Abstract
Several protocols exist for generating megakaryocytes (MKs) and platelets from human induced pluripotent stem cells (hiPSCs) with limited efficiency. We observed previously that mesoderm induction improved endothelial and stromal differentiation. We, therefore, hypothesized that a protocol modification prior to hemogenic endothelial cell (HEC) differentiation will improve MK progenitor (MKP) production and increase platelet output. We further asked if basic media composition affects MK maturation. In an iterative process, we first compared two HEC induction protocols. We found significantly more HECs using the modified protocol including activin A and CHIR99021, resulting in significantly increased MKs. MKs released comparable platelet amounts irrespective of media conditions. In a final validation phase, we obtained five-fold more platelets per hiPSC with the modified protocol (235 ± 84) compared to standard conditions (51 ± 15; p < 0.0001). The regenerative potency of hiPSC-derived platelets was compared to adult donor-derived platelets by profiling angiogenesis-related protein expression. Nineteen of 24 angiogenesis-related proteins were expressed equally, lower or higher in hiPSC-derived compared to adult platelets. The hiPSC-platelet’s coagulation hyporeactivity compared to adult platelets was confirmed by thromboelastometry. Further stepwise improvement of hiPSC-platelet production will, thus, permit better identification of platelet-mediated regenerative mechanisms and facilitate manufacture of sufficient amounts of functional platelets for clinical application.
Highlights
Platelets fulfill essential functions in hemostasis, thrombosis, innate immunity, vascular integrity, and regeneration after injury [1,2]
The total count of CD61+/CD41a+ megakaryocyte progenitors (MKPs) per 1 x 106 input human induced pluripotent stem cells (hiPSCs) was significantly increased to 29 ± 21 × 106 compared to standard conditions with 1.9 ± 1.4 × 106
This study demonstrates that distinct preceding induction by activin A, bone morphogenetic protein 4 (BMP-4), CHIR, and vascular endothelial growth factor (VEGF) in lipid-enriched APEL medium resulted in a significant increase of hiPSCderived platelet production using an otherwise unaltered three-stage standard protocol
Summary
Platelets fulfill essential functions in hemostasis, thrombosis, innate immunity, vascular integrity, and regeneration after injury [1,2]. More than 100 million blood donations worldwide are necessary annually to meet the clinical need for blood products [3]. 1.5 million platelet transfusions are required to prevent bleeding or correct thrombocytopenia of
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