Transfer to the clinic: refining forward programming of hPSCs to megakaryocytes for platelet production in bioreactors

Abstract

The production of in vitro–derived platelets has great potential for transfusion medicine. Here, we build on our experience in the forward programming (FoP) of human pluripotent stem cells (hPSCs) to megakaryocytes (MKs) and address several aspects of the complex challenges to bring this technology to the bedside. We first identify clinical-grade hPSC lines that generate MKs efficiently. We design a bespoke media to maximize both production and maturity of MKs and improve platelet output. Crucially, we transition the lentiviral-based FoP of hPSCs to a nonviral inducible system. We also show how small molecules promote a definitive hematopoiesis phenotype during the differentiation process, thereby increasing the quality of the final product. Finally, we generate platelets using a bioreactor designed to reproduce the physical cues that promote platelet production in the bone marrow. We show that these platelets are able to contribute to both thrombus formation in vitro and have a hemostatic effect in thrombocytopenic mice in vivo.

Conditional CRISPR‐mediated deletion of Lyn kinase enhances differentiation and function of iPSC‐derived megakaryocytes

“…Finally, although recent technological improvements in cell culture technology have enabled several labs to generate large numbers of MKs in vitro, 6,30 the yield and function of the resulting platelets and platelet-like particles leaves much room for improvement. In this study, we genetically modified the iPSC genome to inducibly disrupt Lyn expression with the goal of improving both the yield and the hemostatic effectiveness of the resulting MKs/ platelets.…”

Improving Human Induced Pluripotent Stem Cell-Derived Megakaryocyte Differentiation and Platelet Production

“…This will be tested in the near future before evaluating novel bioreactor-based, more complex and more expensive culture modalities. Earlier this year, an increased number of mature forward programmed MKs and platelets per MK were described after a 24-hour exposure to CHIR during mesoderm commitment [ 15 ]. However, the authors did not use CHIR in their final protocol.…”

“…More recently, pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were recognized as a self-renewing source of all mature cell lineages including blood cells and particularly platelets [ 10 , 11 ]. Established 2D standard protocols for initiating platelet production use feeder-free culture of human iPSCs (hiPSCs) with either direct hemogenic endothelial cell (HEC) induction by bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) [ 12 , 13 , 14 ], or additional transcription factor gene transfer [ 15 ]. Accelerated Wnt-signaling via addition of the glycogen synthase kinase 3β inhibitor CHIR99021 (CHIR) during early mesoderm specification was shown to drive hiPSCs towards definitive hematopoiesis [ 16 , 17 , 18 ].…”

“…Human iPSC sacs were described to permit particularly efficient forward-programmed platelet propagation with a VEGF-only HEC induction protocol [ 23 ]. Several recent protocols took advantage of improved 3D bioreactor strategies [ 13 , 15 , 19 , 21 , 23 ]. A comprehensive overview of 3D MK and platelet culture protocols was published elsewhere [ 24 , 25 ].…”

“…In the majority of the protocols, the specification of hiPSCs towards MKs was obtained via exposure to the morphogen BMP-4 and the growth factor bFGF, with or without VEGF to initiate HEC formation [ 12 , 13 , 14 , 19 , 20 , 21 ]. Mesoderm induction support by CHIR-mediated Wnt signaling may also result in increased MK production [ 15 ].…”