Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34+ cells are functionally active in an ex vivo flow model of thrombosis

Abstract

Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34 ؉ cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 ؋ 10 3 /L). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagenrelated peptide and thrombin receptoractivating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ib␣ (GPIb␣) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo. (Blood. 2009;114:5044-5051) IntroductionHuman platelets are anucleated cells that not only play a crucial role in primary hemostasis and wound repair, but are also particularly important in pathologic conditions such as thrombosis, vascular remodeling, and inflammation. Platelets originate from megakaryocytes (MK) in the bone marrow (BM) by fragmentation of pseudopodial elongations called proplatelets in a process that consumes the entire cytoplasmic content and is tightly regulated by thrombopoietin. 1,2 Human megakaryopoiesis has been studied ex vivo by measuring colony-forming units (CFUs; eg, CFU-MK, CFU-granulocyte, erythrocyte, macrophage, megakaryocyte), 3,4 MK polyploidy state, 5,6 expression of MK markers, 7 and novel genes expressed during MK differentiation. [8][9][10] Unraveling molecular mechanisms involved in megakaryopoiesis and thrombopoiesis is particularly relevant in the light of thrombocytopenia and pancytopenia associated with widespread use of high-dose chemotherapy for treatment of most cancers, also occurring after stem cell transplantation. Therefore, there has been an increasing interest in generating human platelets from MK in culture as well as in developing animal models of human hematopoiesis.Human platelet production has been described from differentiation of CD34 ϩ progenitor cells, isolated from mobilized peripheral blood (PB) or umbilical cord blood (CB), cultured in medium with a cytokine mixture containing thrombopoietin. 5,[11][12][13][14][15] Such produced platelets are functional, as demonstrated in aggregation assays and by expression of P-selectin on the platelet surface or by activation of glycosylphosphatidylinosito...

Myosin-II inhibition and soft 2D matrix maximize multinucleation and cellular projections typical of platelet-producing megakaryocytes

“…S8E). Levels are similar to those reported for human platelets generated in NOD/SCID mice 2 wk after transplantation of CD34 + cells (31). The results thus indicate that transient ex vivo inhibition of NMM-II by the protocol here increases the number of functional human platelets.…”

“…Transplantation of uncultured human cord-blood CD34 + cells in immuno-deficient nonobese diabetic (NOD)/SCID mice has been shown previously to yield sustained generation of human platelets (31), but the intravenous delivery route used to date requires a large number of cells for homing and engraftment. Intrabone marrow transplantation was instead used here to more rapidly expose injected cells to the marrow microenvironment per Fig.…”

Model systems of genetically modified platelets

“…138 The nonobese diabetic/severe combined immunodeficiency mice (NOD.CB17-Prkdc scid /J or NOD/SCID) already allowed stable engraftment of human CD34 ϩ HSCs originating from either mobilized peripheral blood or umbilical cord blood. 95,139,140 The presence of all blood cell types in the bone marrow was confirmed; and although MK represented only a small fraction, successful platelet production was achieved. In addition, these platelets not only get activated in response to known agonists but also incorporated in a (predominantly) mouse thrombus on perfusion of transplanted mouse peripheral blood over a collagen-coated surface.…”

“…In addition, these platelets not only get activated in response to known agonists but also incorporated in a (predominantly) mouse thrombus on perfusion of transplanted mouse peripheral blood over a collagen-coated surface. 139 The current generation of immunodeficient mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ -or NSG) with virtual absence of natural killer cell activity already allows higher engraftment; and with numerous other immunodeficient mouse strains still under development, it is likely to see a novel strain with even better production of human platelets emerging in the ensuing years. 141 Combining this transplantation model with safer and improved delivery and genetic engineering systems can hopefully provide the research community with a small animal model in which transgenic human MKs and platelets can be successfully generated and used in either functional genomics studies, thus aiding in expanding our basic knowledge of platelets, their formation and behavior or as superior models of human pathologies that allow faster and better screening and testing of novel compounds acting to improve platelet production or survival, or to prevent bleeding or thrombosis.…”

Platelet gene therapy corrects the hemophilic phenotype in immunocompromised hemophilia A mice transplanted with genetically manipulated human cord blood stem cells

“…This is a common problem in studies of human thrombocytopoiesis in immunocompromised mouse models, and the cause is still unknown. [28][29][30][31] There was a small population that was positive for both human and mouse platelet markers, which could be due to low level platelet activation during sample processing resulting in human and mouse platelet association.…”