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 6

FLI1-mediated MK/erythrocyte differentiation differed between normal and CAMT iPSCs.

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FLI1-mediated MK/erythrocyte differentiation differed between normal and...
Shown are results for MK and erythrocyte generation from CD34+ HPCs on day 22. (A) Percent CD41+GPA– MKs and CD41–GPA+ erythrocytes derived from normal iPSCs (left) or ESCs (right) and transduced with vehicle or MPL expression vector in the presence of 0, 10, or 100 ng/ml TPO with SCF and EPO. Exogenous MPL expression was assessed based on EGFP fluorescence intensity. Erythropoiesis was enhanced in a TPO/MPL signaling–dependent manner. (B and C) FLI1 expression in CD34+ HPCs before (C) and after (B) differentiation. EGFPhi CD34+ HPCs derived from normal iPSCs and ESCs with or without MPL overexpression, or EGFPlo or EGFPhi CD34+ HPCs from CAMT iPSCs overexpressing MPL, were sorted and cultivated for an additional 8 days. (D) Percent MK and erythrocyte differentiation from CD34+ HPCs derived from normal iPSCs or ESCs overexpressing EGFP alone (vehicle) or FLI1 plus EGFP. (E) Percent MK and erythrocyte differentiation from normal iPSCs or ESCs overexpressing MPL-EGFP and FLI1-RFP. CD34+ HPCs derived from normal iPSCs or ESCs overexpressing MPL were transfected with FLI1-RFP or vector and cultivated for an additional 8 days. Exogenous FLI1 expression was assessed based on RFP expression (shown in the contour plot). FLI1 overexpression attenuated erythrocyte-biased differentiation in normal iPSCs and ESCs. *P < 0.05.