DEFINING THE MEGAKARYOCYTIC LINEAGE TO SUPPORT DEVELOPMENT OF IMPROVED CORD BLOOD MEGAKARYOCYTE EXPANSION PROTOCOLS

Ex vivo expansion of cord blood (CB) megakaryocytes (Mks) has been proposed to shorten the delay in platelet engraftment in patient receiving CB grafts. However, little is known about the subpopulations of the megakaryocyte lineage and which cells provide rapid platelet generation. In preliminary studies using flow cytometry, we have identified 5 subpopulations (34+61-, 34+61+, 34+61++, 34-61++ and 34-61+) that could be separated based upon CD34/CD61 expression and subdivided with CD41a and CD42b. These populations exhibited different morphology and produced platelets at different rates in vitro with early platelet production in the CD34- subpopulations and more overall platelet production with the CD34+ subpopulations. In addition the highest levels of Mk colony formation (CFU-Mk) was obtained with cells expressing CD34 and either CD61- or CD61+. This data suggest that the MK progenitors derive from the CD34+CD61- or CD34+CD61+ population. Furthermore, it demonstrates that development into the MK lineage requires progression of CD61 expression, becoming more mature and losing large CFU-MK colony potential and gaining platelet production. We have isolated CB MNCs and cultured the cells on MSCs for 7 days, in media supplemented with SCF, TPO and FLT3L. We detected significant expansion of the CD34+ subpopulations that produced CFU-MK potential and significant platelet production in vitro. This MK expansion in co-culture with MSCs may provide significant proliferation of early MKs that could be stimulated in vivo to shed platelets and provide faster engraftment times.