Development of mRuby2-Transfected C3H10T1/2 Fibroblasts for Musculoskeletal Tissue Engineering

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 cy...

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Published in:	PloS one 2015-09, Vol.10 (9), p.e0139054-e0139054
Main Authors:	Ker, Dai Fei Elmer, Sharma, Rashmi, Wang, Evelyna Tsi Hsin, Yang, Yunzhi Peter
Format:	Article
Language:	eng
Subjects:	Adipocytes - physiology Adiponectin Aggrecan Alcian Blue - metabolism Alizarin Alkaline phosphatase Alkaline Phosphatase - metabolism American Type Culture Collection Animals Anthraquinones - metabolism Azo Compounds - metabolism Biocompatibility Bioengineering Biomedical materials Bone marrow Calcium Calcium phosphates Cell culture Cell Differentiation Cell proliferation Cells (biology) Chondrocytes - physiology Cloning Coloring Agents - metabolism Cytometry Deoxyribonucleic acid Differentiation (biology) DNA DNA methylation Fibroblasts Fibroblasts - cytology Fibroblasts - physiology Flow Cytometry Fluorescence Gene expression Gene flow Genes Genes, Reporter Genetic aspects Lipase Lipoprotein lipase Luminescent Proteins - genetics Luminescent Proteins - metabolism Mesenchymal Stromal Cells - cytology Mesenchymal Stromal Cells - physiology Mesenchyme Mice Mice, Inbred C3H Musculoskeletal System - cytology Optical Imaging Osteocytes - physiology Polymerase Chain Reaction Progenitor cells Reporter gene Stem cells Tissue engineering Tissue Engineering - methods Transfection Transfection - methods
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Summary:	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.
ISSN:	1932-6203 1932-6203