(229bm) Aggregate-Derived Human Mesenchymal Stem Cells Improve Cell Survival and Neurological Function Recovery in Rats Mcao Stroke Model

Introduction
In the US, stroke is the primary cause of severe disability and the third leading cause of death. To date, the only FDA-approved drug for stroke is tissue plasminogen activator (tPA), a thrombolytic agent that has limited benefits to patients. Originally isolated from bone marrow as the progenitor cells responsible for the repair and regeneration of mesenchymal tissues, human mesenchymal stem cells (hMSCs) have emerged as an important cell source in stroke treatment due to their trophic effects that promote endogenous tissue repair by forming a molecular milieu. However, hMSCs have low cell survival and secretory function post-transplantation, which significantly reduced their therapeutic potency. Cellular damages induced by the microenvironment at the stroke lesion site, such as ischemia, high level of reactive oxygen species (ROS) and inflammation, challenge the survival and function of transplanted hMSCs. While studies have demonstrated that in vitro hMSCs preconditioning, such as hypoxia and aggregation culture, could significantly improve hMSCs survival, migration, and secretory function in vitro, it remains to be determined whether the in vitro beneficial effect could be reflected after transplantation. The objective of this study is to investigate the effects of hMSCs aggregation preconditioning on cell survival and injury recovery in a rat MCAO stroke model. We hypothesize that PI3K/Akt anti-apoptotic pathway activated by aggregation preconditioning play a significant role in enhancing hMSCs survival post-transplantation, and further enhance the trophic effects and neurological function recovery.

Materials and Methods
hMSC aggregates were formed by seeding cells into ultra-low adherent surface plates. After dissociation, aggregate-derived hMSCs were labeled with Bangs iron particles and transplanted to MCAO stroke SD rats by I.V. injection. MRI scanning and behavioral tests were performed for each group.
Survival of aggregate-derived hMSCs was also assessed in vitro under the treatment of ischemia and hydrogen peroxide. To investigate the role of PI3K/Akt anti-apoptosis pathway during aggregation, several PI3K/Akt modulators were used to treat aggregate-derived hMSCs and 2D culture cell. Genes involved in PI3K/Akt pathway were also tested by RT-PCR.

Results
MRI image of the stroke rats with transplantation of aggregate-derived hMSC revealed low cell clearance after one week compared to control group. Aggregate-derived hMSCs lasted longer at lesion site after transplantation. Lesion recovery and neurological function were improved. Histological images showed cell presence and lesion sites after 4 weeks. In vitro ischemic or exogenous ROS challenge of aggregate-derived hMSCs showed higher survival rate compared to the cells from 2D culture. The enhanced survival of aggregate-derived hMSCs was correlated with increased expression of several anti-apoptosis genes involved in PI3K/Akt anti-apoptotic pathway, including Akt, PDK-1, Bcl-2, ERK1/2. PI3K inhibitor could reduce the survival of aggregate-derived hMSCs, which confirmed the significant role of PI3K/Akt signaling pathway in the enhancement of cell survival of aggregate-derived hMSC.

Conclusions
The results of this study demonstrate that aggregation preconditioning of hMSCs enhanced cell survival and functional recovery post-transplantation in MCAO stroke rats. PI3K/Akt pathway is activated in hMSCs aggregates and plays an important role to enhance anti-apoptosis and survival under ischemic and high level ROS conditions. Thus, 3D aggregation culture of hMSCs could be a non-genetic preconditioning method to potentiate hMSC properties for stroke cell therapy.