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Whirling Platelets Away for Transfusion
Leading Edge
Previews Whirling Platelets Away for Transfusion Camelia Iancu-Rubin,1 Ronald Hoffman,1 and Anna Rita Migliaccio2,* 1Tisch
Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, USA of Biomedical and Neuromotorial Sciences, Alma Mater University, Bologna, Italy *Correspondence: [email protected] https://doi.org/10.1016/j.cell.2018.07.018 2Department
With a growing demand for platelet transfusions, large-scale ex vivo platelet production would alleviate the reliance on donors. Now, Ito et al. report that turbulence is an important physical regulator of platelet generation in vivo and can be exploited in a bioreactor to enable clinical scale production of functional platelets starting from human iPSCs. Platelet transfusions are an effective treatment for patients with thrombocytopenia. They are administered prophylactically or at the time of life threatening bleeding events to patients with aplastic anemia, immune causes of thrombocytopenia, sepsis, leukemia, and myelodysplatic syndromes. In addition, thrombocytopenia complicates the clinical course of patients with solid tumors or blood cancers receiving high-dose chemotherapy or those recovering from stem cell transplantations. The demand for platelet transfusions has steadily increased in recent years, straining a platelet supply that is already limited due to its dependency on volunteer donors, short shelf life (5 days), risk of infections, and refractoriness, which is the failure to correct platelet numbers following transfusion (Whitaker and Hinkins, 2011). Approximately 2.2 million platelet units, each containing 3 31011 platelets, are transfused annually in the US alone. This situation has provoked many laboratories to search for alternative sources including systems which allow for ex vivo platelet generation from primary hematopoietic stem/ progenitor cells (HSC/HPCs), embryonic stem cells (hESC), or induced pluripotent stem cells (iPSC) (Avanzi and Mitchell, 2014). Eto and colleagues have previously developed immortalized megakaryocyte cell lines (imMKCL) from iPSC which are capable of differentiating into megakaryocyte progenitors (Nakamura et al., 2014). Despite their ability to expand and mature into megakaryocytes capable of generating functional platelets, the numbers of platelets derived from these progenitors were too low to be clinically useful. For reasons elegantly reviewed elsewhere, the generation of clinically relevant
numbers of platelets ex vivo has represented one of the major obstacles to platelet manufacturing (Lambert et al., 2013). In this issue of Cell, Ito et al. address this obstacle by identifying novel physical and molecular factors which enable large-scale ex vivo platelet production from imMKCL (Ito et al., 2018). The authors built on their previous development of imMKCL by optimizing a two-step culture system which maximized the number of megakaryocyte progenitors generated, enhanced their ability to mature and eliminated the need for mouse feeder cells, a prerequisite in generating clinical products free of xenoantigens. In the past decade, this group and others have made tremendous strides by developing various microfluidic bioreactors to support platelet generation from various megakaryocyte culture systems. Bioreactors have been designed to model flow-dependent physical forces (i.e., shear stress), soluble factors, and cell-to-cell interactions, as well as components of the extracellular matrix and its stiffness, which are known to contribute to human thrombopoiesis in vivo (Thon et al., 2017). Until now, none of these systems has been able to generate sufficient platelet numbers for transfusion. In order to overcome these limitations, Ito et al. explored whether yet undiscovered dynamic factors might be required for platelet production in vivo. The authors imaged murine bone marrow in vivo by two-photon microscopy and particle image velocimetry and found turbulence as a critical force driving platelets shedding megakaryocytes. Turbulence is one of the physical components governing blood dynamics but unlike the laminar (smooth) flow, turbulence has an
