Packaging functionally important plasma proteins into the α-granules of human-induced pluripotent stem cell-derived...

The contents of platelet α-granules arrive via a number of pathways; some are synthesized by megakaryocytes (MKs), for example, von Willebrand factor (VWF), whereas others are endocytosed from plasma, for example, fibrinogen (Fgn) and factor V (FV). Currently, almost all in vitro-induced pluripotent stem cell (iPSC)-derived MKs are generated under serum-free conditions, and their α-granule cargoes lack components that would normally be taken up from plasma during the course of megakaryopoiesis. How this might affect the ability of in vitro-derived platelets to contribute fully to haemostasis is not known. The purpose of this investigation was to examine whether "feeding" human plasma to iPSC-derived MKs might result in loading their α-granules with physiologically important proteins. iPSCs were differentiated to CD41⁺ /CD42b⁺ MKs using a serum-free protocol. The resulting MKs were polyploid, expressed a number of platelet-specific surface receptors, and spread on Fgn or collagen-coated surfaces. Reverse transcription-polymerase chain reaction analysis detected mRNA transcripts for FV and VWF but not Fgn chains. Fluorescence immunocytochemistry and confocal microscopy confirmed constitutive VWF distribution in granule-like structures in MKs cultured under plasma-free conditions, and the granules became positive for Fgn upon incubation with human plasma. iPSC-derived MKs showed a low level of constitutive FV expression that increased dramatically upon incubation with human plasma. Taken together, these data suggest that human iPSC-derived MKs are capable of endocytosing and storing plasma components in their α-granules. Incorporating this methodology into current protocols for producing in vitro-derived MKs should provide novel insights into MK biology and lead to the generation of large numbers of MKs and platelets with improved functionality.