Abstract
BackgroundDifferentiation 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.Methodology/Principal FindingsWe 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 (r2 = 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 CD9hi, SSEA-1− while ESCs are CD9lo, SSEA-1+. Isolation of CD9hi, 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 (r2 = 0.95) and a propensity to differentiate into endothelial-like cells.ConclusionsBy 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.
Highlights
The pluripotency of mouse embryonic stem cells (ESCs) to differentiate into cells from all three germ layers makes ESCs an ideal source of cells for regenerative therapy for many diseases and tissue injuries [1,2]
To derive RoSH-like cells from ESCs, we rationalized that since our derivation of RoSH cell lines was most optimal when using 5.5 to 6.5 dpc mouse embryos[19], 3–6 days old ESC-derived embryoid bodies (EBs) that are developmentally analogous to early post-implantation embryos for the derivation of RoSH-like cells would be optimal for the derivation of RoSH-like cells from ESCs[20]
Significant higher frequencies of these genes (p,0.05) were associated with biological processes that can be broadly classified as neural differentiation, embryo development and metabolism, suggesting that the differentiation potential especially neural differentiation potential of ESC-derived RoSH (E-RoSH) cells was reduced relative to ESCs
Summary
The pluripotency of mouse embryonic stem cells (ESCs) to differentiate into cells from all three germ layers makes ESCs an ideal source of cells for regenerative therapy for many diseases and tissue injuries [1,2] This property of ESCs poses a unique challenge of having to generate therapeutically efficacious quantity of appropriate cell types without being contaminated by potentially deleterious cell types. 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. 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. 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
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