Automated microscopy as a quantitative method to measure differences in adipogenic differentiation in preparations of human mesenchymal stromal cells |
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| Authors: | Jessica L Lo Surdo Bryan A Millis Steven R Bauer |
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| Institution: | 1. FDA/Center for Biologics Evaluation and Research, Division of Cellular and Gene Therapies, Office of Cellular, Tissue, and Gene Therapies, Bethesda, Maryland, USA;2. National Institutes of Health (NIH), National Institute on Deafness and Other Communication Disorders, Section on Structural Cell Biology, Bethesda, Maryland, USA;1. Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, California, USA;2. Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, California, USA;3. Department of Ophthalmology & Vision Science, School of Medicine, University of California, Davis, California, USA;1. O’Brien Institute Department, St Vincent’s Institute of Medical Research, Victoria, Australia;2. Department of Surgery, University of Melbourne, St Vincent’s Hospital Melbourne, Victoria, Australia;3. Department of Physiology, University of Melbourne, Victoria, Australia;4. Centre for Eye Research Australia & Royal Victorian Eye and Ear Hospital, Victoria, Australia;5. Ophthalmology, University of Melbourne, Department of Surgery, Victoria, Australia;1. Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China;2. Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China;3. Stem Cells and Regeneration Program, School of Biomedical Sciences, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China;4. MOE Key Laboratory of Regenerative Medicine, School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China;1. Department of Materials Engineering Science, Osaka University, 1-3, Machikaneyama, Toyonaka, Osaka 560-8531, Japan;2. Department of Basic Medicinal Science, Nagoya University, Furocho, Chiusa-Ku, Aichi 464-8601, Japan;3. Department of Biotechnology, Osaka University, 1-2, Yamadaoka, Suita, Osaka 565-0971, Japan;1. Regenerative Medicine Laboratory, Department of Experimental Medicine, University of Genova, Genova, Italy;2. Molecular and Cell Cardiology Laboratory, CardioCentro Ticino, Lugano, Switzerland;3. Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands;4. Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy;5. General Physiology Laboratory, Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, Pavia, Italy;6. Laboratory of Biochemistry, Biotechnology and Advanced Diagnostic, Myelofibrosis Study Centre, IRCCS Ospedale Policlinico San Matteo, Pavia, Italy;7. Dept. of Obstetrics and Gynecology, Ospedale Policlinico San Martino - IRCCS per l’Oncologia, Genova, Italy;8. Division of Cardiac Surgery, Ospedale Policlinico San Martino - IRCCS per l’Oncologia, Genova, Italy |
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| Abstract: | Background aimsMultipotent 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.MethodsHuman 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.ResultsMSCs 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.ConclusionsWe 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. |
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| Keywords: | adipogenesis bone marrow stromal cell mesenchymal stromal cell microscopy |
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