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Background & Aim Mesenchymal stromal cell (MSC) therapies can be greatly advanced by taking control over the source materials, particularly by selection of tissue-specific connective tissue progenitors (CTPs) with preferred biological performance. This project aims to test the hypothesis that: 1) attributes of a colony founding CTP and the CTP-derived colony can be used to predict biological performance of their culture expanded progeny, and 2) CTP or colony selection based on preferred attributes will improve the reproducibility and quality of in vitro differentiation performance in comparison to traditional unselected multi-clonal expansion strategies. Methods, Results & Conclusion Human articular cartilage (Grade1-2) cells obtained from seven knee arthroplasty patients were cultured in 2-D. Large field of view phase contrast images were acquired daily for 10 days enabling identification of colonies derived from a single CTP. Based on morphology and proliferation in primary culture, 99 colonies were picked using the Cell X™ robot. Of these, the 42 fastest growing clones were expanded to 21 doublings (Db) (2 × 106 cells) for trilineage differentiation assay using Oil Red O, Von Kossa and Alcian Blue staining and RNA sequencing pre and post-differentiation (Fig.1). The mean circularity and diameter of CTP was 0.41 (range:0.05-0.93) and 40.6µm (range:15.9-111) respectively. Colony attributes varied widely with a mean cell density, area per cell and effective proliferation rate (EPR) of 308cells/mm2 (range:92-908), 332.8µm2 (range:240.8-536.6), and 0.79Db/day (range:0.59-1.02) respectively. The clones exhibited wide variation in biological performance: mean EPR at passage 1 (17Db), passage 2 (19Db) and passage 3 (21Db) was 0.19 (range:0.07-0.31), 0.35 (range:0.17-0.77) and 0.28 (range:0.1-0.67) respectively; mean expression of differentiation markers for adipogenesis, osteogenesis and chondrogenesis was 217.4µm2/cell (range:4-1063), 1551.6µm2/cell (range:1-24698) and 1022.4µm2/cell (range:189-3608) respectively. Fig.2 shows the correlation between the attributes of CTPs, colonies, and subsequent biological performance. These results indicate that more circular CTPs had lower P0 EPR, higher P1 EPR and great adipogenesis. Denser colonies had higher P0 EPR, lower P1 EPR, and greater chondrogenesis. An improved understanding of the diverse origins and attributes colony founding CTPs in adult cartilage may enable more precise and rigorous cell fabrication strategies. |
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