Comparative analysis of human ex vivo–generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale

Abstract

• Infused human megakaryocytes release young platelets in the lungs with characteristics similar to donor platelets.• Platelets released from ex vivo-derived megakaryocytes are preactivated and compare poorly to donor platelets.Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo-generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application. (Blood. 2015;125(23):3627-3636)

Understanding platelet generation from megakaryocytes: implications for in vitro–derived platelets

“…These strategies are described in 2 recent publications that have expressed views for the arrival of in vitro-derived platelets in the clinical setting from optimistic 20 to cautiously optimistic. 21 The Lanza group 20 demonstrated good manufacturing practice feasibility of the stem cell-differentiation systems, which is a step forward in the production line of in vitro-generated platelets. They also demonstrated increased megakaryocyte and platelet yields, enabling platelet function to be analyzed by the traditional method of light transmission aggregometry.…”

CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes

“…Building upon this discovery, several groups have developed efficient protocols for differentiating iPSCs to HPCs, 34,35 which can be expanded to megakaryocytes, 36,37 and platelets. [38][39][40] Although still a long way off from producing a transfusable number of platelets, the ability to generate and cryopreserve iPSC-derived megakaryocyte progenitor cells leaves open the possibility of maintaining an inexhaustible source of platelets and their progenitors for diagnostic and investigative applications. We sought to exploit this capability to produce antigenically-distinct megakaryocytes and progenitor cells from genetically customized iPSCs in sufficient quantities for characterization of their plateletspecific alloantigen expression and function by flow cytometry and other diagnostic methods.…”

Megakaryocyte- and megakaryocyte precursor–related gene therapies

“…32,77,78 Current analysis of the field indicates that it is essential to achieve further improvement for in vitro production of platelets (especially to increase the number and quality of platelets for transfusion) to make this strategy clinically feasible. 79 The authors of that article surmised that it may be more advantageous to infuse megakaryocytes (rather than platelets) derived from iPSCs. The megakaryocytes were observed to congregate within murine lungs where the maturation into platelets occurred.…”