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Blood, Vol. 111, Issue 10, 4944-4953, May 15, 2008
Modulation of murine embryonic stem cell–derived CD41+c-kit+ hematopoietic progenitors by ectopic expression of Cdx genes
Blood McKinney-Freeman et al.
111: 4944
Supplemental materials for: McKinney-Freeman et al
Files in this Data Supplement:
- Table S1. Summary of Cdx induction effect on OP9 colony formation/50,000 EB-derived cells (JPG, 55.6 KB)
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- Table S2. Summary of Cdx effect on M3434 colony formation/100,000 EB-derived cells (JPG, 97.2 KB)
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- Table S3. Summary of Cdx induction effect on OP9 colony formation of 1,000 CD41+c-kit+ EB-derived cells (JPG, 63.1 KB)
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- Table S4. Summary of Cdx effect on M3434 colony formation of 1000 CD41+c-kit+ EB-derived cells (JPG, 61.2 KB)
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- Figure S1. Schematic of AinV15 inducible system (JPG, 58.8 KB)
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iCdx1, iCdx2 and iCdx4 ESC lines were created using the AinV15 ESC system depicted in this figure. This system uses CRE-mediated recombination (pLox sites represented as triangles) to insert a cDNA downstream of the Tet response element (TRE) which has been engineered into the HPRT locus upstream of a neomycin selectable marker whose ATG has been removed. Proper insertion of a cDNA restores the neomycin ATG, allowing for the selection of targeted ESC clones via growth in the presence of G418. In this system, the tet-activator (TA) is being constitutively expressed from the ROSA26 locus. Thus, expression of a gene of interest can be induced via the introduction of doxycycline.
- Figure S2. Cdx2 induction in differentiating EBs strongly suppresses hematopoietic progenitor activity without affecting gross morphology of day-6 EBs (JPG, 80.1 KB)
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iCdx2 and Ainv15 EBs cultured with or without doxycycline (0.1–1.0 µg/mL) between day-4 and day-6 of EB differentiation were examined for hematopoietic progenitor activity. Low levels of doxycycline were sufficient to suppressed hematopoietic activity from iCdx2 ESC, while no effect has been observed following high-doses of doxycycline in EB from the parental ESC line Ainv15 (A). Gross morphology of iCdx2 EBs from two independent iCdx2 ESC clones did not show abnormalities after doxycycline exposure (B). Images were acquired on an Olympus IMT-2 and captured with an Olympus DP12 camera at room temperature in culture media at 100x total magnification.
- Figure S3. Cdx1 and Cdx4 expression differentially affect the hematopoietic activity of day-6 EB derived hematopoietic progenitors (JPG, 92.6 KB)
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iCdx1 and iCdx4 EBs were exposed to doxycycline from day-4 to day-6 of differentiation, plated into secondary assays at the indicated concentrations of doxycycline, and then evaluated for OP9 (A) and hematopoietic progenitor colony forming activity (B). In order to pool and average data from multiple experiments (each data point denotes between three and eight replicates) the data is presented as fold change in colony potential relative to non-induced control. iCdx1 EBs were exposed to 0.5 µg/mL doxycycline from day-4 to day-6 of differentiation and then co-cultured with OP9 stroma cells either with (+ dox, Week 1) or without (− dox, Week 1) continued exposure to the indicated concentration of doxycycline for one week of co-culture. Cells co-cultured for one week in the presence of doxycycline (+ dox, Week 1) were then co-cultured for an additional week in the absence of doxycycline (− dox, Week 2). Exposure to doxycycline during Week 1 suppressed the appearance of OP9-colonies, yet the withdrawal of doxycycline during Week 2 allowed appearance of OP9 colonies from these cells. Representative image of colonies re-emerging after the removal of 0.25 µg/mL doxycycline (C). Dose dependent effect of doxycycline on OP9 colony formation (D). Data presented are representative of three independent biological experiments.
- Figure S4. Cdx gene induction selectively affects the hematopoietic potential of CD41+ckit+, but not CD41−c-kit+, CD41+c-kit−, CD41−c-kit− day-6 EB-derived cells (JPG, 114 KB)
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Representative FACS plots, including post-sort purity assessment, of day-6 EBs fractionated on the basis of CD41 and c-kit expression (A). CD41+c-kit+ cells isolated from iCdx1 and iCdx4 day-6 EBs were significantly enriched in OP9 colony-potential relative to unfractionated EB-derived cells. The values in bold represent the relative enrichment in colony activity and are calculated as (Average # colonies/1000 CD41+c-kit+ cells)/(Average # colonies/1000 unfractionated cells). These data are the average of upwards of nine experiments. Induction of ectopic Cdx1 or Cdx4 expression did not significantly modulate the OP9 (C) or hematopoietic progenitor (D) potential of CD41+c-kit−, CD41−c-kit+ or CD41−c-kit− day-6 EB derived cells. These data are presented as fold-change in colony activity relative to non-induced controls for three (C) and two to four (D) independent experiments. Error bars represent the standard error. P values calculated using non-paired, two-tailed, Student’s T test and depicted over bars.
- Figure S5. CD41+c-kit+ EB-derived cells express a distinct Hox profile relative to CD41+c-kit− EB-derived cells (JPG, 67.5 KB)
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Quantitative real time RT-PCR analysis reveals that CD41+c-kit+ day-6 EB-derived cells are enriched in expression of some Hox genes relative to CD41+c-kit− cells. Each data point represents between two and nine independent experiments. Error is presented as standard error.
- Figure S6. Cdx gene expression enhances cluster A and B Hox gene expression of CD41+c-kit+ and CD41+c-kit− EB-derived cells (JPG, 120 KB)
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Quantitative real time RT-PCR analysis reveals that induction of Cdx1 (A) or Cdx4 (B) expression results in the enhancement of select Hox gene expression in CD41+c-kit+ day-6 EB-derived cells relative to CD41+c-kit− cells. Each data point represents the average of between two and four independent experiments. Error bars represent standard error.
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