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 β2-microglobulin (β2m) 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 β2m-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 β2m-KO MKs. In summary, we successfully generated functional iPSCderived platelets in vitro without HLA class I expression by knocking out the β2m gene using paired CRISPR/Cas9 nickases. Platelets are nonnucleated cells that play important roles in thrombosis and haemostasis 1. Patients with thrombocytopenia or platelet dysfunction usually require a platelet transfusion from donors to prevent the risk of life-threatening haemorrhage 2. Because of the rise of ageing populations, the incidence of haematological malignancy, and haematopoietic stem cell transplantation, among many other factors, platelet demand has increased in recent decades around the world 3. In addition to the inadequacy of donors and the costly process of preparing donated platelets 4,5 , transfusion-transmitted infection and the subsequent reaction pose limitations regarding donor platelet transfusion 6. Moreover, recipients may experience inadequate responses to platelet transfusion (platelet transfusion refractoriness) caused by alloimmunity against HLA class I 7-9. Although finding HLA-matched donors or cross-matched platelets could decrease these problems 4,10 , HLA-matched platelet transfusion is still unsuccessful in 20-50% of severely alloimmunized patients 11. The generation of universal platelets would help reduce the incidence of these unfavourable outcomes. β2-microglobulin (β2M) is a light chain member of the HLA class I molecules, and it is necessary for HLA class I assembly and presentation on the cell membrane. Elimination of the β2-microglobulin (β2m) gene leads to the absence of HLA class I expression on the cell surface 12,13. Previous attempts have successfully generated HLA-universal platelets in vitro by disrupting the β2m gene with RNA interference (RNAi) technology in CD34 + progenitor cells and induced pluripotent ste...
Werner mesomelic syndrome (WMS), an autosomal dominant disorder characterized by hypoplastic tibiae, triphalangeal thumbs and polydactyly, is caused by a specific point mutation at the position 404 in zone of polarizing activity regulatory sequence (ZRS). Here we identified two additional families with WMS. All three patients in three generations of Family 1 were found to harbor the same heterozygous 406A>G mutation in ZRS. The fourth patient from Family 2 was a sporadic case with the known 404 point mutation. The novel 406A>G mutation expands mutational spectrum in ZRS causing WMS, provides evidence for a functionally important nucleotide position 406 of ZRS in humans and has implications for genetic counseling.
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