Thrombopoietin knock-in augments platelet generation from human embryonic stem cells

Leisheng Zhang,Pei Su,Xiaobing Zhang,Jiaojiao Guo,Cameron Arakaki,Shixuan Zhao,Hongtao Wang,Mengge Wang,Shuxu Dong,Cuicui Liu,Wen Zhou,Dan Wu,Jiaxi Zhou

doi:10.1186/s13287-018-0926-x

Abstract

BackgroundRefinement of therapeutic-scale platelet 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.MethodsIn this study, we established human embryonic stem cell (hESC) lines containing 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 and function of platelets were analyzed with electronic microscopy and aggregation assay.ResultsThe 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 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.ConclusionsOur 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.

Highlights

Refinement of therapeutic-scale platelet production in vitro will provide a new source for transfusion in patients undergoing chemotherapy or radiotherapy
Our results demonstrate that TPO knock-in in human embryonic stem cell (hESC) enhances early hematopoietic differentiation, megakaryocyte generation, and platelet derivation
To establish the TPO knock-in cell lines, H1 hESCs were transfected with CRISPR plasmids including 717-pEF1-Cas9-wpre-polyA, S479-pU6-AAVS1b, and pD-AAVS1 (HA600)-EF1α-TPO-GFP which express spCas9, sgRNA, and the donor template, respectively [25]

Summary

Introduction

Refinement of therapeutic-scale platelet production in vitro will provide a new source for transfusion in patients undergoing chemotherapy or radiotherapy. Platelets are anucleate and can be irradiated before transfusion, making them safer than other blood cell fractions [3, 4]. We have recently reported the use of a three-dimensional (3D) rotary culture system integrated with biophysical and biochemical signals resulting in significantly augmented megakaryopoiesis and thrombopoiesis. All of these studies have demonstrated that functionally intact platelets are capable of being generated on a large scale from hESCs. the current strategies are inefficient and rely heavily on the addition of a variety of high-dose cytokines, making them unfeasible to produce affordable quantities of platelets from hESCs for transfusion and therapeutic purposes. Replacing expensive cytokines with chemical compounds and/or autonomous production of cytokines by engineered hESCs might facilitate large-scale platelet production from hESCs for clinical purposes

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