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. 2017 Jun 29;129(26):3486-3494.
doi: 10.1182/blood-2017-02-770958. Epub 2017 Apr 21.

FLI1 Level During Megakaryopoiesis Affects Thrombopoiesis and Platelet Biology

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Free PMC article

FLI1 Level During Megakaryopoiesis Affects Thrombopoiesis and Platelet Biology

Karen K Vo et al. Blood. .
Free PMC article

Abstract

Friend leukemia virus integration 1 (FLI1), a critical transcription factor (TF) during megakaryocyte differentiation, is among genes hemizygously deleted in Jacobsen syndrome, resulting in a macrothrombocytopenia termed Paris-Trousseau syndrome (PTSx). Recently, heterozygote human FLI1 mutations have been ascribed to cause thrombocytopenia. We studied induced-pluripotent stem cell (iPSC)-derived megakaryocytes (iMegs) to better understand these clinical disorders, beginning with iPSCs generated from a patient with PTSx and iPSCs from a control line with a targeted heterozygous FLI1 knockout (FLI1+/-). PTSx and FLI1+/- iMegs replicate many of the described megakaryocyte/platelet features, including a decrease in iMeg yield and fewer platelets released per iMeg. Platelets released in vivo from infusion of these iMegs had poor half-lives and functionality. We noted that the closely linked E26 transformation-specific proto-oncogene 1 (ETS1) is overexpressed in these FLI1-deficient iMegs, suggesting FLI1 negatively regulates ETS1 in megakaryopoiesis. Finally, we examined whether FLI1 overexpression would affect megakaryopoiesis and thrombopoiesis. We found increased yield of noninjured, in vitro iMeg yield and increased in vivo yield, half-life, and functionality of released platelets. These studies confirm FLI1 heterozygosity results in pleiotropic defects similar to those noted with other critical megakaryocyte-specific TFs; however, unlike those TFs, FLI1 overexpression improved yield and functionality.

Figures

Figure 1.
Figure 1.
Analysis of FLI1 mRNA and protein levels of FLI1, MPL, and PF4. Day 5 iMegs were selected for CD41a and analyzed using quantitative reverse transcription polymerase chain reaction and western blot. (A) Relative expression of FLI1 mRNA was performed using quantitative reverse transcription polymerase chain reaction compared with WT. Mean ± 1 SEM are shown of 4 separate experiments. P values were calculated using 1-way ANOVA. (B) Western blot analysis of FLI1. Relative expression was compared with WT. Means ± 1 SEM are shown for 4 separate experiments. P values were calculated using 1-way ANOVA. (C) Flow cytometric analysis of MPL. Relative surface MPL expression analyzed from 3 separate experiments using flow cytometry and 1-way ANOVA. (D) Similar to panel B, but measuring PF4.
Figure 2.
Figure 2.
Megacult colony assay and iMeg differentiation in liquid culture. (A) HPCs were plated in a semisolid Megacult colony system and analyzed at the end of the assay for colony count per input HPC. Means ± 1 SEM are shown along with the number of independent experiments performed. Significant P values performed using 1-way ANOVA are shown. (B) HPCs were grown in liquid culture and analyzed at the end of the assay for iMeg numbers. Relative expression to WT iPSC line is shown. Means ± 1 SEM are shown, along with the number of independent experiments performed. P values were calculated using 1-way ANOVA.
Figure 3.
Figure 3.
Percentage of annexinV-CD41+CD42a+CD42b+ iMegs and in vitro–released platelets. Day 5 iMegs and released platelet-like particles were analyzed for surface markers, using flow cytometry. (A-C) iMegs were negative for annexin V and positive for CD41a, CD42a, and CD42b. (D-F) In vitro platelet-like particles positive for CD41a, negative for annexin V, and positive for CD42b. Means ± 1 SEM are shown with n = 3-6 independent experiments per arm. Significant P values were determined using 1-way ANOVA.
Figure 4.
Figure 4.
In vivo iPlt generation is decreased for FLI1-low and increased for FLI1-high lines. (A,B) NSG mice were infused with iMegs, and percentage of human platelets was determined at various points up to 24 hours. Means ± 1 SEM are shown with 4-7 independent experiments per arm. (A, left) Data analyzed per infused iMegs generated from isogenic genome-edited iPSC lines. (Right) Data analyzed per infused iMegs WT iPSCs and the 2 PTSx lines. (B) Same as in panel A, but analyzed per initial HPCs from which the iMegs were prepared. (C-D) Area under the curve (AUC) calculations for iPlt generation either from iMegs (C) or from HPCs (D). Means ± 1 SEM are shown with number of independent experiments per arm shown in each bar. Significant P values were determined using 1-way ANOVA.
Figure 5.
Figure 5.
iPlt half-life is decreased for FLI1-low and increased for FLI1-overexpression lines. Percentage of peak iPlt generation up to 24 hours after iMeg infusion into NSG mice (n = 4-7 independent experiments, same as Figure 4A,D). Half-life is determined by the time at which there is 50% of iPlts compared with peak iPlt numbers.
Figure 6.
Figure 6.
Ex vivo and in vivo functional analyses of iPlts. (A) CD41+ and (B) CD41+CD42b+ iPlt mean fluorescence intensity (MFI) of surface P-selectin before and after thrombin stimulation of iPlts generated at 4 hours after iMeg infusion. Means ± 1 SEM are shown with n = 6 independent experiments per arm. Significance was determined by 1-way ANOVA. (C) Cremaster injuries were induced at 4 hours after iMeg infusion in NSG mice, and fluorescent images were recorded. The numbers reported are of human calcein AM–stained particles incorporated into a growing thrombus after normalization by dividing by the percentage of circulating CD42b+-human platelets as part of the total circulating platelets. Shown are the individual data point and mean ± 1 SEM of experiments from 4 individual mice with up to 6 injuries per mouse. Significance was determined by 1-way ANOVA.

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