FGF-signalling in the differentiation of mouse ES cells towards ...

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The pluripotent stateThe pluripotency of ES cells along with their capability to self-renew indefinitely are the mostimportant characteristics of ES cells. The pluripotent state is characterised morphologically bytightly associated cells growing in rounded clusters. Molecularly, they are characterised by theexpression of markers, some of which are found also in the ICM of the developing embryo. Themost commonly used are Oct4, Nanog, Stage specific embryonic antigen-1 (SSEA-1), Sox2 andAlkaline phosphatase (Solter and Knowles 1978; Pease et al. 1990; Nichols et al. 1998; Avilion etal. 2003; Chambers et al. 2003; Mitsui et al. 2003). To test if ES cells have maintained theirpluripotency over time when in culture, they are evaluated in several ways, each providing a morestringent test but at the same time taking more resources. They can be evaluated by i)morphology; ii) a positive stain with antibodies for the pluripotent markers; iii) subcutaneousinjection in mice and formation of teratomes with cells of all three germ layers; iv) injection intothe ICM of a developing embryo where they give rise to chimaeras with contributions to tissues ofall three germ layers and the germ line (Ohtsuka and Dalton 2008). The last test is considered the‘golden standard’ but is time-consuming and is therefore not routinely carried out except in theestablishment and analysis of newly generated transgenic cell lines or by investigating whether acertain (manipulated) ES cell line is entirely pluripotent.Maintaining the pluripotent state in an ES cell culture is done through prevention ofdifferentiation (or induction of self-renewal properties) and promotion of proliferation. LIF isadded to the culture medium of pluripotent ES cells and is the main factor involved in keepingcells pluripotent (Figure 1-4A). It activates the JAK/STAT3-signalling pathway, resulting intranscription of genes involved in self-renewal, one of these being C-myc (Niwa et al. 1998;Cartwright et al. 2005). LIF also activates the important Phosphoinositol 3 kinase (PI3K), leadingto activation of Ras/ mitogen-activated protein kinase (MAPK) and AKT pathways. The latter isinvolved in relief of C-myc repression by glycogen synthase kinase-3 (GSK-3), which seems to bevery important in maintaining self-renewal capability (Umehara et al. 2007). BMP is a secondimportant factor for maintaining self-renewal, its effect possibly depending on the cultureconditions in which it acts (Ohtsuka and Dalton 2008). BMP activates SMAD1/5/8-signallingleading to expression of Inhibitor of differentiation (Id)-genes which block at least neuraldifferentiation (Figure 1-4A; (Ying et al. 2003a)). BMPs also act by suppressing the p38 MAPK,which would otherwise promote differentiation (Qi et al. 2004; Kunath et al. 2007). FGF4 isexpressed in pluripotent ES cells in an autocrine fashion and the activation of the ERK1/2-signalling cascade by FGF must be suppressed in order to maintain cells in the plupipotent state(Ma et al. 1992; Kunath et al. 2007; Nichols et al. 2009). The LIF-gp130 receptor complex sees tothis.12
Figure 1-4: Key signalling pathways required for maintaining pluripotency and for directed differentiationinto the three germ layers. A) LIF signalling activates JAK–STAT3 and PI3K pathways to induce target genesessential for pluripotency. BMP signals activate SMAD1/5/8-Id gene and suppress p38 MAPK. Activin/ nodalhave been shown to contribute mESCs proliferation but not pluripotency. B) Illustration of signallingdemonstrated induce to ES cell self-renewal and germ layer specification, through the EPL-state. BMP, bonemorphogenetic protein; EPL, early-primitive ectoderm-like; ESCs, embryonic stem cells; FGF, fibroblast growthfactor; ICM, inner cell mass; LIF, leukemia inhibitory factor. Modified from (Ohtsuka and Dalton 2008; Gadueet al. 2005).Recent studies have suggested that epigenetic processes are required for repression ofdevelopmental pathways through the actions of e.g. polycomb-group complex proteins. Howimportant epigenetic control and regulation of the pluripotent state is compared to the addition ofgrowth factors and cytokines is yet to be determined (Niwa 2007).Growing ES cells as a pluripotent culture can be done on a feeder-layer of e.g. mouse embryonicfibroblasts in the presence of serum and LIF. Here, serum contains BMP and feeder cells may besubstituted entirely by LIF, which they contribute to the culture condition (Smith et al. 1988; Yingand Smith 2003). Alternatively, pluripotent ES cells can be maintained in feeder-free serumreplacement media containing LIF, where N2, B27 and BMP4 are added to replace serum ingeneral, and BMP in particular (Ying et al. 2003a).Directed differentiation of ES cellsRemoval of LIF (and BMP4) initiates differentiation of ES cells by increasing ERK activity.FGF5 is up-regulated and pluripotency markers are down-regulated along with PI3K and AKTsignallingpathways (Rathjen et al. 1999; Ohtsuka and Dalton 2008). Intact FGF4-signalling hasbeen shown to be necessary for initiation of differentiation for at least ectoderm and, lessconvincingly, mesoderm lineages (Kunath et al. 2007). As little as a 24-hour pulse of ectopic13
Page 1: PhD thesisCand.scient. Janny Marie
Page 5: ResuméSukkersyge er en sygdom der
Page 9: Table of contents1
Page 12 and 13: ICMinner cell massIdInhibitor of di
Page 14 and 15: cell mass regenerates probably thro
Page 16 and 17: Figure 1-1: Early embryo developmen
Page 18 and 19: Figure 1-3: Regional expression of
Page 25 and 26: There are four membrane-bound FGFRs
Page 27: 2. AimsThe aim of this study was to
Page 30 and 31: Developmental Biology 330 (2009) 28
Page 32 and 33: 288 M. Hansson et al. / Development
Page 50 and 51: Figure S2Figure S2: A subpopulation
Page 52 and 53: Figure S4Figure S4: Expression of T
Page 54 and 55: Figure S6Figure S6: qRT-PCR analyse
Page 56 and 57: epithelium; Cdx2, expressed posteri
Page 58 and 59: Figure 4-4: A high FGF4-concentrati
Page 60 and 61: Figure 4-6: A 3-step protocol does
Page 63 and 64: 5. Paper IIFGFR(IIIc)-activation in
Page 65 and 66: AbstractProgress in embryonic stem
Page 67 and 68: variants in their Ig-like domain II
Page 69 and 70: Cell count and proliferation assayC
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influence on the numbers of Sox17-G
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undifferentiated cells, we found th
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FGF4, 5, FGF8b and FGFR1, are expre
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with EdU-stain (blue sample); and w
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Olsen, S.K., J.Y. Li, C. Bromleigh,
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FiguresFigure 1: Screen for FGFR-is
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Figure 3: Activation of FGFRb or FG
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Opposite, Figure 6: In the absence
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6. General discussionEndoderm diffe
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Overall, the multitude of FGF-signa
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transplantation is the spread of an
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AcknowledgementsThe work presented
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Chambers, I., D. Colby, M. Robertso
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Hawkins, V.J. Wroblewski, D.S. Li,
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Nishikawa, S.I., S. Nishikawa, M. H
Page 105 and 106:
Tanimizu, N., H. Saito, K. Mostov,
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