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

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Figure 4-6: A 3-step protocol does not enhance the numbers of Pdx1-GFP + cells. Pdx1-GFP cells differentiatedfor 5 days in 30 ng/ml activin (Step 1), 2 days in Step 2A-media and 3 days in Step 2B-media (see schematic fordetails). Concentrations used: 0,1 µM RA; 25 ng/ml FGF10; 0,25 µM KAAD-Cyclopamine (Cyc). n = 3 ± S.E.M.is shown.Figure 4-7: Addition of the posteriorizing factor BMP4 in Step 1 does not increase Pdx1-GFP induction. Pdx1-GFP cells differentiated for 5 days in 30 ng/ml activin (Step 1) and 3 days in Step 2-media (see schematic fordetails). Concentrations used: 0,1 or 1 ng/ml BMP4; 0,1 µM RA; 25 ng/ml FGF7; 1 µM SB431542; 50 mg/mlNoggin. n = 3 ± S.E.M. is shown.Posteriorising DE-induction does not increase the posterior foregut cell populationWe decided to look into whether the DE we generated by a high concentration of activin couldbe posteriorized already in Step 1, thus leading to higher numbers of posterior foregutendoderm in Step 2. Based on DE-induction protocols used for mES or hES cells, we addedBMP4 at different concentrations in Step 1 and inhibited this signalling-pathway in Step 2(Candy H.-H. Cho, personal communication; (Morrison et al. 2008; Touboul et al. 2009)). InStep 1, we found that BMP4-concentrations of 0,1 ng/ml on days 1-2 or 1-5, or 1 ng/ml on days1-2 had no inhibitory effect on the percentage of Sox17-GFP + cells formed, whereas higherBMP4-concentrations did (Maria Winzi, unpublished data). Next, we used these concentrationsin Step 1 and added FGF7 and RA ± the BMP4-inhibitor noggin and the ALK4/5/7 inhibitorSB431542. We saw a tendency for increased numbers of Pdx1-GFP + cells when adding the lowconcentration of BMP4 for all 5 days or the high concentration for 2 days (Figure 4-7).However, the percentage of Pdx1-GFP + cells was not significantly higher than when inducing54
DE in medium containing activin only. We therefore conclude that addition of BMP4 has noposteriorising effect on our DE in this system.DiscussionOur protocol for the generation of posterior foregut from an activin-induced DE cell populationis successful as a ‘proof of principle’. We show that addition of posteriorising factors such asRA and FGF induce PDX1-expression in further differentiation of apparently naïve DE cells.The protocol is reproducible between E14, Pdx1-LacZ and Pdx1-GFP cell lines, in which weobtain 2-4% PDX1 + cells on average. However, attempts to increase the efficiency of Pdx1-induction proved difficult and the protocol is therefore not satisfactory for further differentiationinto pancreatic endoderm.It seems that there is a fundamental problem in our protocol setup, as we have had little successin improving the number of PDX1 + cells. One source of problems could be connected to ourculture conditions. We use B27 which contains glucocorticoids that have been shown to inhibitPdx1-expression (Tanimizu et al. 2004). Another issue is timing; we see the highest inductionof Pdx1-GFP + cells after 3 days of culture in Step 2 media, and introducing an extra 2 days ofdifferentiation does not increase the numbers of Pdx1-GFP + cells. However, 3 days seem to bea rather short induction period compared to other protocols. D’Amour and co-workers grewhES cell-derived DE for 4-8 days to differentiate them to posterior foregut (D'Amour et al.2006).The rationales on which we have built our inducing factor-combinations seem reasonable, asstudies performed in hES cells using FGF4 and RA showed an induction of 32% PDX1 + cellsfrom DE-cultures (Johannesson et al. 2009). Also, intermediate concentrations of FGF2 wereshown to induce pancreatic foregut specification, whereas higher concentrations induced amore posterior gut type (Ameri et al. 2010).In conclusion, we show that by addition of RA and FGF we can successfully induce PDX1 +cells, albeit in low numbers. This protocol lays the ground for further optimization, althoughthis may prove difficult in the current culture conditions.55
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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 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 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 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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