Generation and characterization of HLA-universal platelets derived from induced pluripotent stem cells.
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ABSTRACT: Platelet demand has increased around the world. However, the inadequacy of donors, the risk of transfusion-transmitted infections and associated reactions, and the refractory nature of platelet transfusions are among the limitations of allogeneic platelet transfusions. To alleviate these problems, we propose generating platelets in a laboratory that do not induce alloimmunity to human leukocyte antigen (HLA) class I, which is a major cause of immune reaction in platelet transfusion refractoriness. Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) of a healthy Thai woman. We then knocked out the ?2-microglobulin (?2m) gene in the cells using paired CRISPR/Cas9 nickases and sequentially differentiated the cells into haematopoietic stem cells (HSCs), megakaryocytes (MKs) and platelets. Silencing of HLA class I expression was observed on the cell surface of ?2m-knockout iPSCs, iPSC-derived HSCs, MKs and platelets. The HLA-universal iPSC-derived platelets were shown to be activated, and they aggregated after stimulation. In addition, our in vivo platelet survival experiments demonstrated that human platelets were detectable at 2 and 24?hours after injecting the ?2m-KO MKs. In summary, we successfully generated functional iPSC-derived platelets in vitro without HLA class I expression by knocking out the ?2m gene using paired CRISPR/Cas9 nickases.
SUBMITTER: Norbnop P
PROVIDER: S-EPMC7242456 | biostudies-literature | 2020 May
REPOSITORIES: biostudies-literature
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