Hidekazu Nishikii

Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors

Human embryonic stem cells (hESCs) could potentially represent an alternative source for blood transfusion therapies and a promising tool for studying the ontogeny of hematopoiesis. When we cultured hESCs on either C3H10T1/2 or OP-9 cells to facilitate hematopoiesis, we found that exogenous administration of vascular endothelial growth factor promoted the emergence of sac-like structures, which we named embryonic stem cell-derived sacs (ES-sacs). These ES-sacs consisted of multiple cysts demarcated by cellular monolayers that retained some of the properties of endothelial cells. The spherical cells inside ES-sacs expressed primarily CD34, along with VE-cadherin, CD31, CD41a, and CD45, and were able to form hematopoietic colonies in semisolid culture and to differentiate into mature megakaryocytes by day 24 in the presence of thrombopoietin. Apparently, ES-sacs provide a suitable environment for hematopoietic progenitors. Relatively large numbers of mature megakaryocytes could be induced from the hematopoietic progenitors within ES-sacs, which were then able to release platelets that displayed integrin alpha IIb beta 3 activation and spreading in response to ADP or thrombin. This novel protocol thus provides a means of generating platelets from hESCs, which could serve as the basis for efficient production of platelets for clinical transfusion and studies of thrombopoiesis.

Lnk regulates integrin αIIbβ3 outside-in signaling in mouse platelets, leading to stabilization of thrombus development in vivo

The nature of the in vivo cellular events underlying thrombus formation mediated by platelet activation remains unclear because of the absence of a modality for analysis. Lymphocyte adaptor protein (Lnk; also known as Sh2b3) is an adaptor protein that inhibits thrombopoietin-mediated signaling, and as a result, megakaryocyte and platelet counts are elevated in Lnk-/- mice. Here we describe an unanticipated role for Lnk in stabilizing thrombus formation and clarify the activities of Lnk in platelets transduced through integrin alphaIIbbeta3-mediated outside-in signaling. We equalized platelet counts in wild-type and Lnk-/- mice by using genetic depletion of Lnk and BM transplantation. Using FeCl3- or laser-induced injury and in vivo imaging that enabled observation of single platelet behavior and the multiple steps in thrombus formation, we determined that Lnk is an essential contributor to the stabilization of developing thrombi within vessels. Lnk-/- platelets exhibited a reduced ability to fully spread on fibrinogen and mediate clot retraction, reduced tyrosine phosphorylation of the beta3 integrin subunit, and reduced binding of Fyn to integrin alphaIIbbeta3. These results provide new insight into the mechanism of alphaIIbbeta3-based outside-in signaling, which appears to be coordinated in platelets by Lnk, Fyn, and integrins. Outside-in signaling modulators could represent new therapeutic targets for the prevention of cardiovascular events.

Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells

Nishikii et al. 2008. J. Exp. Med. doi:10.1084/jem.20071482 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20071482%26rft_id%253Dinfo%253Apmid%252F18663123%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%