Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.
The development of functional cell populations such as hepatocytes and pancreatic beta cells from embryonic stem cell (ESC) is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ESC differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ESCs with high concentrations of activin A led to the development of a CD25-Foxa3+CD4-Foxa2+ population within 4-5 days of culture. Isolation and characterization of this population showed that it consists predominantly of definitive endoderm that is able to undergo hepatic specification under the appropriate conditions. To develop reagents that can be used for studies on endoderm development from unmanipulated ESCs, from induced pluripotent stem cells, and from the mouse embryo, we generated monoclonal antibodies against the CD25-Foxa3+CD4-Foxa2+ population. With this approach, we identified two antibodies that react specifically with endoderm from ESC cultures and from the early embryo. The specificity of these antibodies enables one to quantitatively monitor endoderm development in ESC differentiation cultures, to study endoderm formation in the embryo, and to isolate pure populations of culture- or embryo-derived endodermal cells.
Gadue
Paul
P
Department of Pathology, Center for Cellular and Molecular Therapeutics; Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Gouon-Evans
Valerie
V
Cheng
Xin
X
Wandzioch
Ewa
E
Zaret
Kenneth S
KS
Grompe
Markus
M
Streeter
Philip R
PR
Keller
Gordon M
GM
D028341:Activins / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015704:CD4 Antigens / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D004707:Endoderm / Q000166:cytology / Q000276:immunology*; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D051542:Hepatocyte Nuclear Factor 3-beta / Q000378:metabolism*; D051543:Hepatocyte Nuclear Factor 3-gamma / Q000378:metabolism*; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism*; D051379:Mice
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; C496503:Foxa2 protein, mouse; C496506:Foxa3 protein, mouse; D053645:Interleukin-2 Receptor alpha Subunit; C422405:activin A; D028341:Activins; D051543:Hepatocyte Nuclear Factor 3-gamma; D051542:Hepatocyte Nuclear Factor 3-beta