Impact of dnMpl expression on the HSC compartment.

<p>(A) Survival curve of primary transplanted mice of the dnMpl or the control groups (experiment 1–3, dnMpl n = 25, control n = 20). Of experiment three, the BM was transplanted into two secondary recipients each (3 dnMpl donors, 2 cd-dnMpl donors, 3 GFP control donors). There was no difference in survival in primary recipients but a significantly reduced survival of secondary recipients (10 dnMpl recipient mice and 6 control recipient mice, p<0.05). (B) Percentage of LSK cells in primary recipients (dnMpl and trCD34 control black filled circles, cd-dnMpl and GFP control in grey filled circles) or in steady state hematopoiesis (wt, <i>Mpl-/-</i>, <i>Thpo-/-</i>). (*p<0.05, Students t-test). (C) Percentage of LSK-CD34 negative cells in primary recipients of GFP control (n = 3) or dnMpl mice (n = 5) (***p<0.005, Students t-test). (D) Histological analysis of the BM of dnMpl and control transplanted mice. Primary (1°) dnMpl chimeric mice had reduced numbers and smaller megakaryocytes similar to <i>Mpl-/-</i> mice. Secondary dnMpl recipient mice (2°) had a hypocellular BM in agreement with the symptoms of BM failure. BM sections were Hematoxylin/Eosin stained and microscopic images were taken at 200x magnification. (E) CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl, cd-dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells (experiment 3). 19 weeks after the first transplantation, two dnMpl, two cd-dnMpl and two GFP mice were infused with a second graft of 2x10⁷ CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 16 weeks. dnMpl conditioned mice allowed the engraftment of a second wt graft without further conditioning. (F) In experiment 5, CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells, however, with intended lower chimerism. 19 weeks after the first transplantation mice were given a second graft of 2 x10⁷ CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 20 weeks. While the two mice exhibiting the highest percentage of dnMpl expression (4 and 14%) allowed stable engraftment of the second graft, the mice with < 1% dnMpl expression in the periphery failed to do so and presented with the low engraftment levels similar to the four GFP control mice.