American Society of Hematology

Figure 2

$Figure 2. GFP marks MIXL1+ cells during the early stages of HESC differentiation. (A) PCR analysis indicates that GFP expression mirrors the wave of expression of endogenous MIXL1. This analysis also shows the progressive down-regulation of the stem-cell marker, OCT4, the transient expression of the primitive streak genes, MIXL1 and BRACHYURY, and activation of genes expressed in endodermal (FOXA2, AFP-alpha fetoprotein, ALBUMIN) and mesodermal (GATA2, CD34) cell types. – RT indicates − reverse transcriptase. This panel is a composite of images for individual ethidium bromide-stained agarose gels for each set of genes. (B) Immunofluorescent images of day 4 MIXL1GFP/w HESCs differentiated in SFM in the presence of either 30 ng/mL BMP4 (top and middle panels) or 100 ng/mL FGF2 (bottom panel). The bottom panel shows that MIXL1GFP/w HESCs differentiated in FGF2 expressed neither GFP nor MIXL1. (C) Sorting and reanalysis experiments examining the relationship between expression of GFP and MIXL1 protein by flow cytometric analysis. The top panel shows the profile of GFP expressing cells in day 4 MIXL1GFP/w EBs. The division between GFP+ (red) and GFP− (black) fractions was based on gates set using MIXL1w/w (HES3) control EBs (inset). The middle panel shows the reanalysis of the sorted populations with the distribution of GFP+ cells and GFP− cells indicated. Endogenous MIXL1 protein (bottom panels), as determined by intracellular flow cytometry with an anti-MIXL1 antibody, is largely restricted to GFP+ cells and excluded from the GFP− cells. The position of gates for intracellular flow cytometry were set with MIXL1GFP/w day 4 EBs differentiated in SFM containing 100 ng/mL FGF2 and stained with the anti-MIXL1 antibody (inset, bottom panel). (D) Graphic representation showing the distribution of MIXL1+ cells between the GFP+ and GFP− sorted populations from 4 separate experiments using 2 independent MIXL1GFP/w HESC lines (P < .001). Error bars represent the SEM. Calculations are shown in Table S3A-C. (E) PCR analysis of the GFP-sorted fractions from C showing both GFP and MIXL1 transcripts are essentially restricted to the GFP+ population.$

GFP marks MIXL1⁺ cells during the early stages of HESC differentiation. (A) PCR analysis indicates that GFP expression mirrors the wave of expression of endogenous MIXL1. This analysis also shows the progressive down-regulation of the stem-cell marker, OCT4, the transient expression of the primitive streak genes, MIXL1 and BRACHYURY, and activation of genes expressed in endodermal (FOXA2, AFP-alpha fetoprotein, ALBUMIN) and mesodermal (GATA2, CD34) cell types. – RT indicates − reverse transcriptase. This panel is a composite of images for individual ethidium bromide-stained agarose gels for each set of genes. (B) Immunofluorescent images of day 4 MIXL1^GFP/w HESCs differentiated in SFM in the presence of either 30 ng/mL BMP4 (top and middle panels) or 100 ng/mL FGF2 (bottom panel). The bottom panel shows that MIXL1^GFP/w HESCs differentiated in FGF2 expressed neither GFP nor MIXL1. (C) Sorting and reanalysis experiments examining the relationship between expression of GFP and MIXL1 protein by flow cytometric analysis. The top panel shows the profile of GFP expressing cells in day 4 MIXL1^GFP/w EBs. The division between GFP⁺ (red) and GFP⁻ (black) fractions was based on gates set using MIXL1^w/w (HES3) control EBs (inset). The middle panel shows the reanalysis of the sorted populations with the distribution of GFP⁺ cells and GFP⁻ cells indicated. Endogenous MIXL1 protein (bottom panels), as determined by intracellular flow cytometry with an anti-MIXL1 antibody, is largely restricted to GFP⁺ cells and excluded from the GFP⁻ cells. The position of gates for intracellular flow cytometry were set with MIXL1^GFP/w day 4 EBs differentiated in SFM containing 100 ng/mL FGF2 and stained with the anti-MIXL1 antibody (inset, bottom panel). (D) Graphic representation showing the distribution of MIXL1⁺ cells between the GFP⁺ and GFP⁻ sorted populations from 4 separate experiments using 2 independent MIXL1^GFP/w HESC lines (P < .001). Error bars represent the SEM. Calculations are shown in Table S3A-C. (E) PCR analysis of the GFP-sorted fractions from C showing both GFP and MIXL1 transcripts are essentially restricted to the GFP⁺ population.

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