Isolation and culture of vascular wall-resident cd34+ stem/progenitor cells

Abstract

Objective: The aim of this study is to observe the maintenance of stem cell properties of purified CD34-positive cells in vessel walls during in vitro expansion. Materials and Methods: Cells that migrated from the adventitial tissues of rat were collected and purified by microbead selection method to obtain CD34+ vascular wall-resident stem (VRS)/progenitor cell (PC). Those purified CD34+ VRS/PCs were evaluated by flow cytometry and immunofluorescent staining. The CD34+ VRS/PCs were continuously cultured until passage P3. Each passaged cell was evaluated by flow cytometry with anti-CD34. Results: After microbead selection, the CD34+ cells reached 88.07% ± 4.36% and these cells expressed neither endothelial (CD31) nor mature smooth muscle cell (smooth muscle-myosin heavy chain and SM22α) markers. Incubation of the purified CD34+ VRS/PCs at a density of 1.5 × 105 cells/100-mm dish, resulted in a gradual reduction of CD34-positive traits when passaged in vitro, starting at P1. Interestingly, the purified primary CD34+ VRS/PCs at a density of 1.0 × 104 cells per 100-mm dish show the traits of colony form growth, and P1 passaged cells were 79.2% ± 2.15% positive for CD34, then gradually lost the traits of CD34-positive cells when passaged in vitro. Conclusions: High purity CD34+ VRS/PCs can be obtained by magnetic bead screening. In vitro, low cell densities contribute to the maintenance of CD34+ VRS/PC traits.

Keywords

• cd34+ vascular wall-resident stem/progenitor cells
• culture
• magnetic bead screening