ABSTRACT
【Objective】 To optimize the existing spin-EB method and promote human induced pluripotent stem cells (hiPSCs) differentiate into megakaryocytes (MKs). 【Methods】 In this study, the initial inoculation amount of hiPSCs was increased from 3 500 cells/well to 8 000 cells/well, and the size of EB was increased. By observing the generation time of EB- hematopoietic cells during differentiation, and detecting the proliferation of CD34+ hematopoietic progenitor cells and CD41+ MKs in different stages, it was studied whether the optimized scheme could promote the differentiation of hiPSCs into hematopoietic progenitor cells(HPCs) and MKs. 【Results】 By increasing the initial inoculation amount of hiPSCs and the size of EB, the differentiation of hiPSCs into HPCs and MKs and the cell production efficiency can be promoted. 【Conclusion】 Our research describes an optimized and repeatable differentiation method, which can produce hematopoietic progenitor cells and mature MKs from hiPSCs in a relatively short time with higher yield. It is of great clinical significance and broad scientific research prospect to continuously optimize the culture scheme of hiPSCs differentiation to produce MKs and platelets in vitro, and to promote large-scale platelet generation in vitro in transfusion medicine.
ABSTRACT
Objective To analyze the genetic stability of master virus seed lots of live attenuated influenza vaccineA/17/California/2009/38(H1N1)andA/17/Perth/09/87(H3N2)strains.Methods The master virus seed lots were inoculated into chicken eggs for subculture.The complete genome of the 2nd, 3rd, 5th and 10th generations of viruses were amplified and sequenced.The genes encoding hemagglu-tinin ( HA) and neuraminidase ( NA) were compared with those of the WHO recommended circulating wild-type virus strains used for vaccine production in northern hemisphere during 2011-2012 influenza season.Six internal genes (PB2, PB1, PA, NP, M and NS) of each virus generation were compared with their master donor virus strain (A/Leningrad/134/17/57) for the evaluation of the genetic stability.Results The muta-tion rates of H1N1 and H3N2 strains after 10 passages were 0.035%and 0.022%, respectively.No muta-tions were found at the critical sites for controling thecold adapted ( ca) , temperature sensitive ( ts) and at-tenuated ( att) phenotypes.Conclusion The live attenuated influenza vaccine strains possessed high genet-ic stability as their tenth generations still shared 99% of homology with the original seed lots.All of the working virus seed lots met the requirements of Pharmacopoeia of the People′s Republic of China ( 2010 edition) .