Mesenchymal Stem Cells (MSC) Display Coordinated Rolling and Adhesion Behaviour on Endothelial Cells under Shear Flow.

Although MSC have been applied in a number of transplantation protocols, the mechanisms which are responsible for their extravasation and tissue specific homing are less well understood. To elucidate the potential of MSC to undergo coordinated interaction with endothelial cells, we investigated MSC under controlled shear flow using a parallel plate flow chamber. MSC were isolated from bone marrow of 6 different donors, depleted of hematopoietic cells by plastic adherence to yield >99% CD45⁻ populations and further expanded up to 10⁶ fold in DMEM/FCS/bFGF over 35 days. Their osteogenic, chondrogenic and adipogenic differentiation potential was maintained during the expansion period. Flow cytometric analysis showed that MSC expressed integrins alpha 1, alpha 4, alpha 5 and beta 1, the chemokine receptor CCR6 and, in a small subpopulation also CXCR4, but that they did not express beta 2 integrin, L-selectin or PSGL-1. During continuous laminar flow on early passage HUVEC at wall shear stresses between 0.1 and 1 dyn/cm², MSC extended cytoplasmic podia towards endothelium, resulting in capturing and subsequent rolling. This resulted in firm adherence of MSC which resisted shear stresses of 2 dyn/cm² for >20min. This was, in a similar way, also observed with peripheral blood mononuclear cells or CD34⁺ primary hematopoietic progenitor cells, whereas erythrocytes and primary endothelial cells did not interact with the HUVEC monolayer. Shear stresses of 2 dyn/cm² and more resulted in a decrease of MSC rolling. Prestimulation of endothelial cells with TNF-alpha increased both MSC rolling (3.7-fold ± 0.6 fold) and firm adhesion (3.3 ± 0.3 fold; means ± SD, n=5; p<0.05). Preincubation of endothelial cells with function-blocking anti-P selectin antibodies reduced the number of rolling MSC by 37.7% ± 5.9% and of firmly adhering MSC by 54.6% ± 11.3% (means ± SD; n=4; p<0.05). Although preincubation with function-blocking antibodies against PSGL-1 did not detectably affect MSC rolling and adhesion, preincubation with O-sialoglycoprotease and with fucosidase but not with neuaminidase significantly reduced MSC rolling and adhesion, indicating that a selectin-type ligand is responsible for the binding. Preincubation of MSC with function-blocking anti-beta 1 integrin antibody also led to a statistically significant reduction of MSC firm adhesion by 21.3% ± 5.7%. Taken together, our results demonstrate that MSC can undergo coordinated rolling and adhesion on endothelium under shear flow involving (i) stimulation in a pro-inflammatory environment, (ii) engagement of P-selectin and a fucosylated ligand on MSC inducing capturing and rolling, and (iii) activation of beta 1 integrins mediating firm adhesion to endothelial cells