AUTHOR=Kumar Bijender , Afshar-Kharghan Vahid , Mendt Mayela , Sackstein Robert , Tanner Mark R. , Popat Uday , Ramdial Jeremy , Daher May , Jimenez Juan , Basar Rafet , Melo Garcia Luciana , Shanley Mayra , Kaplan Mecit , Wan Xinhai , Nandivada Vandana , Reyes Silva Francia , Woods Vernikka , Gilbert April , Gonzalez-Delgado Ricardo , Acharya Sunil , Lin Paul , Rafei Hind , Banerjee Pinaki Prosad , Shpall Elizabeth J. TITLE=Engineered cord blood megakaryocytes evade killing by allogeneic T-cells for refractory thrombocytopenia JOURNAL=Frontiers in Immunology VOLUME=13 YEAR=2022 URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.1018047 DOI=10.3389/fimmu.2022.1018047 ISSN=1664-3224 ABSTRACT=

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.