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

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ICMinner cell massIdInhibitor of differentiationiPSinduced pluripotent stemJAKJanus kinaseJDRF Juvenile diabetes research foundationLIFleukemia inhibitory factorMAPK Mitogen-activated protein kinasem-Cer1 mouse-Cerberus 1MEK a MAP kinasemES cell s mouse embryonic stem cellsMHC major histocompatability complexMixl1 Mix1 homeobox-likeNgn3 Neurogenin 3Nkx6.1 NK homeobox transcription factor 6.1Oct4Octamer-4Otx2 Orthodenticle homeobox 2PCRpolymerase chain reactionPDGFR platelet-derived growth factor receptorPdx1 Pancreatic and duodenal homeobox factor 1PEparietal endodermPLCγ phospholipase CγPPpancreatic polypeptide-producingPI3Kphosphoinosityl 3 kinasePtf1aPancreas-specific transcription factor 1aqPCR quantitative polymerase chain reactionRAretinoic acidRT-PCR reverse transcriptase- polymerase chain reactionS.D.standard deviationS.E.M. standard error of the meanShhSonic hedgehogSMAD proteins modulating the activity of TGFβ ligands; the name is a combination ofthe protein homologs ‘SMA’ (C. elegans) and ‘mosthers againtsdecapentaplegic’ (D. melanogaster)SoxSex determining region Y (SRY)-related HMG boxSPRED Sprouty-related EVH1 proteinSTAT3 signal transducer and activator of transcription 3TBrachyuryTBPTATA-binding proteinTdhThermostable direct hemolysin geneTEtrophectodermTGFβ Transforming growth factor βVEvisceral endodermWHO World Health OrganizationWNT3(a) wingless-type MMTV integration site 3(a)4
1. General introductionDiabetes mellitusDiabetes mellitus (diabetes hereafter) is caused by a lack of insulin-production or insulinresponsiveness,resulting in high blood glucose levels in the patient. There are two types ofdiabetes, type I and type II, resulting in an absolute or a relative lack of β cells, respectively(Donath and Halban 2004). Type II is the most common form of diabetes, accounting for 90 –95% of all cases. The World Health Organization (WHO) estimated a worldwide prevalence of171 million diabetics in 2000 and the prognosis is 336 million people by the year 2030, calling it apandemic. In Denmark alone, 226.000 people (or 5% of the total population) had diabetes in 2006and it is estimated that the Danish health care system spends DKK 22 billion (or app. US$ 4billion) per year on treatment of diabetes and diabetes-related illness (Juvenile Diabetes ResearchFoundation homage). Furthermore, it is estimated that the disease costs Denmark an extra DKK 9billion per year due to loss-of-production.In type I patients, the disease is a result of an auto-immune attack on the insulin-producing β cells,resulting in the loss of β cell mass, followed by dependence on insulin-treatment for the patient(see (Lernmark and Falorni 1998; Madsen 2005) for reviews). This dependency is life-long, as theβ cell mass does not regenerate to levels where it can sustain the body’s need for insulin. Theonset of type I diabetes is due to genetic and/ or environmental factors, but a comprehensiveknowledge of the aetiology of the disease is still lacking despite intense studies thereof. Alongwith the primary disease which is treated with insulin injections, patients develop severesecondary complications such as blindness, kidney failure, and amputations due to chronicvascular defects caused by their blood-glucose levels being irregular and difficult to stabilize.In type II patients, a gradual insulin resistance of the peripheral tissues leads to an increase in βcell mass as a compensation for this condition (Rhodes 2005). This condition is partly reversible,but will ultimately lead to type I diabetes and insulin-dependence if not treated. Type II diabetes ismainly caused by genetic predisposition and environmental factors such as obesity, physicalinactivity, excessive calorie intake and aging (Ling and Groop 2009).Cell replacement therapy in diabetesAlthough there is no cure for diabetes at present, research focusing on therapeutic treatment isenvisioned as a palliative treatment or maybe even a cure for the disease. This section will focuson therapies directed against type I diabetes.In 2000, Shapiro and co-workers published the Edmonton protocol, as it is now referred to,providing proof of principle for curing diabetes by transplanting donor islets of Langerhans andre-establishing eu-glycaemia in seven patients suffering from type I (Shapiro et al. 2000).However, 85% of islet recipients needed insulin treatment after a 5-year period (Ryan et al. 2005).The obstacles of auto-immunity along with the scarcity of islet donor material, has directed focustowards other sources of β cell material to put to use in a similar treatment.In type I diabetics, there is evidence of a continuous β cell regeneration taking place (Meier et al.2005). However, attempts to regenerate the β cell mass are most likely overruled by the autoimmuneattack, the gross result being no insulin-production from the islets of Langerhans. Thereis evidence that β-cells can be generated from existing cells, but it is unclear whether this isthrough replication of existing β cells or by re-differentiation of other pancreatic cells such as ductcells or even from hepatic cells (Bouwens and Pipeleers 1998; Yang et al. 2002a; Dor et al. 2004;Hardikar 2004; Xu et al. 2008; Borowiak and Melton 2009). It is debated whether the pancreashosts a pancreatic endocrine stem cell that can be stimulated to proliferate (Madsen 2005). Bypartial pancreatectomy or cellophane wrapping of the pancreas, it has been demonstrated that β5
Page 1: PhD thesisCand.scient. Janny Marie
Page 5: ResuméSukkersyge er en sygdom der
Page 9: Table of contents1
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 20 and 21: The pluripotent stateThe pluripoten
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
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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
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Cell count and proliferation assayC
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influence on the numbers of Sox17-G
Page 73 and 74:
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,
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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