Anti-apoptotic increases megakaryocyte proplatelet formation in cultures of human cord blood.

Apoptosis is a recognized limitation to generating large numbers of megakaryocytes in culture. The genes responsible have been rigorously studied in mice, but are poorly characterized in human culture systems. As CD34-positive () cells isolated from human umbilical vein cord blood were differentiated into megakaryocytes in culture, two distinct cell populations were identified by flow cytometric forward and side scatter: larger size, lower granularity (LLG), and smaller size, higher granularity (SHG). The LLG cells were CD41a CD42a phosphatidylserine, had an electron microscopic morphology similar to mature bone marrow megakaryocytes, developed proplatelets, and displayed a signaling response to platelet agonists. The SHG cells were CD41aCD42aphosphatidylserine, had a distinctly apoptotic morphology, were unable to develop proplatelets, and showed no signaling response. Screens of differentiating megakaryocytes for expression of 24 apoptosis genes identified as a novel candidate megakaryocyte apoptosis regulator. Lentiviral overexpression decreased megakaryocyte apoptosis, increased CD41a LLG cells, and increased proplatelet formation by 58%. An association study in 154 healthy donors identified a significant positive correlation between platelet number and platelet mRNA levels. This finding was consistent with the observed increase in platelet-like particles derived from cultured megakaryocytes over-expressing also induced small, but significant increases in thrombin-induced platelet-like particle αIIbβ3 activation and P-selectin expression. Thus, restrains apoptosis in cultured megakaryocytes, promotes proplatelet formation, and is associated with platelet number. is a novel target for improving megakaryocyte and platelet yields in culture systems.