1021 – DIFFERENTIATION ROUTE DETERMINES THE FUNCTIONAL OUTPUTS OF ADULT MEGAKARYOPOIESIS

One of the breakthroughs in hematopoietic stem cell (HSC) field over the last decade is the discovery of two alternative differentiation routes from primitive HSCs to mature megakaryocytes: one through the stepwise hematopoietic hierarchy (stepwise route), and the other by direct differentiation (direct route). This raises a fundamental question of the physiological importance of two alternative differentiation routes for megakaryopoiesis. A major challenge in addressing this question is the lack of fate-mapping systems that distinguish the two differentiation routes.

This work was initiated by designing genetic systems that distinguished the direct and stepwise differentiation routes for hematopoiesis. We found that Cd48-Dre specifically and constitutively marked all haematopoietic cells on the stepwise differentiation route. A combination of KitcreER, Cd48dre and Rosa26loxp-STOP-loxp-rox-loxp-ZsGreen-STOP-rox-tdTomato allowed inducible and simultaneous fate-mapping of haematopoietic stem and progenitor cells on the direct and stepwise differentiation routes.

We mapped the turnover rates and differentiation kinetics of each branch of the hematopoietic hierarchy. We found that megakaryocytes were produced through the two routes with comparable kinetics and quantity under homeostasis. Single-cell RNA-sequencing of the fate-mapped megakaryocytes revealed that the direct and stepwise routes contributed to the niche-supporting and immune megakaryocytes respectively, but contributed to the platelet-producing megakaryocytes together. Consistent with this, megakaryocytes generated through different routes displayed different activities in vitro and in vivo. Chemotherapy preferentially enhanced megakaryopoiesis through the direct route, whereas inflammation preferentially enhanced megakaryopoiesis through the stepwise route. In summary, our work links the differentiation route to the cellular heterogeneity of adult megakaryocytes. Alternative differentiation routes result in variable combinations of functionally distinct megakaryocyte subpopulations poised for different physiological demands.