Contributions
Abstract: S143
Type: Oral Presentation
Presentation during EHA23: On Friday, June 15, 2018 from 12:30 - 12:45
Location: Room A9
Background
Transfusion of donor-derived platelets is commonly employed to treat patients with thrombocytopenia, while current supply system confronts challenges including limited donors, short shelf life of platelet products, the risk of bacterial and viral contamination and transfusion refractoriness. These limitations have motivated the development of alternative donor-independent platelet sources to meet the growing need of clinical transfusion. Human induced pluripotent stem cells (iPSCs) have been proposed as a potential source of megakaryocytes (MKs) and platelets to meet the demands of clinical transfusion.
Aims
Our group previously established immortalized megakaryocyte cell lines (imMKCLs) derived from iPSCs as cryopreservable master cells that can be robustly expanded and then produce platelets upon maturation (Nakamura et al, Cell Stem Cell, 2014). However, the low platelet yield and requirement of mouse somatic feeder cells are the major hurdles that must be overcome before clinical realization. This study aimed to efficiently identify small molecules that could facilitate in vitro platelet generation from imMKCLs under the feeder-free culture condition.
Methods
We herein established a genetically modified imMKCL that express the β1-tubulin-Venus reporter to identify optimal feeder-free culture condition by monitoring in vitro MK maturation in a high throughput manner. The Venus transgene was inserted downstream of the TUBB1 locus in imMKCLs using CRISPR/Cas9 technology, and the expression was visualized by Venus fluorescence intensity. The imMKCL reporter line enabled visible real-time studies under a confocal quantitative image system. This reporter line was then employed for a high throughput screening (HTS) to identify compounds that significantly improved the efficiency of platelet production.
Results
The HTS of five thousand compounds successfully identified several compounds from diverse categories (i.e. a WNT inhibitor and a FLT3 inhibitor) that facilitate in vitro MK maturation and platelet release from imMKCLs under feeder-free conditions. The in vitro thrombopoietic effects of candidate compounds was also confirmed in two common MK lineages, one was cord blood derived CD34+ cells and another was iPSC directly derived MKs. In addition, candidate compounds improved in vivo recovery of thrombopoiesis in two mouse thrombocytopenia models, which were induced by irradiation or anti-GPIbα antibody administration. Given that WNT and FLT3 inhibitors are used to treat leukemia and cancers, that often manifest thrombocytopenia due to the diseases themself or as a result of chemotherapy. This finding may also facilitate clinical settings of cancer therapy.
Interestingly, according to a luciferase aryl hydrocarbon receptor (AhR) reporter gene assay, most of these compounds, including a WNT signaling inhibitor, facilitated thrombopoiesis by antagonizing AhR signaling, in accordance with the crucial role of AhR inhibition in MK maturation reported by some groups. Others promoted thrombopoiesis independently of AhR signaling.
Conclusion
In summary, we have established a comprehensive, real-time monitoring system for MK maturation based on β1-tubulin expression, which thereby allows to identify novel small molecule inducers of MK maturation and platelet production through HTS as well as to perform more in depth studies of thrombopoiesis.
Session topic: 33. Platelets disorders
Keyword(s): Megakaryocyte, Platelet, thrombopoiesis
Abstract: S143
Type: Oral Presentation
Presentation during EHA23: On Friday, June 15, 2018 from 12:30 - 12:45
Location: Room A9
Background
Transfusion of donor-derived platelets is commonly employed to treat patients with thrombocytopenia, while current supply system confronts challenges including limited donors, short shelf life of platelet products, the risk of bacterial and viral contamination and transfusion refractoriness. These limitations have motivated the development of alternative donor-independent platelet sources to meet the growing need of clinical transfusion. Human induced pluripotent stem cells (iPSCs) have been proposed as a potential source of megakaryocytes (MKs) and platelets to meet the demands of clinical transfusion.
Aims
Our group previously established immortalized megakaryocyte cell lines (imMKCLs) derived from iPSCs as cryopreservable master cells that can be robustly expanded and then produce platelets upon maturation (Nakamura et al, Cell Stem Cell, 2014). However, the low platelet yield and requirement of mouse somatic feeder cells are the major hurdles that must be overcome before clinical realization. This study aimed to efficiently identify small molecules that could facilitate in vitro platelet generation from imMKCLs under the feeder-free culture condition.
Methods
We herein established a genetically modified imMKCL that express the β1-tubulin-Venus reporter to identify optimal feeder-free culture condition by monitoring in vitro MK maturation in a high throughput manner. The Venus transgene was inserted downstream of the TUBB1 locus in imMKCLs using CRISPR/Cas9 technology, and the expression was visualized by Venus fluorescence intensity. The imMKCL reporter line enabled visible real-time studies under a confocal quantitative image system. This reporter line was then employed for a high throughput screening (HTS) to identify compounds that significantly improved the efficiency of platelet production.
Results
The HTS of five thousand compounds successfully identified several compounds from diverse categories (i.e. a WNT inhibitor and a FLT3 inhibitor) that facilitate in vitro MK maturation and platelet release from imMKCLs under feeder-free conditions. The in vitro thrombopoietic effects of candidate compounds was also confirmed in two common MK lineages, one was cord blood derived CD34+ cells and another was iPSC directly derived MKs. In addition, candidate compounds improved in vivo recovery of thrombopoiesis in two mouse thrombocytopenia models, which were induced by irradiation or anti-GPIbα antibody administration. Given that WNT and FLT3 inhibitors are used to treat leukemia and cancers, that often manifest thrombocytopenia due to the diseases themself or as a result of chemotherapy. This finding may also facilitate clinical settings of cancer therapy.
Interestingly, according to a luciferase aryl hydrocarbon receptor (AhR) reporter gene assay, most of these compounds, including a WNT signaling inhibitor, facilitated thrombopoiesis by antagonizing AhR signaling, in accordance with the crucial role of AhR inhibition in MK maturation reported by some groups. Others promoted thrombopoiesis independently of AhR signaling.
Conclusion
In summary, we have established a comprehensive, real-time monitoring system for MK maturation based on β1-tubulin expression, which thereby allows to identify novel small molecule inducers of MK maturation and platelet production through HTS as well as to perform more in depth studies of thrombopoiesis.
Session topic: 33. Platelets disorders
Keyword(s): Megakaryocyte, Platelet, thrombopoiesis