iPSC-derived mesenchymal stem cells attenuate cerebral ischemia-reperfusion injury by inhibiting inflammatory signaling and oxidative stress

Abstract

Induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) hold great promise as a cell source for transplantation into injured tissues to alleviate inflammation. However, the therapeutic efficacy of iMSC transplantation for ischemic stroke remains unknown. In this study, we evaluated the therapeutic effects of iMSC transplantation on brain injury after ischemia-reperfusion using a rat transient middle cerebral artery occlusion model and compared its therapeutic efficacy with that of bone marrow mesenchymal stem cells (BMMSCs). We showed that iMSCs and BMMSCs reduced infarct volumes after reperfusion and significantly improved motor function on days 3, 7, 14, 28, and 56 and cognitive function on days 28 and 56 after reperfusion compared with the vehicle group. Furthermore, immunological analyses revealed that transplantation of iMSCs and BMMSCs inhibited microglial activation and expression of proinflammatory cytokines and suppressed oxidative stress and neuronal cell death in the cerebral cortex at the ischemic border zone. No difference in therapeutic effect was observed between the iMSC and BMMSC groups. Taken together, our results demonstrate that iMSC therapy can be a practical alternative as a cell source for attenuation of brain injury and improvement of neurological function because of the unlimited supply of uniform therapeutic cells.

Keywords: BMMSC; MSC; focal cerebral ischemia model rats; iMSC; iPSC; intravenous administration; ischemic stroke; mesenchymal stem cell; mesenchymal stromal cell; stem cell therapy.

Graphical abstract

Figure 1

Characterization of induced mesenchymal stem cells (iMSCs) and bone marrow mesenchymal stem cells (BMMSCs) (A) Cell morphology of BMMSCs and iMSCs. Scale bar, 200 μm. (B) Surface marker analyses of iMSCs using flow cytometry. The blue histogram represents the isotype control, and the red overlay represents each antigen. The MSC-positive markers CD90, CD73, and CD105 were measured. (C) Comparison of the differentiation potential between BMMSCs and iMSCs. MSCs were differentiated into osteogenic (left), chondrogenic (center), and adipogenic (right) lineages and stained by alizarin red for osteocytes, Alcian blue for chondrocytes, and oil red O for adipocytes. Scale bar, 200 μm.

Figure 2

Reduction of infarct volume by administration of BMMSCs and iMSCs (A) 2,3,5-Triphenyl tetrazolium chloride (TTC) staining of rat brains in the vehicle-treated (left), BMMSC-treated (center), and iMSC-treated (right) groups. Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (tMCAO) and then injected intravenously with BMMSCs, iMSCs, or PBS. The infarct volumes were analyzed by TTC staining 3 days after tMCAO. In TTC staining, normal tissue is stained red, and the infarcted area is unstained. Six sections from one rat brain divided into sections of 2 mm each are shown. One representative rat brain per vehicle, BMMSC, and iMSC group is displayed. (B) Quantitative analysis of the infarct volumes 3 days post tMCAO in each group. The data presented are the means and SDs (∗p < 0.05, n = 5 for each group).

Figure 3

Improvement of neurological score, motor function, and cognitive function by administration of BMMSCs and iMSCs (A and B) Abnormal posture (A) and hemiparesis (B) assessment 3, 7, 14, 28, and 56 days after tMCAO in vehicle-, BMMSC-, and iMSC-transplanted reperfusion groups. Boxplots indicate the median and IQR, and whiskers indicate the maximum and minimum values (∗p < 0.05, ∗∗p < 0.01; n = 8 for each group). (C and D) Rotarod performance (C) and forelimb grip strength (D) assessment 3, 7, 14, 28, and 56 days after tMCAO in vehicle-, BMMSC-, and iMSC-transplanted reperfusion groups. Data are presented as mean ± SD (∗p < 0.05, ∗∗p < 0.01; n = 8 for each group). (E) Improvement of spatial working memory 28 and 56 days after tMCAO. Shown is the alternation rate in the Y-maze spontaneous test in the vehicle-, BMMSC-, and iMSC-treated groups. Data are presented as mean ± SD (∗p < 0.05, ∗∗p < 0.01; n = 8 for each group).

