Congenital amegakaryocytic thrombocytopenia iPS cells exhibit defective MPL-mediated signaling
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
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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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 2

Disease phenotype of CAMT iPSCs was seemingly restored by MPL supplementation.

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Disease phenotype of CAMT iPSCs was seemingly restored by MPL supplement...
(A) In vitro differentiation protocol using CAMT iPSCs transduced with MPL. CAMT iPSCs were transduced using a pMY retroviral vector harboring EGFP and MPL or EGFP alone and then selected as EGFP+ populations. Using the indicated cytokines, CAMT iPSC–derived EGFP+ CD34+ HPCs overexpressing MPL were subjected to in vitro differentiation toward the MK or erythrocyte lineage or colony formation. Scale bar: 100 μm. (B and C) Numbers of MKs and platelets (B) cultured in SCF, TPO, and heparin for 8–10 days and erythrocytes (C) cultured with SCF (50 ng/ml), TPO (10 ng/ml), and EPO (6 U/ml) for 8 days. Cells were cultured without or with supplemental MPL to induce overexpression (O/E). (D) Colony-forming potential for myeloid lineage in normal iPSC–derived CD34+ HPCs, and CAMT iPSC–derived EGFP+ CD34+ HPCs with or without MPL supplementation, in MethoCult H4434 semisolid medium containing TPO (50 ng/ml), SCF, EPO, IL-3, and GM-CSF. M, macrophage; N, neutrophil; N+M, neutrophil and macrophage; E, erythrocyte. *P < 0.05.