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Blood, Vol. 111, Issue 4, 1876-1884, February 15, 2008
Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak–like cells and enables isolation of primitive hematopoietic precursors
Blood Davis et al.
111: 1876
Supplemental materials for: Davis et al
Files in this Data Supplement:
- Table S1. Primers used to identify MIXL1GFP/w HESCs (PDF, 83.3 KB)
- Table S2. Primers used for RT-PCR analysis (PDF, 40 KB)
- Table S3. Analysis of MIXL1+ cells in the GFP+ and GFP− sorted populations shown in Figure 2D (PDF, 58 KB) -
(A) Proportion of GFP+, GFP− and MIXL1+ cells in unsorted differentiating MIXL1GFP/w HESCs and the frequency of MIXL1+ cells in the GFP+ and GFP− sorted fractions. Data is shown as the percentage of cells that were MIXL1+ as determined by intracellular flow cytometry using an antibody recognising endogenous MIXL1 protein. The proportion of cells that expressed GFP was determined by flow cytometric analysis. (B). Distribution of MIXL1+ cells between GFP+ and GFP− sorted fractions. The value for each fraction was derived by multiplying the proportion of MIXL1+ cells (data columns 4 and 5 of supplementary Table 3A) by the % of cells for each fraction (data columns 2 and 3 of supplementary Table 3A). The sum of the sorted fractions for each experiment is shown in the last data column. (C) Proportion of MIXL1+ cells present in the GFP+ versus GFP− fractions. The proportion of MIXL1+ cells found within each fraction was calculated from data shown in Table S3B by dividing the numbers in data columns 1 and 2 by data column 3. The result of this calculation is the proportion of MIXL1+ cells in a given differentiation that can be found in the GFP+ versus GFP− fractions. The graph shown in figure 2D was derived from this table.
- Table S4. Distribution of clonogenic cells in sorted fractions of d4 differentiated MIXL1GFP/w cells (PDF, 16.5 KB) -
Data is shown as CFC/2 × 104 cells plated for the unsorted cells and for each sorted fraction. The proportion of cells in each fraction determined from the flow cytometry profile of unsorted cells is shown as a percentage (% of cells).
- Table S5. Sum of calculated colony distribution in each sorted fraction (PDF, 46.6 KB) -
Data is shown as CFC/2 × 104 cells plated. The value for each fraction was derived by multiplying the CFC number by the % of cells for each fraction (data in Table S3). The sum of the sorted fractions for each experiment is shown in the first data column.
- Table S6. Progenitor yield from sorted fractions compared to CFC yield from unsorted cells (PDF, 13.9 KB) -
The sum of the CFC in the sorted fractions (sum of sorted fractions, see Table S4) was compared with the number of CFC/2 × 104 cells from the unsorted cells to calculate the CFC recovery after flow cytometric sorting.
- Table S7. CFC distribution in sorted fractions (PDF, 13.8 KB) -
The distribution of CFC found within each sorted fraction was calculated from data shown in Table S4 by dividing the CFC found in each sorted fraction by the sum of CFC in the sorted fractions and expressing the result as a percentage.
- Figure S1. Flow cytometric analysis of the HES3 derivative MIXL1GFP/w 17.26 showing uniform expression of the surface markers E-CAD and TRA-1-60 (A) and of the transcription factor OCT4 (B) (JPG, 108 KB)
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Numbers indicate the fraction of cells in the corresponding quadrant. C) Chromosome preparation of the same clone showing a normal female karyotype.
- Figure S2 (JPG, 138 KB)
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(A) Q-PCR analysis of MIXL1 on the samples shown in Figure 2E. Levels of relative gene expression were calculated as described in Pick et al22. (B) Time course of differentiating MIXL1GFP/w (cl. 7) HESCs shows GFP expression correlates with MIXL1 expression prior to d6, as determined by intracellular flow cytometric analysis. While MIXL1 was not detected after d6, GFP+ cells were still observed at d10, although GFP mRNA was not present. This illustrates the longer half-life of the GFP protein compared with that of MIXL1.
- Figure S3. Time course analysis of a second independently derived MIXL1GFP/w HESC line (JPG, 197 KB)
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GFP expression was detected when cells were differentiated in SFM containing 50 ng/ml BMP4. The expression profile of GFP, E-CAD and PDGFR is similar to that shown in Figure 3D. The proportion of MIXL1+ cells, as well as the level of intensity, was increased when the media was supplemented with 100 ng/ml Activin A at day 1.
- Figure S4 (JPG, 191 KB)
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(A) An example of how the region statistics relating to the proportion of cells expressing GFP and the surface markers (in this case PDGFR) were calculated for Figure 3D. GFP-expressing cells were first identified on a plot of FL-1 (GFP) versus FL-2 (PE). The position of gates were set relative to those for FGF2 differentiated cells which do not express MIXL1 or GFP. GFP+ (red) and GFP− (black) cells displayed on the FL-1(GFP) versus FL-4 (APC) plots were analysed separately for expression of PDGFR. The absolute percentages were calculated for all four possible fractions by multiplying the proportion of PDGFR+/− cells in each region by the percentage of cells that were either GFP− or GFP+.
(B) Expression of PDGFR and CD34 on d4 spin EBs differentiated in BVS medium, shows that PDGFR expression antedates expression of CD34.
- Figure S5. A typical experiment showing the gating strategy for cell sorting of GFP and PDGFRα expressing subfractions used for colony assays (Figure 4) and Q-PCR analysis (supplementary figures 6A and B) (JPG, 237 KB)
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GFP+ cells were first identified using the FL1 (GFP) versus FL3 (propidium iodide, PI) gates. The GFP+ and GFP− subfractions were then further divided on the basis of PDGFR expression.
- Figure S6. Q-PCR analysis of gene expression in differentiation day 4 cellular subfractions isolated on the basis of GFP and PDGFRα expression (JPG, 231 KB)
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Data was compiled from 3 separate experiments (except for E-CAD). Levels of relative gene expression were calculated as described in Pick et al22. Error bars represent the standard error of the mean. In panel B, gene expression ratios were calculated by dividing the value obtained for the GFP+PDGFR+ fraction by those obtained for the GFP-PDGFR− fraction.
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