Inducible Gata1 suppression expands megakaryocyte-erythroid progenitors from embryonic stem cells
Ji-Yoon Noh, Shilpa Gandre-Babbe, Yuhuan Wang, Vincent Hayes, Yu Yao, Paul Gadue, Spencer K. Sullivan, Stella T. Chou, Kellie R. Machlus, Joseph E. Italiano Jr., Michael Kyba, David Finkelstein, Jacob C. Ulirsch, Vijay G. Sankaran, Deborah L. French, Mortimer Poncz, Mitchell J. Weiss
Ji-Yoon Noh, Shilpa Gandre-Babbe, Yuhuan Wang, Vincent Hayes, Yu Yao, Paul Gadue, Spencer K. Sullivan, Stella T. Chou, Kellie R. Machlus, Joseph E. Italiano Jr., Michael Kyba, David Finkelstein, Jacob C. Ulirsch, Vijay G. Sankaran, Deborah L. French, Mortimer Poncz, Mitchell J. Weiss
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Brief Report Hematology

Inducible Gata1 suppression expands megakaryocyte-erythroid progenitors from embryonic stem cells

Abstract

Transfusion of donor-derived platelets is commonly used for thrombocytopenia, which results from a variety of clinical conditions and relies on a constant donor supply due to the limited shelf life of these cells. Embryonic stem (ES) and induced pluripotent stem (iPS) cells represent a potential source of megakaryocytes and platelets for transfusion therapies; however, the majority of current ES/iPS cell differentiation protocols are limited by low yields of hematopoietic progeny. In both mice and humans, mutations in the gene-encoding transcription factor GATA1 cause an accumulation of proliferating, developmentally arrested megakaryocytes, suggesting that GATA1 suppression in ES and iPS cell–derived hematopoietic progenitors may enhance megakaryocyte production. Here, we engineered ES cells from WT mice to express a doxycycline-regulated (dox-regulated) shRNA that targets Gata1 transcripts for degradation. Differentiation of these cells in the presence of dox and thrombopoietin (TPO) resulted in an exponential (at least 1013-fold) expansion of immature hematopoietic progenitors. Dox withdrawal in combination with multilineage cytokines restored GATA1 expression, resulting in differentiation into erythroblasts and megakaryocytes. Following transfusion into recipient animals, these dox-deprived mature megakaryocytes generated functional platelets. Our findings provide a readily reproducible strategy to exponentially expand ES cell–derived megakaryocyte-erythroid progenitors that have the capacity to differentiate into functional platelet-producing megakaryocytes.

Authors

Ji-Yoon Noh, Shilpa Gandre-Babbe, Yuhuan Wang, Vincent Hayes, Yu Yao, Paul Gadue, Spencer K. Sullivan, Stella T. Chou, Kellie R. Machlus, Joseph E. Italiano Jr., Michael Kyba, David Finkelstein, Jacob C. Ulirsch, Vijay G. Sankaran, Deborah L. French, Mortimer Poncz, Mitchell J. Weiss

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Figure 2

Physiological restoration of Gata1 optimizes megakaryocytic maturation.

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Physiological restoration of Gata1 optimizes megakaryocytic maturation. ...
Megakaryocytes were generated from G1ME2 cells after dox withdrawal, from FL progenitors after culture in TPO, and from G1ME cells after retroviral transfer of Gata1 cDNA (10). (A) Quantification of megakaryocyte-expressed mRNAs. Increased Gata2 expression in G1ME cells at day 6 likely represents outgrowth of uninfected cells (n = 3–6 experiments). (B) Western blotting for VWF, GATA1, and PF4. Representative of 3 experiments. (C) CD42b+ cell surface expression measured by flow cytometry (n = 4–6 experiments). (D) Megakaryocyte DNA content at the optimal times after initiation of megakaryocyte differentiation: G1ME2 cells, 5 days; FL, 6 days; G1ME cells, 4 days. Megakaryocyte percentages with more than 4 N DNA are indicated in the upper left corners of the panels. Mean ± SEM, 3 experiments. (E) Megakaryocytes generated from G1ME2 cells 5 days after dox withdrawal were injected intravenously into mice expressing human αIIb (hCD41) in place of both endogenous mouse (mCD41) genes. G1ME2-derived platelets (mCD41+) were distinguished from endogenous platelets (hCD41+) by species-specific antibodies. (F) Platelet forward scatter (FSC) distribution of G1ME2 cell–derived and endogenous platelets. (G) Approximately equal numbers of G1ME2 or G1ME cell–derived megakaryocytes were injected intravenously, and their circulating platelet progeny were quantified (n = 4 experiments). *P < 0.05 vs. preinfusion group by Student’s t test. Mean ± SEM. (H) FL-derived megakaryocytes or normal mouse platelets (PLTs) were injected intravenously, and circulating donor platelets were quantified (n = 3 experiments). The numbers of infused megakaryocytes for each experiment are shown in Supplemental Table 1.