A system to enrich for primitive streak-derivatives, definitive endoderm and mesoderm, from pluripotent cells in culture.
Two lineages of endoderm develop during mammalian embryogenesis, the primitive endoderm in the pre-implantation blastocyst and the definitive endoderm at gastrulation. This complexity of endoderm cell populations is mirrored during pluripotent cell differentiation in vitro and has hindered the identification and purification of the definitive endoderm for use as a substrate for further differentiation. The aggregation and differentiation of early primitive ectoderm-like (EPL) cells, resulting in the formation of EPL-cell derived embryoid bodies (EPLEBs), is a model of gastrulation that progresses through the sequential formation of primitive streak-like intermediates to nascent mesoderm and more differentiated mesoderm populations. EPL cell-derived EBs have been further analysed for the formation of definitive endoderm by detailed morphological studies, gene expression and a protein uptake assay. In comparison to embryoid bodies derived from ES cells, which form primitive and definitive endoderm, the endoderm compartment of embryoid bodies formed from EPL cells was comprised almost exclusively of definitive endoderm. Definitive endoderm was defined as a population of squamous cells that expressed Sox17, CXCR4 and Trh, which formed without the prior formation of primitive endoderm and was unable to endocytose horseradish peroxidase from the medium. Definitive endoderm formed in EPLEBs provides a substrate for further differentiation into specific endoderm lineages; these lineages can be used as research tools for understanding the mechanisms controlling lineage establishment and the nature of the transient intermediates formed. The similarity between mouse EPL cells and human ES cells suggests EPLEBs can be used as a model system for the development of technologies to enrich for the formation of human ES cell-derived definitive endoderm in the future.
School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia, Australia.
Svetlana
S
Vassilieva
Hwee Ngee
HN
Goh
Kevin X
KX
Lau
James N
JN
Hughes
Mary
M
Familari
Peter D
PD
Rathjen
Joy
J
Rathjen
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D058732:Embryoid Bodies / Q000648:ultrastructure*; D004707:Endoderm / Q000648:ultrastructure*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D006735:Horseradish Peroxidase / Q000493:pharmacokinetics; D008648:Mesoderm / Q000648:ultrastructure*; D051379:Mice; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D039904:Pluripotent Stem Cells / Q000378:metabolism / Q000648:ultrastructure*; D054240:Primitive Streak / Q000648:ultrastructure*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D006735:Horseradish Peroxidase