A new in vitro model for stem cell differentiation and interaction.
Development involves an interplay between various cell types from their birth to their disappearance by differentiation, migration, or death. Analyzing these interactions provides insights into their roles during the formation of a new organism. As a study tool for these interactions, we have created a model based on embryoid bodies (EBs) generated from mouse embryonic stem (mES) cells, which can be used to visualize the differentiation of mES cells into specific cell types while at the same time allowing controlled removal of this same cell population using an enzyme-prodrug approach. Cell-specific expression of Cre induces a switch of EGFP expression to LacZ. Furthermore, it leads to the expression of nitroreductase (NTR), which in combination with the prodrug CB1954 induces apoptosis. Here, we validate this model by showing expression of LacZ and NTR after Cre-mediated recombination. Additionally we show, as an example, that we can target the endothelial cells in EBs using the Tie-2 promoter driving Cre. Ablating Cre-expressing cells by adding CB1954 to the culture led to an abrogated vascular formation. This system can easily be adapted to determine the fate and interaction of many different cell types provided that there is a cell-type-specific promoter available.
Smits
Anke M
AM
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
van den Hengel
Lisa G
LG
van den Brink
Stieneke
S
Metz
Corina H
CH
Doevendans
Pieter A
PA
Goumans
Marie-José
MJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001388:Aziridines / Q000008:administration & dosage; D002450:Cell Communication; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D053595:Embryonic Stem Cells / Q000166:cytology*; D042783:Endothelial Cells / Q000166:cytology; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D019426:Integrases / Q000235:genetics; D007763:Lac Operon; D051379:Mice; D008954:Models, Biological; D009601:Nitroreductases / Q000235:genetics; D011401:Promoter Regions, Genetic; D014162:Transfection
D001388:Aziridines; D049452:Green Fluorescent Proteins; C100099:tretazicar; D009601:Nitroreductases; C045073:Cre recombinase; D019426:Integrases