irregular path creating small whirlpools. Ito et. al. made a critical observation: turbulent forces were present around proplatelet-bearing megakaryocytes (i.e., actively producing platelets) but not around resting megakaryocytes, suggesting that these whirling forces could enable platelet release. With this critical information in hand, they simulated agitated flow systems in various devices and established the optimal physical parameters (i.e., shear stress, strain rate, vorticity, turbulent energy, and energy dissipation), which were replicated into a liquid culture bioreactor named VerMES. In a series of elegant experiments, they scaled up imMKCL-derived cultures in the new VerMES bioreactor (i.e., from 0.3 to 2.4 to 8L) and demonstrated successful generation of 1-1.3 3 1011 platelets that possessed in vitro and in vivo functional properties similar to those of donorderived platelets. This value is comparable to the minimal clinical dose of 1.1-31011-1.3-31011platelets/m2 of body surface area (Kumar et al., 2015) which for an average-sized adult patient is 1.6-1.9 m2. Thus, by introducing turbulence as new physical parameter, the authors were able to generate near-clinical scale numbers of platelets. Remarkably, Ito et al. discovered that turbulence induces cultured megakaryocytes to secrete several soluble factors including insulin factor binding protein 2 (IGFBP2), macrophage migration inhibitory factor (MIF), and nardilysin (NRDC). These factors enhanced ex vivo platelet shedding, both in the microfluidic and turbulence VerMES systems. The authors found that NRDC, a zinc-dependent endopeptidase, is a novel thrombopoietic factor which plays a dual role: first, at the
Cell 174, July 26, 2018 ª 2018 Elsevier Inc. 503
intracellular level by interacting with HDAC6 and tubulin thereby influencing platelet biogenesis; second, at the extracellular level by facilitating platelet shedding during shear stress. These observations warrant future studies which should clarify if such events are also relevant for primary megakaryocyte and in vivo thrombopoiesis. This study represents a milestone in addressing scalability as the major concern in the field of ex vivo platelet manufacturing. Several advantages further support the use of iPSCs as a source for platelet production including virtually unlimited supply, cryopreservability of imMKCLs, and the potential for creating universal HLA null platelets to address immune complications. The fact that nucleus-free final products are amenable to irradiation eliminates the legitimate concerns associated with the tumorigenic potential of iPSC. Equally important, however, are the challenges that have yet to be faced in order for platelets made using this technology to become a viable choice for transfusion. In fact, when compared to donor-derived platelets, platelets derived from the three imMKCL lines varied in size, level of baseline activation, and survival in vivo. Therefore, efforts should be made to identify optimal sources for future clinical development. While it is anticipated that these concerns might eventually be addressed, imMKCL-generated platelet products will encounter identical limitations as donorderived platelets: they require storage at room temperature and have short lifespan. These issues are compounded by
504 Cell 174, July 26, 2018
the long production time (26 days), which will not solve immediate platelet demand in the case of emergencies. Another significant issue is the production cost under Good Manufacturing Procedures as compared to a cost of $500-600 for an apheresis unit in the US. Alternative approaches are being developed to generate transfusable megakaryocyte cell product (Fong et al., 2016; Wang et al., 2015), which might be more practical. These latter approaches do not encounter the technological difficulties associated with ex vivo generation of platelets because they use the lungs and/or bone marrow as the body’s own ‘‘bioreactors’’ to replenish platelet numbers from infused megakaryocytes in thrombocytopenic patients. The path to the clinic for such megakaryocyte cell products might be shorter than that of imMKCL-generated platelets as their safety and tolerability have been already demonstrated in Phase I trials (Xi et al., 2013). In summary, the study by Ito et al. advances our understanding of thrombopoiesis by discovering new physical and biological factors responsible for platelet release and stimulates the race to the clinic for megakaryocyte- and/or platelet-based cell products as alternatives to donor-based platelet transfusion. REFERENCES Avanzi, M.P., and Mitchell, W.B. (2014). Ex vivo production of platelets from stem cells. Br. J. Haematol. 165, 237–247. Fong, H., Mosoyan, G., Patel, A., Hoffman, R., Tong, J., and Iancu-Rubin, C. (2016). Preclinical
