Isolation of vascular endothelial cells from intact and injured murine brain cortex-technical issues and pitfalls in FACS analysis of the nervous tissue.

Abstract

CNS endothelial cells (CNS-ECs), one of the main non-neural CNS cell populations, play a vital role in physiology, pathology, and regeneration of the nervous system. Therefore, there is an urgent need to enhance our knowledge on their biology to elucidate mechanisms responsible for the blood brain barrier function in normal and pathological conditions, interaction between brain endothelium and neural stem cells in the neurogenic niche, the paracrine processes in the brain and spinal cord, etc. Here, we described a novel, simple, and efficient protocol for isolation of endothelial, vessel-forming cells from the murine CNS, which is based on Sca-1 expression. Using this newly described protocol we were able to detect and sort viable, highly pure CNS-ECs with minimal contamination by cells of non-endothelial origin. This method will increase the availability of CNS-ECs for in vitro research. Moreover, we compared phenotype of CNS-ECs isolated from neonatal mice and adult intact and injured brain looking for the cells of endothelial precursor characteristic, such as those found in the bone marrow and circulating in the bloodstream after organ injuries. We have found that neonatal brain capillaries contain proportion of endothelial precursor cells (Sca-1(+) , CD45(-) , c-Kit(+) ). Such precursors were also found in adult brain cortex, both in intact and injured brain. Finally, we discuss several crucial technical issues concerning CNS tissue preparation for flow cytometry analysis and cell sorting as well as nonspecific antibody binding caused by inflammatory microglia/macrophages which should be avoided in order to reliable isolation of pure CNS cells for downstream procedures including cell transplantation-based translational studies.