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

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involved in WNT-signalling and maintenance of the pluripotent state (Chang et al. 2010). Thus,here it seems that a high cell density may inhibit differentiation, which has also been suggestedby Smith and co-workers (Smith et al. 1992). Similarly, we propose inhibition by high celldensities in the FGF4 –/– ES cell culture, and suggest low seeding densities for optimaldifferentiation.DE patterningIt seems rather symptomatic that we successfully reach 40-60% SOX17 + DE cells, but havelimited success in further patterning this DE to PDX1-expressing posterior foregut. We saw aninduction of PDX1-expressing cells when adding RA and FGF (and Cyclopamine), but nevermore than 2-4% on average. Competing groups have successfully obtained 32% PDX1 + cells inhES cell cultures by using some of the same posterior foregut-inducing factors, basing theirscientific approach on the same hypotheses as we do (Johannesson et al. 2009; Ameri et al.2010). Therefore the reason for our inefficient Pdx1-GFP induction shall possibly be soughtelsewhere.The Pdx1-GFP cell line we use has shown a nice expression pattern within the posterior foregutregion when injected into mouse blastocysts to form chimaeras (Tino Klein, unpublished data).The cell line works in vivo and is pluripotent, and we therefore expect it to also work in vitro.One point could be that we have optimized the DE-induction step in the Sox17-GFP and not thePdx1-GFP cell line. Data from hES cells indicate that the outcome of a differentiation protocolvaries much between cell lines (D'Amour et al. 2006; Mfopou et al. 2010). If this is also thecase for mES cells, we will have to redo optimization of DE-induction in our Pdx1-GFP cellline to have the best starting material. However, our protocol induces 2-4% Pdx1 + cells in E14,Pdx1-LacZ and Pdx1-GFP cell lines, showing robustness of the protocol.A developmental-based explanation is that the DE we have is simply not the ‘correct’ one.Using our protocol, we get many SOX2 + cells, suggesting that the DE we have after 5 days inhigh concentrations of activin may be somehow pre-patterned to respond to patterning factorspredominantly by induction of anterior foregut, marked by SOX2.Finally, there could be one or more components in our basic medium or medium supplementsthat inhibit differentiation. This problem could be overcome by changing the basic medium,medium supplements and the culture dish coating individually to decipher which may beinhibitory.Cell replacement therapy as a future cure for TIDMES cells are not the only source of β cells or β-like cells envisioned as material for futuretransplantation in the treatment or even cure for diabetes. Some perspectives of the variousalternatives are discussed below.Xeno-transplantationXeno-transplantation of islets of Langerhans from pig to primate is being investigated as atreatment for type I diabetes. The use of pigs is promising, as their vascular physiology issimilar to that of humans and they are relatively cheap to breed. Furthermore, the so-calledmini-pigs weighing app. 120 kg are similar to humans in organ and body sizes and can beinbred to homozygosity at e.g. the porcine major histocompatibility complex (MCH). The latterholds a great potential for manipulation to create customized donor organs/ cells and overcomesome of the problems of immune-responses normally seen in transplantation. This could bedone by introducing porcine MHC genes into the bone marrow of the recipient human inducingmixed chimaerism thereby introducing immunological tolerance to the xenograft (Hoerbelt andMadsen 2004). Alternatively, pigs could be genetically manipulated not to express geneproducts to which the recipient immune system reacts. Of major concern in xeno-86
transplantation is the spread of animal diseases to humans. Some of the risk factors may beeliminated by breeding homozygous mini-pigs in controlled environments.EpiSCs, hESCs and iPSCsIt has recently been shown that epiblast stem cells (epiSCs) derived from post-implantationmouse blastocysts show characteristics of hES cells in their need for pluripotency-maintainingfactors activin and FGF2 (Brons et al. 2007; Tesar et al. 2007; Vallier et al. 2009). Also, theirresponse to differentiation-inducing factors is more similar to hES cells than what is seen formES cells (Vallier et al. 2009). This close resemblance to hES cells may make epiSCs a bettermodel for studying differentiation, as extrapolation of knowledge to the hES cell field mayprove easier and more valuable. One major advantage is that existing ES cell lines can beconverted into epiSCs without new derivation from mouse embryos (Guo et al. 2009), makingalready established transgenic cell lines readily transferable by a low work load. This may holdgreat potential for better inter-species protocol transfer between epiSCs and hES cells.In 2006, the Yamanaka-group showed that mature somatic cells, i.e. skin fibroblasts, can beinduced to achieve an ES cell-like phenotype, i.e. become pluripotent and are reported tobehave in the same way as mES or hES cells upon differentiation. These induced pluripotentstem (iPS) cells were generated by introduction of four transcription factors Oct4, Sox2, Klf4,and C-myc (Takahashi and Yamanaka 2006). This was done in mice, and the protocol has sincebeen modified in several ways and has been transferred to human cells (Takahashi et al. 2007;Yamanaka 2009). iPS cells hold the potential for development of patient-specific pluripotentstem cell lines, which can be differentiated into any cell type of choice. They thereforerepresent a source of transplantable cells, which eliminates the need for immune-suppressingagents to a large degree. Although this is a very positive future application, in reality it mayprove much too expensive for actual treatment. iPS cells will likely be important tools formodelling of and investigating the aetiology of (inherited) human diseases, which are notdiscovered until the disease state is complete. For instance, type I diabetes is normally notdiscovered until patients suffer from high blood glucose levels at which time point their β cellmass is practically obsolete (Maehr et al. 2009). Whether iPS cells will serve as material for cellreplacement-therapies is still to be seen. Of major concern is to ensure that the genomicreprogramming of the cells is complete, a trait believed necessary for the cells to adopt thecorrect fate upon exposure to differentiation-inducing conditions (Yamanaka 2009). Also,teratoma formation from fully reprogrammed iPS cells cannot be avoided so far, making themunsuited for treatment in humans at this point. In general, avoiding teratoma-formation fromdifferentiated cell populations is a major concern in transplantation. It is not acceptable to curefor instance diabetes but at the same time induce a cancerous condition, and as long as this riskexists with cell therapy-protocols, they will not be approved for treatment.Generation of β cells from existing cell sources in the pancreasThe presence of a pancreatic stem cell, which has clonogenic potential, is multipotent and canbe induced to generate insulin-producing cells in vitro has been suggested in both mice andhumans (Ramiya et al. 2000; Seaberg et al. 2004; Zhao et al. 2007). However, it is speculatedthat these cells only show such stem cell-like properties due to the in vitro culture conditions(Baeyens and Bouwens 2008). A more convincing in vivo study showed the presence of isletprecursors that could be activated upon serious tissue injury by the so-called partial ductligation,in which facultative multipotent progenitor cells in the ductal lining differentiate andproliferate into functional β cells (Xu et al. 2008).An alternative approach is to generate β cells by reprogramming of existing endocrine orexocrine cells in the pancreas. Following pancreatectomy to a mild or severe degree (70% and95% respectively), regeneration of β cell mass is achieved through either replication of existingβ cells or through neogenesis of precursor cells in addition to replication (Dor et al. 2004;Bouwens and Rooman 2005). Exocrine ductal cells show convincing potential as they seemable to contribute to glucose-responsive β cells through reprogramming (Baeyens and Bouwens87
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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 42 and 43: 298 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 and 88: Opposite, Figure 6: In the absence
Page 89 and 90: 6. General discussionEndoderm diffe
Page 91: Overall, the multitude of FGF-signa
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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