Preclinical Development of a Cryopreservable Megakaryocyte Cell Product from Cord Blood Derived Hematopoietic Stem Cells

Platelet (PTL) transfusions are currently the most effective treatment for patients with thrombocytopenia. Demand for PTL transfusions has steadily increased in recent years, straining a PTL supply that is already limited due to dependency on volunteer donors, short shelf life, risk of infections, and alloimmunization. This dilemma has stimulated the search for alternative approaches for generating PTLs ex vivo from different sources of hematopoietic stem cells (HSCs). Although PTLs have been successfully generated in cultures initiated with primary human CD34⁺ cells and pluripotent stem cells, the generation of a clinically relevant PTL product ex vivo faces significant obstacles due to scalability, reproducibility and shelf life. We propose an alternative approach to overcome such obstacles by developing a cryopreservable cell product consisting of megakaryocytes (MK) that can produce PTL in vivo after transfusion into patients.

Umbilical cord blood units (CBU) are FDA-approved, readily available sources for allogeneic HSC for transplantation in patients with various blood disorders. Our method utilizes a previously developed two-step culture system of megakaryopoiesis from CB CD34⁺ cells to generate an MK culture composed of defined MK populations: CD34⁺/CD41⁺/CD42b^- MK precursors (MKP), immature CD34^-/CD41⁺/CD42b^- MK (iMK) and mature CD34^-/CD41⁺/CD42b⁺ MK (mMK). While robust, the yield of MKs obtained in these cultures is restricted due to limited numbers of HSCs in CB. Our group has recently demonstrated that the numbers of CB CD34⁺ can be significantly expanded by epigenetic reprogramming following treatment with valproic acid (VPA). Here, we report the integration and optimization of HSC expansion with MK differentiation in order to generate a clinically relevant MK cell product. We tested 20 different culture conditions in which CD34⁺ cells were cultured for 5 to 8 days in the absence or presence of VPA in serum-free media with various cytokines to allow for HSC expansion. The resulting HSC pool is cultured for additional 4 to 7 days in MK differentiation/maturation media. The overall yield of CD41⁺ MKs obtained ranged from 8 to 33 MK per input CD34⁺ cell expanded in the presence of cytokines alone (n=10; mean 19.8 MK) and from 9 to 34 MK per input CD34⁺ cell expanded in the presence of cytokines plus VPA (n=10; mean 20.7 MK). Given that up to 2x10⁶ CD34⁺ cells can be isolated from one CBU, it is anticipated that a culture yielding 28 or more MK per one CD34⁺ cell would generate over 56x10⁶ MK or the equivalent of 7x10⁵ CD41⁺ MK/kg/body weight for infusion into an 80 kg recipient. The culture conditions resulting in a yield of 28 or more MK per one CD34⁺ cell input are currently optimized to further maximize the fraction of MK generated which currently varies between 15-57% of culture. The predominant sub-population of MK resulted in these conditions consists of mMKs, regardless of VPA treatment. However, in the presence of VPA, the cultures contain a greater number of assayable CFU-MKs as compared to cytokines alone. Furthermore, preliminary studies suggest that transplantation of ex vivo generated MK leads to detectable human CD41⁺ cells into the BM and human PTL into the PB of NSG recipient mice. These results indicate that a MK cell product derived from CB HSCs expanded by VPA comprises not only mMK and iMK capable of immediate PTL release but also MKP and HPCs which are capable of sustained MK and PTL production. Another major advantage of a transfusion product composed of nucleated MKs is the possibility of storage by cryopreservation. Due to the fragility of mMK, we tested the cryopreservation of heterogeneous and purified MK cultures. Viability of cryopreserved MK cultures post-thaw was between 68.4-70% and no changes in the MK phenotype. Studies are ongoing to test the ex vivo and in vivo functionality of the cryopreserved MKs. In summary, starting with expanded CB HSC we created a cryopreservable cell product composed of different MK sub-populations within the MK hierarchy which is being developed into a clinically relevant therapy for treatment of thrombocytopenia.