Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterised by microthrombocytopenia, complex immunodeficiency, autoimmunity, and haematologic malignancies. It is caused by mutations in the gene encoding WAS protein (WASP), a regulator of actin cytoskeleton and chromatin structure in various blood cell lineages. The molecular mechanisms underlying microthrombocytopenia caused by WASP mutations remain elusive. Murine models of WASP deficiency exhibited only mild thrombocytopenia with normal-sized platelets. Here we report on the successful generation of induced pluripotent stem cell (iPSC) lines from two patients with different mutations in WASP (c.1507T>A and c.55C>T). When differentiated into early CD34+ haematopoietic and megakaryocyte progenitors, the WAS-iPSC lines were indistinguishable from the wild-type iPSCs. However, all WAS-iPSC lines exhibited defects in platelet productionin vitro. WAS-iPSCs produced platelets with more irregular shapes and smaller sizes. Immunofluorescence and electron micrograph showed defects in cytoskeletal rearrangement, F-actin distribution, and proplatelet formation. Proplatelet defects were more pronounced when using culture systems with stromal feeders comparing to feeder-free culture condition. Overexpression of WASP in the WAS-iPSCs using a lentiviral vector improved proplatelet structures and increased the platelet size. Our findings substantiate the use of iPSC technology to elucidate the disease mechanisms of WAS in thrombopoiesis.

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...

DNA methylation of specific genome locations contributes to the distinct functions of multicellular organisms. DNA methylation can be governed by RNA-dependent DNA methylation (RdDM). RdDM is carried out by endogenous small-RNA-guided epigenomic editing complexes that add a methyl group to a precise DNA location. In plants, the Argonaute 4 (AGO4) protein is one of the main catalytic components involved in RdDM. Although small interfering RNA or short hairpin RNA has been shown to be able to guide DNA methylation in human cells, AGO protein-regulated RdDM in humans has not yet been evaluated. This study aimed to identify a key regulatory AGO protein involved in human RdDM by bioinformatics and to explore its function in RdDM by a combination of AGO4 knockdown, Alu small interfering RNA transfection, AGO4-expressing plasmid transfection, chromatin immunoprecipitation, cell-penetrating peptide-tagged AGO4 combined Alu single-guide RNA transfection, and methylation analyses. We found that first, human AGO4 showed stronger genome-wide association with DNA methylation than AGO1–AGO3. Second, endogenous AGO4 depletion demethylated DNA of known AGO4 bound loci. Finally, exogenous AGO4 de novo methylated the bound DNA sequences. Therefore, we discovered that AGO4 plays a role in human RdDM.

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Stem cells are promising cell source for treatment of multiple diseases as well as myocardial infarction. Rabbit model has essentially used for cardiovascular diseases and regeneration but information on establishment of induced pluripotent stem cells ( iPSCs) and differentiation potential is fairly limited. In addition, there is no report of cardiac differentiation from iPSCs in the rabbit model. In this study, we generated rabbit iPSCs by reprogramming rabbit fibroblasts using the 4 transcription factors (OCT3/4, SOX2, KLF4, and c-Myc). Three iPSC lines were established. The iPSCs from all cell lines expressed genes ( OCT3/ 4, SOX2, KLF4 and NANOG) and proteins ( alkaline phosphatase, OCT-3/ 4 and SSEA-4) essentially described for pluripotency (in vivo and in vitro differentiation). Furthermore, they also had ability to form embryoid body ( EB) resulting in three-germ layer differentiation. However, ability of particular cell lines and cell numbers at seeding markedly influenced on EB formation and also their diameters. The cell density at 20,000 cells per EB was selected for cardiac differentiation. After plating, the EBs attached and cardiac-like beating areas were seen as soon as 11 days of culture. The differentiated cells expressed cardiac progenitor marker FLK1 (51±1.48%) on day 5 and cardiac troponin-T protein (10.29±1.37%) on day 14. Other cardiac marker genes (cardiac ryanodine receptors (RYR2 ) , α-actinin and PECAM1) were also expressed. This study concluded that rabbit iPSCs remained their in vitro pluripotency with capability of differentiation into mature-phenotype cardiomyocytes. However, the efficiency of cardiac differentiation is still restricted.