Development of a Cryopreservable Megakaryocyte Cell Product from Cord Blood Derived Hematopoietic Stem Cells. Blood 128, 3859. Ito, Y., Nakamura, S., Tomohiro, S., Naoshi, S., Kato, Y., Ohno, M., Sakuma, S., Ito, K., Kumon, H., Hirose, H., et al. (2018). Turbulence activates platelet biogenesis to enable clinical scale manufacturing from human iPS cells. Cell 174, this issue, 636–648. Kumar, A., Mhaskar, R., Grossman, B.J., Kaufman, R.M., Tobian, A.A., Kleinman, S., Gernsheimer, T., Tinmouth, A.T., and Djulbegovic, B.; AABB Platelet Transfusion Guidelines Panel (2015). Platelet transfusion: a systematic review of the clinical evidence. Transfusion 55, 1116–1127, quiz 1115. Lambert, M.P., Sullivan, S.K., Fuentes, R., French, D.L., and Poncz, M. (2013). Challenges and promises for the development of donor-independent platelet transfusions. Blood 121, 3319–3324. Nakamura, S., Takayama, N., Hirata, S., Seo, H., Endo, H., Ochi, K., Fujita, K., Koike, T., Harimoto, K., Dohda, T., et al. (2014). Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells. Cell Stem Cell 14, 535–548. Thon, J.N., Dykstra, B.J., and Beaulieu, L.M. (2017). Platelet bioreactor: accelerated evolution of design and manufacture. Platelets 28, 472–477. Wang, Y., Hayes, V., Jarocha, D., Sim, X., Harper, D.C., Fuentes, R., Sullivan, S.K., Gadue, P., Chou, S.T., Torok-Storb, B.J., et al. (2015). Comparative analysis of human ex vivo-generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale. Blood 125, 3627–3636. Whitaker, B.I., and Hinkins, S. (2011). The 2011 National Blood Collection and Utilization Survey Report (Washington, DC, USA: The United States Department of Health and Human Services). Xi, J., Zhu, H., Liu, D., Nan, X., Zheng, W., Liu, K., Shi, W., Chen, L., Lv, Y., Yan, F., et al. (2013). Infusion of megakaryocytic progenitor products generated from cord blood hematopoietic stem/progenitor cells: results of the phase 1 study. PLoS ONE 8, e54941.
Previews Whirling Platelets Away for Transfusion Camelia Iancu-Rubin,1 Ronald Hoffman,1 and Anna Rita Migliaccio2,* 1Tisch
Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, USA of Biomedical and Neuromotorial Sciences, Alma Mater University, Bologna, Italy *Correspondence: [email protected] https://doi.org/10.1016/j.cell.2018.07.018 2Department
With a growing demand for platelet transfusions, large-scale ex vivo platelet production would alleviate the reliance on donors. Now, Ito et al. report that turbulence is an important physical regulator of platelet generation in vivo and can be exploited in a bioreactor to enable clinical scale production of functional platelets starting from human iPSCs. Platelet transfusions are an effective treatment for patients with thrombocytopenia. They are administered prophylactically or at the time of life threatening bleeding events to patients with aplastic anemia, immune causes of thrombocytopenia, sepsis, leukemia, and myelodysplatic syndromes. In addition, thrombocytopenia complicates the clinical course of patients with solid tumors or blood cancers receiving high-dose chemotherapy or those recovering from stem cell transplantations. The demand for platelet transfusions has steadily increased in recent years, straining a platelet supply that is already limited due to its dependency on volunteer donors, short shelf life (5 days), risk of infections, and refractoriness, which is the failure to correct platelet numbers following transfusion (Whitaker and Hinkins, 2011). Approximately 2.2 million platelet units, each containing 3 31011 platelets, are transfused annually in the US alone. This situation has provoked many laboratories to search for alternative sources including systems which allow for ex vivo platelet generation from primary hematopoietic stem/ progenitor cells (HSC/HPCs), embryonic stem cells (hESC), or induced pluripotent stem cells (iPSC) (Avanzi and Mitchell, 2014). Eto and colleagues have previously developed immortalized megakaryocyte cell lines (imMKCL) from iPSC which are capable of differentiating into megakaryocyte progenitors (Nakamura et al., 2014). Despite their ability to expand and mature into megakaryocytes capable of generating functional platelets, the numbers of platelets derived from these progenitors were too low to be clinically useful. For reasons elegantly reviewed elsewhere, the generation of clinically relevant
