TY - JOUR A1 - Chen, Chunguang A1 - Rawat, Divya A1 - Samikannu, Balaji A1 - Bender, Markus A1 - Preissner, Klaus T. A1 - Linn, Thomas T1 - Platelet glycoprotein VI‐dependent thrombus stabilization is essential for the intraportal engraftment of pancreatic islets JF - American Journal of Transplantation N2 - Platelet activation and thrombus formation have been implicated to be detrimental for intraportal pancreatic islet transplants. The platelet‐specific collagen receptor glycoprotein VI (GPVI) plays a key role in thrombosis through cellular activation and the subsequent release of secondary mediators. In aggregometry and in a microfluidic dynamic assay system modeling flow in the portal vein, pancreatic islets promoted platelet aggregation and triggered thrombus formation, respectively. While platelet GPVI deficiency did not affect the initiation of these events, it was found to destabilize platelet aggregates and thrombi in this process. Interestingly, while no major difference was detected in early thrombus formation after intraportal islet transplantation, genetic GPVI deficiency or acute anti‐GPVI treatment led to an inferior graft survival and function in both syngeneic mouse islet transplantation and xenogeneic human islet transplantation models. These results demonstrate that platelet GPVI signaling is indispensable in stable thrombus formation induced by pancreatic islets. GPVI deficiency resulted in thrombus destabilization and inferior islet engraftment indicating that thrombus formation is necessary for a successful intraportal islet transplantation in which platelets are active modulators. KW - basic (laboratory) research / science KW - coagulation and hemostasis KW - graft survival KW - islet transplantation KW - molecular biology Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224471 VL - 21 SP - 2079 EP - 2089 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 -