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

Abstract

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 cellderived 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, ESsacs 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 ␣IIb␤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. (Blood. 2008;111:5298-5306)

Generation of T Cells from Human Embryonic Stem Cell-Derived Hematopoietic Zones

“…1C). Similar structures were recently described as ES-sacs and were shown to contain precursors for megakaryocytes (11). We could demonstrate by flow cytometric analysis the presence of CD34 ϩ CD43 ϩ HPC cells within the blood island-like structures that were mechanically picked up from the hESC/OP9 cultures ( Fig.…”

“…During the course of these experiments we noted the formation of endothelium-lined cell clumps, reminiscent of blood islands during embryonic development, which we termed hematopoietic zones (HZs). These structures have since been described by two other groups and were shown to contain HPC with self renewing capacity and HPC capable of generating massive amounts of megakaryocytes (5,11). In this study, we report that these HZs generate large numbers of T cells when transferred on OP9-DL1 cells in the presence of growth factors.…”

“…Takayama et al (11) designated similar hematopoietic sites as ES-sacs. The ES-sacs were obtained after coincubation of hESC with OP9 or C3H101/2 stromal cells and appeared around day 15 of culture.…”

Platelet bioreactor-on-a-chip

“…Two major quantitative roadblocks persist in the development of donorindependent PLTs for therapeutic use: (1) generating sufficient numbers (;3 3 10 8 ) of human MKs to support the production of 1 PLT transfusion unit (;3 3 10 11 PLTs); and (2) generating physiological numbers of functional human PLTs (;10 3 ) per MK. 18 The development of human embryonic stem cell cultures (hESC), 44,46 and more recently, hiPSC 47 (Feng Q et al, manuscript submitted 2014) offer a physiologically relevant and potentially unlimited source of progenitor cells that can be differentiated into human MKs in vitro to address the first quantitative roadblock. 48 Indeed, because PLTs are anucleate, PLT bioreactor-derived units could be irradiated prior to infusion, addressing concerns that cellular products derived from hESC or hiPSCs could be oncogenic or teratogenic.…”

Induced pluripotent stem cell (iPSCs) and their application in immunotherapy

“…To this end, with the availability of hiPSCs, the next step of obtaining HSCs/HPCs from hiPSCs can now be achieved. Since various hematopoietic-derived cell types have been successfully induced from human ESCs (hESCs), including red blood cells, [13][14][15][16] monocytes, 17 dendritic cells (DCs), 18 megakaryocytes 19 and lymphocytes, 20 it is anticipated that hiPSCs can also differentiate into hematopoietic cells using similar methods previously applied to hESCs.…”