Vasculogenesis, angiogenesis and local blood flow recovery after embryonic stem cells transplantation in the ischemic brain

Abstract

Stroke is a vascular disease in which brain cell death is initiated by a great reduction in blood flow. Only a few minutes of markedly lowered local blood flow (focal ischemia) may injure neurons and non-neuronal cells (e.g., endothelial cells and astrocytes) and lead to their death. Vasculogenesis is a process in which angioblasts differentiated into endothelial cells to form a primitive capillary network; whereas angiogenesis is the sprouting of capillaries from pre-existing blood vessels. Both mechanisms may contribute to neovascularization during embryogenesis and repair of damaged tissues in adults. The multi-potent embryonic stem (ES) cells can differentiate into neural cells and endothelial cells in vitro and in vivo. The multi-potentiality of ES cells provides a potential strategy for repairing the damaged brain after ischemic stroke. To examine the feasibility of ES cell transplantation in the ischemic brain and functional benefits of neovascularization, this study explored differentiations of transplanted ES cells in a rodent focal ischemia model. Transient focal cerebral ischemia was induced in adult male animals by 30-min occlusion of middle cerebral artery. Mouse ES cells were differentiated down neural lineages using the retinoic acid 4-/4+ protocol. To be able to identify ES cells after transplantation, ES cells were genetically marked with green fluorescent protein GFP or pre-labeled with BrdU and Hoechst. ES cell transplantation was performed 7 days after ischemia. Differentiation of ES cells was demonstrated by specific antibodies including MAP2, neurofilament, NeuN for neuron-like cells, GFAP for astrocytes, NG-2 for oligodendrocytes, Glut-1 and CD-31 for endothelial cells. Transplanted ES cells survived, differentiated and form functional structure in the ischemic core and penumbra regions. ES cell-derived endothelial cells formed new vesicular structures 7–14 days after transplantation. Local blood flow measured by Laser Doppler restored in the former ischemic region. Stroke animals received ES cell transplantation showed improved neurological and behavioral function 20–30 days after the transplantation. Our study demonstrates potential strategy of using ES cell transplantation to repair damaged brain tissues for reconstruction of neurovascular networks. The restoration of local blood supply and neuronal activities supports long-term functional benefits of ES cell transplantation after stroke (See Figure 1).