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. 2020 Mar 30;13(1):127-141.
doi: 10.15283/ijsc19111.

Neurogenin-1 Overexpression Increases the Therapeutic Effects of Mesenchymal Stem Cells Through Enhanced Engraftment in an Ischemic Rat Brain

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Free PMC article

Neurogenin-1 Overexpression Increases the Therapeutic Effects of Mesenchymal Stem Cells Through Enhanced Engraftment in an Ischemic Rat Brain

Gyu-Hee Kim et al. Int J Stem Cells. .
Free PMC article

Abstract

Background and objectives: Stem cell therapy is a promising strategy for treating neurological diseases but its effectiveness is influenced by the route of administration and the characteristics of the stem cells. We determined whether neural induction of mesenchymal stem cells (MSCs) was beneficial when the cells were delivered intra-arterially through the carotid artery.

Methods and results: MSCs were neurally induced using a retroviral vector expressing the neurogenic transcription factor neurogenin-1 (Ngn1). The LacZ gene encoding bacterial β-galactosidase was used as a control. Ischemic stroke was induced by transluminal occlusion of the middle cerebral artery and 3 days later the MSCs were delivered intra-arterially through the internal carotid artery. Magnetic resonance imaging analysis indicated that compared to MSCs expressing LacZ (MSCs/LacZ), MSCs expressing Ngn1 (MSCs/Ngn1) exhibited increased recruitment to the ischemic region and populated this area for a longer duration. Immunohistochemical analysis indicated that compared to MSCs/LacZ, MSCs/Ngn1 more effectively alleviated neurological dysfunction by blocking secondary damage associated with neuronal cell death and brain inflammation. Microarray and real-time PCR analysis indicated that MSCs/Ngn1 exhibited increased expression of chemotactic cytokine receptors, adherence to endothelial cells, and migration ability.

Conclusions: Neural induction with Ngn1 increases the homing ability of MSCs, enhancing their engraftment efficiency in the ischemic rat brain. Intra-arterial delivery of neurally induced MSCs/Ngn1 3 days after ischemic injury blocks neuronal cell death and inflammation, and improves functional recovery. Thus, intra-arterial administration of stem cells with neural properties may be a novel therapy for the treatment of ischemic stroke.

Keywords: Intra-arterial; Mesenchymal stem cell; Neural induction; Neurogenin-1; Stroke.

Conflict of interest statement

Potential Conflict of Interest

D.J. and H.S-K. are employees of CelleBrain Ltd. H.S-K. is a founder of CelleBrain Ltd.

