The long-term fate of mesenchymal stem cells labeled with magnetic resonance imaging-visible polymersomes in cerebral ischemia

Abstract

Understanding the long-term fate and potential mechanisms of mesenchymal stem cells (MSCs) after transplantation is essential for improving functional benefits of stem cell-based stroke treatment. Magnetic resonance imaging (MRI) is considered an attractive and clinically translatable tool for longitudinal tracking of stem cells, but certain controversies have arisen in this regard. In this study, we used SPION-loaded cationic polymersomes to label green fluorescent protein (GFP)-expressing MSCs to determine whether MRI can accurately reflect survival, long-term fate, and potential mechanisms of MSCs in ischemic stroke therapy. Our results showed that MSCs could improve the functional outcome and reduce the infarct volume of stroke in the brain. In vivo MRI can verify the biodistribution and migration of grafted cells when pre-labeled with SPION-loaded polymersome. The dynamic change of low signal volume on MRI can reflect the tendency of cell survival and apoptosis, but may overestimate long-term survival owing to the presence of iron-laden macrophages around cell graft. Only a small fraction of grafted cells survived up to 8 weeks after transplantation. A minority of these surviving cells were differentiated into astrocytes, but not into neurons. MSCs might exert their therapeutic effect via secreting paracrine factors rather than directing cell replacement through differentiation into neuronal and/or glial phenotypes.

Keywords: green fluorescence protein; ischemic stroke; magnetic resonance imaging; mesenchymal stem cells; polymersome; superparamagnetic iron oxide nanoparticles.

Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1

Schematic diagram of the synthesis of polymersomes, cell labeling, cell transplantation, MRI tracking, and long-term fate of grafted GFP-MSCs. Abbreviations: IL, interleukin; MSC, mesenchymal stem cell; MRI, magnetic resonance imaging; PEI, poly(-etherimide); PDLLA, poly(d,l-lactide); SPIO, superparamagnetic iron oxide.

Figure 2

Efficacy of cell labeling. Notes: In vitro MRI shows that GFP-MSCs labeled with SPION-loaded polymersomes have an obvious hypointense signal on T1WI, T2WI, and T2*WI, and decreased T2 value compared with unlabeled GFP-MSCs (A). Prussian blue staining shows abundant blue-stained particles inside GFP-MSCs labeled with SPION-loaded polymersomes (B), but no blue-stained particles in unlabeled GFP-MSCs (C) (Bar =100 µm). TEM shows numerous, dense, iron particles in the cytosol (arrows) of GFP-MSCs labeled with SPION-loaded polymersomes (D), whereas no such iron particles are found in unlabeled GFP-MSCs (E) (Bar =200 nm). Abbreviations: MSC, mesenchymal stem cell; MRI, magnetic resonance imaging; SPION, superparamagnetic iron oxide nanoparticle.

Figure 3

In vitro cytotoxicity of cell labeling. Notes: Graphs show no significant differences in the cell viability (A, n=6), apoptosis rate (B, n=3) and intracellular ROS level (C, n=6) at 24 h after GFP-MSCs labeling with SPION-loaded polymersomes compared with unlabeled GFP-MSCs. Confocal laser microscopy shows that a few GFP-MSCs labeled with SPION-loaded polymersomes (D) and unlabeled GFP-MSCs (E) are ROS positive (arrows). Bar =100 µm. Abbreviations: MSC, mesenchymal stem cell; ROS, reactive oxygen species; SPION, superparamagnetic iron oxide nanoparticle.

Figure 4

Therapeutic effects of transplanted MSCs. Notes: Longitudinal coronal T2WI shows that the infarcted hemisphere in animals grafted with polymersome-labeled GFP-MSCs (A), unlabeled GFP-MSCs (B), and PBS (C) exhibited hyperintense signals. Graphs show the measured infarct volume (D) and modified neurologic severity scores (E) among the three groups. *p<0.05 between labeled GFP-MSCs and PBS; #p<0.05 between unlabeled GFP-MSCs and PBS. Abbreviations: mNSS, modified neurological severity score; MSC, mesenchymal stem cell; PBS, phosphate-buffered saline.

