Supplementary MaterialsAdditional file 1: Additional Information. microparticles performed. (PDF 85 kb) 13287_2018_926_MOESM6_ESM.pdf (86K) GUID:?93D72030-355B-4494-A4B0-89E4D65C56A2 Data Availability StatementAll data generated or analyzed Peptide5 during this study are included in this published article and its supplementary information files. Meanwhile, the datasets used and analyzed during the current study are also available from your corresponding author on affordable request. Abstract Background Refinement of therapeutic-scale platelet Peptide5 production in vitro will provide a new source for transfusion in patients undergoing chemotherapy or radiotherapy. However, procedures for cost-effective and scalable platelet generation remain to be established. Methods In this study, we established human embryonic stem cell (hESC) lines made up of knock-in of thrombopoietin (TPO) via CRISPR/Cas9-mediated genome editing. The expression and secretion of TPO was detected by western blotting and enzyme-linked immunosorbent assay. Then, we tested the potency for hematopoietic differentiation by coculturing the cells with mAGM-S3 cells and measured the generation of CD43+ and CD45+ hematopoietic progenitor cells (HPCs). The potency for megakaryocytic differentiation and platelet generation of TPO knock-in hESCs were further detected by measuring the expression of CD41a and CD42b. The morphology Peptide5 and function of platelets were analyzed with electronic microscopy and aggregation assay. Results The TPO gene was successfully inserted into the AAVS1 locus of the hESC genome and two cell lines with stable TPO expression and secretion were established. TPO knock-in exerts minimal effects on pluripotency but enhances early hematopoiesis Peptide5 and generation of more HPCs. More importantly, upon its knock-in, TPO augments megakaryocytic differentiation and platelet generation. In addition, the platelets derived from hESCs in vitro are functionally and morphologically comparable to those found in peripheral blood. Furthermore, TPO knock-in can partially replace the large quantities of extrinsic TPO necessary for megakaryocytic differentiation and platelet generation. Conclusions Our results demonstrate that autonomous production of cytokines in hESCs may become a powerful approach for cost-effective and large-scale platelet generation in translational medicine. Electronic supplementary material The online version of this article (10.1186/s13287-018-0926-x) contains supplementary material, which is available to authorized users. in hESCs. To derive platelets on a large level, Nakamura et al.  successfully established immortalized megakaryocyte progenitor cell lines (imMKCLs) from hESC-derived hematopoietic progenitors via overexpression. We have recently reported the use of a three-dimensional (3D) rotary culture system integrated with biophysical and biochemical signals resulting in significantly augmented Peptide5 megakaryopoiesis and thrombopoiesis. All TNFSF4 of these studies have exhibited that functionally intact platelets are capable of being generated on a large level from hESCs. However, the current strategies are inefficient and rely greatly around the addition of a variety of high-dose cytokines, thus making them unfeasible to produce affordable quantities of platelets from hESCs for transfusion and therapeutic purposes. Thus, replacing expensive cytokines with chemical compounds and/or autonomous production of cytokines by designed hESCs might facilitate large-scale platelet production from hESCs for clinical purposes. Thrombopoietin (TPO) is the main regulator of megakaryopoiesis and platelet production  and is currently regarded as a principal panhematopoietic cytokine . The dysregulation of TPO/c-MPL expression prospects to hematological disorders, and c-MPL agonists or TPO mimics have been shown to be effective treatments in patients with thrombocytopenia [15, 16]. TPO?/? or c-MPL?/? mice develop normally but.