title: A molecular signature for purified definitive endoderm guides differentiation and isolation of endoderm from mouse and human embryonic stem cells.
abstract: Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as liver, pancreas, and intestine. However, purification of DE in mammals has not been achieved, limiting the molecular "definition" of endoderm, and hindering our understanding of DE development and attempts to produce endoderm from sources such as embryonic stem (ES) cells. Here, we describe purification of mouse DE using fluorescence-activated cell sorting (FACS) and mice harboring a transgene encoding enhanced green fluorescent protein (eGFP) inserted into the Sox17 locus, which is expressed in the embryonic endoderm. Comparison of patterns of signaling pathway activation in native mouse DE and endoderm-like cells generated from ES cells produced novel culture modifications that generated Sox17-eGFP⁺ progeny whose gene expression resembled DE more closely than achieved with standard methods. These studies also produced new FACS methods for purifying DE from nontransgenic mice and mouse ES cell cultures. Parallel studies of a new human SOX17-eGFP ES cell line allowed analysis of endoderm differentiation in vitro, leading to culture modifications that enhanced expression of an endoderm-like signature. This work should accelerate our understanding of mechanisms regulating DE development in mice and humans, and guide further use of ES cells for tissue replacement.
authors.affiliation: Department of Developmental Biology, Stanford University School of Medicine, Stanford, Califorina, USA.
authors.forename: Pei
authors.initials: P
authors.lastname: Wang
authors.forename: Kristen D
authors.initials: KD
authors.lastname: McKnight
authors.forename: David J
authors.initials: DJ
authors.lastname: Wong
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Rodriguez
authors.forename: Takuya
authors.initials: T
authors.lastname: Sugiyama
authors.forename: Xueying
authors.initials: X
authors.lastname: Gu
authors.forename: Amar
authors.initials: A
authors.lastname: Ghodasara
authors.forename: Kun
authors.initials: K
authors.lastname: Qu
authors.forename: Howard Y
authors.initials: HY
authors.lastname: Chang
authors.forename: Seung K
authors.initials: SK
authors.lastname: Kim
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D019485:Bone Morphogenetic Proteins / Q000502:physiology; D002454:Cell Differentiation; D002469:Cell Separation; D002478:Cells, Cultured; D016000:Cluster Analysis; D018920:Coculture Techniques; D004622:Embryo, Mammalian / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D004707:Endoderm / Q000166:cytology* / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000502:physiology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D020411:Oligonucleotide Array Sequence Analysis; D055760:SOXF Transcription Factors / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D059467:Transcriptome; D014212:Tretinoin / Q000502:physiology
chemical_list: D000943:Antigens, Differentiation; D019485:Bone Morphogenetic Proteins; D055760:SOXF Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014212:Tretinoin; D005346:Fibroblast Growth Factors