Cell Stem Cell
In Translation Personalized Platelet Transfusions: One Step Closer to the Clinic Ji-Yoon Noh,1 Mitchell J. Weiss,1,2 and Mortimer Poncz1,2,* 1Division
of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2014.03.012 2Department
In this issue of Cell Stem Cell, Nakamura et al. (2014) use human pluripotent stem cells to generate immortalized megakaryocyte progenitor cell lines that can be cryopreserved, expanded in vitro, and induced to undergo terminal maturation and platelet release. This strategy advances efforts to produce clinically relevant numbers of tissue-culture-derived platelets for transfusion therapies. Platelets are a central component of the blood clotting system. Many diseases and modern medical treatments cause reduced platelet numbers (thrombocytopenia), so demands for transfusions of these cells are on the rise. Current human-donor-derived platelets are limited in supply, have a short shelf life, and present infectious risks related to donor transmission or the requirement for room temperature storage, which promotes bacterial proliferation. In vitro hematopoietic differentiation of human pluripotent stem cells (PSCs), including induced pluripotent stem cells and embryonic stem cells, represents a potential new source of megakaryocytes (Mks) and platelets for transfusion therapies. In principle, it should be possible to manufacture unlimited PSC-derived supplies of platelets with robust biological activity and a long circulating half-life. Two challenges exist in this field, both of which are addressed in this issue of Cell Stem Cell by Nakamura et al. (2014): (1) the generation of Mks and their platelet progeny from pluripotent stem cells is inefficient, and (2) the biological activities of such platelets have not been well tested or compared to standard donor platelets. Previous efforts to optimize Mk yield from PSCs have had limited success (Lu et al., 2011; Takayama et al., 2008). In the current study, Nakamura et al. (2014) developed a novel approach based on their prior observation that overexpression of the MYC proto-oncogene via retroviral transduction expands PSC-derived Mk progenitors (Takayama et al., 2010). They hypothesized that subsequent suppression of the MYC transgene would trigger maturation of the expanded Mks with
platelet release. To test this, the authors used a doxycycline-regulated promoter to express a chimeric version of MYC fused to a ligand-dependent destabilization domain whose activity is blocked by the small molecule Shield-1 (Banaszynski et al., 2006). Thus, addition of doxycycline and Shield-1 enhanced MYC expression, while removal of these drugs produced the opposite effect, allowing for precise control of MYC protein levels. The authors combined this strategy with doxycyclineregulated coexpression of BMI1, a polycomb complex component, that represses MYC-regulated apoptotic factors INK4A/ ARF (Oguro et al., 2006). This combination of ectopically expressed transcription factors stimulated Mk precursor proliferation for up to 8 weeks with terminal differentiation and platelet release ensuing after withdrawal of doxycycline and Shield-1. To suppress cell death associated with caspase activation, the authors coexpressed the anti-apoptotic protein BCLXL along with doxycycline-regulated MYC and BMI1. Two resultant clones, described as immortalized Mk cell lines (imMKCL), exhibited increased longevity with continuous expansion in culture for up to 20 weeks. Upon suppression of the immortalizing genes under standard culture conditions, imMKCs matured into Mks that released platelet-like particles resembling endogenous platelets in many respects, including in ultrastructure, surface antigen expression, activation upon exposure to the agonist thrombin, and binding to immobilized von Willebrand factor in a flow chamber system. Moreover, imMKCL-derived platelets bound laser-injured blood vessel walls and contributed to blood clots after
