title: Tuberin and PRAS40 are anti-apoptotic gatekeepers during early human amniotic fluid stem-cell differentiation.
abstract: Embryoid bodies (EBs) are three-dimensional multicellular aggregates allowing the in vitro investigation of stem-cell differentiation processes mimicking early embryogenesis. Human amniotic fluid stem (AFS) cells harbor high proliferation potential, do not raise the ethical issues of embryonic stem cells, have a lower risk for tumor development, do not need exogenic induction of pluripotency and are chromosomal stable. Starting from a single human AFS cell, EBs can be formed accompanied by the differentiation into cells of all three embryonic germ layers. Here, we report that siRNA-mediated knockdown of the endogenous tuberous sclerosis complex-2 (TSC2) gene product tuberin or of proline-rich Akt substrate of 40 kDa (PRAS40), the two major negative regulators of mammalian target of rapamycin (mTOR), leads to massive apoptotic cell death during EB development of human AFS cells without affecting the endodermal, mesodermal and ectodermal cell differentiation spectrum. Co-knockdown of endogenous mTOR demonstrated these effects to be mTOR-dependent. Our findings prove this enzyme cascade to be an essential anti-apoptotic gatekeeper of stem-cell differentiation during EB formation. These data allow new insights into the regulation of early stem-cell maintenance and differentiation and identify a new role of the tumor suppressor tuberin and the oncogenic protein PRAS40 with the relevance for a more detailed understanding of the pathogenesis of diseases associated with altered activities of these gene products.
authors.affiliation: Institute of Medical Genetics, Medical University of Vienna, Währinger Strasse 10, Vienna 1090, Austria.
authors.forename: Christiane
authors.initials: C
authors.lastname: Fuchs
authors.forename: Margit
authors.initials: M
authors.lastname: Rosner
authors.forename: Helmut
authors.initials: H
authors.lastname: Dolznig
authors.forename: Mario
authors.initials: M
authors.lastname: Mikula
authors.forename: Nina
authors.initials: N
authors.lastname: Kramer
authors.forename: Markus
authors.initials: M
authors.lastname: Hengstschläger
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000653:Amniotic Fluid / Q000166:cytology*; D017209:Apoptosis; D002454:Cell Differentiation; D002460:Cell Line; D045744:Cell Line, Tumor; D015536:Down-Regulation; D058732:Embryoid Bodies / Q000166:cytology / Q000502:physiology; D005855:Germ Layers / Q000166:cytology; D006801:Humans; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000502:physiology*; D034741:RNA, Small Interfering; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C484523:AKT1S1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D034741:RNA, Small Interfering; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases