First published online 4 July 2006
doi: 10.1242/jcs.03038
Journal of Cell Science 119, 3039-3046 (2006)
Published by The Company of Biologists 2006
The survival of differentiating embryonic stem cells is dependent on the SCF-KIT pathway
Anu Bashamboo1,
A. Helen Taylor1,
Kay Samuel2,
Jean-Jacque Panthier3,
Anthony D. Whetton4 and
Lesley M. Forrester1,*
1 John Hughes Bennett Laboratory, Edinburgh Cancer Centre, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK
2 Scottish National Blood Transfusion, John Hughes Bennett Laboratory, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK
3 Mouse functional Genetics, Institut Pasteur, 25 Rue du Dr Roux, 75015 Paris, France
4 Stem Cell and Leukaemia Proteomics Laboratory, Faculty of Medical and Human Sciences, University of Manchester, Christie Hospital, Manchester, M20 9BX, UK
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Fig. 1. Kit-targeting scheme showing a partial map of a mouse genomic DNA fragment containing Kit exon 1 of wild-type allele (A) and the resultant targeted alleles, KitW-lacZ allele (B) and KitW-FKB (C). The BamHI and Kpn sites mark the 5'and 3' ends of the targeting vector, respectively. (D) Multiplex PCR analysis of DNA isolated from Kit+/+ (lane 1), KitW-lacZ/+ lane 2) and KitW-lacZ/W-lacZ (lane 3) cell lines. The 1.4 and 1.8 kb fragments represent the wild-type and WlacZ alleles respectively. (E) Southern blot analysis of EcoR1-digested DNA isolated from KitW-lacZ/W-lacZ (lane 1), KitW-lacZ/+ (lane 2), Kit+/+ (lane 3) and KitW-lacZ/W-FKB (lane 4) targeted ES cell clones. The 9.1, 6.4 and 2.4 EcoRI restriction fragments represent the wild-type, KitW-lacZ and KitW-FKB alleles respectively. (F) Flow cytometric analysis of Kit+/+, KitW-lacZ/+ and KitW-lacZ/W-FKB cell lines using a TC-conjugated anti-mouse KIT monoclonal antibody showing no KIT expression in the KitW-lacZ/W-FKB cell line. Restriction enzymes: B, BamHI; EI, EcoRI; EV, EcoRV; K, KpnI.
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Fig. 2. (A) Morphology of ES cells growing in the presence of 100 U/ml LIF. (B) Growth rate of the different cell lines during routine passage expressed as the total number of cells present 2 days after plating 1x106 cells. The solid bars within the individual scatter represent the median of six independent passages. (C) Total number of colonies produced from Kit+/+ (+/+), KitW-lacZ/+ (+/-), KitW-lacZ/W-lacZ (-/-) and KitW-lacZ/W-FKB (-/FKB) ES cells 5 days after plating at low density in varying concentrations of LIF. Colonies were scored as stem cells (SC), differentiated (DIFF) or mixed (MIX) according to their morphology and AP-staining profile. The results shown are representative examples of several independently derived clones that were all assayed three times in duplicate. (D) The combined number of mixed and differentiated colonies produced from Kit+/+ (+/+), KitW-lacZ/+ (+/-), KitW-lacZ/W-lacZ (-/-) and KitW-lacZ/W-FKB (-/FKB) ES cells 5 days after plating at low density in 100 U/ml LIF. Values shown represent the mean (± s.e.m.) of four independent experiments performed in duplicate. (E) Number of colonies produced from wild-type ES cells after plating at low density in varying concentrations of LIF in the presence (+) or absence (-) of the monoclonal anti-KIT antibody, ACK2.
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Fig. 3. (A) Flow cytometric analysis of PI-stained Kit+/+ and KitW-lacZ/W-FKB cells at different times after LIF withdrawal. The increase in the subG-phase fraction and decrease in G0-G1 and G2-phase fraction was observed in KitW-lacZ/W-FKB cells 3 days after induction of differentiation. (B) Ethidium-bromide-stained genomic DNA isolated from undifferentiated (d0) Kit+/+ (+) and KitW-lacZ/W-FKB (-) ES cells and 1, 2, 2.5, 3 and 3.5 days after LIF withdrawal. (C) AnnexinV (x axis) and PI (y axis) staining of Kit+/+ and KitW-lacZ/W-FKB ES cells grown in the presence of LIF. The numbers shown in each quadrant represent the percentage of cells staining for Annexin only (red), PI only (green) and both Annexin and PI (magenta). (D) Western blot analysis of protein lysates isolated from undifferentiated (d0) Kit+/+ (+) and KitW-lacZ/W-FKB (-) ES cells and four days (d4) after LIF withdrawal. Blots were sequentially probed with an antibody specific to BCL2 protein then an anti-GAPDH antibody as a loading control.
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Fig. 4. (A) Growth rates of Kit+/+ and KitW-lacZ/W-FKB cells in the presence and absence of AP20187 during routine passage, expressed as the total number of cells present 2 days after plating 1x106 cells. The solid bars within the individual scatters represents the median of six independent passages. (B) Total numbers of stem cell (SC), differentiated (DIFF) or mixed (MIX) colonies produced from Kit+/+ (+/+), KitW-lacZ/+ (+/-), KitW-lacZ/W-lacZ (-/-) and KitW-lacZ/W-FKB (-/FKBKit) in 100 or 0 U/ml of LIF in the presence of AP20187. The results shown are representative examples of three assays carried out in duplicate. (C) Combined number of mixed and differentiated colonies produced from Kit+/+ (+/+), KitW-lacZ/+ (+/-), KitW-lacZ/W-lacZ (-/-) and KitW-lacZ/W-FKB (-/FKB) ES cells 5 days after plating at low density in 100 U/ml LIF and 10 nM AP20187. The graphs show the mean (± s.e.m.) calculated from three independent experiments carried out in duplicate. (D) Western blot analysis of protein lysates isolated from Kit+/+, KitW-lacZ/+, KitW-lacZ/W-lacZ and KitW-lacZ/W-FKB ES cells grown in the presence (+) or absence (-) of AP20187 as indicated. Blots were sequentially probed with an antibody specific to KIT phosphotyrosine 730 (pY730) then an anti-GAPDH (GAPDH) antibody as a loading control.
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© The Company of Biologists Ltd 2006
