139x Filetype PPT File size 0.55 MB Source: wd.vghtpe.gov.tw
ABSTRACT • Embryonic stem (ES) cells are typically derived from the inner cell mass of the preimplantation blastocyst and can both self-renew and differentiate into all the cells and tissues of the embryo. Because they are pluripotent, ES cells have been used extensively to analyze gene function in development via gene targeting. The embryonic stem cell is also an unsurpassed starting material to begin to understand a critical, largely inaccessible period of development. If their differentiation could be controlled, they would also be an important source of cells for transplantation to replace cells lost through disease or injury or to replace missing hormones or genes. Traditionally, ES cells have been differentiated in suspension culture as embryoid bodies, named because of their similarity to the early postimplantation-staged embryo. Unlike the pristine organization of the early embryo, differentiation in embryoid bodies appears to be largely unpatterned, although multiple cell types form. It has recently been possible to separate the desired cell types from differentiating ES cells in embryoid bodies by using cell-type-restricted promoters driving expression of either antibiotic resistance genes or fluorophores such as EGFP. In combination with growth factor exposure, highly differentiated cell types have successfully been derived from ES cells. Recent technological advances such as RNA interference to knock down gene expression in ES cells are also producing enriched populations of cells and elucidating gene function in early development. FIG. 1. Development of the early mouse embryo from the E2.5 morula to the E6.5 gastrula... Primitive endoderm Epiblast O'Shea, K. S. Biol Reprod 2004;71:1755-1765 Biology of Reproduction Copyright ©2004 Society for the Study of Reproduction STEM CELL CHARACTERISTICS • The basic characteristics of a stem cell population: pluripotency, self-renewal. • transcription factors, maintain self-renewal and to inhibit differentiation, • STAT3, by the binding of leukemia inhibitory factor (LIF) to gp130 receptors. • Gene targeting experiments have demonstrated that neither STAT3, LIF, nor gp130 is required to maintain the inner cell mass, although Stat3 –/– embryos die around E6.5, and gp130 –/– embryos die after E12.5. • the essential POU homeodomain protein OCT4, the variant homeodomain containing protein NANOG, the SRY family member Sox2, Foxd3 (previously Genesis) a member of the forkhead winged-helix family, and possibly Wnt signaling. Oct-4 • The Oct4 promoter contains a retinoic acid (RA) -responsive repressor element, such that exposure of ES cells to the morphogen/teratogen retinoic acid inhibits Oct4 expression and allows expression of lineage-specific transcription factors present in ES cells. The Oct4 promoter also contains a proximal element that drives Oct4 expression in the epiblast that is downregulated at gastrulation, and a germ cell-specific distal enhancer that is not active in the epiblast. Germ cell nuclear factor, an orphan nuclear receptor, binds to the distal enhancer to restrict expression to the germ cell lineage. By using this distal promoter to drive expression of EGFP to ES cells, then selecting those cells, it has recently been possible to differentiate oocytes from mouse ES cells in vitro [Hubner er al., 2003]. • OCT4 interacts with a number of other factors, including the SRY-related HMG box containing transcription factor SOX2, E1A protein, the transcriptional coactivator Utf1, and the Forkhead box protein FOXD3. • Several downstream targets of Oct4 have been identified, including the extracellular matrix protein osteopontin, which may promote the lateral migration of primitive endoderm to line the trophoblast as well as initiate expression of endoderm lineage- specific gene expression. Other targets include Hand1 and Fgf4, which are expressed in early trophectoderm; Fbx15, which is expressed in ES cells and later in testis; and Zfp42, also known as Rex1. NANOG • The recently identified pluripotency factor Nanog is a member of the homeobox family of DNA-binding transcription factors. Two human genes and a single mouse gene with three alternative splice variants have now been identified. Nanog is first expressed in the compacted morula, in the ICM, then in the proximal epiblast at the location of the future primitive streak. Nanog is downregulated at gastrulation in mesoderm and endoderm, remaining in the epiblast to E8. Nanog is present in ES cells, primordial germ, and EG cells and can be detected by reverse transcription-PCR in adult tissues as well. • Interestingly, NANOG, STAT3, and OCT4 appear to affect both overlapping and independent events. • However, Nanog is expressed in Oct4 –/– embryos, suggesting that they act in parallel pathways. • Nanog null embryos and ES cells form extraembryonic endoderm at the expense of epiblast. Thus, like Oct4, Nanog appears to regulate both self-renewal and inhibit differentiation. • Nanog appears to be critical at slightly later stages of development because deletion does not affect early stages of trophoblast differentiation (as is the case for Oct4) and it is expressed in the epiblast until E8, remaining in some adult tissues. Like Oct4, it appears that Nanog may regulate differentiation by transcriptional repression of genes that promote differentiation. In the case of Nanog, the enhancers of both Gata6 and Rex1/Zfp42 contain NANOG binding sites. Interestingly, NANOG contains an amino terminal region of homology to SMAD4, suggesting that NANOG in the epiblast may play a role in controlling TGFß signaling.
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