numbers of platelets ex vivo has represented one of the major obstacles to platelet manufacturing (Lambert et al., 2013). In this issue of Cell, Ito et al. address this obstacle by identifying novel physical and molecular factors which enable large-scale ex vivo platelet production from imMKCL (Ito et al., 2018). The authors built on their previous development of imMKCL by optimizing a two-step culture system which maximized the number of megakaryocyte progenitors generated, enhanced their ability to mature and eliminated the need for mouse feeder cells, a prerequisite in generating clinical products free of xenoantigens. In the past decade, this group and others have made tremendous strides by developing various microfluidic bioreactors to support platelet generation from various megakaryocyte culture systems. Bioreactors have been designed to model flow-dependent physical forces (i.e., shear stress), soluble factors, and cell-to-cell interactions, as well as components of the extracellular matrix and its stiffness, which are known to contribute to human thrombopoiesis in vivo (Thon et al., 2017). Until now, none of these systems has been able to generate sufficient platelet numbers for transfusion. In order to overcome these limitations, Ito et al. explored whether yet undiscovered dynamic factors might be required for platelet production in vivo. The authors imaged murine bone marrow in vivo by two-photon microscopy and particle image velocimetry and found turbulence as a critical force driving platelets shedding megakaryocytes. Turbulence is one of the physical components governing blood dynamics but unlike the laminar (smooth) flow, turbulence has an
irregular path creating small whirlpools. Ito et. al. made a critical observation: turbulent forces were present around proplatelet-bearing megakaryocytes (i.e., actively producing platelets) but not around resting megakaryocytes, suggesting that these whirling forces could enable platelet release. With this critical information in hand, they simulated agitated flow systems in various devices and established the optimal physical parameters (i.e., shear stress, strain rate, vorticity, turbulent energy, and energy dissipation), which were replicated into a liquid culture bioreactor named VerMES. In a series of elegant experiments, they scaled up imMKCL-derived cultures in the new VerMES bioreactor (i.e., from 0.3 to 2.4 to 8L) and demonstrated successful generation of 1-1.3 3 1011 platelets that possessed in vitro and in vivo functional properties similar to those of donorderived platelets. This value is comparable to the minimal clinical dose of 1.1-31011-1.3-31011platelets/m2 of body surface area (Kumar et al., 2015) which for an average-sized adult patient is 1.6-1.9 m2. Thus, by introducing turbulence as new physical parameter, the authors were able to generate near-clinical scale numbers of platelets. Remarkably, Ito et al. discovered that turbulence induces cultured megakaryocytes to secrete several soluble factors including insulin factor binding protein 2 (IGFBP2), macrophage migration inhibitory factor (MIF), and nardilysin (NRDC). These factors enhanced ex vivo platelet shedding, both in the microfluidic and turbulence VerMES systems. The authors found that NRDC, a zinc-dependent endopeptidase, is a novel thrombopoietic factor which plays a dual role: first, at the
Cell 174, July 26, 2018 ª 2018 Elsevier Inc. 503
