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The current global platelet supply is often insufficient to meet all the transfusion needs of patients, in particular for those with alloimmune thrombocytopenia. To address this issue, we have developed a strategy employing a combination of approaches to achieve more efficient production of functional megakaryocytes (MKs) and platelets collected from cord blood (CB)-derived CD34+ hematopoietic cells. This strategy is based on ex-vivo expansion and differentiation of MKs in the presence of bone marrow niche-mimicking mesenchymal stem cells (MSCs), together with two other key components: (1) To enhance MK polyploidization, we used the potent pharmacological Rho-associated coiled-coil kinase (ROCK) inhibitor, KD045, resulting in liberation of increased numbers of functional platelets both in-vitro and in-vivo; (2) To evade HLA class I T-cell-driven killing of these expanded MKs, we employed CRISPR-Cas9-mediated β-2 microglobulin (β2M) gene knockout (KO). We found that coculturing with MSCs and MK-lineage-specific cytokines significantly increased MK expansion. This was further increased by ROCK inhibition, which induced MK polyploidization and platelet production. Additionally, ex-vivo treatment of MKs with KD045 resulted in significantly higher levels of engraftment and donor chimerism in a mouse model of thrombocytopenia. Finally, β2M KO allowed MKs to evade killing by allogeneic T-cells. Overall, our approaches offer a novel, readily translatable roadmap for producing adult donor-independent platelet products for a variety of clinical indications.

Cytomegalovirus (CMV) remains a major cause of morbidity following allogeneic hematopoietic stem cell transplant. Natural killer cells expressing NKG2C have been shown to play a role in the immune surveillance of human CMV. We studied NKG2C copy number in the donor graft and the risk of CMV reactivation after double umbilical cord blood transplantation (DUCBT) in 100 CMV seropositive DUCBT recipients and their corresponding cord blood (CB) grafts (n = 200). In the setting of DUCBT, the combined graft may contain 0–4 functional copies of NKG2C gene. Sixteen patients received a combined graft with 1 or 2 NKG2C copies and 84 patients were recipients of a combined graft with 3 or 4 NKG2C copies. The 6-month cumulative incidence of CMV reactivation for the two groups was 93.7 and 58.4%, respectively (p = 0.0003). In multivariate analysis, low NKG2C copies in the graft was an independent predictor of CMV reactivation (HR = 2.72, CI = 1.59–4.64; p < 0.0001). Our study points to an important role for donor NKG2C for protection against CMV reactivation after DUCBT. These novel findings may help identify patients at a higher risk of CMV reactivation after DUCBT. Donor NKG2C genotype may be used as a potential criterion in the algorithm for graft selection for DUCBT.