TY - JOUR A1 - Dütting, Sebastian A1 - Gaits-Iacovoni, Frederique A1 - Stegner, David A1 - Popp, Michael A1 - Antkowiak, Adrien A1 - van Eeuwijk, Judith M.M. A1 - Nurden, Paquita A1 - Stritt, Simon A1 - Heib, Tobias A1 - Aurbach, Katja A1 - Angay, Oguzhan A1 - Cherpokova, Deya A1 - Heinz, Niels A1 - Baig, Ayesha A. A1 - Gorelashvili, Maximilian G. A1 - Gerner, Frank A1 - Heinze, Katrin G. A1 - Ware, Jerry A1 - Krohne, Georg A1 - Ruggeri, Zaverio M. A1 - Nurden, Alan T. A1 - Schulze, Harald A1 - Modlich, Ute A1 - Pleines, Irina A1 - Brakebusch, Cord A1 - Nieswandt, Bernhard T1 - A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis JF - Nature Communications N2 - Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V. KW - megakaryocytes KW - blood platelets KW - regulatory circuit downstream KW - glycoprotein Ib Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170797 VL - 8 IS - 15838 ER - TY - JOUR A1 - Stegner, David A1 - van Eeuwijk, Judith M.M. A1 - Angay, Oğuzhan A1 - Gorelashvili, Maximilian G. A1 - Semeniak, Daniela A1 - Pinnecker, Jürgen A1 - Schmithausen, Patrick A1 - Meyer, Imke A1 - Friedrich, Mike A1 - Dütting, Sebastian A1 - Brede, Christian A1 - Beilhack, Andreas A1 - Schulze, Harald A1 - Nieswandt, Bernhard A1 - Heinze, Katrin G. T1 - Thrombopoiesis is spatially regulated by the bone marrow vasculature JF - Nature Communications N2 - In mammals, megakaryocytes (MKs) in the bone marrow (BM) produce blood platelets, required for hemostasis and thrombosis. MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Through imaging of MKs in the intact BM, here we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. By combining in vivo two-photon microscopy and in situ light-sheet fluorescence microscopy with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche. As MKs do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts. KW - bone marrow KW - megakaryocytes KW - thrombopoiesis Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170591 VL - 8 IS - 127 ER - TY - JOUR A1 - Cullmann, Katharina A1 - Jahn, Magdalena A1 - Spindler, Markus A1 - Schenk, Franziska A1 - Manukjan, Georgi A1 - Mucci, Adele A1 - Steinemann, Doris A1 - Boller, Klaus A1 - Schulze, Harald A1 - Bender, Markus A1 - Moritz, Thomas A1 - Modlich, Ute T1 - Forming megakaryocytes from murine‐induced pluripotent stem cells by the inducible overexpression of supporting factors JF - Research and Practice in Thrombosis and Haemostasis N2 - Background Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low. Objectives To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA‐binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre–B‐cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc). Methods To avoid off‐target effects, we generated iPSCs carrying the reverse tetracycline‐responsive transactivator M2 (rtTA‐M2) in the Rosa26 locus and expressed the factors from Tet‐inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation. Results Overexpression of GATA1 and Pbx1 increased MK output 2‐ to 2.5‐fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro–generated platelets were functional in spreading on fibrinogen or collagen‐related peptide. Conclusion We demonstrate that the use of rtTA‐M2 transgenic iPSCs transduced with Tet‐inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production. KW - genetic modification KW - iPS cells KW - megakaryocytes KW - retroviral vectors KW - Tet‐inducible system Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224565 VL - 5 IS - 1 SP - 111 EP - 124 ER - TY - JOUR A1 - Wagner, Nicole A1 - Mott, Kristina A1 - Upcin, Berin A1 - Stegner, David A1 - Schulze, Harald A1 - Ergün, Süleyman T1 - CXCL12-abundant reticular (CAR) cells direct megakaryocyte protrusions across the bone marrow sinusoid wall JF - Cells N2 - Megakaryocytes (MKs) release platelets into the lumen of bone marrow (BM) sinusoids while remaining to reside within the BM. The morphogenetic events of this complex process are still not fully understood. We combined confocal laser scanning microscopy with transmission and serial block-face scanning electron microscopy followed by 3D-reconstruction on mouse BM tissue sections. These analyses revealed that MKs in close vicinity to BM sinusoid (BMS) wall first induce the lateral retraction of CXCL12-abundant reticular (CAR) cells (CAR), followed by basal lamina (BL) degradation enabling direct MK-sinusoidal endothelial cells (SECs) interaction. Subsequently, an endothelial engulfment starts that contains a large MK protrusion. Then, MK protrusions penetrate the SEC, transmigrate into the BMS lumen and form proplatelets that are in direct contact to the SEC surface. Furthermore, such processes are induced on several sites, as observed by 3D reconstructions. Our data demonstrate that MKs in interaction with CAR-cells actively induce BMS wall alterations, including CAR-cell retraction, BL degradation, and SEC engulfment containing a large MK protrusion. This results in SEC penetration enabling the migration of MK protrusion into the BMS lumen where proplatelets that are adherent to the luminal SEC surface are formed and contribute to platelet release into the blood circulation. KW - megakaryocytes KW - microvasculature KW - CXCL12-abundant reticular (CAR)-cells Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234180 SN - 2073-4409 VL - 10 IS - 4 ER -