Establishing clonal cell lines with endothelial-like potential from CD9(hi), SSEA-1(-) cells in embryonic stem cell-derived embryoid bodies.
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, self-renewing 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.


RESULTS
We describe here a method for the isolation of clonal lineage-restricted 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 (r(2) = 0.93) while that between E-RoSH 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 CD9(hi), SSEA-1(-) while ESCs are CD9(lo), SSEA-1(+). Isolation of CD9(hi), SSEA-1(-) cells that constituted 1%-10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r(2) = 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.
Lian
Qizhou
Q
Department of Surgery, National University of Singapore, Singapore; Genome Institute of Singapore, Singapore.
Yeo
Keng Suan
KS
Que
Jianwen
J
Tan
Eileen Khia Way
EK
Yu
Fenggang
F
Yin
Yijun
Y
Salto-Tellez
Manuel
M
Menshawe El Oakley
Reida
R
Lim
Sai-Kiang
SK
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods; D002999:Clone Cells; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000276:immunology* / Q000378:metabolism / Q000637:transplantation; D042783:Endothelial Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D020869:Gene Expression Profiling; D016256:Lewis X Antigen / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D020411:Oligonucleotide Array Sequence Analysis; D060245:Tetraspanin 29
D015703:Antigens, CD; C554870:Cd9 protein, mouse; D016256:Lewis X Antigen; D008562:Membrane Glycoproteins; D060245:Tetraspanin 29