Generation and characterization of HLA-universal platelets derived from induced pluripotent stem cells

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 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...

Generation of universal and hypoimmunogenic human pluripotent stem cells

“…Table 2 summarizes some studies that have generated universal and hypoimmunogenic hPSCs. 5,11,[19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]…”

“…Several investigators have designed hPSCs in which B2M is knocked out ( Table 2). 5,[19][20][21][22][23][24][25][26][27] This is because the B2M protein forms a heterodimer with HLA class I proteins and is required for HLA class I expression on the cell surface ( Figure 2A). 11 Knocking out the B2M gene can restrict an immune response from cytotoxic CD8 + T cells by depleting all HLA class I molecules (HLA-A, -B, -C, -E, -F and -G), although B2M-knockout hPSCs become sensitive to natural killer (NK) cell-mediated killing because they lack the missing-self response ( Figure 2B(a)).…”

“…11 Knocking out the B2M gene can restrict an immune response from cytotoxic CD8 + T cells by depleting all HLA class I molecules (HLA-A, -B, -C, -E, -F and -G), although B2M-knockout hPSCs become sensitive to natural killer (NK) cell-mediated killing because they lack the missing-self response ( Figure 2B(a)). 19 Norbnop et al 25 platelets are generated from a natural HSC-derived megakaryocyte.…”

“…HLA-universal platelets by disrupting the B2M gene using transcription activator-like effector nuclease (TALEN) engineering in hiPSCs 22,25 or RNA interference (RNAi) engineering in hiPSCs and CD34 + HSC progenitor cells. 24,25,36,37 Petrus-Reurer et al 20 generated B2M-knockout hESCs, class II major histocompatibility complex transactivator (CIITA)-knockout hESCs, and both B2M-and CIITA-knockout hESCs using CRISPR/ which B2M was knocked out or suppressed, but the edited cells escaped lysis by NK cells. 5,11,23,26,27 Using an HLA-1 light-chain B2M short hairpin RNA (shRNA), Residual HLA expression in HLA-silenced cells can protect against NK cell-mediated lysis.…”

Disease modeling and stem cell immunoengineering in regenerative medicine using CRISPR/Cas9 systems

“…Precise CRISPR-based gene editing strategies have been used to efficiently target the exons of the MHC class I HLA-A, HLA-B, and HLA-C genes [225] . Various strategies have reduced HLA class I gene expression through knockout of B2M [226] , [227] , [228] , [229] , [230] . However, those cells could be targeted by host NK cells due to their deficiency of class I genes.…”