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

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.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0926-x) contains supplementary material, which is available to authorized users.

JNKi- and DAC-programmed mesenchymal stem/stromal cells from hESCs facilitate hematopoiesis and alleviate hind limb ischemia

“…Human umbilical blood mononuclear cells were isolated from blood of healthy donors by standard Ficoll density gradient centrifugation as we previously reported [15]. The isolated cells were used for UCB-CD34 + HSC enrichment by magnetic cell sorting.…”

“…Enrichment and purification of platelets were performed by centrifugation at 800×g for 10 min as we previously reported [15]. Before further morphological and functional assay, platelets were resuspended in 1 × CSG buffer containing 1 μM prostaglandin E1 (PGE1, Sigma) and maintained at room temperature.…”

“…To estimate the proliferation capacity of MSCs, 1 × 10 4 hESC-MSCs and hBM-MSCs were respectively collected and seeded into 3-cm 2 plates as we previously reported [13,15]. Cells were cultured for 7 days with replacement of culture medium every 3.5 days.…”

“…For flow cytometry analysis of the phenotype of MPCs or hESC-MSCs, cells were collected and stained with antibodies in dark for 30 min as we previously reported [13,15]. The antibodies used in this study were PE-conjugated CD29, PE-conjugated CD31, APC-conjugated CD34, FITC-conjugated CD45, APC-conjugated CD41a, PEconjugated CD42b, PE-conjugated CD44 (BD Pharmigen), APC-conjugated CD105 (BioLegend), PE-conjugated CD106, PerCP-cy5.5-conjugated CD73, and FITCconjugated CD90 (eBioscience).…”

“…Human embryonic stem cells (hESCs), together with human induced pluripotent stem cells (hiPSCs), belong to human pluripotent stem cells (hPSCs) [14,15]. These cell sources have incomparable self-renewal capacity and the potential to differentiate into more than 200 cell types including MSCs [16,17].…”