title: Induction and selection of Sox17-expressing endoderm cells generated from murine embryonic stem cells.
abstract: Embryonic stem (ES) cells offer a valuable source for generating insulin-producing cells. However, current differentiation protocols often result in heterogeneous cell populations of various developmental stages. Here we show the activin A-induced differentiation of mouse ES cells carrying a homologous dsRed-IRES-puromycin knock-in within the Sox17 locus into the endoderm lineage. Sox17-expressing cells were selected by fluorescence-assisted cell sorting (FACS) and characterized at the transcript and protein level. Treatment of ES cells with high concentrations of activin A for 10 days resulted in up to 19% Sox17-positive cells selected by FACS. Isolated Sox17-positive cells were characterized by defini- tive endoderm-specific Sox17/Cxcr4/Foxa2 transcripts, but lacked pluripotency-associated Oct4 mRNA and protein. The Sox17-expressing cells showed downregulation of extraembryonic endoderm (Sox7, Afp, Sdf1)-, mesoderm (Foxf1, Meox1)- and ectoderm (Pax6, NeuroD6)-specific transcripts. The presence of Hnf4α, Hes1 and Pdx1 mRNA demonstrated the expression of primitive gut/foregut cell-specific markers. Ngn3, Nkx6.1 and Nkx2.2 transcripts in Sox17-positive cells were determined as properties of pancreatic endocrine progenitors. Immunocytochemistry of activin A-induced Sox17-positive embryoid bodies revealed coexpression of Cxcr4 and Foxa2. Moreover, the histochemical demonstration of E-cadherin-, Cxcr4-, Sox9-, Hnf1β- and Ngn3-positive epithelial-like structures underlined the potential of Sox17-positive cells to further differentiate into the pancreatic lineage. By reducing the heterogeneity of the ES cell progeny, Sox17-expressing cells are a suitable model to evaluate the effects of growth and differentiation factors and of culture conditions to delineate the differentiation process for the generation of pancreatic cells in vitro.
authors: Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben, Germany.
authors: Insa S
authors: IS
authors: Schroeder
authors: Sabine
authors: S
authors: Sulzbacher
authors: Tobias
authors: T
authors: Nolden
authors: Joerg
authors: J
authors: Fuchs
authors: Judith
authors: J
authors: Czarnota
authors: Ronny
authors: R
authors: Meisterfeld
authors: Heinz
authors: H
authors: Himmelbauer
authors: Anna M
authors: AM
authors: Wobus
mesh_terms: D028341:Activins / Q000494:pharmacology; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D019070:Cell Lineage / Q000187:drug effects / Q000235:genetics; D002469:Cell Separation / Q000379:methods*; D058732:Embryoid Bodies / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D004707:Endoderm / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D004848:Epithelium / Q000187:drug effects / Q000196:embryology / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D024002:HMGB Proteins / Q000378:metabolism*; D000090322:Homeobox Protein Nkx-2.2; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D055760:SOXF Transcription Factors / Q000378:metabolism*; D013997:Time Factors
chemical_list: D015415:Biomarkers; D024002:HMGB Proteins; D000090322:Homeobox Protein Nkx-2.2; D008164:Luminescent Proteins; C000715627:Nkx2-2 protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D012333:RNA, Messenger; D055760:SOXF Transcription Factors; C511606:Sox17 protein, mouse; C422405:activin A; C413663:fluorescent protein 583; D028341:Activins