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.
Takayama
Naoya
N
Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
Nishikii
Hidekazu
H
Usui
Joichi
J
Tsukui
Hiroko
H
Sawaguchi
Akira
A
Hiroyama
Takashi
T
Eto
Koji
K
Nakauchi
Hiromitsu
H
D001792:Blood Platelets / Q000166:cytology* / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism; D018613:Microscopy, Confocal; D015539:Platelet Activation / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D042461:Vascular Endothelial Growth Factor A