intracellular level by interacting with HDAC6 and tubulin thereby influencing platelet biogenesis; second, at the extracellular level by facilitating platelet shedding during shear stress. These observations warrant future studies which should clarify if such events are also relevant for primary megakaryocyte and in vivo thrombopoiesis. This study represents a milestone in addressing scalability as the major concern in the field of ex vivo platelet manufacturing. Several advantages further support the use of iPSCs as a source for platelet production including virtually unlimited supply, cryopreservability of imMKCLs, and the potential for creating universal HLA null platelets to address immune complications. The fact that nucleus-free final products are amenable to irradiation eliminates the legitimate concerns associated with the tumorigenic potential of iPSC. Equally important, however, are the challenges that have yet to be faced in order for platelets made using this technology to become a viable choice for transfusion. In fact, when compared to donor-derived platelets, platelets derived from the three imMKCL lines varied in size, level of baseline activation, and survival in vivo. Therefore, efforts should be made to identify optimal sources for future clinical development. While it is anticipated that these concerns might eventually be addressed, imMKCL-generated platelet products will encounter identical limitations as donorderived platelets: they require storage at room temperature and have short lifespan. These issues are compounded by
504 Cell 174, July 26, 2018
the long production time (26 days), which will not solve immediate platelet demand in the case of emergencies. Another significant issue is the production cost under Good Manufacturing Procedures as compared to a cost of $500-600 for an apheresis unit in the US. Alternative approaches are being developed to generate transfusable megakaryocyte cell product (Fong et al., 2016; Wang et al., 2015), which might be more practical. These latter approaches do not encounter the technological difficulties associated with ex vivo generation of platelets because they use the lungs and/or bone marrow as the body’s own ‘‘bioreactors’’ to replenish platelet numbers from infused megakaryocytes in thrombocytopenic patients. The path to the clinic for such megakaryocyte cell products might be shorter than that of imMKCL-generated platelets as their safety and tolerability have been already demonstrated in Phase I trials (Xi et al., 2013). In summary, the study by Ito et al. advances our understanding of thrombopoiesis by discovering new physical and biological factors responsible for platelet release and stimulates the race to the clinic for megakaryocyte- and/or platelet-based cell products as alternatives to donor-based platelet transfusion. REFERENCES Avanzi, M.P., and Mitchell, W.B. (2014). Ex vivo production of platelets from stem cells. Br. J. Haematol. 165, 237–247. Fong, H., Mosoyan, G., Patel, A., Hoffman, R., Tong, J., and Iancu-Rubin, C. (2016). Preclinical
Development of a Cryopreservable Megakaryocyte Cell Product from Cord Blood Derived Hematopoietic Stem Cells. Blood 128, 3859. Ito, Y., Nakamura, S., Tomohiro, S., Naoshi, S., Kato, Y., Ohno, M., Sakuma, S., Ito, K., Kumon, H., Hirose, H., et al. (2018). Turbulence activates platelet biogenesis to enable clinical scale manufacturing from human iPS cells. Cell 174, this issue, 636–648. Kumar, A., Mhaskar, R., Grossman, B.J., Kaufman, R.M., Tobian, A.A., Kleinman, S., Gernsheimer, T., Tinmouth, A.T., and Djulbegovic, B.; AABB Platelet Transfusion Guidelines Panel (2015). Platelet transfusion: a systematic review of the clinical evidence. Transfusion 55, 1116–1127, quiz 1115. Lambert, M.P., Sullivan, S.K., Fuentes, R., French, D.L., and Poncz, M. (2013). Challenges and promises for the development of donor-independent platelet transfusions. Blood 121, 3319–3324. Nakamura, S., Takayama, N., Hirata, S., Seo, H., Endo, H., Ochi, K., Fujita, K., Koike, T., Harimoto, K., Dohda, T., et al. (2014). Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells. Cell Stem Cell 14, 535–548. Thon, J.N., Dykstra, B.J., and Beaulieu, L.M. (2017). Platelet bioreactor: accelerated evolution of design and manufacture. Platelets 28, 472–477. Wang, Y., Hayes, V., Jarocha, D., Sim, X., Harper, D.C., Fuentes, R., Sullivan, S.K., Gadue, P., Chou, S.T., Torok-Storb, B.J., et al. (2015). Comparative analysis of human ex vivo-generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale. Blood 125, 3627–3636. Whitaker, B.I., and Hinkins, S. (2011). The 2011 National Blood Collection and Utilization Survey Report (Washington, DC, USA: The United States Department of Health and Human Services). Xi, J., Zhu, H., Liu, D., Nan, X., Zheng, W., Liu, K., Shi, W., Chen, L., Lv, Y., Yan, F., et al. (2013). Infusion of megakaryocytic progenitor products generated from cord blood hematopoietic stem/progenitor cells: results of the phase 1 study. PLoS ONE 8, e54941.