Figure 4

Evaluation of microglial activity and proinflammatory cytokine levels in the ischemic side of the brain (A) Schematic of the cortical IBZ. The red-lined square on the brain map shows the cortical IBZ. The black area represents the ischemic lesion. (B) Iba-1 staining in rat brain sections treated with vehicle (left), BMMSCs (center), and iMSCs (right). Scale bar, 100 μm. (C) Quantification of Iba-1-positive cells in the rat brain with or without MSC treatment. Data are presented as mean ± SD (∗∗p < 0.01, n = 5 for each group). (D) TNF-α staining in rat brain sections treated with vehicle (left), BMMSCs (center), and iMSCs (right). Scale bar, 100 μm. (E) Quantification of TNF-α-positive cells in the rat brain with or without MSC treatment. Data are presented as mean ± SD (∗p < 0.05; n = 5 for each group). (F and G) Quantification of IL-1β (F) and IL-6 (G) expression in ischemic hemisphere extracts. Data are presented as mean ± SD (∗p < 0.05, n = 5 for each group). Sprague-Dawley rats were treated with tMCAO and injected intravenously with BMMSCs, iMSCs, or PBS. Three days after tMCAO, brain sections were stained with anti-Iba-1 or anti-TNF-α antibodies. The ischemic hemisphere extracts were also analyzed using ELISA.

Figure 5

Localization of TNF-α expression in the ischemic side of the brain (A) Localization of TNF-α (green) and NeuN (red) expression in rat brain sections treated with vehicle (left vertical column), BMMSCs (center vertical column), and iMSCs (right vertical column). Scale bar, 100 μm. Nuclei were stained in blue with DAPI. Merge overlays of three fluorescence images are shown in the bottom row. (B) Quantification of TNF-α-positive neurons in the rat brain. Data are shown as mean ± SD (∗p < 0.05, n = 6 for each group).

Figure 6

Evaluation of oxidative stress in the ischemic side of the brain (A and C) 4-Hydroxynonenal (4-HNE) (A) and 8-hydroxy-20-deoxyguanosine (8-OHdG) (C) staining in rat brain sections treated with vehicle (left), BMMSCs (center), and iMSCs (right). Scale bar, 100 μm. (B and D) Quantification of 4-HNE-positive (B) and 8-OHdG-positive (D) cells in the rat brain with or without MSC treatment. Data are presented as mean ± SD (∗∗p < 0.01, n = 5 for each group). (E) Total SOD activity in ischemic hemisphere extracts. Data are presented as mean ± SD (∗p < 0.05, n = 5 for each group). SD rats were treated with tMCAO and injected intravenously with BMMSCs, iMSCs, or PBS. Three days after tMCAO, brain sections were stained with anti-4-HNE or anti-8-OHdG antibodies. The ischemic hemisphere extracts were also analyzed using a SOD assay kit.

Figure 7

Evaluation of apoptosis and neuronal degeneration in the cortical ischemic boundary zone (A) Apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL) staining (top panels) in the vehicle (left vertical column), BMMSC (center vertical column), and iMSC groups (right vertical column). Nuclei were stained in blue with DAPI (center panels). Merge overlays of two fluorescence images are shown in the bottom panels. (B) Quantification of TUNEL-positive neurons in the rat brain. Data are shown as mean ± SD (∗p < 0.05, n = 6 for each group). (C) Fluoro-Jade C (FJC) staining in rat brain sections treated with vehicle (left), BMMSCs (center), and iMSCs (right). Scale bar, 100 μm. (D) Quantification of FJC-positive cells in the rat brain with or without MSC treatment. Data are presented as mean ± SD (∗p < 0.05, n = 6 for each group). Sprague-Dawley rats were treated with tMCAO and injected intravenously with BMMSCs, iMSCs, or PBS. FJC staining was performed 3 days after tMCAO.

Figure 8

Biodistribution and quantification of engrafted MSCs Shown are biodistribution and quantification of viable BMMSCs (n = 7) and iMSCs (n = 8) 72 h after tMCAO using Alu-based real-time PCR. Values represent the amount of human DNA in 100 ng of total DNA per organ sample. Data are presented as mean ± SD (∗p < 0.01).