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Introduction: Cell-based therapies for stroke remain under development and there is a lack of pre-clinical large animal data defining optimal dosing and delivery methods. Whereas safety and efficacy of intra-arterial (IA) mesenchymal stem cells (MSC) have been demonstrated in rodent models, large animal model are lacking, hampering advancement of the field and limiting mechanistic insights. Methods: An endovascular canine reversible Middle Cerebral Artery Occlusion (MCAO) model using a retractable platinum coil for 60-120 min was established. At 48-hour post-MCAO, allogeneic male canine MSCs (10-80 million) were delivered using a microcatheter in the ipsilateral upper cervical internal carotid artery of female canine subjects. Serial MRIs and neurological deficit scoring (NDS) were performed over 30 days. Animals were euthanized at15-30 d post-MCAO and brains were harvested for histology and molecular analyses. Results: There was a dose-dependent reduction of infarct volume on MRI associated with improved neurological scoring and corticospinal tract caliber on Diffusion Tensor Imaging over the range of 10, 20 and 40 million MSCs vs. PBS control. This dose-range was safe and did not result in worsening of stroke volume or behavioral outcomes. In contrast, 80 million IA MSCs led to a worsened neurological score immediately following the injection and an increase in the infarct volume at 4 days post injection. Conclusion: Together these findings establish a successful preclinical canine model of stroke in which dose-dependent efficacy of IA MSC therapy is demonstrated to reduce stroke size and augment DTI. These findings have important clinical implications in designing early-stage trials of IA delivery in humans.

Introduction: Pre-clinical studies have shown great promise in the use of cell-based therapies for stroke. Intra-arterial (IA) delivery directs the cells to the injured brain, is minimally invasive and widely available, making it attractive for clinical translation. Our group and others have shown the safety and efficacy of IA Mesenchymal Stem Cell (MSC) treatment in a rodent stroke model at 24 hours post-reperfusion. The optimal timing of a single dose of IA MSC is unknown. Also, whether an additional dose will lead to greater benefit and the optimal timing of the second dose need to be determined for successful translation of IA MSC to clinic. The objective of this study is to determine if dual dose IA-MSC treatment is superior to single dose and find the optimal timing for the second dose. Methods: We used a reversible middle cerebral artery occlusion (rMCAO) model for stroke in rats. IA-MSCs were administered using a PE-10 catheter at 1, 2 and 4 day after stroke as single dose. Based on the results of the first experiment, dual doses were given at 1-4, 1-6 and 1-15 day. Behavioral analysis of rotarod, neurological deficit scoring and stroke volume were determined at 30 days after treatment. Results: Single dose IA-MSC at 1day was superior (p=0.02, 0.01, 0.04) to single dose given on day 2 and no benefit was seen when given on day 4. Among the dual dose groups, 1-6 day after stroke showed significant improvement in neurodeficit score and stroke volume (p=0.007, p=0.03). However, the percentage increase in improvement in 1-6day (dual) was much higher than that observed in 1day (single) group (Fig 1). Detailed results will be presented at the meeting. Conclusion: The results of our study indicate that 1day after stroke is optimal for single dose of IA-MSC. An additional dose at 6days after stroke enhances the effect of IA-MSC in aiding symptomatic recovery and stroke lesion reduction. In summary, our findings contribute to understanding of the benefit of dual dose of IA MSC for translation to the clinic.

Background: Recent preclinical studies of stroke recovery have explored the possibility of utilizing exosomes (ES) instead of complete cells as therapeutic agents. ES are endosomal origin small-membrane vesicles with a size of 40 to 100nm in diameter, secreted from many types of cells including mesenchymal stem cells (MSCs). ES-encapsulated transfer of miRNAs promotes neurite remodeling and functional recovery of stroke in rats. The optimal route of delivery of ES in stroke is not established. While a few studies have shown efficacy of intra-venous exosomes administration for stroke, there is one study exploring intra-arterial exosome therapy (IAX) with single dose of 100ug exosomes. We propose intra-arterial delivery of exosomes in a dose escalation study to establish safety and early efficacy. Methods: Female ovariectomized Sprague-Dawley rats were exposed to MCAo for 90 min. Rats were treated with IAX (10, 50 and 100ug/0.5mL), IAMSCs (1x10 5 cells/0.5mL) or IA phosphate-buffered saline (IAPBS) at 1 day (1D) after MCAo. To test neurological and motor function, the standardized neurobehavioral test battery and the rotarod test were performed. The mean duration (in seconds) on the device was recorded from 3 rotarod measurements. The rats were tested at pre-surgery, 7, 15 and 30 days post-MCAo. Results: There was no neurological worsening or mortality post IA exosome delivery at any dose tier. The Neurological deficit score of IAX 10ug treatment group at POD 7, 15 and 30, and IAX 50ug treatment at POD 15 were significantly improved in comparison to IA PBS. IAX 10ug treatment group showed trend of improvement in motor coordination compared to the IA PBS/MSC/higher doses of exosome treatment groups. Conclusions: The Neurological deficit scores of IAX 10ug treatment group were significantly improved in comparison to IA PBS. Lower dose than previously published might be most effective, and noninferior to IA-MSCs.

