In vitro and in vivo megakaryocyte differentiation of fresh and ex-vivo expanded cord blood cells: rapid and transient megakaryocyte reconstitution.

Megakaryocyte (Mk) engraftment is often poor and delayed after cord blood (CB) transplantation. Ex vivo manipulations of the cells that will be infused may be a way to achieve better Mk engraftment. In this study we investigated the ability of different hematopoietic growth factor combinations to generate large numbers of Mk cells ex vivo.To find the best cytokine combination capable of generating large numbers of Mks, baseline CB CD34+ (bCD34+) cells and CD34+ and CD34- cells, immunoselected after 4 weeks of expansion with thrombopoietin (TPO), stem cell factor (SCF) and Flt-3 ligand (FL) (eCD34+, eCD34-), were further cultured in the presence of different cytokine combinations (containing interleukin(IL)-3, SCF, TPO and IL-6). To evaluate Mk reconstitution in vivo, Mk-committed cells, generated during 10 days of in vitro culture, were injected into NOD/SCID mice and the kinetics of human platelet production was evaluated.TPO and SCF together were found to be sufficient to generate large numbers of Mk cells (3 +/- 0.40 x 10(6)/1 x 10(5) input bCD34+ cells) from bCD34+ cells; the addition of IL-3 and IL-6 did not further increase Mk production (3.5 +/- 0.63 x 10(6)/1 x 10(5) input bCD34+ cells). In contrast only one cytokine combination (IL-3+SCF+IL-6+TPO) induced a large Mk production from eCD34+ and eCD34- cells (0.16 +/- 0.04 x 10(6)/1 x 10(5) input eCD34+ cells and 0.035 x 10(6) +/- 0.012 x 106/1 x 10(5) input eCD34- cells, respectively). In mice injected with Mk-committed cells derived from bCD34+ or eCD34+ cells, human platelets were first detected on day 3 and disappeared after 4 weeks; in mice injected with MK-committed cells derived from eCD34- cells, human platelets peaked at day 3, but disappeared quickly.Fast Mk-engraftment can be obtained by in vitro selective lineage-commitment of baseline and ex vivo expanded CB cells.