Study On The Effect And Mechanism Of Dynamic Culture Mode On The Large-scale Preparation Of Megakaryocytes Derived From Human Embryonic Stem Cell

Platelet infusion is an effective method for the prevention and treatment of thrombocytopenia,and has been widely used in purpura,tumor chemoradiotherapy and adjuvant therapy after hematopoietic stem cell transplantation.Due to the limited number of donors,short shelf life and the risk of contamination by pathogenic microorganisms,the need of platelets(PLTs)supply has been barely met for a long time.Megakaryocytes(MKs)for cell therapy have been mainly prepared by stem cells differentiation strategy in vitro.Among all kinds of stem cells,human pluripotent stem cells have strong self-renewal ability and multi-differentiation potential,and are ideal seeds for preparation of MKs and PLTs products in vitro.Several researches have successfully constructed efficient preparation schemes for MKs derived from human pluripotent stem cells.However,an ongoing challenge has been to establish scalable culture systems to maximize the yield of stem cell-derived MKs which could release the pressure of PLTs supply.Methods:(1)We first tested the dynamic conditions,including baffled-flow and unbaffledflow culture and different rotary speeds,for the culturation of megakaryopoiesis in vitro derived from human embryonic stem cells(h ESCs).We then evaluated the features of the induced MKs and calculated the yield of MKs from h ESCs under different cultured conditions.(2)Flow cytometry,RT-q PCR and giemsa staining were used to analyze the biological characteristics of megakaryocytes derived from human embryonic stem cells in dynamic cultured mode.The biological functions of the differentiated MKs were further defined by polyploidy detection and in vitro PLTs production experiments.(3)We examined the role of the specific dynamic culture conditions on megakaryocytic fate commitment during early hematopoiesis process.RNA sequencing technique was used to dissect the potential mechanism of the differentiational bias of MKs derived from h ESCs by the specific dynamic conditions.Results:We defined a dynamic culture system consisting of baffled flasks and an orbital rotator at a speed of 95 rpm that could remarkably facilitate megakaryopoieisis and increase the yield of platelet-producing MKs from h ESCs within 12-day induction period.MKs generated from h ESCs highly express MK-related genes(PF4,THBS1,FLI1,NFE2)and proteins(CD41a,CD61,CD42b),and have typical MKs morphological and ultrastructural characteristics,which can further differentiate into >16N ploidy MKs,proplatelets and PLTs.Notably,the specific dynamic culture manner significantly enhanced endothelium to hematopoietic transition and early hematopoiesis.More importantly,MKs fate was significantly facilitated by the specific dynamic manner during the early hematopoiesis process.Mechanistically,this dynamic culture manner significantly enhanced mitochondria function and promoted the production of more ATP via oxidative phosphorylation(OXPHOS)pathway,promoted the expression of MK-related genes(MPL,RUNX1,PF4 and THBS1),and causing the differentiation skewing of h ESCs towards megakryopoiesis.Conclusion:We defined a specific dynamic culture system with baffled-flow manner that enabled mass production of MKs from h ESCs within 12 days.This study could aid in the automatic and scalable production of MKs from stem cells using baffled-flow bioreactors and could also assist in the manufacturing of h ESCs-derived MKs and platelets products.