@article{CullmannJahnSpindleretal.2021, author = {Cullmann, Katharina and Jahn, Magdalena and Spindler, Markus and Schenk, Franziska and Manukjan, Georgi and Mucci, Adele and Steinemann, Doris and Boller, Klaus and Schulze, Harald and Bender, Markus and Moritz, Thomas and Modlich, Ute}, title = {Forming megakaryocytes from murine-induced pluripotent stem cells by the inducible overexpression of supporting factors}, series = {Research and Practice in Thrombosis and Haemostasis}, volume = {5}, journal = {Research and Practice in Thrombosis and Haemostasis}, number = {1}, doi = {10.1002/rth2.12453}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-224565}, pages = {111 -- 124}, year = {2021}, abstract = {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.}, language = {en} } @article{WagnerMottUpcinetal.2021, author = {Wagner, Nicole and Mott, Kristina and Upcin, Berin and Stegner, David and Schulze, Harald and Erg{\"u}n, S{\"u}leyman}, title = {CXCL12-abundant reticular (CAR) cells direct megakaryocyte protrusions across the bone marrow sinusoid wall}, series = {Cells}, volume = {10}, journal = {Cells}, number = {4}, issn = {2073-4409}, doi = {10.3390/cells10040722}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-234180}, year = {2021}, abstract = {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.}, language = {en} } @article{DuettingGaitsIacovoniStegneretal.2017, author = {D{\"u}tting, Sebastian and Gaits-Iacovoni, Frederique and Stegner, David and Popp, Michael and Antkowiak, Adrien and van Eeuwijk, Judith M.M. and Nurden, Paquita and Stritt, Simon and Heib, Tobias and Aurbach, Katja and Angay, Oguzhan and Cherpokova, Deya and Heinz, Niels and Baig, Ayesha A. and Gorelashvili, Maximilian G. and Gerner, Frank and Heinze, Katrin G. and Ware, Jerry and Krohne, Georg and Ruggeri, Zaverio M. and Nurden, Alan T. and Schulze, Harald and Modlich, Ute and Pleines, Irina and Brakebusch, Cord and Nieswandt, Bernhard}, title = {A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis}, series = {Nature Communications}, volume = {8}, journal = {Nature Communications}, number = {15838}, doi = {10.1038/ncomms15838}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170797}, year = {2017}, abstract = {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.}, language = {en} } @article{StegnervanEeuwijkAngayetal.2017, author = {Stegner, David and van Eeuwijk, Judith M.M. and Angay, Oğuzhan and Gorelashvili, Maximilian G. and Semeniak, Daniela and Pinnecker, J{\"u}rgen and Schmithausen, Patrick and Meyer, Imke and Friedrich, Mike and D{\"u}tting, Sebastian and Brede, Christian and Beilhack, Andreas and Schulze, Harald and Nieswandt, Bernhard and Heinze, Katrin G.}, title = {Thrombopoiesis is spatially regulated by the bone marrow vasculature}, series = {Nature Communications}, volume = {8}, journal = {Nature Communications}, number = {127}, doi = {10.1038/s41467-017-00201-7}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170591}, year = {2017}, abstract = {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.}, language = {en} }