Activin alters the kinetics of endoderm induction in embryonic stem cells cultured on collagen gels.
Embryonic stem cell-derived endoderm is critical for the development of cellular therapies for the treatment of disease such as diabetes, liver cirrhosis, or pulmonary emphysema. Here, we describe a novel approach to induce endoderm from mouse embryonic stem (mES) cells using fibronectin-coated collagen gels. This technique results in a homogeneous endoderm-like cell population, demonstrating endoderm-specific gene and protein expression, which remains committed following in vivo transplantation. In this system, activin, normally an endoderm inducer, caused an 80% decrease in the Foxa2-positive endoderm fraction, whereas follistatin increased the Foxa2-positive endoderm fraction to 78%. Our work suggests that activin delays the induction of endoderm through its transient precursors, the epiblast and mesendoderm. Long-term differentiation displays a twofold reduction in hepatic gene expression and threefold reduction in hepatic protein expression of activin-treated cells compared with follistatin-treated cells. Moreover, subcutaneous transplantation of activin-treated cells in a syngeneic mouse generated a heterogeneous teratoma-like mass, suggesting that these were a more primitive population. In contrast, follistatin-treated cells resulted in an encapsulated epithelial-like mass, suggesting that these cells remained committed to the endoderm lineage. In conclusion, we demonstrate a novel technique to induce the direct differentiation of endoderm from mES cells without cell sorting. In addition, our work suggests a new role for activin in induction of the precursors to endoderm and a new endoderm-enrichment technique using follistatin.
The Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Natesh
N
Parashurama
Yaakov
Y
Nahmias
Cheul H
CH
Cho
Daan
D
van Poll
Arno W
AW
Tilles
François
F
Berthiaume
Martin L
ML
Yarmush
D028341:Activins / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D003094:Collagen; D016895:Culture Media, Serum-Free; D017931:DNA Primers / Q000235:genetics; D004627:Embryonic Induction / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism / Q000637:transplantation; D004707:Endoderm / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D038681:Follistatin / Q000494:pharmacology; D005782:Gels; D015870:Gene Expression / Q000187:drug effects; D005855:Germ Layers / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008648:Mesoderm / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016895:Culture Media, Serum-Free; D017931:DNA Primers; D038681:Follistatin; D005782:Gels; D028341:Activins; D003094:Collagen