Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Mol Cell Proteomics. 2012 Aug;11(8):303-16. doi: 10.1074/mcp.M112.018135. Epub 2012 Apr 6.
Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.
通过核重编程诱导多能性状态为再生医学开辟了一个新时代。然而，诱导多能干细胞 (iPSC) 系之间的异质性和不同的分化潜力是限制其在疾病建模、药物发现和患者治疗中应用的复杂因素。因此，迫切需要开发非诱变的高通量方法，以区分不同质量的潜在 iPSC 系。为了解决这个问题，我们应用了一种高度特异的化学蛋白质组学靶向策略，用于从头发现细胞表面 N-糖蛋白，以增加多能干细胞表面暴露蛋白和可及表位的知识库。我们报告了在四个胚胎干细胞和 iPSC 系上鉴定了 500 个细胞表面蛋白，并证明了该资源在含有 oct4-GFP 表达盒的小鼠成纤维细胞中的生物学意义，该表达盒在重编程细胞中活跃。这些结果与免疫表型、细胞分选和功能分析一起表明，这些新鉴定的表面标记面板可用于从异质重编程培养物中分离 iPSC 并分离功能不同的干细胞亚群。
