title: Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells.
abstract: Embryonic stem cells (ESCs) could potentially compensate for the lack of blood platelets available for use in transfusions. Here, we describe a new method for generating mouse ESC-derived platelets (ESPs) that can contribute to hemostasis in vivo. Flow cytometric sorting of cells from embryoid bodies on day 6 demonstrated that c-Kit(+) integrin alpha IIb (alpha IIb)(+) cells, but not CD31(+) cells or vascular endothelial cadherin(+) cells, are capable of megakaryopoiesis and the release of platelet-like structures by day 12. alpha IIb beta 3-expressing ESPs exhibited ectodomain shedding of glycoprotein (GP)Ibalpha, GPV, and GPVI, but not alpha IIb beta 3 or GPIb beta. ESPs showed impaired alpha IIb beta 3 activation and integrin-mediated actin reorganization, critical events for normal platelet function. However, the administration of metalloproteinase inhibitors GM6001 or TAPI-1 during differentiation increased the expression of GPIb alpha, improving both thrombogenesis in vitro and posttransfusion recovery in vivo. Thus, the regulation of metalloproteinases in culture could be useful for obtaining high-quality, efficacious ESPs as an alternative platelet source for transfusions.
authors.affiliation: Division of Stem Cell Therapy, Center for Stem Cell and Regenerative Medicine, The Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Nishikii
authors.forename: Koji
authors.initials: K
authors.lastname: Eto
authors.forename: Noriko
authors.initials: N
authors.lastname: Tamura
authors.forename: Koichi
authors.initials: K
authors.lastname: Hattori
authors.forename: Beate
authors.initials: B
authors.lastname: Heissig
authors.forename: Taisuke
authors.initials: T
authors.lastname: Kanaji
authors.forename: Akira
authors.initials: A
authors.lastname: Sawaguchi
authors.forename: Shinya
authors.initials: S
authors.lastname: Goto
authors.forename: Jerry
authors.initials: J
authors.lastname: Ware
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Nakauchi
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D066298:In Vitro Techniques; D039661:Integrin beta3 / Q000378:metabolism; D008533:Megakaryocytes / Q000166:cytology / Q000201:enzymology; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D034061:Thrombopoiesis / Q000187:drug effects / Q000502:physiology; D013927:Thrombosis / Q000209:etiology
chemical_list: D004151:Dipeptides; D039661:Integrin beta3; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D019038:Platelet Glycoprotein GPIb-IX Complex; D040201:Platelet Membrane Glycoprotein IIb; D011480:Protease Inhibitors; D019009:Proto-Oncogene Proteins c-kit; D045726:Metalloproteases