TY - JOUR
T1 - Engineered cord blood megakaryocytes evade killing by allogeneic T-cells for refractory thrombocytopenia
AU - Kumar, Bijender
AU - Afshar-Kharghan, Vahid
AU - Mendt, Mayela
AU - Sackstein, Robert
AU - Tanner, Mark R.
AU - Popat, Uday
AU - Ramdial, Jeremy
AU - Daher, May
AU - Jimenez, Juan
AU - Basar, Rafet
AU - Melo Garcia, Luciana
AU - Shanley, Mayra
AU - Kaplan, Mecit
AU - Wan, Xinhai
AU - Nandivada, Vandana
AU - Reyes Silva, Francia
AU - Woods, Vernikka
AU - Gilbert, April
AU - Gonzalez-Delgado, Ricardo
AU - Acharya, Sunil
AU - Lin, Paul
AU - Rafei, Hind
AU - Banerjee, Pinaki Prosad
AU - Shpall, Elizabeth J.
N1 - Publisher Copyright:
Copyright © 2022 Kumar, Afshar-Kharghan, Mendt, Sackstein, Tanner, Popat, Ramdial, Daher, Jimenez, Basar, Melo Garcia, Shanley, Kaplan, Wan, Nandivada, Reyes Silva, Woods, Gilbert, Gonzalez-Delgado, Acharya, Lin, Rafei, Banerjee and Shpall.
PY - 2022/9/20
Y1 - 2022/9/20
N2 - 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.
AB - 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.
KW - beta2 microglobin
KW - cord blood
KW - megakaryocyte
KW - platelet
KW - rho-associated coiled coil-containing protein kinase
KW - thrombocytopaenia
UR - http://www.scopus.com/inward/record.url?scp=85139211284&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85139211284&partnerID=8YFLogxK
U2 - 10.3389/fimmu.2022.1018047
DO - 10.3389/fimmu.2022.1018047
M3 - Article
C2 - 36203567
AN - SCOPUS:85139211284
SN - 1664-3224
VL - 13
JO - Frontiers in immunology
JF - Frontiers in immunology
M1 - 1018047
ER -