Probing Multiple Transplant Delivery Routes of CD+34 Stem Cells for Promoting Behavioral and Histological Benefits in Experimental Ischemic Stroke

Abstract

Stroke is a leading cause of death in the US and around the world but with limited treatment options. Survivors often present with long-term cognitive and neurological deficits. Stem cell-based therapy has emerged as a potential treatment for stroke. While stem cell transplantation in stroke has reached clinical trials, mostly safety outcomes have been reported with efficacy readouts warranting more studies. In an effort to optimize the stem cell regimen for stroke, here we conducted vis-a-vis comparison of different routes of transplantation, namely, intracerebral, intraarterial, and intranasal delivery of expanded human CD34 + stem cells, called ProtheraCytes, in the established stroke model of transient middle cerebral artery occlusion (MCAO) using adult Sprague-Dawley rats. After adjusting for the dose and subacute timing of cell delivery, animals were randomly assigned to receive either ProtheraCytes or vehicle. Motor and neurological assays from days 7 to 28 post-stroke revealed significant functional recovery across all 3 delivery routes of ProtheraCytes compared to vehicle-treated stroke rats. Additionally, ProtheraCytes-transplanted stroke rats displayed significantly reduced infarct size and cell loss in the peri-infarct area coupled with enhanced neurogenesis and angiogenesis compared to vehicle-treated stroke rats. These results highlight the safety and efficacy of transplanting ProtheraCytes, including via the minimally invasive intranasal route, in conferring robust and stable behavioral and histological positive outcomes in experimental stroke.

Keywords: angiogenesis; cell delivery route; cell transplantation; cerebral ischemia; functional recovery; neurogenesis.

Graphical Abstract

Stem cell-derived extracellular vesicles promote recovery of ischemic cells. The present study demonstrates a novel mechanism of action mediating stem cell-induced therapeutic effects in ischemic stroke. Whereas the long-standing view of stem cell repair of the stroke brain implicates cell replacement, our findings implicates a process of by-stander effects, whereby stem cells release extracellular vesicles (EVs), in particular CD-63-labeled EVs, which appear to target the vasculature and enhance vascular endothelial growth factor zlevels, while dampening inflammation (Iba-1 cells) and increasing neural cell proliferation (DCX). Altogether, these reparative processes rescue the neurovascular unit via a multi-pronged mechanism involving EV release, vasculogenesis, anti-inflammation, and neurogenesis.

Figure 1.

A timeline of the experimental procedures. Stem cell transplantation was performed on day 3. Behavior testing was performed on day −1 (baseline), 0, 7, 14, and 28. Middle cerebral arterial occlusion (MCAO) was performed on day 0.

Figure 2.

Behavioral tests. (a) Motor activity revealed by EBST. MCAO + cell groups displayed significantly less asymmetry on days 7, 14, and 28 (*P < .05, **P < .01, ****P < .0001). (b) Motor activity revealed by cylinder test. MCAO + cell groups demonstrated significantly more use of impaired forelimb (*P < .05, **P < .01, ***P < .001, ****P < .0001). (c) Motor activity revealed by Grip Strength. MCAO + cell groups presented significantly less impaired paw grasp (*P < .05, **P < .01, ***P < .001). (d) Motor activity revealed by balance beam. MCAO + cell showed significantly better motor coordination during beam walks (**P < .01, ***P < .001).

Figure 3.

Infarct area. Nissl staining for coronal brain sections showing infarct areas (black outline) of MCAO + Cell IA, MCAO + Cell IN, MCAO + Cell IC, MCAO + Media IA, MCAO + Media IN, MCAO + Media IC. MCAO + Cell groups displayed significantly smaller infarcts (*P < .05, **P < .01, ***P < .001, ****P < .0001).

Figure 4.

Peri-infarct area. Nissl staining for quantitative analysis of live cells (black arrow heads) in the peri-infarct area for MCAO + Cell IA, MCAO + Cell IN, MCAO + Cell IC, MCAO + Media IA, MCAO + Media IN, MCAO + Media IC. MCAO + Cell groups showed significantly more living cells in the peri-infarct (*P < .05, **P < .01, ****P < .0001). Scale bar = 50 µm.

Figure 5.

Inflammation. Iba-1 double stained with CD34 to measure inflammatory activity. MCAO + Cell IC had significantly less Iba-1 positive cells than MCAO + media IN, MCAO + media IA, and MCAO + media IC groups (**P < 0.01). Treatment groups had significantly more CD34 expression (***P < .001) with MCAO + Cell IC having the highest cell count within the treatment group (*P < .05; ***P < .001). Scale bar = 50 µm.

Figure 6.

Neurogenesis. DCX double stained with CD34 measured neurogenesis differences between groups. All treatment groups MCAO + Cell IA, MCAO + Cell IN, and MCAO + Cell IC had significantly higher DCX expression than control groups MCAO + Media IA, MCAO + Media IN, and MCAO + Media IC (**P < .01). Treatment groups had significantly more CD34 expression (**P < .01). MCAO + Cell IN displayed significantly lower CD34 expression compared to MCAO + Cell IC (*P < .05). Scale bar = 50 µm.

Figure 7.

Angiogenesis. VEGFr1 double stained with CD34 quantified angiogenesis activity. MCAO + Cell IC and MCAO + Cell IA had significantly higher VEGFr1 expression than all MCAO + Media groups (**P < .01). MCAO + Cell IC had significantly more VEGFr1 positive cell counts than MCAO + Cell IA (*P < .05) and MCAO + Cell IN (**P < .01). MCAO + Cell IN had significantly less angiogenesis activity when compared to MCAO + Cell IA (*P < .05). CD34 expression was significantly higher in all treatment groups than in all media groups (**P < .01). Scale bar = 50 µm and 10 µm.

Figure 8.

Extracellular vesicles. CD63 staining was used as a marker to quantify extracellular vesicles and healthy stem cell activity. All treatment groups MCAO + Cell IA, MCAO + Cell IN, and MCAO + Cell IC had significantly higher CD63 positive stain count than control groups MCAO + Media IA, MCAO + Media IN, and MCAO + Media IC (*p < 0.05). Scale bar = 50 µm.

Figure 9.

ProtheraCytes protect against in vitro stroke model of oxygen glucose deprivation. (A) Trypan blue. ANOVA revealed significant treatment effects. Post hoc Bonferonni’s tests revealed significant differences in cell survival, with primary neurons exposed to OGD and cocultured with ProtheraCytes rescuing against OGD-induced cell death significantly better than primary neurons subjected to OGD. (B) MTT. Similarly, ANOVA showed significant treatment effects, with post hoc Bonferonni’s tests detecting significant differences in metabolic activity, again with primary neurons exposed to OGD and cocultured with ProtheraCytes reducing the OGD-induced metabolic impairment significantly better than primary neurons subjected to OGD. (C) CD63. Analysis of exosome marker expression revealed that ProtheraCytes express the exosomal marker CD63, which was not detectable in the standard medium. Statistical significance is depicted as follows: *P < .05; **P < .01; ***P < .001.