PB0723 - Development of a fully in vitro approach to generation of megakaryocytes and platelets from mouse embryonic stem cells

Background: Much of our understanding of platelet function derives from genetic studies in mouse model systems, using targeted gene deletion. There is now a need for more refined genetic models, in which point deletions or modifications are made, to understand roles of protein domains in platelet function, and to mimic human disease mutations. There is, therefore, a need for large scale generation of genetically modified platelets in vitro. Here we present data showing the ability to generate megakaryocytes (MK) and platelets from a genetically modifiable source in murine embryonic stem cells.

Aims: To generate megakaryocytes from ES-R1 embryonic mouse stem cells and subsequently generate large numbers of functional platelets.

Methods: Mouse embryonic stem cells (ES-R1) were transformed into megakaryocytes using a forward programming method. Ploidy, granularity and surface markers were assessed by FACS, confocal and TEM imaging. Platelet generation was assessed in the ex vivo heart-lung system we recently developed and functionality of these generated platelets was assessed by FACS for P-selectin expression and activation of integrin aIIbb3.

Results: FACS analysis of generated megakaryocytes showed cells became CD41a and CD42d positive as well as having increased ploidy comparable to that of bone marrow-derived controls. TEM and confocal imaging showed cells to be granular and confirmed their polyploid nature. Following passage ex vivo through a mouse heart-lung preparation, forward programmed MKs were able to generate platelets in greater numbers than bone marrow-derived controls, by approx. 1000 greater per MK. Generated platelets were also functional, showing agonist-stimulated P-selectin expression and activation of integrin aIIbb3.

Conclusion(s): We conclude it is possible to generate megakaryocytes from mouse embryonic stem cells, that have comparable features to those derived from bone marrow and generate large numbers of functional platelets. The genetic tractability of ES cells establishes a process for generating large numbers of genetically modified platelets.