Large Scale Manufacturing of GMP Grade Cord Blood Derived Megakaryocytes for Chemotherapy Induced Thrombocytopenia

Introduction: A reliable and sustainable supply of platelets remains a critical challenge in managing thrombocytopenic patients, with over 2 million platelet units transfused annually in the United States alone. This demand is common in patients undergoing treatments such as chemotherapy or bone marrow transplantation, who frequently require platelet transfusions due to the short life span of platelets. It can be further exacerbated by immune refractoriness to platelet transfusions. Currently, hospitals rely heavily on adult donor apheresis to meet this need; however, the short storage life of platelets and donor availability limit this supply. We thus developed a method of large-scale ex vivo production of good manufacturing practice (GMP) grade platelet and platelet progenitor cells from CD34+ cord blood (CB) cells co-cultured with mesenchymal stem cells (MSCs) derived from CB using hollowfiber flow-based bioreactors. Our approach aims to establish a continuous and renewable source of platelets from the large global cord blood bank inventories.

Methods: We developed a dynamic protocol for the large-scale production of expanded megakaryocyte (MKs) and platelets in vitro, employing a coculture system with CB MSCs, the addition of dynamic concentrations of MK differentiation factors, and chemical inhibition of Rho-associated protein kinase (ROCK). Cord blood and CB MSCs were obtained from the MDACC Cord Blood and MSC Banks, respectively. Our manufacturing process consists of three phases: the first phase involves the co-culturing fresh CB-derived CD34+ cells (enriched to >90% using MACS enrichment columns, Milteny Biotec) with monolayers of CB MSCs cells for 3 days, using GMP media supplemented with MK early differentiation cytokines. During the second phase, the expanded CD34+ cells and MK progenitors were cultured using GMP media supplemented with late differentiation factors including high concentrations of thrombopoietin (TPO) for 7 days. At day 10, the cells were transferred to the hollowfiber bioreactor, where they were co-cultured for 9 days with MSCs in GMP media supplemented with MK late differentiation factors and a lower concentration of TPO. In the final phase, the MKs were treated with a ROCK inhibitor for 4 days, to promote further MK maturation and release of platelets. On Day 23, the bioreactor was harvested, and the final product consisting of platelets and expanded MKs were washed, analyzed by flow cytometry and infused into xenogeic NSG mice.

Results: After a 23 day culture, our final product contains 113 x 10⁶ ±29.2 megakaryocytes (CD41+CD61+) and 0.4-0.9 x 10¹¹ platelets. The cultured MK were polyploid, and the platelets demonstrated functional equivalence to control human platelets, as confirmed by in vitro aggregation studies. To evaluate the in vivo efficacy and functionality of the product, we transfused a combination of 10⁶ megakaryocytes and 20 x 10⁶ platelets into sublethally irradiated NSG mice. Post-transfusion assessments revealed a significant reduction in the tail vein bleeding time 24 hours later. Moreover, human platelet production was detected in the peripheral blood of the mice up to 2 weeks following the transfusion, suggesting that the administered MKs were actively producing platelets in vivo. Importantly, the product was well-tolerated with no observed toxicity in the mice. Our results highlight the clinical feasibility of this approach. In addition to MSC co-culture and ROCK inhibition, the novelty of this approach includes the transfusion of a product containing both megakaryocytes and platelets, offering a promising strategy for addressing platelet shortages and improving patient outcomes.

Conclusions: We standardized a GMP-compliant protocol for the manufacture of megakaryocytes and platelets from CD34+ derived from Cord Blood. Large-scale expansion of functional MKs and platelets can be generated efficiently in the bioreactor system for clinical use. Clinical trials to evaluate this new product are planned.