title: Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering.
abstract: 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.
authors.affiliation: Department of Orthopaedic Surgery, Stanford University, Stanford, California, United States of America.
authors.forename: Dai Fei Elmer
authors.initials: DF
authors.lastname: Ker
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, California, United States of America.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Sharma
authors.affiliation: Department of Material Science and Engineering, Stanford University, Stanford, California, United States of America.
authors.forename: Evelyna Tsi Hsin
authors.initials: ET
authors.lastname: Wang
authors.affiliation: Department of Orthopaedic Surgery, Stanford University, Stanford, California, United States of America; Department of Bioengineering, Stanford University, Stanford, California, United States of America; Department of Material Science and Engineering, Stanford University, Stanford, California, United States of America.
authors.forename: Yunzhi Peter
authors.initials: YP
authors.lastname: Yang
mesh_terms: D017667:Adipocytes / Q000502:physiology; D000423:Alcian Blue / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000880:Anthraquinones / Q000378:metabolism; D001391:Azo Compounds / Q000378:metabolism; D002454:Cell Differentiation; D019902:Chondrocytes / Q000502:physiology; D004396:Coloring Agents / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000502:physiology; D005434:Flow Cytometry; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000502:physiology; D051379:Mice; D008809:Mice, Inbred C3H; D009141:Musculoskeletal System / Q000166:cytology*; D061848:Optical Imaging; D010011:Osteocytes / Q000502:physiology; D016133:Polymerase Chain Reaction; D023822:Tissue Engineering / Q000379:methods*; D014162:Transfection / Q000379:methods
chemical_list: D000880:Anthraquinones; D001391:Azo Compounds; D004396:Coloring Agents; D008164:Luminescent Proteins; C010078:alizarin; D000469:Alkaline Phosphatase; C011049:oil red O; D000423:Alcian Blue