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HSCI Retreat 2020 Abstract 17

Chemical Genetics Screen Identifies Vinca Alkaloids as Promoters of Platelet Formation

Arunoday Bhan,*1,2 Niharika Kareddy,3 Emma Forde,3 Bruce Furie,4 Glenn Merrill-Skoloff,4 Christian Skorik,1,2,5 Anjali Jha,1,2,6 John Atwater,1,2 Trevor Bingham,1,2 Leonard I. Zon,1,2,7–9 George Q. Daley,1,2,7,9 Andrew Frelinger,2 and Thorsten M. Schlaeger1,2,7
1 Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA 
2 Stem Cell Program, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA 
3 Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Center for Platelet Research Studies, Boston, MA, USA 
4 Beth Israel Deaconess Medical Center, Division of Hemostasis and Thrombosis, Boston, MA, USA 
5 Stem Cell Technologies Inc., Harvard University, Boston, MA, USA 
6 Harvard T.H. Chan School of Public Health, Harvard University, Boston, MA, USA 
7 Harvard Stem Cell Institute, Harvard University, Boston, MA, USA 
8 Howard Hughes Medical Institute, Harvard University, Boston, MA, USA 
9 Stem Cell and Regenerative Biology Department, Harvard University, Boston, MA, USA
* Presenting and corresponding author: arunoday.bhan@childrens.harvard.edu 

Submitted: Jun 11, 2020; Published online: Jul 27, 2020

 

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ABSTRACT

Platelet function is critical to hemostasis, and millions of platelet units are perfused every year to manage thrombocytopenia. Supply shortages, functional variability, risk of contamination, as well as histo-incompatibility issues are inherent to conventional donor platelets, but these limitations could ultimately be overcome by transfusion of human induced pluripotent stem cell (hiPSC)-derived platelets engineered to lack problematic antigens. However, in vitro megakaryocyte (MK) differentiation and platelet production systems generally suffer from low yields. We developed imaging platforms for assessing maturation phenotypes in hiPSC-derived and conditionally immortalized megakaryocytic cells (imMKCLs). Single-cell time-course imaging revealed that imMKCLs maturation is heterogeneous, with only a minority producing large quantities of platelet-like particles. Through a 1536 well-plate based chemical genetics screen we identified vincristine and vinblastine as compounds that promote maturation and the formation of pro-platelet-like extensions by most imMKCLs. Vinca alkaloids have been associated with thrombocytosis, but they have not previously been shown to promote MK maturation. MK maturation and platelet production yields could also be augmented by addition of CCT137690, an Aurora Kinase inhibitor that promotes polyploidization and that synergizes with vincristine in boosting imMKCL terminal differentiation and platelet production. Application of both compounds in a vertical wheel bioreactor system allowed production of > 90 platelets/imMKCL or > 10e11 (~1 unit) platelets per 10-liter culture, a ~22.5 fold improvement over our starting conditions. The bioreactor-produced platelets are structurally and functionally similar to conventional human donor platelets as judged by size, morphology, ultrastructure, resting and agonist stimulated surface expression of relevant biomarkers as well as platelet aggregation. Mouse in vivo studies demonstrate that transfused bioreactor-produced human platelets participate in thrombus formation and reduce bleeding times in vascular injury models.