Research ReportInvolvement of PI3K and ROCK signaling pathways in migration of bone marrow-derived mesenchymal stem cells through human brain microvascular endothelial cell monolayers
Graphical abstract
In vitro studies of MSC transendothelial migration revealed that MSC transendothelial migration was associated with the PI3K and ROCK pathways. Interestingly, a ROCK inhibitor (Y27632) or adenovirus-mediated interference with ROCK expression in MSC significantly increased MSC transendothelial migration. These results indicate that genetically modified mesenchymal stem cells that are able to penetrate the BBB can be constructed for cell therapy of CNS disease.
Introduction
Bone marrow-derived mesenchymal stem cells (MSC) have the potential to differentiate into mesenchymal lineage cells (Makino et al., 1999, Matsushita et al., 2011); additionally, they can be experimentally induced to undergo unorthodox differentiation into neural and myogenic cells (Bianco et al., 2001). As such, MSC represent an important paradigm and an easily available source for potential therapeutic use in neurological diseases (Lee et al., 2010, Danielyan et al., 2009, Inoue et al., 2003, Li et al., 2002). Considering their possible use in cell therapy, injection of cells into the venous blood (Ali et al., 2012) is a routine medical procedure (Sokolova et al., 2007) that is often used because it is less invasive than local administration (Olson et al., 2012).
The primary structural basis of the integrity of the blood–brain barrier (BBB) is the presence of tight junctions between microvascular endothelial cells throughout the brain; these junctions regulate the paracellular passage of cells, molecules, and ions. The entry of circulating cells into the central nervous system (CNS) requires, firstly, the passage of the cells across the vascular endothelial cell layer. It has been reported that peripheral blood T lymphocytes (Man et al., 2007) and small-cell lung cancer cells (Li et al., 2006) can “open” the tight junction of human brain microvascular endothelial cells (HBMEC) and trigger subsequent transendothelial migration. MSCs are capable of transendothelial migration through ES cell-derived endothelial cell monolayers with the disruption of tight junction (Schmidt et al., 2006). However, the defined molecular mechanisms involved in MSC transmigration through the BBB remain unknown.
In the present study, we employed an in vitro BBB model reported previously (Man et al., 2007, Li et al., 2006) to study the mechanism underlying MSC transendothelial migration and found that the phosphatidylinositol 3-kinase (PI3K)/Akt and Rho/ROCK (Rho kinase) pathways of MSC are involved in the transmigration process.
Section snippets
MSC trigger tight junction disassembly in HBMEC monolayers
HBMEC monolayers cultured on Transwell inserts have been used as an in vitro BBB model (Man et al., 2007, Li et al., 2006) to study the mechanism of transendothelial migration in cells in suspension by measuring the cells that crawl through the membrane to the lower chamber. However, adherent MSC attached to the bottom of the porous membrane instead of remaining in the medium in the lower chamber. By referring to the adherent breast cancer cell migration model (Lee et al., 2004), we developed
Discussion
According to Dharmasaroja, the therapeutic potential of MSC in various pathological conditions of the CNS has been explored (Dharmasaroja, 2009). Although transplantation of MSC through intravenous injection represents a minimally invasive strategy (Li et al., 2002), the delivery of cells into the brain is limited due to the existence of the BBB. A better understanding of the mechanisms underlying MSC transendothelial migration will be helpful in designing successful cellular therapies for CNS
Cell culture
Rat MSC were prepared as described by Tropel et al. (2004). Briefly, male Wistar rats were sacrificed by cervical dislocation; the femurs were aseptically dissected, repeatedly flushed with MSC medium containing DMEM-LG, 10% fetal bovine serum (FBS, Hyclone) and 2 mM glutamine, and plated in a culture flask (Corning Costar). Twenty-four hours later, the non-adherent cells were removed, and the adherent cells were cultured for 14 days as passage 0. MSC at passages 3–8 were used in the
Acknowledgments
The authors are grateful to Drs. Monique Stins and Kwang Sik Kim (Department of Pediatrics, John Hopkins University School of Medicine) for providing HBMEC. This work was supported by the Innovation Team Program Foundation of Liaoning Province (2006T131, LT2011011), Liaoning Province Doctoral Startup Fund (20061035), National Natural Science Foundation of China (30970120, 31171291) and National Program on Key Basic Research Project (2013CB531003).
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These authors contributed equally to this work.