Bystander Effect Fuels Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells to Quickly Attenuate Early Stage Neurological Deficits After Stroke

Abstract

: Present therapies for stroke rest with tissue plasminogen activator (tPA), the sole licensed antithrombotic on the market; however, tPA's effectiveness is limited in that the drug not only must be administered less than 3-5 hours after stroke but often exacerbates blood-brain barrier (BBB) leakage and increases hemorrhagic incidence. A potentially promising therapy for stroke is transplantation of human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSCs). To date, the effects of iPSCs on injuries that take place during early stage ischemic stroke have not been well studied. Consequently, we engrafted iPSC-NSCs into the ipsilesional hippocampus, a natural niche of NSCs, at 24 hours after stroke (prior to secondary BBB opening and when inflammatory signature is abundant). At 48 hours after stroke (24 hours after transplant), hiPSC-NSCs had migrated to the stroke lesion and quickly improved neurological function. Transplanted mice showed reduced expression of proinflammatory factors (tumor necrosis factor-α, interleukin 6 [IL-6], IL-1β, monocyte chemotactic protein 1, macrophage inflammatory protein 1α), microglial activation, and adhesion molecules (intercellular adhesion molecule 1, vascular cell adhesion molecule 1) and attenuated BBB damage. We are the first to report that engrafted hiPSC-NSCs rapidly improved neurological function (less than 24 hours after transplant). Rapid hiPSC-NSC therapeutic activity is mainly due to a bystander effect that elicits reduced inflammation and BBB damage.

Significance: Clinically, cerebral vessel occlusion is rarely permanent because of spontaneous or thrombolytic therapy-mediated reperfusion. These results have clinical implications indicating a much extended therapeutic window for transplantation of human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSCs; 24 hours after stroke as opposed to the 5-hour window with tissue plasminogen activator [tPA]). In addition, there is potential for a synergistic effect by combining hiPSC-NSC transplantation with tPA to attenuate stroke's adverse effects.

Keywords: Blood-brain barrier; Cellular therapy; Induced pluripotent stem cells; Inflammation; Neural stem cell; Stem cell transplantation; Stroke.

Figure 1.

Behavioral deficits in MCAO/R mice ameliorated by hiPSC-NSC transplantation. (A): Forepaw adhesive removal tests show improvement with hiPSC-NSCs: mean adhesive removal times are significantly shorter than those for nontransplanted MCAO/R mice (n = 10). (B): Beam walk tests show improvement with hiPSC-NSCs: beam walking times are significantly shorter compared with those of nontransplanted MCAO/R mice (n = 9). (C): Rotarod tests show improvement with hiPSC-NSCs: rod-remaining times for transplanted mice were significantly longer (n = 10). Four velocities (10, 15, 20, and 25 rotations per minute) were used, with three trials performed at each velocity. Durations at all speeds are summed; data are presented as a percentage relative to sham or MCAO/R mice. ∗, p < .05, ∗∗∗, p < .001, ∗∗∗∗, p < .0001 vs. sham group; ##, p < .01, ###, p < .001, ####, p < .0001 vs. MCAO/R group. Data are expressed as mean ± SEM. Abbreviations: hiPSC-NSC, human induced pluripotent stem cell-derived neural stem cell; MCAO/R, middle cerebral artery occlusion with subsequent reperfusion; Tx, transplantation.

Figure 2.

hiPSC-NSCs rapidly migrated into the site of stroke injury. (A–D): Experimental timeline. (A): MCAO/R was performed in C57BL/6J mice at time 0, and hiPSC-NSCs were transplanted into the ipsilesional hippocampus 24 hours after MCAO/R. Outcomes were assessed at indicated intervals. (B): Representative images of hiPSC-NSCs immunostained with anti-Sox2 (red) and anti-Nestin (green) in culture. Scale bar = 30 μm. (C): Triphenyl tetrazolium chloride staining of sham, MCAO/R, and hiPSC-NSC-engrafted mouse brains (red, viable tissue; white, infarct). Sections from the same animal are shown from anterior to posterior (top to bottom). Scale bar = 2 mm. (D): Quantification of infarct volume. Data are expressed as mean ± SEM (n = 3; ∗∗∗, p < .001 vs. sham group. (E–I): Brain diagram. (E): Injection sites (arrowheads); hiPSC-NSC-disseminated areas (red dots). (F–I): Human iPSC-NSCs were identified with human-specific antibody (STEM121) against human cytoplasmic protein (green, arrowheads) and human neural stem cell marker hNestin (red, arrows) on brain slices 24 hours after transplantation. Three sampling sites are shown in Fig. 2E (insets, higher magnification). Scale bar = 30 μm (10 μm, inset). Abbreviations: Contra, contralesional; DAPI, 4′,6-diamidino-2-phenylindole; hiPSC-NSC, human induced pluripotent stem cell-derived neural stem cell; Ipsi, ipsilesional; MCAO/R, middle cerebral artery occlusion with subsequent reperfusion; RT-PCR, reverse transcriptase-polymerase chain reaction; Tx, transplantation.

