Human Multipotential Bone Marrow Stem Cells Exert Immunomodulatory Effects, Prevent Splenic Contraction, and Enhance Functional Recovery in a Rodent Model of Ischemic stroke Bing Yang MD, Jason Hamilton PhD, Roger Strong MS, XiaoPei Xi BS, Robert Mays PhD, Sean I. Savitz MD Neurology Department, University of Texas Medical School at Houston

Disclosure Supported by a grant from Athersys to study MultiStem® in animal models of stroke

Background There are no FDA proven therapies to enhance recovery from stroke Stem cells offers a promising direction Major mechanisms: paracrine effects such as immunomodulation Emerging role for the spleen in stroke: Activey contributes to ongoing injury: release inflammatory cytokines and immune cells MultiStem® is an adherent, human pluripotent adult stem cell product derived from bone marrow. Hypothesis: Multistem enhances recovery after stroke by immunomodulatory mechanisms involving the spleen

Experiments: Animals: Long-evan Rats, Male, 300-320g Ischemic Stroke Model: MCAo/CCAo for 3 hrs Cells dosing and route: 1.2x107/kg, iv Timing: cells infusion at 24 hours after MCAo Behavior test: Cylinder test Endpoints: behavior tests up to 28 days after MCAo, lesion sizes at 28 days; serum cytokines level up to 3 day; spleen mass changes at 3 day; IL-10 gene level changes at 3 day; and splenocytes changes at 3 day;

* * * * MultiStem® cell significantly improve the recovery at 28 day after MCAo, compared with saline group. *p<0.05. n=12 each group

Inflammatory Cytokine Changes IL-6, IL-1b and IL-10 increased in the serum after stroke MultiStem® significantly reduced IL-6 and IL-1b and increased IL-10 at 3 days after treatment Saline n=10; MultiStem® n=12; Sham n=10

Spleen mass changes at 3 day MAPC Vehicle * p<0.05 p=0.014 ** p<0.01 p=0.005 MEAN (mg) 838.5 636.9 815.2 SD 144.5 140.8 115.2 n 12 11 10 MCAO Sham Multistem Vehicle Vehicle Multistem restores the splenic mass

At 72 hrs after treatment, TUNEL positive cells were found in the white pulp of spleen. 100x Capsule sinusoid 200x MCAO+Multistem sinusoid Sinusoid 200x MCAO+Vehicle

There are less TUNEL positive cells found in Multistem treated animals vs. Vehicle-treated animals (n=3 per group, respectively) MCAO+MAPC MAPC Vehicle MEAN 18 49.33 SD 6.63 15.15 p=0.010 400x MCAO+Vehicle 400x

IL-10 and IL-4 Gene Expression in spleen * * *p<0.05 compared with MCAo

Conclusion: MultiStem® enhances recovery from stroke in a rodent model. The mechanisms may involve upregulating anti-inflammatory pathways and downregulating pro-inflammatory pathways. The spleen may be a potential target of Multistem’s immunomodulatory effects.

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