Insights Into Thrombopoiesis From Infused Human Megakaryocytes Into Mice

Thrombopoiesis is the process by which megakaryocytes (Megs) release platelets (Plts), but issues remain as to the detailed in vivo mechanisms underlying this process. We now report new insights into this process by studying infused human Megs into immunocompromized NOD/SCID, gamma-interferon deleted (NSG) mice. Prior in situ microscopy has suggested that Megs release varied-size cytoplasmic fragments up to whole Megs in size into the medullary vascular space. Other studies have suggested that at least a portion of thrombopoiesis occurs by Megs lodged in the lungs. We previously infused ex vivo-generated murine Megs into mice and found that these Megs become entrapped in the animals’ lungs, and in <1.5 hrs, release functional Plts (termed here “Meg-Plts”) that have a similar half-life as infused mouse donor-derived Plts (termed here “Donor-Plts”). To better understand the biology of thrombopoiesis, we have infused ex vivo-generated human Megs into NSG mice. These studies replicated many of the observations seen with infused murine Megs: Human Megs were entrapped in the lungs with delayed release of human Meg-Plts, and these Meg-Plts had the same half-life as infused human Donor-Plts. Human Plts differ from murine Plts in size so this parameter was analyzed following infusion of human Megs using forward cell scatter analysis. We noted that 10 mins post-infusion, the Meg-Plt size range was wide and displayed a non-bell-shaped distribution. This distribution was in contrast to the tight bell-shaped curves seen for the endogenous murine Plts and for infused human Donor-Plts. However, by 3 hrs post-human Meg infusion - at the time of peak Meg-Plt counts - the human Meg-Plts now displayed an identical bell-shaped distribution curve as infused human Donor-Plt. The smaller, human Meg-Plts had disappeared. The size and distribution of these Meg-Plts then remained near identical to Donor-Plts for the remaining portion of the 48 hr post-infusion study. However, after impairing macrophage clearance in NSG recipient mice with clodronate-ladened liposome infusion, the small Meg-Plts did not disappear and were present at 48 hrs. Using thiazole orange (TO) to stain platelets for RNA content, we noted that ∼70% of all Meg-Plts were initially TO⁺ compared to the steady-state of ∼10% for mouse endogenous platelets. This high TO⁺ state decreased to near 10% by 24 hrs post-infusion. Up to ∼6 hrs, all of the large Meg-Plts were TO⁺, while the smaller-sized Meg-Plts were predominantly TO^-. Unless the mice were treated with clodronate-ladened liposomes, these TO^-, small Meg-Plts disappeared before 6 hrs. In conclusion, these data support that ex vivo-generated human Megs release physiologic platelets in the pulmonary vascular bed of NSG mice with the same size range/distribution and survival as infused human Donor-Plts. Mean Meg-Plt size depends on the species of origin of the infused Megs rather than on the species of the recipient animal. We did not detect large Meg cytoplasmic fragments that underwent further size reduction although our technique may not be capable of detecting small numbers of such fragments or the small size changes that would accompany platelet maturation from preplatelets. Our data also suggest that Megs generated in culture release a wide size range of non-physiologic Plt-like particles that when infused are cleared rapidly by macrophages.