Conclusions
We developed a simple, stepwise, 3D and completely xeno-free/feeder-free/transgene-free induction system for the generation of MKs from hESCs. hESC-derived MKs were shown to have typical MK characteristics and PLT formation ability. This study further enhances the clinical applications of MKs or PLTs derived from pluripotent stem cells.
Methods
We defined an optimal, stepwise and completely xeno-free culture protocol for the generation of MKs from human embryonic stem cells (hESCs). To generate MKs from hESCs on a large scale, we improved the monolayer induction manner to define three-dimensional (3D) and sphere-like differentiation systems for MKs by using a special polystyrene CellSTACK culture chamber.
Results
The 3D manufacturing system could efficiently generate large numbers of MKs from hESCs within 16-18 days of continuous culturing. Each CellSTACK culture chamber could collect on an average 3.4 × 108 CD41+ MKs after a three-stage orderly induction process. MKs obtained from hESCs via 3D induction showed significant secretion of IL-8, thrombospondin-1 and MMP9. The induced cells derived from hESCs in our culture system were shown to have the characteristics of MKs as well as the function to form proPLTs and release PLTs. Furthermore, we generated clinically applicable MKs from clinical-grade hESC lines and confirmed the biosafety of these cells. Conclusions: We developed a simple, stepwise, 3D and completely xeno-free/feeder-free/transgene-free induction system for the generation of MKs from hESCs. hESC-derived MKs were shown to have typical MK characteristics and PLT formation ability. This study further enhances the clinical applications of MKs or PLTs derived from pluripotent stem cells.