Large‐scale generation of megakaryocytes from human embryonic stem cells using transgene‐free and stepwise defined suspension culture conditions

Abstract

Objectives Ex vivo engineered production of megakaryocytes (MKs) and platelets (PLTs) from human pluripotent stem cells is an alternative approach to solve shortage of donor‐donated PLTs in clinics and to provide induced PLTs for transfusion. However, low production yields are observed and the generation of clinically applicable MKs and PLTs from human pluripotent stem cells without genetic modifications still needs to be improved. Materials and 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.

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

“…However, the authors did not use CHIR in their final protocol. While preparing our manuscript, we realized that another group also included CHIR in their MK differentiation protocol from human embryonic stem cells (hESCs), producing a 0.6-fold higher MK yield to 7.3 ± 3.0 MKs/ hESC in an approach using suspension cell stack culture chambers compared to monolayer induction [ 35 ].…”

Generation of Rh D‐negative blood using CRISPR/Cas9

“…The in vitro generation of erythrocytes comprises four phases: (I) mesoderm induction, (II) hemogenic endothelium (HE) commitment, (III) haematopoietic cell emergence and erythroid differentiation and (IV) erythrocyte maturation. Cells were differentiated into mesoderm and subsequently subjected to HE using a previously published protocol with modifications 20,21 …”

Ricolinostat promotes the generation of megakaryocyte progenitors from human hematopoietic stem and progenitor cells

“…Single-cell suspensions were stained with cell surface antigens in phosphate-buffered saline (PBS) at 4 °C for 40 min. The stained cells were analyzed or sorted using BD FACSCalibur™ (BD Biosciences, Franklin Lakes, NJ, USA), and the data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA) [ 16 ]. The sources of antibodies are listed in Additional file 1 : Table S1.…”

“…Several types of stem cells, including human pluripotent stem cells and cord blood (CB)-derived hematopoietic stem and progenitor cells (HSPCs), can differentiate into MKs and platelets [ 9 , 13 – 19 ]. Clinically applicable MKs and platelets from human pluripotent stem cells without gene modification are currently being evaluated [ 16 ]. Recently, expandable MK cell lines induced by overexpression of several genes in human pluripotent stem cells have been manufactured and exhibited the capacity for scalable platelet generation [ 9 , 20 ].…”