Elimination of tumorigenic stem cells from differentiated progeny and selection of definitive endoderm reveals a Pdx1+ foregut endoderm stem cell lineage.
Embryonic stem cell (ESC) derivatives offer promise for generating clinically useful tissues for transplantation, yet the specter of producing tumors in patients remains a significant concern. We have developed a simple method that eliminates the tumorigenic potential from differentiated ESC cultures of murine and human origin while purifying lineage-restricted, definitive endoderm-committed cells. A three-stage scheme utilizing magnetic bead sorting and specific antibodies to remove undifferentiated ESCs and extraembryonic endoderm cells, followed by positive selection of definitive endoderm cells on the basis of epithelial cell adhesion molecule (EpCAM) expression, was used to isolate a population of EpCAM(+)SSEA1(-)SSEA3(-) cells. Sorted cells do not form teratomas after transplantation into immunodeficient mice, but display gene and protein expression profiles indicative of definitive endoderm cells. Sorted cells could be subsequently expanded in vitro and further differentiated to express key pancreas specification proteins. In vivo transplantation of sorted cells resulted in small, benign tissues that uniformly express PDX1. These studies describe a straightforward method without genetic manipulation that eliminates the risk of teratoma formation from ESC differentiated derivatives. Significantly, enriched populations isolated by this method appear to be lineage-restricted definitive endoderm cells with limited proliferation capacity.
University of Wisconsin–Madison School of Medicine and Public Health and Wisconsin Institute of Medical Research, Department of Surgery, Division of Transplantation, 600 Highland Avenue, Madison, WI 53792, USA.
Brenda
B
Kahan
Joseph
J
Magliocca
Fabiola
F
Merriam
Nathan
N
Treff
Melisa
M
Budde
Jeffrey
J
Nelson
Victoria
V
Browning
Benjamin
B
Ziehr
Jon
J
Odorico
D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000378:metabolism; D002454:Cell Differentiation; D019070:Cell Lineage; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D004707:Endoderm / Q000166:cytology*; D041981:Gastrointestinal Tract / Q000166:cytology* / Q000196:embryology*; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D016256:Lewis X Antigen / Q000378:metabolism; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D055746:Stage-Specific Embryonic Antigens / Q000378:metabolism; D013724:Teratoma / Q000378:metabolism / Q000473:pathology; D015534:Trans-Activators / Q000378:metabolism*
D015295:Antigens, Tumor-Associated, Carbohydrate; D018398:Homeodomain Proteins; D016256:Lewis X Antigen; D055746:Stage-Specific Embryonic Antigens; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; C052924:stage-specific embryonic antigen-3