intravenous injection into immunodeficient mice that were irradiated to reduce endogenous platelet counts. Of particular importance for producing large numbers of standardized cells for clinical applications, imMKCLs could be cryopreserved and then reestablished with retained proliferation and differentiation potential. Overall, this study advances the field by showing that carefully titrated, reversible modulation of transcriptional pathways can arrest PSC-derived Mk progenitors for expansion and subsequent manipulation to form platelets. Potential medical applications include use of human PSCderived platelets to treat thrombocytopenia and engineering these platelets to deliver bioactive proteins to sites of blood vessel injury for enhancing or inhibiting clot formation (Neyman et al., 2008). Like all interesting studies, this one identifies new future challenges; in this instance, they’re related to optimizing the yield and quality of imMKCL-derived platelets for clinical utility. For example, the imMKCLs released 3–10 platelets per cell, which is similar to reported yields from human-PSC-derived Mks (Lu et al., 2011), but this is in sharp contrast to normal endogenous Mks, which release 2,000–10,000 platelets in vivo (Kaufman et al., 1965). The authors calculate that imMKCLs cultured according to current methods could theoretically generate 1011 platelets (one donor unit) within 5 days with 25–50 l of medium. This represents a major advance in PSC-derived platelet yields, but it remains prohibitively expensive compared to the cost of human donor platelets. Moreover, the imMKCLderived platelets were predominantly smaller, contained fewer granules, and
Cell Stem Cell 14, April 3, 2014 ª2014 Elsevier Inc. 425
Cell Stem Cell
In Translation showed increased baseline activation and reduced response to agonists in vitro compared to normal donor-derived platelets. These differences may reflect aberrant megakaryopoiesis caused by ectopic expression of MYC, BMI1, and BCL-XL, or alternatively, they may be intrinsic to the embryonic nature of PSC-derived Mks/ platelets, which exhibit distinct properties compared to those formed postnatally (Tober et al., 2007). Thus, innovative new in vitro differentiation strategies for PSCs that more accurately reproduce adult-bone-marrow-type hematopoiesis may enhance the yield and functionality of Mk/platelet progeny. Additionally, culture adaptations to more closely resemble the hydrodynamic conditions and cellular microenvironment of normal Mks may improve the quantity and quality of imMKCL-derived platelets. In this regard, an in vitro microfluidic system described by their group (Nakagawa et al., 2013) may be useful.
In conclusion, the current study by Nakamura et al. represents an important advance toward generating large numbers of PSC-derived platelets and creates a new standard to judge future approaches. The next major challenge for supplanting human-donor-derived platelets with those synthesized ex vivo are to improve yields even further and generate a product whose intrinsic properties—including size, life span, reactivity, and hemostatic properties—more closely resemble those of the cells that we wish to replace. REFERENCES Banaszynski, L.A., Chen, L.C., Maynard-Smith, L.A., Ooi, A.G., and Wandless, T.J. (2006). Cell 126, 995–1004. Kaufman, R.M., Airo, R., Pollack, S., and Crosby, W.H. (1965). Blood 26, 720–731. Lu, S.J., Li, F., Yin, H., Feng, Q., Kimbrel, E.A., Hahm, E., Thon, J.N., Wang, W., Italiano, J.E.,
426 Cell Stem Cell 14, April 3, 2014 ª2014 Elsevier Inc.
Cho, J., and Lanza, R. (2011). Cell Res. 21, 530–545. Nakagawa, Y., Nakamura, S., Nakajima, M., Endo, H., Dohda, T., Takayama, N., Nakauchi, H., Arai, F., Fukuda, T., and Eto, K. (2013). Exp. Hematol. 41, 742–748. Nakamura, S., Takayama, N., Hirata, S., Seo, H., Endo, H., Ochi, K., Fujita, K.I., Koike, T., Harimoto, K.I., Dohda, T., et al. (2014). Cell Stem Cell 14, this issue, 535–548. Neyman, M., Gewirtz, J., and Poncz, M. (2008). Blood 112, 1101–1108. Oguro, H., Iwama, A., Morita, Y., Kamijo, T., van Lohuizen, M., and Nakauchi, H. (2006). J. Exp. Med. 203, 2247–2253. Takayama, N., Nishikii, H., Usui, J., Tsukui, H., Sawaguchi, A., Hiroyama, T., Eto, K., and Nakauchi, H. (2008). Blood 111, 5298–5306. Takayama, N., Nishimura, S., Nakamura, S., Shimizu, T., Ohnishi, R., Endo, H., Yamaguchi, T., Otsu, M., Nishimura, K., Nakanishi, M., et al. (2010). J. Exp. Med. 207, 2817–2830. Tober, J., Koniski, A., McGrath, K.E., Vemishetti, R., Emerson, R., de Mesy-Bentley, K.K., Waugh, R., and Palis, J. (2007). Blood 109, 1433–1441.
