Assessment of the green florescence protein labeling method for tracking implanted mesenchymal stem cells

Abstract

Although green fluorescent protein (GFP) labeling is widely accepted as a tracking method, much remains uncertain regarding the retention of injected GFP-labeled cells implanted in ischemic organs. In this study, we evaluate the effectiveness of GFP for identifying and tracking implanted bone marrow-mesenchymal stem cells (BM-MSCs) and the effect of GFP on the paracrine actions of these cells. MSCs isolated from rat femur marrow were transduced with a recombinant adenovirus carrying GFP. After transplantation of the GFP-labeled BM-MSCs into the infarct zone of rat hearts, the survival, distribution, and migration of the labeled cells were analyzed at 3, 7, 14, and 28 days. To evaluate the effect of GFP on the paracrine actions of BM-MSCs, Western blot analysis was performed to detect the expression of vascular endothelial growth factor (VEGF), b fibroblast growth factor (b FGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinases-2 (MMP-2). GFP was successfully expressed by BM-MSCs in vitro. At 14 days after cell transplantation the GFP-positive cells could not be detected via confocal microscopy. By using a GFP antibody, distinct GFP-positive cells could be seen and quantitative analysis showed that the expression volume of GFP was 6.42 ± 0.92 mm 3 after 3 days, 1.24 ± 0.76 mm 3 after 7 days, 0.33 ± 0.03 mm 3 after 14 days, and 0.09 ± 0.05 mm 3 after 28 days. GFP labeling did not adversely affect the paracrine actions of BM-MSCs. GFP labeling could be used to track MSC distribution and their fate for at least 28 days after delivery to rat hearts with myocardial infarction, and this stem cell tracking strategy did not adversely affect the paracrine actions of BM-MSCs.

Activities of MSCs Derived from Transgenic Mice Seeded on ADM Scaffolds in Wound Healing and Assessment by Advanced Optical Techniques

“…To understand this process in detail and to ensure safety and efficacy, it is crucial to detect the fate and distribution of MSCs delivered to wound sites. GFP has already become the most suitable biomarker for tracking MSCs due to its biocompatibility, safety, nontoxicity and high detectability [36, 37]. GFP-tagged MSCs derived from transfection methods have been commonly used as donor cell sources in previous studies [36, 38, 39].…”

“…GFP has already become the most suitable biomarker for tracking MSCs due to its biocompatibility, safety, nontoxicity and high detectability [36, 37]. GFP-tagged MSCs derived from transfection methods have been commonly used as donor cell sources in previous studies [36, 38, 39]. Previous results demonstrated that GFP-tagged MSCs could be traced for 4 weeks after surgical treatment [36].…”

“…GFP-tagged MSCs derived from transfection methods have been commonly used as donor cell sources in previous studies [36, 38, 39]. Previous results demonstrated that GFP-tagged MSCs could be traced for 4 weeks after surgical treatment [36]. In consideration of the above-mentioned findings, we used this strategy in our previous work [30] and found that almost no GFP fluorescent signals could be detected at 14 days after transplantation.…”

Immunogenicity of allogeneic mesenchymal stem cells transplanted via different routes in diabetic rats

“…MSCs were also characterized by differentiation toward adipogenic and osteogenic lineages using previously described protocols. 13 The purified MSCs at passage (P)3 were transduced with a lentiviral vector carrying a GFP reporter gene as described previously. 14,15 Immune antigen expression in MSCs The immunophenotype (MHC I, MHC II, CD40, CD80, CD86) of MSCs was examined by flow cytometry on a FACSCanto II (BD Pharmingen, San Diego, CA, USA) with specific phycoerythrin-conjugated monoclonal antibodies (eBioscience, San Diego, CA, USA) and performed as previously described.…”

The Current ‘State of Play’ of Regenerative Medicine in Horses: What the Horse Can Tell the Human

“…Studies of the effects of stem cell implantation in partially transected infraspinatus tendons in sheep [80] have demonstrated that MSCs had a beneficial effect on induced tendon pathology. Such effects were observed not only when injected acutely, but also in the face of established degenerative change, although there was no effect of MSCs on the actual lesion site.…”