Figure 5

In vivo MRI of the grafted MSCs. Notes: Longitudinal coronal T2*WI show a persistent hypointense area (arrows) in the striatum of animals grafted with polymersome-labeled GFP-MSCs (A). These hypointense signal areas were obviously reduced at 2 weeks, and then gradually reduced but remained until 8 weeks after transplantation. At 2 weeks after transplantation, a spotty hypointense area was found in the ipsilateral corpus callosum (arrow head). No obvious hypointense signal was observed in animals grafted with unlabeled GFP-MSCs (B) and PBS (C). Graphs show the hypointense signal volume (D) and signal–contrast ratio (E) on T2WI and T2*WI in animals grafted with polymersome-labeled GFP-MSCs. #p<0.05. Abbreviations: MSC, mesenchymal stem cell; PBS, phosphate-buffered saline.

Figure 6

Apoptosis and survival of GFP-MSCs at implantation site. Notes: TUNEL analysis shows that apoptotic cells peaked at 1 week, and then decreased over time in animals grafted with labeled GFP-MSCs (A), unlabeled GFP-MSCs (B), and PBS (C). Graph show the percentages of TUNEL-positive cells in animals grafted with labeled and unlabeled GFP-MSCs (D). Graph shows the number of apoptotic GFP-MSCs in animals grafted with labeled and unlabeled GFP-MSCs (E). Graph shows the percentage of viable GFP-positive cells in animals grafted with labeled and unlabeled GFP-MSCs (F). *p<0.05 between labeled GFP-MSCs and PBS; #p<0.05 between unlabeled GFP-MSCs and PBS; Bar =15 µm. Abbreviations: MSC, mesenchymal stem cell; PBS, phosphate-buffered saline.

Figure 7

Relationship between SPIONs and macrophages. Notes: Representative photomicrographs of iron and CD11b co-staining show that many iron+/CD11b+ cells (dark blue cells) are present in the injection site (A) and in the corpus callosum (B) in animals grafted with polymersome-labeled GFP-MSCs. Graphs show the percentages of iron+/CD11b+ cells in the injection site (C) and corpus callosum (D). *p<0.05. Bar =25 µm. Abbreviations: MSC, mesenchymal stem cell; SPIONs, superparamagnetic iron oxide nanoparticles.

Figure 8

Histology of the grafted cells in the injection site. Notes: Fluorescence immunostaining micrographs show that GFP-MSCs remained in the injection site in animals grafted with polymersome-labeled GFP-MSCs and unlabeled GFP-MSCs. Only a few surviving GFP-MSCs differentiated into GFAP-positive astrocytes (arrows), but not into NeuN-positive neurons. A large number of cells were phagocytized by macrophages (arrows) (A). Graphs show the numbers of GFAP+GFP+ (B), CD11b+GFP+ (C), and the percentage of GFP-MSCs (D) in animals grafted with polymersome-labeled GFP-MSCs and unlabeled GFP-MSCs. Bar =15 µm. Abbreviation: MSC, mesenchymal stem cell.

Figure 9

Histology of migrating cells in the corpus callosum. Notes: Fluorescence immunostaining micrographs show that a small amount of GFP-MSCs migrated to the corpus callosum in animals treated with polymersome-labeled cells and unlabeled cells. Only a few surviving GFP-MSCs were differentiated into GFAP-positive astrocytes (arrows) and were phagocytized by macrophages (arrows), but no cells differentiated into NeuN-positive neurons (A). Graphs show the numbers of GFAP+GFP+ (B), CD11b+GFP+ (C), and GFP-MSCs (D) in animals grafted with polymersome-labeled GFP-MSCs and unlabeled GFP-MSCs. Bar =15 µm. Abbreviation: MSC, mesenchymal stem cell.

Figure 10

ELISA of grafted MSCs. Notes: Graphs show the concentration levels of BDNF (A), GDNF (B), VEGF (C), and IL-6 (D) in animals grafted with polymersome-labeled GFP-MSCs, unlabeled GFP-MSCs, and PBS. *p<0.05 between labeled GFP-MSCs and PBS; #p<0.05 between unlabeled GFP-MSCs and PBS. Abbreviations: IL, interleukin; MSC, mesenchymal stem cell; PBS, phosphate-buffered saline.