Neuroprotective effects of human umbilical cord mesenchymal stem cells (Neuroncell-EX) in a rat model of ischemic stroke are mediated by immunomodulation, blood-brain barrier integrity, angiogenesis, and neurogenesis

Abstract

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are a potential off-the-shelf product for acute ischemic stroke. This study explored the underlying mechanism of Cytopeutics® hUC-MSCs (Neuroncell-EX) as well as its feasibility and efficacy at two different doses: 2 × 10⁶ cells per rat and 4 × 10⁶ cells/rat in middle cerebral artery occlusion (MCAO) ischemic stroke model for 28 d. Modified neurological severity score (mNSS) and rotarod tests were evaluated at days 1, 4, 7, and 14. Transforming growth factor-beta 1 (TGF-β1), interleukin-1 receptor antagonist (IL-1Ra), and vascular endothelial growth factor (VEGF) were evaluated by enzyme-linked immunosorbent assay (ELISA) at days 4 and 28. Immunohistochemistry expression of aquaporin-4 (AQP4) and neuronal protein marker (NeuN) were performed at days 4 and 28, respectively. Both doses of Neuroncell-EX showed significant lower mNSS scores at days 7 and 14 compared to stroke control. Both Neuroncell-EX groups showed significant longer latency time at day 7, with only 4 × 10⁶ cells/rat group having significant longer time at day 14 than stroke control. At both time points, the 2 × 10⁶ cells/rat group had significantly higher TGF-β1 and IL-1Ra levels, with significantly increased TGF-β1 only observed in 4 × 10⁶ cells/rat group at day 4 compared to stroke control. The VEGF levels were significantly lower at day 4 but then significantly increased at day 28 in both Neuroncell-EX groups than stroke control. AQP4 expression was significantly higher in stroke control compared to healthy control at day 4. Both doses of Neuroncell-EX showed significantly higher NeuN expression compared to stroke control at day 28. There is a weak correlation between TGF-β1 with VEGF and inversely with AQP4. These results suggest that Neuroncell-EX is feasible and effective in promoting functional recovery and neuroprotection in ischemic rats, potentially through immunomodulation, angiogenesis, and neurogenesis mechanisms.

Keywords: Angiogenesis; Immunomodulation; Ischemic stroke; Neurogenesis; Neuroinflammation; hUC-MSCs.

Figure 1.

(A) Schematic experimental procedure with the number of animals per group for each assessment. (B) Time course of body weight changes exposed to 90 min of MCAO until the end of the study. Two-way ANOVA followed by post hoc Dunnett’s test was performed to compare multiple groups. Data are presented as mean ± SEM where *** indicates p < 0.001, **** indicates p < 0.0001 (stroke control versus healthy control), and # indicates p < 0.05 (Neuroncell-EX-treated groups versus stroke control). AQP4, aquaporin-4; BW, body weight; ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry; IL-1Ra, interleukin-1 receptor antagonist; IV, intravenous; mNSS, modified neurological severity score; MCAO, middle cerebral artery occlusion; NeuN, neuronal protein marker; TGF-β1, transforming growth factor-beta 1; VEGF, vascular endothelial growth factor; SEM, standard error of the mean.

Figure 2.

Effects of Neuroncell-EX on the recovery of neurological function in post-stroke rats. Neurological function assessments were performed using (A) mNSS and (B) rotarod at days 1, 4, 7, and 14 after MCAO induction. Two-way ANOVA followed by post hoc Dunnett’s test was performed to compare multiple groups. Data are presented as mean ± SEM, where * indicates p < 0.05, ** indicates p < 0.01, and **** indicates p < 0.0001 comparing stroke control with healthy control and # indicates p < 0.05, ## indicates p < 0.01, ### indicates p < 0.001, #### indicates p < 0.0001 comparing Neuroncell-EX-treated groups with stroke control. Error bars on each value represent SEM. MCAO, middle cerebral artery occlusion; mNSS, modified neurological severity score; SEM, standard error of the mean.

Figure 3.

Effect of Neuroncell-EX on the selected markers in serum levels including (A) TGF-β1, (B) IL-1Ra, and (C) VEGF. Data were compared between two groups using Student’s t-test. Data are presented as mean ± SEM where * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001 comparing stroke control with healthy control and # indicates p < 0.05, ## indicates p < 0.01, ### indicates p < 0.001, and #### indicates p < 0.0001 comparing Neuroncell-EX-treated groups with stroke control. IL-1Ra, interleukin-1 receptor antagonist; MCAO, middle cerebral artery occlusion; SEM, standard error of the mean; TGF-β, transforming growth factor-beta 1; VEGF, vascular endothelial growth factor.

Figure 4.

Effect of Neuroncell-EX on the expression of AQP4 in rats across all four groups. (A) Representative images of AQP4 immunostaining analysis in the rat brain sections at day 4 following MCAO. AQP4-positive expression is stained with brown in the neuropil (marked by a white star) and perivascular areas (marked by a black arrow). The scale bars represent 200 μm. (B) The scores of AQP4-positive expression were compared between two groups using Student’s t-test. Data are presented as mean ± SEM where * indicates p < 0.05 comparing stroke control and healthy control groups. AQP4, aquaporin-4; MCAO, middle cerebral artery occlusion.

Figure 5.

Effect of Neuroncell-EX on the expression of neuronal protein marker NeuN in the MCAO rat model. (A) Representative images of NeuN immunostaining analysis in the rat brain sections at day 28 following MCAO. NeuN-positive expression were stained with brown, marked by red arrow. The scale bars represent 200 μm for micrographs. (B) Comparison of the mean number of NeuN-positive cells in high power field (magnification 200 ×) from 4 random fields across groups. One-way ANOVA followed by post hoc Dunnett’s test was performed for comparison of multiple groups. Data are presented as mean ± SEM, where * indicates p < 0.05 comparing stroke control to healthy group and # indicates p < 0.05, ## indicates p < 0.01 comparing Neuroncell-EX-treated groups to stroke control. SEM, standard error of the mean; MCAO, middle cerebral artery occlusion.