Megakaryocytopoiesis detected from liquid culture stimulated by megakaryocyte growth and development factor: difference between bone marrow and mobilised peripheral blood

Peripheral blood progenitor cell transplants (PBPCT) have shown to allow faster neutrophil and platelet recovery than bone marrow transplants (BMT). This difference may be due to the difference in the frequency or the intrinsic properties of megakaryocyte progenitors in bone marrow (BM) and mobilised peripheral blood leukapheresis samples (mPB). In addition, the presence of accessory cells may also augment the megakaryocytic potential of the graft. To compare the megakaryocytic potential of BM and mPB, we studied the production of megakaryocytic cells in liquid culture stimulated by megakaryocyte growth and development factor (MGDF). Cells were cultured for 7, 14 or 21 days (37 C; 5% CO2; humidified air) in Opti-MEM supplemented by 30ng/mL MGDF and 10% fetal calf serum. The cells were analysed by flow cytometry after dual immunofluorescence labelling. We have investigated the nature of the progenitors by comparing cell cultures of unfractionated or enriched CD34+ cells fromBM (purity = 83 ±14%; n = 19) or mPB (purity = 71 ±25%; n = 11). Under the stimulation of MGDF. the proliferation of cells is not restricted to the megakaryocyte lineage and the unilineage proliferation of >90% reported by other groups was not observed. A well defined population of megakaryocytic cells expressing high levels of platelet specific antigen (CDG1hi) but not the monocyte marker (CD14neg) was characterised as the output of megakaryocytopoiesis. Since we have demonstrated in clonogenic assay that all megakaryocyte colony forming activity is included in the CD34+ population, we defined megakaryocytic potential as the output number of megakaryocytic cells (0061hiCD14neg) per CD34+ cells input, and the results are as follow: Unfractionated Enriched CD34+ Day 7 Day 14 Day 7 Day 14 BM 0.45 ± 0.56(n=6) 0.36 ± 0.48(n=10) 0.30 (n=5) 0.31± 0.44(n=15) mPB 0.40 ± 0.27(n=5) 2.17± 1.39 (n=5) 0.09 ± 0.10 (n=4) 0.17 ± 0.27(n=4) Using unfractionated samples, the megakaryocytic potential of BM was similar to that in mPB after 7 days in culture. However, after 14 days, the megakaryocytic potential of mPB was significantly higher than that of BM, which remained unchanged. After Day 14, the number of megakaryocytic cells declined rapidly and only very few were detected on Day 21. Progenitors enriched by CD34+ selection from BM responded similarly as in the unfractionated samples. Interestingly, the response of enriched CD34+ from mPB was significantly lower than that of2unfractionated samples. Although the megakaryocytic potential of enriched CD34+ from mPB was slightly lower than that from BM, the range of responses overlap considerably and the difference between the two enriched population is inconclusive. This observation suggested two possibilities: (1) some cells other than CD34+ cells in mPB have megakaryocytic potential, or (2) the accessory cells in unfractionated mPB augmented the megakaryocytic potential of CD34+ progenitors. We have excluded the possibility that there is a substantial number of megakaryocytic cells in the starting preparation which may enhance the measured output. It is concluded that the presented method of measuring megakaryocytic potential is effective in analysing differences between stem cell sources, and that mPB derived accessory cells may contribute to the increased megakaryocyte potential and accelerated platelet recovery observed after PBPCT.