Figure 3.

Proinflammatory gene expression reduced by hiPSC-NSC transplantation. (A): Reduced inflammatory marker expression in the ipsilesional hemisphere of hiPSC-NSC-transplanted MCAO/R brains. Reverse transcriptase-polymerase chain reaction was used to measure inflammatory gene expression, then normalized to that of GAPDH. Transcript levels of proinflammatory cytokines TNF-α, IL-6, IL-1β, and ICAM-1 and VCAM-1 are elevated in the MCAO/R brains and downregulated in transplanted brains compared with MCAO/R control brains. ∗, p < .05, ∗∗, p < .01, ∗∗∗, p < .001 vs. sham mice; #, p < .05, ##, p < .01, ###, p < .001 vs. MCAO/R mice. (B): Chemokines MCP-1 and MIP-1α are dramatically elevated in MCAO/R brains and significantly downregulated in hiPSC-NSC-transplanted brains. ∗, p < .05, ∗∗, p < .01, ∗∗∗, p < .001, ∗∗∗∗, p < .0001 vs. sham; #, p < .05, ##, p < .01, ###, p < .001, ####, p < .0001 vs. MCAO/R. Data are expressed as mean ± SEM (n = 4). Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hiPSC-NSC, human induced pluripotent stem cell-derived neural stem cell; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; MCAO/R, middle cerebral artery occlusion with subsequent reperfusion; MCP-1, monocyte chemotactic protein 1; MIP-1α, macrophage inflammatory protein 1α; TNF-α, tumor necrosis factor α; Tx, transplantation; VCAM-1, vascular cell adhesion molecule 1.

Figure 4.

Blood-brain barrier leakage lessened by hiPSC-NSC transplantation. (A): Western blot shows mouse IgG levels in ipsilesional cortex in sham, MCAO/R, and transplanted mice. (B): Quantification of panel A (n = 3; ∗, p < .05, ∗∗∗, p < .001 vs. sham group; #, p < .05 vs. MCAO/R group). (C): Brain diagram. Two sampling sites are shown (rectangles). (D): Spatial distribution of intravenously administered Texas Red-dextran 70 kDa (red) in stroke’s contralateral (Di, Diii) and ipsilateral (Dii, Div) cortical regions with or without hiPSC-NSC transplantation. Reduced extravasation was observed in hiPSC-NSC-transplanted brains. Scale bar = 10 μm (10 μm, inset). (E): Distribution of engrafted hiPSC-NSCs (STEM121-positive, green) around the blood vessels (CD31-positive endothelial cells in red). Scale bar = 10 μm. (F): Western blot analysis of MMP-9 protein levels in ipsilesional cortex. (G): Quantification of panel F (n = 3; ∗∗∗, p < .001 vs. sham group; ##, p < .01 vs. MCAO/R group). (H): Zymography assay shows MMP-9 activity in the ipsilesional cortex. (I): Quantification of panel H (n = 3; ∗, p < .05, ∗∗∗∗, p < .0001 vs. sham group; ###, p < .001 vs. MCAO/R group). (J): Western blot analysis of ZO-1. (K): Quantification of panel J (n = 3; ∗, p < .05 vs. sham group; #, p < .05 vs. MCAO/R group). Data are expressed as mean ± SEM. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; hiPSC-NSC, human induced pluripotent stem cell-derived neural stem cell; MCAO/R, middle cerebral artery occlusion with subsequent reperfusion; MMP, matrix metalloproteinase; Tx, transplantation; ZO-1, zonula occludens-1.

Figure 5.

Microglial activation reduced by hiPSC-NSC transplantation. Immunofluorescence staining for microglial marker Iba-1 (red) and STEM121, an hNSC marker (green). (A): Sham mice, (B): MCAO/R mice, (C): Transplanted mice. Microglial activation increased 48 hours after MCAO/R, whereas transplanted hiPSC-NSCs suppressed activation 24 hours after transplantation (arrowhead, transplantation site). Sampling regions (rectangles). Infarct (gray region). (D): Quantification of Iba-1-positive active microglia per area (0.15 mm²) in mouse groups (n = 4; ∗∗∗, p < .001 vs. sham; ###, p < .001 vs. MCAO/R). (E): Representative Western blot shows BDNF expression in ipsilesional hemisphere. (F): Quantification of panel E (n = 3; ∗∗∗, p < .001 vs. sham; ###, p < .001 vs. MCAO/R). Data expressed as mean ± SEM. Abbreviations: BDNF, brain-derived neurotrophic factor; DAPI, 4′,6-diamidino-2-phenylindole; hiPSC-NSC, human induced pluripotent stem cell-derived neural stem cell; MCAO/R, middle cerebral artery occlusion with subsequent reperfusion; Tx, transplantation.