Pre‐clinical development of a cryopreservable megakaryocytic cell product capable of sustained platelet production in mice

Abstract

BACKGROUND Platelet (PLT) transfusions are the most effective treatments for patients with thrombocytopenia. The growing demand for PLT transfusion products is compounded by a limited supply due to dependency on volunteer donors, a short shelf‐life, risk of contaminating pathogens, and alloimmunization. This study provides preclinical evidence that a third‐party, cryopreservable source of PLT‐generating cells has the potential to complement presently available PLT transfusion products. STUDY DESIGN AND METHODS CD34+ hematopoietic stem/progenitor cells derived from umbilical cord blood (UCB) units were used in a simple and efficient culture system to generate a cell product consisting of megakaryocytes (MKs) at different stages of development. The cultures thus generated were evaluated ex vivo and in vivo before and after cryopreservation. RESULTS We generated a megakaryocytic cell product that can be cryopreserved without altering its phenotypical and functional capabilities. The infusion of such a product, either fresh or cryopreserved, into immune‐deficient mice led to production of functional human PLTs which were observed within a week after infusion and persisted for 8 weeks, orders of magnitude longer than that observed after the infusion of traditional PLT transfusion products. The sustained human PLT engraftment was accompanied by a robust presence of human cells in the bone marrow (BM), spleen, and lungs of recipient mice. CONCLUSION This is a proof‐of‐principle study demonstrating the creation of a cryopreservable megakaryocytic cell product which releases functional PLTs in vivo. Clinical development of such a product is currently being pursued for the treatment of thrombocytopenia in patients with hematological malignancies.

Towards Reduction or Substitution of Cytotoxic DMSO in Biobanking of Functional Bioengineered Megakaryocytes

“…Yet, here the premature MK progenitors were cryopreserved, which could be potentially less challenging due to a less complex state of the membrane in comparison to mature MKs, and would require subsequent maturation of MK progenitors after thawing prior to potential application in a patient. The 10% DMSO-based CryoStor medium also provided sufficient recovery after cryopreservation for MKs generated from CD34+ umbilical cord blood cells [ 69 ]. In this case, MK generation approach is donor dependent and limited to proliferation capacity of primary umbilical cord cells.…”

“…Though transfusion of cryopreserved PLTs have been shown in experimental clinical trials already in the late 1970s, up to now cryopreserved PLTs are not routinely applied in transfusion medicine practice [ 71 ]. Thus, in terms of biobanking strategy for clinical application, it seems more feasible to focus on rather cryopreserving MKs or MK-precursors, then the PLTs per se [ 7 , 13 , 69 ].…”

Generation and manipulation of human iPSC-derived platelets

“…Interestingly, because of the unique ability of these ASCLs to produce endogenous TPO through transferrin stimulation, TPO does not need to be added to the media. Meanwhile, Patel et al reported a scaled-up induction of cryopreservable megakaryocytes from human cord blood HSCs through an optimized culture condition [78]. While these induced megakaryocytes have not produced platelets at the clinically required quantity and quality, the findings may contribute to improved ex vivo platelet production.…”

Generation of HLA Universal Megakaryocytes and Platelets by Genetic Engineering

“…In the case of pluripotent stem cells, such as ESCs or iPSCs, the process of cytokine-induced MK differentiation includes the following steps: 1) culturing and expansion of initial cell line, 2) mesoderm induction, 3) forwarding the cell fate into hematopoietic progenitors, and, finally, 4) induction of megakaryopoiesis and thrombopoiesis. Cytokine-induced differentiation showed to be a robust method enabling the possibility to upscale the process in bioreactor systems and delivering MKs and PLTs that show functionality in vivo ( 32 , 47 , 56 , 60 , 63 , 64 ).…”