Figures

Fig. 1
Fig. 1
Comparison of the gene expression profile of mesenchymal stem cells expressing LacZ (MSCs/LacZ) and mesenchymal stem cells expressing neurogenin 1 (MSCs/Ngn1). (A) Expression of neurogenin 1 (Ngn1) in MSCs/Ngn1 was verified by real-time PCR analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. (B) Quantitative assessment of Ngn1 mRNA expression in MSCs/Ngn1. MSCs/LacZ were used as negative controls for transduction. (C) Relative mRNA expression of chemokine receptors and transforming growth factor beta (TGFβ) in MSCs/LacZ and MSCs/Ngn1. (D) Microarray analysis showing differentially expressed genes with a fold change >1.5 in MSCs/Ngn1 compared to MSCs/LacZ. (E) Gene Ontology (GO) enrichment analysis revealed that the key GO terms were leukocyte migration, cell motility, and locomotion. Data are presented as mean±S.E. Statistically significant differences between MSCs/Ngn1 and MSCs/LacZ are indicated (**p<0.01, ***p<0.001; Student’s t-test).
Fig. 2
Fig. 2
Enhanced adhesion and migration activity of mesenchymal stem cells expressing neurogenin 1 (MSCs/Ngn1). (A) Schematic diagram showing the experimental procedures of the adhesion and transwell migration assays. (B) Transwell migration assay showing Hoechst-stained mesenchymal stem cells expressing LacZ (MSCs/LacZ) and MSCs/Ngn1 in the bottom layer. (C) Quantification of Hoechst-stained cells that migrated to the bottom compartment containing the brain ischemic extract (IE) over a 4 h period. (D) Florescence image showing green fluorescent protein- (GFP-) positive MSCs/LacZ and MSCs/Ngn1 adhering to IE-stimulated bEnd.3 cells. (E) Quantification of GFP-positive cells following a 4 h adhesion assay. At least four random fields were used to obtain the number of adherent cells. Data are presented as mean±S.E. from three independent experiments. Statistically significant differences between MSCs/Ngn1 and MSCs/LacZ are indicated (*p<0.05, **p<0.01, ***p<0.001; Student’s t-test).
Fig. 3
Fig. 3
Enhanced engraftment of mesenchymal stem cells expressing neurogenin 1 (MSCs/Ngn1) in the acute ischemic brain. (A) Prussian blue (PB) staining of superparamagnetic iron oxide (SPIO)-labeled mesenchymal stem cells expressing LacZ (MSCs/LacZ) and MSCs/Ngn1 in vitro. (B) T2* magnetic resonance imaging was used to assess the distribution and engraftment of intra-arterially injected MSCs/LacZ in the ischemic brain over a period of 25 days after transplantation. (C) Quantification of engrafted MSCs/LacZ and MSCs/Ngn1 in the ischemic hemisphere by green fluorescent protein (GFP) fluorescence 4 h and 1 day after injection. The data were obtained from 10 serial sections of 3 independent animals and are presented as mean per section±S.E. (***p<0.001 compared to MSCs/LacZ group; Student’s t-test). (D) GFP-positive cells were prominent in the vascular lumen 4 h after transplantation, and were found in the perivascular area of the brain parenchyma in the MSCs/Ngn1 group. (E) Human Alu-specific PCR analysis revealed enhanced engraftment of MSC/Ngn1 compared to MSCs/LacZ 1 day after transplantation. Experiment was conducted in three animals per group and the data are presented as mean±standard error of the mean (S.E.). (***p<0.001 compared to right hemisphere of MSCs/LacZ group; Student’s t-test). (F) A standard curve with human Alu-specific PCR was used to extrapolate the amount of human DNA in the rat brain. (G) Prussian blue staining revealed SPIO-labeled MSCs/Ngn1 25 days after injection (blank arrowheads). Immunostaining for human mitochondria antigen (hMT) or ED1 along with Prussian blue staining revealed MSCs/Ngn1 or cells engulfed by phagocytic cells in the penumbra 25 days after injection, respectively (filled arrowheads).
Fig. 4
Fig. 4
Neuroprotective effects of mesenchymal stem cells expressing neurogenin 1 (MSCs/Ngn1 cells). (A) Low magnification light microscopy showing cortical and striatal ischemic penumbra region used for quantification of terminal deoxynucleotidyl transferase dUTP nick end labeling- (TUNEL-), ED1- and neuronal nuclei- (NeuN-) positive cells. (B) Representative photographs of TUNEL staining and ED1 immunostaining 4 days after cell transplantation and NeuN immunostaining 25 days after cell transplantation. Quantification of TUNEL-, ED1, and NeuN- positive cells are illustrated in (C∼E), respectively. Results from three animals are presented as mean±S.E. (*p<0.05, ***p<0.001 compared to saline group and #p<0.05, ##p<0.01 compared to MSCs/LacZ group; One-way Anova).
Fig. 5
Fig. 5
Improved functional recovery and tissue integrity following mesenchymal stem cells expressing neurogenin 1 (MSCs/Ngn1) transplantation. Behavioral performance in the rotarod (A) and adhesive removal tests (B) up to 28 days after ischemia. Data were collected from at least five animals per group and are presented as mean±S.E. (C) Representative magnetic resonance image showing the infarcted area. (D) Quantification of magnetic resonance images from days 2∼28 reveals the infarct volume as mean±S.E. (*p<0.05, ***p<0.001 compared to saline group and #p<0.05, ##p<0.01 compared to MSCs/LacZ group; One-way Anova).

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