In Translation Personalized Platelet Transfusions: One Step Closer to the Clinic Ji-Yoon Noh,1 Mitchell J. Weiss,1,2 and Mortimer Poncz1,2,* 1Division
of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2014.03.012 2Department
In this issue of Cell Stem Cell, Nakamura et al. (2014) use human pluripotent stem cells to generate immortalized megakaryocyte progenitor cell lines that can be cryopreserved, expanded in vitro, and induced to undergo terminal maturation and platelet release. This strategy advances efforts to produce clinically relevant numbers of tissue-culture-derived platelets for transfusion therapies. Platelets are a central component of the blood clotting system. Many diseases and modern medical treatments cause reduced platelet numbers (thrombocytopenia), so demands for transfusions of these cells are on the rise. Current human-donor-derived platelets are limited in supply, have a short shelf life, and present infectious risks related to donor transmission or the requirement for room temperature storage, which promotes bacterial proliferation. In vitro hematopoietic differentiation of human pluripotent stem cells (PSCs), including induced pluripotent stem cells and embryonic stem cells, represents a potential new source of megakaryocytes (Mks) and platelets for transfusion therapies. In principle, it should be possible to manufacture unlimited PSC-derived supplies of platelets with robust biological activity and a long circulating half-life. Two challenges exist in this field, both of which are addressed in this issue of Cell Stem Cell by Nakamura et al. (2014): (1) the generation of Mks and their platelet progeny from pluripotent stem cells is inefficient, and (2) the biological activities of such platelets have not been well tested or compared to standard donor platelets. Previous efforts to optimize Mk yield from PSCs have had limited success (Lu et al., 2011; Takayama et al., 2008). In the current study, Nakamura et al. (2014) developed a novel approach based on their prior observation that overexpression of the MYC proto-oncogene via retroviral transduction expands PSC-derived Mk progenitors (Takayama et al., 2010). They hypothesized that subsequent suppression of the MYC transgene would trigger maturation of the expanded Mks with
platelet release. To test this, the authors used a doxycycline-regulated promoter to express a chimeric version of MYC fused to a ligand-dependent destabilization domain whose activity is blocked by the small molecule Shield-1 (Banaszynski et al., 2006). Thus, addition of doxycycline and Shield-1 enhanced MYC expression, while removal of these drugs produced the opposite effect, allowing for precise control of MYC protein levels. The authors combined this strategy with doxycyclineregulated coexpression of BMI1, a polycomb complex component, that represses MYC-regulated apoptotic factors INK4A/ ARF (Oguro et al., 2006). This combination of ectopically expressed transcription factors stimulated Mk precursor proliferation for up to 8 weeks with terminal differentiation and platelet release ensuing after withdrawal of doxycycline and Shield-1. To suppress cell death associated with caspase activation, the authors coexpressed the anti-apoptotic protein BCLXL along with doxycycline-regulated MYC and BMI1. Two resultant clones, described as immortalized Mk cell lines (imMKCL), exhibited increased longevity with continuous expansion in culture for up to 20 weeks. Upon suppression of the immortalizing genes under standard culture conditions, imMKCs matured into Mks that released platelet-like particles resembling endogenous platelets in many respects, including in ultrastructure, surface antigen expression, activation upon exposure to the agonist thrombin, and binding to immobilized von Willebrand factor in a flow chamber system. Moreover, imMKCL-derived platelets bound laser-injured blood vessel walls and contributed to blood clots after
