Isolation of mesenchymal stem cells (MSCs) from green fluorescent protein positive (GFP+) transgenic rodents: the grass is not always green(er).
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.
Harting
Matthew T
MT
Department of Pediatric Surgery and Trauma Research Center, University of Texas Medical School, Houston, Texas 77030, USA.
Jimenez
Fernando
F
Cox
Charles S
CS
D000818:Animals; D030801:Animals, Genetically Modified; D001374:Azacitidine / Q000494:pharmacology; D002454:Cell Differentiation / Q000502:physiology; D002469:Cell Separation / Q000379:methods*; D019175:DNA Methylation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D012377:Rodentia
D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D001374:Azacitidine