied between cell lines, ranging from 0.5% to 13.6% differentiation at P3. Adipogenic capacity decreased significantly with increasing passage. MSC cell surface marker analysis revealed no changes caused by passaging or donor differences.
  
  
  CONCLUSIONS
  We measured adipogenic differentiation on a per-cell basis with high precision and accuracy with the use of automated fluorescence microscopy. We correlated these findings with other quantitative bioassays to better understand the role of donor variability and passaging on CFU, cell size and adipogenic differentiation capacity in vitro. These quantitative approaches provide valuable tools to measure MSC quality and measure functional biological differences between donors and cell passages that are not revealed by conventional MSC cell surface marker analysis.
authors.affiliation: FDA/Center for Biologics Evaluation and Research, Division of Cellular and Gene Therapies, Office of Cellular, Tissue, and Gene Therapies, Bethesda, Maryland, USA.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Lo Surdo
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Millis
authors.forename: Steven R
authors.initials: SR
authors.lastname: Bauer
mesh_terms: D017667:Adipocytes / Q000166:cytology; D050156:Adipogenesis; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D045164:Mesenchymal Stem Cell Transplantation / Q000379:methods*; D059630:Mesenchymal Stem Cells; D008853:Microscopy; D010012:Osteogenesis / Q000235:genetics
keywords: adipogenesis; bone marrow stromal cell; mesenchymal stromal cell; microscopy