Human adipose multilineage-differentiating stress-enduring cells on treatment of ischemic stroke in rats

Abstract

BACKGROUND: Mesenchymal stem cells have shown good therapeutic effects in ischemic stroke, while the role of multilineage-differentiating stress-enduring (Muse) cells isolated from adipose-derived mesenchymal stem cells in ischemic stroke needs further study. OBJECTIVE: To explore the neurorestorative effect of intravenous administration of adipose Muse cells on ischemic stroke in rats. METHODS: The Muse cells expressing stage-specific embryonic antigen 3 were sorted by magnetic beads after long-term (4 hours) trypsin incubation of human adipose-derived mesenchymal stem cells. The middle cerebral artery occlusion model was established in rats. After successful modeling, the adipose-derived mesenchymal stem cell group and adipose-derived Muse group were injected with 200 μL adipose-derived mesenchymal stem cell suspension or adipose Muse cell suspension (containing 2×10^5 cells) via the tail vein, while the saline group received 200 μL saline. Behavioral scores were assessed at days 3 and 7 after transplantation. Hematoxylin-eosin staining was used to observe brain tissue damage at day 3. Immunofluorescence was used to detect microtubule-associated protein 2 and Ki67 expression in the damaged area. TUNEL staining was used to observe apoptosis. Western blot was used to detect growth-associated protein 43 and Bcl-2/Bax protein expression. RESULTS AND CONCLUSION: After magnetic bead sorting, flow cytometry showed that the expression rate of stage-specific embryonic antigen 3 in positively sorted cells was as high as 80%. At day 7 after transplantation, compared with the saline group, the neurological deficit score was reduced in both the adipose-derived mesenchymal stem cell group and the adipose-derived Muse group (P < 0.05). At day 3, compared with the adipose-derived mesenchymal stem cell group, the adipose-derived Muse group showed a lower neurological deficit score (P < 0.05). Hematoxylin-eosin staining showed reduced inflammatory response and vacuolation in the cerebral cortex of rats in both cell-treated groups, and immunofluorescence for microtubule-associated protein 2 showed that the adipose-derived Muse group had more obvious inhibition of neuronal loss. TUNEL and Ki67 staining showed reduced apoptosis and increased proliferation in the damaged area in both cell-treated groups. Western blot results showed that the Bcl-2/Bax ratio and growth-associated protein 43 expression were increased in both cell-treated groups, and the adipose-derived Muse group was superior to the adipose-derived mesenchymal stem cell group in inhibiting apoptosis. These results indicate that both adipose-derived mesenchymal stem cells and adipose-derived Muse cells promote neural repair in rats, with adipose-derived Muse cells playing a better role in inhibiting apoptosis.

1. Introduction

Stroke includes ischemic and hemorrhagic stroke. In China, 70%-80% of strokes are ischemic. The main goal of treatment for ischemic stroke is to restore blood flow as soon as possible after symptom onset. The main methods are intravenous thrombolysis and endovascular intervention, both of which have strict therapeutic time windows, causing most patients to miss the optimal treatment opportunity [1-2]. Therefore, it is particularly important to actively seek methods to improve the prognosis of ischemic stroke.

In recent years, stem cell therapy has achieved satisfactory results in preclinical and clinical studies [3-4]. Currently, most stem cells used for transplantation are mesenchymal stem cells [5-6], and their role in treating stroke has been widely studied [7-8]. Mesenchymal stem cells can promote neuronal survival, inhibit inflammatory responses, and improve neurological function through paracrine effects (such as releasing growth factors and exosomes) [9-10]. However, traditional mesenchymal stem cells have limitations in neural differentiation potential and targeted repair ability. Therefore, how to enhance the neural repair efficacy of mesenchymal stem cells through biological modification or combined intervention strategies [11-14] remains a key and difficult point in current research.

Multilineage-differentiating stress-enduring (Muse) cells were discovered and reported by the Japanese scientist KURODA's research group in 2010 [15]. These cells express stage-specific embryonic antigen 3 (SSEA3) and have the ability to differentiate into cell lineages of all three germ layers. Muse cells can be isolated from various tissues such as skin and bone marrow [16-17], and have good repair potential in wound scarring, myocardial infarction, and nerve injury [18-20]. Since the content of Muse cells in the body is extremely low, how to efficiently obtain a sufficient number of cells for clinical translation [21] has become a key bottleneck restricting their clinical application. In previous work, we compared the biological characteristics of Muse cells sorted from adipose tissue and umbilical cord tissue-derived mesenchymal stem cells. The results showed that long-term trypsin digestion (4 h) of adipose-derived mesenchymal stem cells significantly improved the enrichment efficiency of Muse cells [22]. Based on this, to further explore the therapeutic potential of adipose-derived Muse cells in a rat model of ischemic stroke, this study transplanted adipose-derived Muse cells via tail vein injection into rats with middle cerebral artery occlusion (MCAO).