Abstract
Megakaryocytes (MKs), rare hematopoietic cells in adult bone marrow, produce platelets that are critical to vascular hemostasis and wound healing. Ex vivo generation of MKs from human induced pluripotent stem cells (hiPSCs) provides a renewable cell source of platelets for treating thrombocytopenic patients and allows a better understanding of MK/platelet biology. The key requirements in this approach include developing a robust and consistent method for the production of functional progeny cells, such as MKs from hiPSCs, and minimizing risk and variation due to the animal-derived products in cell cultures. Here, we describe an efficient system to generate MKs from hiPSCs under a feeder-free and xeno-free condition, in which all the animal-derived products were eliminated. Several crucial reagents were evaluated and replaced with FDA-approved pharmacological reagents, including romiplostim (Nplate®, a thrombopoietin analog), Oprelvekin (recombinant IL-11), and Plasbumin (human albumin). This basic and defined differentiation system provides a platform for our future effort in investigation of regulatory factors and protocol optimization toward generating large numbers of platelets ex vivo.
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References
George JN. Platelets. Lancet. 2000;355(9214):1531–9.
Kaushansky K. The molecular mechanisms that control thrombopoiesis. J Clin Invest. 2005;115(12):3339–47.
Stroncek DF, Rebulla P. Platelet transfusions. Lancet. 2007;370(9585):427–38.
Tober J, et al. The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of definitive hematopoiesis. Blood. 2007;109(4):1433–41.
Shultz LD, et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol. 2005;174(10):6477–89.
Ramirez PA, Wagner JE, Brunstein CG. Going straight to the point: intra-BM injection of hematopoietic progenitors. Bone Marrow Transplant. 2010;45(7):1127–33.
Choi ES, et al. Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional. Blood. 1995;85(2):402–13.
de Sauvage FJ, et al. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature. 1994;369(6481):533–8.
Drachman JG, Griffin JD, Kaushansky K. The c-Mpl ligand (thrombopoietin) stimulates tyrosine phosphorylation of Jak2, Shc, and c-Mpl. J Biol Chem. 1995;270(10):4979–82.
Bunting S, et al. Normal platelets and megakaryocytes are produced in vivo in the absence of thrombopoietin. Blood. 1997;90(9):3423–9.
Fox N, et al. Thrombopoietin expands hematopoietic stem cells after transplantation. J Clin Invest. 2002;110(3):389–94.
Avecilla ST, et al. Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis. Nat Med. 2004;10(1):64–71.
Tian X, et al. Bioluminescent imaging demonstrates that transplanted human embryonic stem cell-derived CD34(+) cells preferentially develop into endothelial cells. Stem Cells. 2009;27(11):2675–85.
Takayama N, Eto K. In vitro generation of megakaryocytes and platelets from human embryonic stem cells and induced pluripotent stem cells. Methods Mol Biol. 2012;788:205–17.
Lu SJ, et al. Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice. Cell Res. 2011;21(3):530–45.
Takayama N, et al. Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells. J Exp Med. 2010;207(13):2817–30.
Gaur M, et al. Megakaryocytes derived from human embryonic stem cells: a genetically tractable system to study megakaryocytopoiesis and integrin function. J Thromb Haemost. 2006;4(2):436–42.
Pick M, et al. Generation of megakaryocytic progenitors from human embryonic stem cells in a feeder- and serum-free medium. PLoS ONE. 2013;8(2):e55530.
Ono Y, et al. Induction of functional platelets from mouse and human fibroblasts by p45NF-E2/Maf. Blood. 2012;120(18):3812–21.
Nakamura S, et al. Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells. Cell Stem Cell. 2014;14(4):535–48.
Ng ES, et al. Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood. 2005;106(5):1601–3.
Chen G, et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011;8(5):424–9.
Hulse WL, Gray J, Forbes RT. Evaluating the inter and intra batch variability of protein aggregation behaviour using Taylor dispersion analysis and dynamic light scattering. Int J Pharm. 2013;453(2):351–7.
Yahata T, et al. Functional human T lymphocyte development from cord blood CD34+ cells in nonobese diabetic/Shi-scid, IL-2 receptor gamma null mice. J Immunol. 2002;169(1):204–9.
Hiramatsu H, et al. Complete reconstitution of human lymphocytes from cord blood CD34+ cells using the NOD/SCID/gammacnull mice model. Blood. 2003;102(3):873–80.
Ye Z, et al. Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders. Blood. 2009;114(27):5473–80.
Chou BK, et al. Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures. Cell Res. 2011;21(3):518–29.
Chou BK, et al. A facile method to establish human induced pluripotent stem cells from adult blood cells under feeder-free and xeno-free culture conditions: a clinically compliant approach. Stem Cells Transl Med. 2015.
Civin CI, et al. Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo. Blood. 1996;88(11):4102–9.
Michelson AD. Flow cytometry: a clinical test of platelet function. Blood. 1996;87(12):4925–36.
Michelson AD, Furman MI. Laboratory markers of platelet activation and their clinical significance. Curr Opin Hematol. 1999;6(5):342–8.
Cheng L, et al. Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells. J Cell Physiol. 2000;184(1):58–69.
Himburg HA, et al. Pleiotrophin regulates the retention and self-renewal of hematopoietic stem cells in the bone marrow vascular niche. Cell Rep. 2012;2(4):964–75.
Acknowledgments
This work was partially supported in part by grants from NIH (U01 HL107446 and 2R01 HL-073781) and Maryland State Stem Research Cell Fund (2012-MSCRFII-0124).
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Li, Y., Wang, Y., Cheng, L., Wang, Z.Z. (2015). Derivation of Megakaryocytes and Platelets from Human Pluripotent Stem Cells. In: Cheng, T. (eds) Hematopoietic Differentiation of Human Pluripotent Stem Cells. SpringerBriefs in Stem Cells, vol 6. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-7312-6_3
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DOI: https://doi.org/10.1007/978-94-017-7312-6_3
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