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

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Figure S2: A few areas of SOX7 + cells in the FGF4 –/– cell line after DE-induction. Cells were differentiated for 5days in 30 ng/ml activin w/wo 5 ng/ml FGF4 and stained for SOX17, E-cadherin, SOX7 and the nuclear stainDAPI. The yellow frame indicates area blown up and shown to the right (red signal boosted). Representativeimages are shown for each condition, except for FGF4 –/– cells in activin alone which shows a SOX7 + area in theculture. White scale bar: 100 µm; yellow scale bar: 50 µm.82
6. General discussionEndoderm differentiationBasic culture conditionsOur culture system is based on serum and feeder-free conditions by means of gelatine-coatedculture flasks and the N2, B27, BMP4 and LIF supplements. Remnants of serum and feedershave been removed by growing cells for 3 passages, i.e. 6 days, under these conditions prior toexperimental setup. In the presence of LIF and BMP4 cells do not differentiate, but it is hard todetermine the effect of trace amounts of BMP4 on the very early differentiation in the culture.However, such differentiation would most likely be opposed by trace amounts of LIF andtherefore not have any practical effect especially when cells are differentiated using highconcentrations of growth factors.A recent study showed that inhibition of FGFRs, MEK and GSK3 through addition of the smallmolecule inhibitors SU5402, PD184352 and Chir99021 respectively, could maintain cellspluripotent over time (Ying et al. 2008). This protocol was refined by using the stronger MEKinhibitorPD0325901 in combination with Chir99021 (Nichols et al. 2009). Thus, LIF andBMP4 are not necessary for maintenance of pluripotency, as inhibition of the MAPK-pathwayand GSK3 is sufficient. This protocol may be preferred in the future to avoid maintenance ofthe pluripotent state through addition of e.g. BMP4, a potent inducer of mesodermdifferentiation.‘Mesendoderm’: Does it exist?Nodal and BMP4, both members of the TGFβ superfamily are expressed in the PS and ExE,respectively, and act in opposite directions to induce DE and mesoderm. These two germ layersderive from the same population of epiblast cells migrating through the PS during gastrulation.Cells moving through the PS are bipotent and are collectively termed the mesendoderm beforethey are further differentiated into mesoderm and endoderm (Lawson et al. 1991; Kinder et al.2001; Rodaway and Patient 2001). This mesendoderm population seems much conserved, as itis found from Caenorhabditis elegans (C. elegans) through Xenopus laevis (Xenopus) tozebrafish (Rodaway et al. 1999; Rodaway and Patient 2001). A bipotent mesendoderm cellpopulation has also been shown in mES cell work where a pool of cells expressing T orGSC/FOXA2/PDGFRα, i.e. a mesendoderm population, had the potential to form bothmesoderm and endoderm dependent on the growth factors presented (Kubo et al. 2004; Tada etal. 2005). Indeed, we found that by using GFP-reporter cell lines for the PS markers T, Gsc andMixl1, we could analyse the effect of anteriorising and posteriorising TGFβ- and WNTsignallingin the early differentiation steps.Although the mesendoderm cell population is well-established from C. elegans to mousedevelopment, it is challenged by a recent study. By use of single-cell lineage tracing, theauthors claim that endoderm and surface ectoderm segregate during gastrulation, whereas apool of bipotent neuromesoderm persists through all stages of axis elongation (Tzouanacou etal. 2009). If these observations hold true, it has implication for ES cell differentiation towardscells of the DE lineage, as it will then be better to obtain a pool of cells expressing DE markersonly, and not a combination of DE and mesoderm markers at early stages.Other groups and we have shown derivation of DE cells from a population of cells expressingapp. 40 – 70% mesendoderm markers (Tada et al. 2005). Whether these DE cells derive from amesendoderm population or rather from an endoderm population directly, is difficult toestablish without the use of lineage tracing during differentiation. Also, any contribution ofsignals from randomly differentiated cells to the forming DE may prove important, but are asyet not studied in depth. All in all this mesendoderm population may prove not to have apractical limitation to ES cell differentiation in vitro as long as the DE is properly established.83
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PhD thesisCand.scient. Janny Marie
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ResuméSukkersyge er en sygdom der
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Table of contents1
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ICMinner cell massIdInhibitor of di
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cell mass regenerates probably thro
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Figure 1-1: Early embryo developmen
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Figure 1-3: Regional expression of
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The pluripotent stateThe pluripoten
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There are four membrane-bound FGFRs
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2. AimsThe aim of this study was to
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Developmental Biology 330 (2009) 28
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288 M. Hansson et al. / Development
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Page 38 and 39: 294 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
Page 71 and 72: influence on the numbers of Sox17-G
Page 73 and 74: undifferentiated cells, we found th
Page 75 and 76: FGF4, 5, FGF8b and FGFR1, are expre
Page 77 and 78: with EdU-stain (blue sample); and w
Page 79 and 80: Olsen, S.K., J.Y. Li, C. Bromleigh,
Page 81 and 82: FiguresFigure 1: Screen for FGFR-is
Page 83 and 84: Figure 3: Activation of FGFRb or FG
Page 87: Opposite, Figure 6: In the absence
Page 91 and 92: Overall, the multitude of FGF-signa
Page 93 and 94: transplantation is the spread of an
Page 96: AcknowledgementsThe work presented
Page 99 and 100: Chambers, I., D. Colby, M. Robertso
Page 101 and 102: Hawkins, V.J. Wroblewski, D.S. Li,
Page 103 and 104: Nishikawa, S.I., S. Nishikawa, M. H
Page 105 and 106: Tanimizu, N., H. Saito, K. Mostov,
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