Isolation of Mesenchymal Stem Cells (MSCs) from Green Fluorescent Protein Positive (GFP + ) Transgenic Rodents: The Grass Is Not Always Green(er)

Abstract

Cellular therapy is under intense basic science and clinical investigation as a therapeutic intervention. One of the challenges lies in tracking these cells in vivo. While there are many ways to label and track cells—each with strengths and weaknesses—the green fluorescent protein (GFP) is a reporter gene commonly employed. We report a significant and consistent reduction in the expression of GFP with the culture of mesenchymal stem cells (MSCs) isolated from the bone marrow of GFP⁺ transgenic rodents. After MSC isolation and immunophenotype characterization, along with co-localization with GFP, MSCs were evaluated for GFP expression through flow cytometry and fluorescent microscopy, revealing that only 50% of the cells expressed GFP. Differentiation of the cells to adipocytes did not alter the GFP expression significantly. Incubation with an anti-GFP antibody increased the fluorescent intensity of the GFP-expressing and some of the GFP nonexpressing cells. Incubation of MSCs with a histone deacetylase inhibitor, trichostatin A, did not significantly alter GFP expression, while incubation with a DNA demethylation reagent, 5-azacytidine, increased GFP expression, suggesting that epigenetic modification by DNA methylation may play a role in GFP expression among MSCs.