Isolation of the side population from neurogenic niches enriches for endothelial cells.

Alena Kalinina,Catherine Gnyra,Vera Tang,Yingben Xue,Diane C Lagace

doi:10.1371/journal.pone.0250752

Abstract

In stem cell research, DNA-binding dyes offer the ability to purify live stem cells using flow cytometry as they form a low-fluorescence side population due to the activity of ABC transporters. Adult neural stem cells exist within the lateral ventricle and dentate gyrus of the adult brain yet the ability of DNA-binding dyes to identify these adult stem cells as side populations remains untested. The following experiments utilize the efflux of a DNA-binding dye, Vyrbant DyeCycle Violet (DCV), to isolate bona fide side populations in the mouse dentate gyrus and subventricular zone (SVZ), and test their sensitivity to ABC transporter inhibitors. A distinct side population was found in both the adult lateral ventricle and dentate gyrus using DCV fluorescence and forward scatter instead of the conventional dual fluorescence approach. These side populations responded strongly to inhibition with the ABC transporter antagonists, verapamil and fumitremorgin C. The majority of the cells residing in the side populations of dentate gyrus and SVZ were characterized by their expression of CD31. Additionally, at least 90% of all CD31+ cells found in the dentate gyrus and SVZ were negative for the hematopoietic marker CD45, leading to the hypothesis that the CD31+ cells in the side population were endothelial cells. These findings, therefore, suggest that the side population analysis provides an efficient method to purify CD31-expressing endothelial cells, but not adult neural stem cells.

Highlights

DNA-binding dyes have been perpetually used in flow cytometry and fluorescence-activated cell-sorting (FACS) paradigms to identify cancer stem cells [1, 2]
Since cluster of differentiation 31 (CD31) is a marker of hematopoietic cells, we examined whether CD31+ cells coexpressed CD45, a ubiquitous cell antigen expressed on the surface of hematopoietic cells, including monocytes [34, 35], and which has been used in flow cytometry together with CD31 to exclude blood cells [28, 36]
The fairly small overlap in CD31 and CD45 markers suggests that the vast majority of the CD31-expressing DCV-effluxing cells in the side populations of the subventricular zone (SVZ) and DG are endothelial cells rather than blood cells. This series of experiments was used to examine whether a side population can be identified in primary cells of the dentate gyrus and SVZ using DCV, a live-cell-permeable DNA-binding dye, in flow cytometry

Summary

Introduction

DNA-binding dyes have been perpetually used in flow cytometry and fluorescence-activated cell-sorting (FACS) paradigms to identify cancer stem cells [1, 2] This has included the use of dyes such as Hoechst 33342 [3, 4] and, more recently, Vybrant DyeCycleViolet (DCV), which is less toxic to stem cells [5,6,7]. In these assays, live stem cells are identified as a side population that has low dual fluorescence intensity in both blue- and red-shifted spectra due to the activity of ABC transporters, which can efflux the DNA-binding dyes.

Methods

Results

Conclusion