Lian Q, Yeo K, Que J, Tan E, Yu F, et al. (2006) Establishing Clonal Cell Lines with Endothelial-Like Potential from CD9hi, SSEA-1− Cells in Embryonic Stem Cell-Derived Embryoid Bodies. PLoS ONE 1(1): e6. doi:10.1371/journal.pone.0000006
Abstract:
Background. Differentiation of embryonic stem cells (ESCs) into specific cell types with minimal risk of teratoma formation
could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, selfrenewing
lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack
of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in
turn, their isolation. Methodology/Principal Findings. We describe here a method for the isolation of clonal lineagerestricted
cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based
methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with
endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs). Despite originating
from different mouse strains, RoSH and E- RoSH lines have similar gene expression profiles (r2 = 0.93) while that between ERoSH
and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased
propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate
efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH
cells are CD9hi, SSEA-12 while ESCs are CD9lo, SSEA-1+. Isolation of CD9hi, SSEA-12 cells that constituted 1%–10% of EB-derived
cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r2 = 0.95) and a propensity to
differentiate into endothelial-like cells. Conclusions. By combining empirical and rational evidence-based methods, we
identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells with
endothelial-like potential from mouse ESCs.