By Robert Flaumenhaft, MD, PhD

2008-11-01

Dr. Flaumenhaft indicated no relevant conflicts of interest.

Nishikii H, Eto K, Tamura N, et al. Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells. J Exp Med. 2008;205:1917-27.

More than 10 million units of platelets are transfused in the United States annually. Platelet supplies presently rely on volunteer donations, which are subject to regional and seasonal variation and shortages. Cold storage of platelets dramatically reduces their half-life in the circulation. Since they cannot be refrigerated, donated platelets can only be stored for five days owing to the risk of bacterial contamination from transfusion. Alternative sources of platelet production include large-scale expansion of CD34⁺ cells and platelet production from embryonic stems cells (ESCs). An advantage of using ESCs for purposes of generating platelets is that these cells have the potential to proliferate indefinitely in culture. Since platelets are anucleate, they can be irradiated before transfusion to prevent transfusion of undifferentiated ESCs. The theoretical potential is limitless. However, technical barriers to generating functional platelets from ESCs have so far prevented widespread implementation of this technology.

Nishikii, et al. have addressed a significant difficulty in platelet production from ESCs. They described a two-stage approach for producing platelets from mouse ESCs. During the first stage, ESCs were induced for embryoid body formation in liquid culture. Selected cells were subsequently co-cultured with stromal cells in the second stage. Culture supernatants contained proplatelets and platelet-sized particles that displayed an open canalicular system as well as multiple dense and α-granules. Evaluation of major surface proteins on ESC-derived platelets demonstrated αIIb, GPIbß, and GPIX levels similar to those of fresh mouse platelets. However, GPIbα, GPV, and GPVI were substantially reduced in platelets derived from ESCs. In contrast, megakaryocytes derived from ESCs had normal levels of GPIbα. These observations raised the possibility that GPIbα, GPV, and GPVI are shed during maturation in co-culture.

Metalloproteinases belonging to the ADAM family cleave platelet GPIbα, GPV, and GPVI. To determine whether inhibition of metalloproteinase activity prevented loss of GPIbα, GPV, and GPVI from ESC-derived platelets, Nishikii, et al. added GM6001, a potent metalloproteinase inhibitor, to the co-culture system. Incubation with GM6001 restored GPIbα, GPV, and GPVI expression, indicating that metalloproteinase-mediated shedding caused loss of these surface proteins during cell culture.

Inhibition of metalloproteinase activity improved several aspects of ESC-derived platelet function. Signaling through αIIbß3 was enhanced in ESC-derived platelets cultured in the presence of GM6001 compared with those that were not. Thrombus formation occurring when whole blood flowed over a collagen matrix at physiological shear rates was markedly enhanced in ESC-derived platelets incubated with GM6001. In addition, incubation with GM6001 substantially increased the survival of ESC-derived platelets when infused into recipient mice. These results demonstrate the importance of inhibiting platelet surface receptor shedding in producing functional ESC-derived platelets.

The demand for platelet transfusion has continued to rise, prompting efforts to devise novel strategies of platelet production and storage. Production of platelets from ESCs offers a theoretically limitless supply of platelets. Yet considerable technical barriers hinder the progress toward efficient production of functional ESC-derived platelets. Nishikii, et al. demonstrate that metalloproteinase activity that occurs during co-culture results in loss of critical platelet surface proteins. Inhibition of metalloproteinase activity results in the production of platelets with enhanced functionality and longer survival in vivo. Although these studies do not solve all the technical problems associated with large-scale production of functional ESC-derived platelets, they do shed an important barrier to accomplishing this objective.

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