Qualitative and Quantitative Comparison of the Proteome of Erythroid Cells Differentiated from Human iPSCs and Adult Erythroid Cells by Multiplex TMT Labelling and NanoLC-MS/MS

Induced pluripotent stem cells (iPSC) are an attractive progenitor source for the generation of in vitro blood products. However, before iPSC-derived erythroid cells can be considered for therapeutic use their similarity to adult erythroid cells must be confirmed. We have analysed the proteome of erythroid cells differentiated from the iPSC fibroblast derived line (C19) and showed they express hallmark RBC proteins, including all those of the ankyrin and 4.1R complex. We next compared the proteome of erythroid cells differentiated from three iPSC lines (C19, OCE1, OPM2) with that of adult and cord blood progenitors. Of the 1989 proteins quantified <3% differed in level by 2-fold or more between the different iPSC-derived erythroid cells. When compared to adult cells, 11% of proteins differed in level by 2-fold or more, falling to 1.9% if a 5-fold threshold was imposed to accommodate slight inter-cell line erythropoietic developmental variation. Notably, the level of >30 hallmark erythroid proteins was consistent between the iPSC lines and adult cells. In addition, a sub-population (10–15%) of iPSC erythroid cells in each of the iPSC lines completed enucleation. Aberrant expression of some cytoskeleton proteins may contribute to the failure of the majority of the cells to enucleate since we detected some alterations in cytoskeletal protein abundance. In conclusion, the proteome of erythroid cells differentiated from iPSC lines is very similar to that of normal adult erythroid cells, but further work to improve the induction of erythroid cells in existing iPSC lines or to generate novel erythroid cell lines is required before iPSC-derived red cells can be considered suitable for transfusion therapy.

诱导多能干细胞（induced pluripotent stem cells, iPSC）是体外制备血液制品的极具吸引力的祖细胞来源。然而，若要将iPSC分化所得的红系细胞应用于临床治疗，需先验证其与成人红系细胞的相似性。本研究分析了源自iPSC成纤维细胞系（C19）的分化红系细胞的蛋白质组（proteome），结果显示其表达红细胞标志性蛋白，涵盖锚蛋白（ankyrin）与4.1R复合物的全部组分。随后，我们比较了3株iPSC系（C19、OCE1、OPM2）分化得到的红系细胞的蛋白质组，以及成人与脐带血祖细胞的蛋白质组。在定量检测的1989种蛋白质中，不同iPSC来源红系细胞间表达水平差异达2倍及以上的蛋白质占比不足3%。与成人红系细胞相比，该差异占比为11%；若将差异阈值提升至5倍以抵消不同细胞系红系发育过程中的轻微差异，该占比可降至1.9%。值得注意的是，超过30种标志性红系蛋白的表达水平在各iPSC系与成人细胞间保持一致。此外，每株iPSC系中均有10%~15%的红系细胞完成了脱核（enucleation）过程。部分细胞骨架蛋白的异常表达可能是多数细胞无法完成脱核的诱因，本研究检测到了细胞骨架蛋白丰度的部分异常改变。综上，iPSC系分化所得红系细胞的蛋白质组与正常成人红系细胞高度相似，但仍需开展进一步研究，要么优化现有iPSC系的红系细胞诱导效率，要么构建新型红系细胞系，方可使iPSC来源红细胞满足输血治疗的应用要求。