title: Megakaryocytic differentiation of mouse embryonic stem cells via coculture with immortalized OP9 stromal cells.
abstract: Established from the calvaria of newborn macrophage colony-stimulating factor (M-CSF)-deficient mice, OP9 is a stromal cell line that used as a feeder layer to support the in vitro differentiation of pluripotent stem cells into various hematopoietic lineage cells, including granulocytes, erythrocytes, lymphocytes, and megakaryocytes. However, as a primary culture cell line, OP9 can be used as stromal cells for only 1 month. Therefore, to obtain functional OP9 cells, numerous M-CSF-deficient newborn mice must be sacrificed. These limitations in some ways restrict the application of OP9 cells in longterm and largescale experiments. In this study, we used human papillomavirus 16 E6 and E7 genes to generate immortalized OP9 stromal cells, designated I-OP9 cells, and then tested their ability to support the megakaryocytic differentiation of pluripotent stem cells in vitro. I-OP9 cells have similar morphology and properties as do parental OP9 cells, and, as expected, have an extended lifespan and can support megakaryocytic differentiation. Our data suggest that the method used in this study, including establishing I-OP9 cells, enables the possibility to enlarge and lengthen the scale of the experiment and, more critically, provides a humanistic approach for preparing stromal cells that support the hematopoietic differentiation of pluripotent stem cells in vitro.
authors.affiliation: Institute of Medical Sciences, Tzu-Chi University, Hualien, Taiwan.
authors.forename: Po-Kong
authors.initials: PK
authors.lastname: Chen
authors.affiliation: Institute of Medical Sciences, Tzu-Chi University, Hualien, Taiwan.
authors.forename: Guan-Ling
authors.initials: GL
authors.lastname: Lin
authors.affiliation: Institute of Medical Sciences, Tzu-Chi University, Hualien, Taiwan; Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien, Taiwan.
authors.forename: Hsin-Hou
authors.initials: HH
authors.lastname: Chang
authors.affiliation: Institute of Medical Sciences, Tzu-Chi University, Hualien, Taiwan; Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien, Taiwan. Electronic address: dssun@mail.tcu.edu.tw.
authors.forename: Der-Shan
authors.initials: DS
authors.lastname: Sun
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002471:Cell Transformation, Neoplastic / Q000378:metabolism / Q000473:pathology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D004622:Embryo, Mammalian / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006410:Hematopoiesis; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000502:physiology; D008297:Male; D008533:Megakaryocytes / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D016688:Mice, Inbred NOD; D018345:Mice, Knockout; D016513:Mice, SCID; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D016173:Macrophage Colony-Stimulating Factor
keywords: Immortalization; Megakaryocytic differentiation; OP9 stromal cells