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Blood, Vol. 111, Issue 1, 122-131, January 1, 2008
Wnt signaling promotes hematoendothelial cell development from human embryonic stem cells
Blood Woll et al.
111: 122
Supplemental materials for: Woll et al
Files in this Data Supplement:
- Figure S1. Evaluation of human specific primers utilized for RT-PCR analysis (JPG, 55 KB)
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cDNA from mouse embryonic fibroblasts (MEF), S17 stromal cells (S17) and M210-B4 stromal cells (M210) was prepared and analyzed for expression of indicated genes by RT-PCR using conditions as in Table 1. An appropriate positive control (pos) was also analyzed for each sample.
- Figure S2. Quantification of CFCs (JPG, 18.8 KB)
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hESCs were differentiated for 14 days and CFCs were quantified from the unsorted cells (pre), or sorted phenotypic populations of CD34brightCD31+Flk1+, CD34dimCD31+Flk1−, CD34+CD31−Flk1+ and CD34−CD31−Flk1− cells, as indicated.
- Figure S3. Limiting dilution analysis of endothelial and hematopoietic progenitor cells (JPG, 74.9 KB)
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Sorted CD34brightCD31+Flk1+, CD34dimCD31+Flk1− and CD34+CD31−Flk1− cells were analyzed by limiting dilution under both hematopoietic and endothelial cell growth conditions. Both hematopoietic and endothelial cells were observed from CD34brightCD31+Flk1+ cells, whereas other cell populations appeared more committed to one lineage only.
- Figure S4. Measurement of Wnt expression and DKK activity (JPG, 75.8 KB)
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(A) Expression of Wnt proteins and Frizzled receptors was evaluated by RT-PCR from (lane 1) undifferentiated hESCs, (lane 2) hESCs differentiated by stromal cell co-culture for 14 days, (lane 3) CD34+ hESC-derived cells and (lane 4) CD34+CD45+ hESC-derived cells. Water was utilized as a negative control (lane 5). (B) Recombinant human DKK1 was serially diluted in 293 control medium and ability to inhibit Wnt signaling in hESCs transfected with Wnt-responsive reporter gene was measured (■), with extrapolated line between measurement as indicated. Activity of DKK1-conditioned media was then determined. Solid line with arrow indicates DKK1 conditioned medium mixed as described in materials and methods, dotted line with arrow indicates DKK1 conditioned medium diluted 1:5 before use. Based on linear regression, 293 DKK1-conditioned medium had a DKK1-concentration between 350-500ng/mL. (C) hESCs were plated in 24-well plate with confluent monolayer of M210 stromal cells and cultured in control medium (○), DKK1-conditioned medium (■),and control medium supplemented with 400ng/mL recombinant human DKK1 (♦). Flow cytometric analysis for CD34+, CD34+CD31+, CD34+Flk1+ and CD31+Flk1+ cells is shown. CD34+CD45+ cells were analyzed at day 17 of differentiation.
- Figure S5. Immuostaining for Wnt1 expression in M210 stromal cell transfected with Wnt1 and GFP (JPG, 95.7 KB)
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Left panel showns M210-GFP control stromal cells and right panel shows M210-Wnt1 stromal cells stained for Wnt1 expresssion. Wnt1 was detected by immunostaining with goat anti-Wnt1 primary antibody and Alexa Fluor 647 donkey anti-goat IgG secondary antibody. Images were acquired on a Zeiss Axioscope 2 plus microscope with 20×/0.60 objective. Images were photographed with Lasersharp2000 Emulation 5.2 (Bio-Rad, Hercules, CA) software and processed with Adobe Photoshop 7.0.
- Figure S6. Q-RT-PCR analysis for expression of CYCLIN-D1 (JPG, 65 KB)
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Expression of CYCLIN-D, a downstream target gene of Wnt signaling, in hESCs allowed to differentiate in conditions which inhibit (DKK1, ■) or activate (Wnt1, ●) Wnt signaling, as compared to hESCs cultured in control conditions. All samples were adjusted to GAPDH expression.
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