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.
View: Text | PDF
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

×

Figure 1

Dox-regulated suppression of Gata1 generates self-renewing MEPs from ES cells.

Options: View larger image (or click on image) Download as PowerPoint
Dox-regulated suppression of Gata1 generates self-renewing MEPs from ES ...
(A) Murine ES cells containing dox-inducible Gata1 shRNA or control transgenes were differentiated into embryoid bodies for 6 days, disaggregated, and cultured with dox, TPO, and SCF. Cumulative numbers of nonadherent cells are plotted versus time (n = 4 experiments). (B) Flow cytometry showing expression of KIT and CD41 in G1ME2 cells. (C) Semiquantitative RT-PCR showing induction of Gata1 mRNA after dox withdrawal (n = 3 experiments). *P < 0.05 versus +dox group by Student’s t test. Mean ± SEM. (D) Western blotting for GATA1 protein after dox removal. (E) May-Grünwald Giemsa–stained G1ME2 cells after culture in TPO following dox removal. Scale bars: 20 μm. (F) Acetylcholinesterase expression. Scale bars: 20 μm. (G) Phase contrast microscopy showing proplatelet formation by G1ME2-derived megakaryocytes 6 days after dox removal. Original magnification, ×400. Scale bar: 20 μm. (H) Proplatelet formation by G1ME2-derived megakaryocytes illustrated by anti–β1-tubulin immunofluorescence microscopy. Original magnification, ×900. Scale bar: 10 μm. All representative data are shown from 3 to 4 experiments.