Inducible Gata1 suppression expands megakaryocyte-erythroid progenitors from embryonic stem cells
Ji-Yoon Noh, … , Mortimer Poncz, Mitchell J. Weiss
Ji-Yoon Noh, … , Mortimer Poncz, Mitchell J. Weiss
Published May 11, 2015
Citation Information: J Clin Invest. 2015;125(6):2369-2374. https://doi.org/10.1172/JCI77670.
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Brief Report Hematology Stem cells

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 3

G1ME2-derived megakaryocytes produce functional platelets in vivo.

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G1ME2-derived megakaryocytes produce functional platelets in vivo. The c...
The cremaster muscle artery injury model (20). (A) Calcein AM loaded, BSA-purified G1ME2 cell–derived megakaryocytes (0.5 to 1 × 106 cells) were infused into C57BL/6 mice. Thereafter, a cremaster arteriole was laser pulsed and monitored by video microscopy. Incorporation of G1ME2-derived platelets (green) into a thrombus after injury is shown. Representative of 10 experiments. 4 to 7 separate laser injuries were performed for each experiment. (B–D) 0.5 to 1 × 106 megakaryocytes (B and C) or 1 × 107 washed mouse platelets (D) were incubated with or without platelet inhibitor drugs and tested for adherence to fresh laser-induced thrombi (n = 3–6 experiments). PGE1, 1 μM; ASA, 1 mM. (E) Alexa Fluor 647–conjugated anti–P-selectin antibody was infused after G1ME2-derived megakaryocytes and before laser injury. Left panels: confocal fluorescent video microscopy of thrombi. Endogenous calcein–, P-selectin+ platelets are blue. The arrows show 2 G1ME2-derived platelets visualized over time by single particle tracking. These platelets were green initially (calcein+ P-selectin–), then became cyan (green + blue) following P-selectin induction. Representative images are shown from 3 independent experiments. Scale bar: 10 μm. Right: calcein (green lines) and P-selectin expression (blue lines) versus time after laser injury in platelets 1 and 2 from the left panels.