intravenous injection into immunodeficient mice that were irradiated to reduce endogenous platelet counts. Of particular importance for producing large numbers of standardized cells for clinical applications, imMKCLs could be cryopreserved and then reestablished with retained proliferation and differentiation potential. Overall, this study advances the field by showing that carefully titrated, reversible modulation of transcriptional pathways can arrest PSC-derived Mk progenitors for expansion and subsequent manipulation to form platelets. Potential medical applications include use of human PSCderived platelets to treat thrombocytopenia and engineering these platelets to deliver bioactive proteins to sites of blood vessel injury for enhancing or inhibiting clot formation (Neyman et al., 2008). Like all interesting studies, this one identifies new future challenges; in this instance, they’re related to optimizing the yield and quality of imMKCL-derived platelets for clinical utility. For example, the imMKCLs released 3–10 platelets per cell, which is similar to reported yields from human-PSC-derived Mks (Lu et al., 2011), but this is in sharp contrast to normal endogenous Mks, which release 2,000–10,000 platelets in vivo (Kaufman et al., 1965). The authors calculate that imMKCLs cultured according to current methods could theoretically generate 1011 platelets (one donor unit) within 5 days with 25–50 l of medium. This represents a major advance in PSC-derived platelet yields, but it remains prohibitively expensive compared to the cost of human donor platelets. Moreover, the imMKCLderived platelets were predominantly smaller, contained fewer granules, and
Cell Stem Cell 14, April 3, 2014 ª2014 Elsevier Inc. 425
Cell Stem Cell
In Translation showed increased baseline activation and reduced response to agonists in vitro compared to normal donor-derived platelets. These differences may reflect aberrant megakaryopoiesis caused by ectopic expression of MYC, BMI1, and BCL-XL, or alternatively, they may be intrinsic to the embryonic nature of PSC-derived Mks/ platelets, which exhibit distinct properties compared to those formed postnatally (Tober et al., 2007). Thus, innovative new in vitro differentiation strategies for PSCs that more accurately reproduce adult-bone-marrow-type hematopoiesis may enhance the yield and functionality of Mk/platelet progeny. Additionally, culture adaptations to more closely resemble the hydrodynamic conditions and cellular microenvironment of normal Mks may improve the quantity and quality of imMKCL-derived platelets. In this regard, an in vitro microfluidic system described by their group (Nakagawa et al., 2013) may be useful.
In conclusion, the current study by Nakamura et al. represents an important advance toward generating large numbers of PSC-derived platelets and creates a new standard to judge future approaches. The next major challenge for supplanting human-donor-derived platelets with those synthesized ex vivo are to improve yields even further and generate a product whose intrinsic properties—including size, life span, reactivity, and hemostatic properties—more closely resemble those of the cells that we wish to replace. REFERENCES Banaszynski, L.A., Chen, L.C., Maynard-Smith, L.A., Ooi, A.G., and Wandless, T.J. (2006). Cell 126, 995–1004. Kaufman, R.M., Airo, R., Pollack, S., and Crosby, W.H. (1965). Blood 26, 720–731. Lu, S.J., Li, F., Yin, H., Feng, Q., Kimbrel, E.A., Hahm, E., Thon, J.N., Wang, W., Italiano, J.E.,
426 Cell Stem Cell 14, April 3, 2014 ª2014 Elsevier Inc.
Cho, J., and Lanza, R. (2011). Cell Res. 21, 530–545. Nakagawa, Y., Nakamura, S., Nakajima, M., Endo, H., Dohda, T., Takayama, N., Nakauchi, H., Arai, F., Fukuda, T., and Eto, K. (2013). Exp. Hematol. 41, 742–748. Nakamura, S., Takayama, N., Hirata, S., Seo, H., Endo, H., Ochi, K., Fujita, K.I., Koike, T., Harimoto, K.I., Dohda, T., et al. (2014). Cell Stem Cell 14, this issue, 535–548. Neyman, M., Gewirtz, J., and Poncz, M. (2008). Blood 112, 1101–1108. Oguro, H., Iwama, A., Morita, Y., Kamijo, T., van Lohuizen, M., and Nakauchi, H. (2006). J. Exp. Med. 203, 2247–2253. Takayama, N., Nishikii, H., Usui, J., Tsukui, H., Sawaguchi, A., Hiroyama, T., Eto, K., and Nakauchi, H. (2008). Blood 111, 5298–5306. Takayama, N., Nishimura, S., Nakamura, S., Shimizu, T., Ohnishi, R., Endo, H., Yamaguchi, T., Otsu, M., Nishimura, K., Nakanishi, M., et al. (2010). J. Exp. Med. 207, 2817–2830. Tober, J., Koniski, A., McGrath, K.E., Vemishetti, R., Emerson, R., de Mesy-Bentley, K.K., Waugh, R., and Palis, J. (2007). Blood 109, 1433–1441.
