Development of mRuby2-Transfected C3H10T1-2 Fibroblasts for Musculoskeletal Tissue EngineeringReport as inadecuate




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Mouse C3H10T1-2 fibroblasts are multipotent, mesenchymal stem cell MSC-like progenitor cells that are widely used in musculoskeletal research. In this study, we have established a clonal population of C3H10T1-2 cells stably-transfected with mRuby2, an orange-red fluorescence reporter gene. Flow cytometry analysis and fluorescence imaging confirmed successful transfection of these cells. Cell counting studies showed that untransfected C3H10T1-2 cells and mRuby2-transfected C3H10T1-2 cells proliferated at similar rates. Adipogenic differentiation experiments demonstrated that untransfected C3H10T1-2 cells and mRuby2-transfected C3H10T1-2 cells stained positive for Oil Red O and showed increased expression of adipogenic genes including adiponectin and lipoprotein lipase. Chondrogenic differentiation experiments demonstrated that untransfected C3H10T1-2 cells and mRuby2-transfected C3H10T1-2 cells stained positive for Alcian Blue and showed increased expression of chondrogenic genes including aggrecan. Osteogenic differentiation experiments demonstrated that untransfected C3H10T1-2 cells and mRuby2-transfected C3H10T1-2 cells stained positive for alkaline phosphatase ALP as well as Alizarin Red and showed increased expression of osteogenic genes including alp, ocn and osf-1. When seeded on calcium phosphate-based ceramic scaffolds, mRuby2-transfected C3H10T1-2 cells maintained even fluorescence labeling and osteogenic differentiation. In summary, mRuby2-transfected C3H10T1-2 cells exhibit mRuby2 fluorescence and showed little-to-no difference in terms of cell proliferation and differentiation as untransfected C3H10T1-2 cells. These cells will be available from American Type Culture Collection ATCC; CRL-3268™ and may be a valuable tool for preclinical studies.



Author: Dai Fei Elmer Ker , Rashmi Sharma , Evelyna Tsi Hsin Wang , Yunzhi Peter Yang

Source: http://plos.srce.hr/



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