Human iPSC-derived platelets generated in alginate scaffolds exhibit functional haemostasis in vitro and in vivo

The manufacture of platelets from induced pluripotent stem cells (iPSCs) in a cost-effective, controlled and reproducible manner is a critical challenge for transfusion and regenerative medicine applications. Here we employ a high throughput, combinatorial screening method (CombiCult®) to discover new efficient protocols for generation of mature megakaryocytes (MKs) and platelets from human iPSC (hiPSC) lines. The CombiCult® platform was adapted to encapsulate hiPSCs cells in alginate by electro-spraying and cell differentiation was initiated in alginate beads, circumventing the need for stromal inducer cells, extracellular matrix materials and the process of embryoid body (EB) formation. Both feeder-dependent and feeder-free iPSC lines were multiplexed in one screen, allowing discovery of multiple serum-free feeder-free protocols that direct MK differentiation from diverse cell lines. These protocols provide high yield, high purity and low-cost methods for MK production compatible with large-scale suspension format bioreactor systems. Platelets produced by these methods (iPLT) phenotypically and functionally resemble human donor platelets following in vitro activation by platelet agonists. Furthermore, following transfusion in NOD-SCID mice, resting iPLT showed no evidence of pulmonary thrombo-embolism or heightened tissue accumulation up to 72 h post infusion and exhibited similar responses to donor platelets in a murine model of pulmonary platelet aggregation.