Background: Despite the efficacy of thrombectomy for large vessel occlusion acute ischemic stroke (AIS) , ~50% of patients have significant residual deficits. Pre-clinical data on intra-arterially (IA) administered mesenchymal stem cells (MSCs) in stroke are promising and this approach is attractive for clinical application. While there is a concern for micro-occlusion with IA delivery due to the large size of MSCs, a dose of 1 x 10 5 MSCs given 24-48 hr in a rodent reperfusion middle cerebral artery occlusion (rMCAo) model has been shown to be safe and effective. As per STAIR recommendations, we performed a dose-escalation (DE) study of IA-MSCs in a large animal stroke model. Methods: An endovascular canine rMCAo model using retractable platinum coil for 60-120 min was established. At 48 hr post-rMCAo, allogeniec canine MSCs were delivered using a 0.0165” microcatheter in the ipsilateral upper cervical internal carotid artery in escalating doses (based on proportion of rodent to canine total cerebral blood volume). Serial MRIs and neurological deficit scoring (NDS) were performed over 30 days. Animals were euthanized at 15-30 d post-rMCAo and brains were harvested. Results: Female canines (n=13), age 12-36 months, weighing 22-26 kg received IA MSCs ranging from 5-80 x 10 6 (M). At doses of 5-40 M IA-MSCs no neurological worsening was observed. Serial NDS and stroke volume on MRI showed no increase post-IA-MSCs and actually showed progressive reduction. A higher numerical reduction was seen in the 10-40 M groups compared to 5 M. However, in the one canine receiving 80 M IA-MSCs, there was significant worsening of the MCA-area infarction and NDS due to microembolization at this higher dose. Gross examinations and histopathology of brain tissue were consistent with ischemia. The brain of a canine receiving 80 M cells showed differentially aged areas of necrosis supporting two ischemic events. Neuroblasts, doublecortin-positive cells, and neovascularization were observed in canine brains suggesting regenerative mechanisms. Conclusions: These data suggest that IA-MSCs are safe in a large animal model up to 40 M IA-MSCs and is the maximum tolerated dose in this DE study. Furthermore, our data suggests that up to 40 M IA-MSCs may be promising in exploring efficacy in AIS.

Background: Cell therapy is emerging as a promising novel treatment for ischemic stroke. Intra-arterial (IA) mesenchymal stem cells (MSCs) delivery in ischemic stroke has a high potential for clinical translation. Recently, we demonstrated the safety and efficacy of IA delivery of MSCs at 24h in a reversible middle cerebral artery occlusion (rMCAo) rodent model. Given the trophic mechanism of action of cell therapy in stroke, a second dose of cells may be beneficial. However, it is unclear if a second IA-MSCs administration is safe and efficacious. Therefore, we aimed to evaluate administration of two doses of IA-MSCs in the rodent stroke model. Methods: Female ovariectomized Sprague–Dawley rats were exposed to MCAo for 90 min. Rats were treated with IA-MSCs (1x10 5 cells) or phosphate-buffered saline (PBS) at 1 and 6 days (1D-6D) after MCAo. To test neurological and motor function, the standardized neurobehavioral test battery and the rotarod test were performed. The mean duration (in seconds) on the device was recorded from 3 rotarod measurements. The rats were tested at 7, 15 and 30 days after MCAo. Rats were sacrificed at 30 days for infarct volume measurement using histology. Results: There were no neurological worsening or mortality seen in either treatment group. We observed significant reduction in infarct volume in 1D-6D MSCs group (21 ± 15mm 3 ; n=5) compared to the PBS-treated group (86 ± 19 mm 3 ; n=8, p Conclusions: Double dose IA-MSCs at 1D-6D post rMCAo is safe and reduces ischemic brain injury in female rats, with a trend towards functional improvement.

Background: Cell therapy is emerging as a promising treatment for stroke. We recently we demonstrated the efficacy of intra-arterial delivery of MSCs (IA MSCs) at 24h after a reversible middle cerebral artery occlusion (MCAo). Our study also identified a maximum tolerated dose of MSCs that could be delivered IA without compromising middle cerebral artery flow. The IA approach avoids first-pass trapping of MSCs in the lungs and liver as seen with intravenous delivery. Intra-arterial treatment is also minimally invasive and widely available in clinical practice and thus appealing for clinical translation. Since majority of ischemic strokes in women occur after onset of menopause it is crucial that we test the efficacy of IA MSCs in reproductively senescent females consistent with STAIR recommendations. We aimed to validate the efficacy of IA MSCs in reproductively senescent female rats. Methods: Retired breeder female (9-11 months; 280-350 g) Sprague-Dawley rats showing estrous acyclicity were exposed to MCAo (90 min). A day later, rats were treated with IA MSCs (1x10^5 cells) or phosphate-buffered saline (PBS). MSCs or PBS treated rats were sacrificed at 28-30 days for infarct volume measurement using histology. To test motor function, the rotarod test was performed. Rats were trained for 3 consecutive days for the rotarod test before undergoing the MCAo procedure. The mean duration (in seconds) on the device was recorded from 3 rotarod measurements 1 day before surgery. The rats were tested at 1, 7, 15 and 28-30 days after MCAo. Results: We observed significantly lower mean infarct volume in the MSC-treated group (12 ± 3 mm3; n=6) compared to the PBS-treated group (29 ± 7 mm3; Mean ± SEM; n=4, p