Umbilical cord blood (UCB) provides a rich source of hematopoietic stem cells (HSCs) for transplantation after high dose chemotherapy. However, delayed platelet recovery severely limits its application as a treatment for adult patients. Ex vivo expansion of megakaryocyte (Mk) progenitors followed by their transfusion has been suggested as a potential solution to shorten the time for platelet recovery. Efforts have also been made to generate culture derived platelets for transfusion into thrombocytopenic patients. Previous work has shown that HSC proliferation and differentiation can be influenced by glycosaminoglycans (GAGs). In this work, we studied the effect of various GAGs like heparin, dermatan sulfate, hyaluronan and heparan sulfate on the process of megakaryopoiesis. To better mimic the signaling dynamics of matrix bound GAGs in vivo, we covalently immobilized GAGs onto chitosan. Freshly isolated CD34+ HSCs from UCB were seeded onto 24 well plates coated with GAG162 derivatized chitosan membranes. Controls consisted of uncoated and chitosan coated polystyrene surfaces. At first, we evaluated the effect of immobilized GAGs on Mk expansion in serum vs. serum free culture medium supplemented with FL, TPO and SCF. Our results indicated that higher rates of Mk proliferation was observed in the serum free culture and immobilized GAGs further enhanced Mk expansion. Our next study involved examining the effect of immobilized GAGs on the entire process of megakaryopoiesis. Studies included evaluating the effect of immobilized GAGs on Mk expansion, apoptosis, ploidy and platelet release. In this study, we employed serum free culture medium supplemented with TPO only. Our results revealed that immobilized GAGs reduce Mk apoptosis during the early time points, promote cell viability and enhance overall total cell and Mk cell proliferation. Specifically, sulfated GAG surfaces showed significantly higher total and Mk cell proliferation (p < 0.05) compared to both the controls. Mks generated in our culture had a ploidy level below 8N on all surfaces and GAG had no effect on Mk ploidy. Platelet production was significantly (p < 0.05) enhanced on the dermatan sulfate and heparan sulfate surfaces compared to both the controls and the number of platelets correlated closely with the number of apoptotic Mks on all surfaces. Finally, we designed and tested the effectiveness of a three dimensional bioreactor culture system on the expansion of Mks and production of platelets. Our preliminary data suggest that this bioreactor culture system has significant potential to enhance Mk expansion and platelet production
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