Functional Platelet Generation from Engineered Megakaryocyte Cell Line Derived from Adult CD34+Hematopoietic Cells Using a Modified Rotating Bed Bioreactor

Introduction: Platelet concentrates are crucial for managing severe thrombocytopenia, particularly prevalent in onco-haematology. Currently, platelets are sourced solely from blood donations, but their short shelf life (5-7 days) creates significant supply challenges, particularly during health crises or holiday periods. To address these limitations, we have developed a method to generate platelets from an engineered megakaryocytic cell line, derived from adult CD34⁺hematopoietic cells, using a modified rotating bed bioreactor.

Method: CD34⁺hematopoietic cells were isolated from G-CSF-mobilized peripheral blood from adult donors. These cells were transduced with over 40 different gene combinations, controlled by doxycycline. Cells were cultured in serum-free media for expansion and differentiated into megakaryocytes (MKs) in the absence of doxycycline. Derived megakaryocytes were characterized by flow cytometry for key surface markers, including GPIIIa, GPIIb, GPIb, and GPVI. For platelet production, cells were transferred to HemostOD’s modified rotating bed reactor containing microstructures in a 200 mL bioreactor, where they were spun at 900 RPM for two hours. The resulting platelet-like particles were analysed for surface marker expression (GPIIIa, GPIb), membrane integrity using CalceinAM, and functionality through convulxin and thrombin agonist-induced fibrinogen binding via flow cytometry.

Results: CD34⁺cells transduced with specific homeobox gene family members exhibited long-term culture capability (up to 4 months) and megakaryocytic differentiation potential. The megakaryocytes generated from the cell bulk expressed GPIIIa⁺GPIb⁺(16.6%±3) and GPVI⁺GPIIb⁺(8.4%±3.3) markers and are polyploids. Platelet-like particles generated in HemostOD’s bioreactor were intact and functional, showing GPIIIa⁺GPIb⁺CalceinAM⁺expression and fibrinogen binding upon activation with convulxin and thrombin (39.35%±15.54), respectively.

Conclusion: In this study, we successfully established a megakaryocytic cell line derived from adult CD34+haematopoietic cells that supports megakaryocytic differentiation in culture. Moreover, megakaryocytes produced from these transduced cells were able to generate functional platelets using a modified rotating bed bioreactor system.