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.