Nose-to-brain delivery of human muse cells enhances structural and functional recovery in the murine ischemic stroke model

Abstract

Muse cells are endogenous, non-tumorigenic, pluripotent-like stem cells already applied to clinical trials based on intravenous injection. They can selectively home to the post-infarct area, replenish apoptotic neural cells by phagocytosis-induced differentiation, and enhance functional recovery. The effect of nose-to-brain delivery of Muse cells on cerebral infarct was examined. Permanent middle cerebral artery occlusion model BALB/c mice received intranasal administration of either human Muse cells (6.0 × 10⁴ cells), high-dose human-mesenchymal stem cells (MSCs) (1.6 × 10⁶ cells), low-dose human-MSCs (6.0 × 10⁴ cells), or vehicle at 7 days after onset. An accelerated rotarod test and a histological assessment were done. The vehicle- or low-dose MSC groups showed no significant improvement in the rotarod test. In the high-dose MSC group, motor function was transiently recovered, but the therapeutic effect disappeared thereafter. The Muse group continuously improved motor function, with statistical significance to the other groups. The engraftment of administered cells in the peri-infarct area was the highest in the Muse group, while few cells were detected in other groups. 63.6 ± 8.5% and 26.2 ± 3.0% of Muse cells were positive for NeuN and GSTpi, respectively. Intranasal administration of Muse cells might be a viable approach to improving functional recovery with less invasiveness after ischemic stroke.

Keywords: Intranasal administration; Ischemic stroke; Mesenchymal stem cell; Muse cell.

Fig. 1

Rotarod treadmill performance. A line graph shows the temporal profile of functional recovery in Muse cell- (red), high-dose MSC- (green), low-dose MSC- (blue), and vehicle-treated mice (black) subjected to permanent middle cerebral artery occlusion. *, P < 0.05; **, P < 0.01 vs. pretreatment.

Fig. 2

Effects of cell therapy with vehicle, low-dose MSCs, high-dose MSCs, and Muse cells on motor dysfunction. A bar graph shows the Rotarod treadmill performance in Muse cell- (red), high-dose MSC- (green), low-dose MSC- (blue), and vehicle-treated mice (black), respectively. *, P < 0.05; **, P < 0.01.

Fig. 3

Fluorescent staining for detecting human mitochondria-positive cells. (A) A diagram demonstrates the distribution of human mitochondria-positive cells. Green plots represent the human mitochondria-positive cells in the brain. The engrafted cells are densely distributed in the peri-infarct area. The black square indicates the location of magnified fluorescence immunohistochemistry images shown in Panel B. (B) On fluorescence immunohistochemistry for the muse brain harvested at 84 days after the onset of ischemia, the human mitochondria-positive cells were found in the Muse group, but were very sparse in the high-dose and low-dose MSC groups. None of them were observed in the vehicle group. (C) Semi-quantitative analysis of engrafted cells in the infarct brain harvested at 84 days after the onset of ischemia. A line graph shows the number of human mitochondria-positive cells per slice in Muse cell- (black circles), high-dose MSC- (gray circles), and low-dose MSC-treated mice (white circles) subjected to permanent middle cerebral artery occlusion, respectively. ** and ***, P < 0.01 and P < 0.001, Muse group vs. high-dose MSC group; ^†††, P < 0.001, Muse group vs. low-dose MSC group.

Fig. 4

Differentiation of Muse cells in the infarct brain. (A) On multi-color fluorescence immunohistochemistry, the human-mitochondria-positive Muse cells were doubly positive for NeuN (red, upper; arrowhead) and GSTpi (red, middle; arrowhead), but not for GFAP (red, lower; arrowhead). The white square shows the magnified images of the cell indicated by the arrowheads. Scale bar = 50 μm. h-Mito, human-mitochondria. (B) A bar graph shows the percentages of NeuN-, GSTpi-, and GFAP-positive cells in human-mitochondria-positive cells in the Muse group. ND, not detected.