Congenital amegakaryocytic thrombocytopenia iPS cells exhibit defective MPL-mediated signaling
Shinji Hirata, … , Shinji Kunishima, Koji Eto
Shinji Hirata, … , Shinji Kunishima, Koji Eto
Published August 1, 2013
Citation Information: J Clin Invest. 2013;123(9):3802-3814. https://doi.org/10.1172/JCI64721.
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Research Article Hematology

Congenital amegakaryocytic thrombocytopenia iPS cells exhibit defective MPL-mediated signaling

Abstract

Congenital amegakaryocytic thrombocytopenia (CAMT) is caused by the loss of thrombopoietin receptor–mediated (MPL-mediated) signaling, which causes severe pancytopenia leading to bone marrow failure with onset of thrombocytopenia and anemia prior to leukopenia. Because Mpl–/– mice do not exhibit the human disease phenotype, we used an in vitro disease tracing system with induced pluripotent stem cells (iPSCs) derived from a CAMT patient (CAMT iPSCs) and normal iPSCs to investigate the role of MPL signaling in hematopoiesis. We found that MPL signaling is essential for maintenance of the CD34+ multipotent hematopoietic progenitor (MPP) population and development of the CD41+GPA+ megakaryocyte-erythrocyte progenitor (MEP) population, and its role in the fate decision leading differentiation toward megakaryopoiesis or erythropoiesis differs considerably between normal and CAMT cells. Surprisingly, complimentary transduction of MPL into normal or CAMT iPSCs using a retroviral vector showed that MPL overexpression promoted erythropoiesis in normal CD34+ hematopoietic progenitor cells (HPCs), but impaired erythropoiesis and increased aberrant megakaryocyte production in CAMT iPSC–derived CD34+ HPCs, reflecting a difference in the expression of the transcription factor FLI1. These results demonstrate that impaired transcriptional regulation of the MPL signaling that normally governs megakaryopoiesis and erythropoiesis underlies CAMT.

Authors

Shinji Hirata, Naoya Takayama, Ryoko Jono-Ohnishi, Hiroshi Endo, Sou Nakamura, Takeaki Dohda, Masanori Nishi, Yuhei Hamazaki, Ei-ichi Ishii, Shin Kaneko, Makoto Otsu, Hiromitsu Nakauchi, Shinji Kunishima, Koji Eto

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Figure 3

Essential role for MPL signaling in MPP maintenance and MEP development.

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Essential role for MPL signaling in MPP maintenance and MEP development....
(A) Putative in vitro differentiation from CD34+CD43+CD41a–GPA– MPPs into CD41a+GPA+ MEPs, and subsequently into MKs/erythrocytes. MPPs or MEPs were cultured with SCF, TPO, and EPO for 2–8 days. (B–D) Representative flow cytometric plots for MPPs derived from normal (B) or CAMT (D) iPSCs, and for MEPs derived from normal iPSCs (C). (E) Numbers of MEPs (cells/well) were plotted to illustrate the kinetics of MEP development from MPPs. MPL expression was required for MEP development. (F) Time-dependent loss of MPPs derived from normal and CAMT iPSCs, with or without MPL overexpression, in the presence of SCF, TPO, and EPO. Reduction kinetics were calculated as the absolute number of MPPs. The data indicated the critical role of MPL expression in MPPs. (G) Kinetics of CD43+ hematopoietic cell generation from the MPPs in F. (H) Percentage of individual cell cycle phases in the MPPs in F on day 2 of culture. Increase in sub-G1 (reflecting apoptosis) and decrease in S/G2/M (reflecting proliferation) were observed in MPPs derived from CAMT iPSCs. These results suggest that MPL deficiency has a significant effect on apoptosis among progenitors. *P < 0.05.