Megakaryocyte Colony Assay

mutation in a hemopoietic stem cell, burst-forming unit erythroid (BFU-E) colonies, colony-forming unit granulocyte-macrophage (CFU-GM), and colonyforming unit-eosinophil (CFU-Eo) were grown from a blood group 0 patient with a typical Tn syndrome displaying two distinct populations (Tn+ and Tn-) of platelets, granulocytes, and erythrocytes. A large number of colonies was observed. Individual colonies were studied with a fluorescent conjugate of Helix pomatia agglutinin (HPA). A sizeable fraction of each of the erythroid and granulocytic colonies appeared to consist exclusively of either HPA-positive or HPA-negative cells, thereby demonstrating the clonal origin of those exhibiting the Tn marker. Similar results were obtained from a second patient. These findings establish that the HPA labeling of Tn cells is an accurate marker permitting assessment of the clonality of the human megakaryocyte (MK) colony assay. For the study of MK cultures a double-staining procedure using the HPA lectin and a monoclonal antiplatelet antibody (J-15) was applied in situ to identify all MK constituting a colony. Our results, obtained in studies of 133 MK colonies, provide definitive evidence that the human MK colony assay is clonal because all MK colonies were exclusively composed of Tn+ and TnReceived for publication 24 September 1981 and in revised form 28 December 1981. MK. Furthermore, the distribution of MK within a single colony was shown to be seminormal with a mean at 6 MK, isolated MK typically being absent in culture. Comparison of the proportion of mature Tn+ cells in blood with their respective Tn+ progenitors has also shown that no proliferative advantage occurs after the commitment; because Tn polyagglutinability is an acquired disorder, then the expansion of the Tn+ clone must occur either during the proliferative stage of the pluripotent stem cell or during the commitment itself. This study therefore affords evidence that a blood group antigen plays a role in the differentiation of a pluripotent stem cell.