Kammers Kai, Taub Margaret A, Ruczinski Ingo, Martin Joshua, Yanek Lisa R, Frazee Alyssa, Gao Yongxing, Hoyle Dixie, Faraday Nauder, Becker Diane M, Cheng Linzhao, Wang Zack Z, Leek Jeff T, Becker Lewis C, Mathias Rasika A
Division of Biostatistics and Bioinformatics, Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
Department of Biostatistics, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America.
PLoS One. 2017 Jan 20;12(1):e0167794. doi: 10.1371/journal.pone.0167794. eCollection 2017.
Previously, we have described our feeder-free, xeno-free approach to generate megakaryocytes (MKs) in culture from human induced pluripotent stem cells (iPSCs). Here, we focus specifically on the integrity of these MKs using: (1) genotype discordance between parent cell DNA to iPSC cell DNA and onward to the differentiated MK DNA; (2) genomic structural integrity using copy number variation (CNV); and (3) transcriptomic signatures of the derived MK lines compared to the iPSC lines. We detected a very low rate of genotype discordance; estimates were 0.0001%-0.01%, well below the genotyping error rate for our assay (0.37%). No CNVs were generated in the iPSCs that were subsequently passed on to the MKs. Finally, we observed highly biologically relevant gene sets as being upregulated in MKs relative to the iPSCs: platelet activation, blood coagulation, megakaryocyte development, platelet formation, platelet degranulation, and platelet aggregation. These data strongly support the integrity of the derived MK lines.
此前，我们已经描述了一种无饲养层、无异种成分的方法，用于从人诱导多能干细胞（iPSC）在培养中生成巨核细胞（MK）。在此，我们特别关注这些MK的完整性，方法如下：（1）亲代细胞DNA与iPSC细胞DNA以及后续分化的MK DNA之间的基因型不一致性；（2）使用拷贝数变异（CNV）评估基因组结构完整性；（3）将衍生的MK系与iPSC系的转录组特征进行比较。我们检测到基因型不一致的发生率非常低；估计为0.0001%-0.01%，远低于我们检测方法的基因分型错误率（0.37%）。在随后传递给MK的iPSC中未产生CNV。最后，我们观察到相对于iPSC，MK中高度生物学相关的基因集上调：血小板活化、血液凝固、巨核细胞发育、血小板形成、血小板脱颗粒和血小板聚集。这些数据有力地支持了衍生的MK系的完整性。
