Robust detection of undifferentiated iPSC among differentiated cells
Robust detection of undifferentiated iPSC among differentiated cells
复制标题
DOI：
10.1038/s41598-020-66845-6
复制
发表时间：
2020-06-24
影响因子：
4.6
通讯作者：
Taniguchi, Hideki
中科院分区：
文献类型：
--
作者：
Sekine, Keisuke;Tsuzuki, Syusaku;Taniguchi, Hideki
人类诱导多能干细胞（iPSC）技术的最新进展表明，iPSC在再生医学中的应用是一个更接近现实的问题。许多挑战阻碍了iPSC在许多组织的发育和治疗各种疾病中的应用。治疗应用中的一个关键问题是要移植到患者体内的细胞产品的安全性。在这里，我们提出了一种新的方法，用于检测所有三种生殖谱系的定向分化细胞中残留的未分化iPSC。从单细胞RNA序列数据中选择在未分化iPSC中特异性和高度表达的标记基因，以进行分化细胞产物中残留未分化细胞的稳健和灵敏的检测。ESRG（胚胎干细胞相关）、CNMD（软骨调节蛋白）和SFRP 2（分泌型卷曲相关蛋白2）与残留未分化细胞的实际量密切相关，可用于使用qPCR以高度灵敏的方式检测残留细胞。此外，这些标记物可用于检测来自各种分化细胞的残留未分化细胞，包括内胚层谱系的肝细胞和胰腺细胞、中胚层谱系的内皮细胞和间充质细胞以及外胚层谱系的神经细胞。我们的方法有利于稳健的验证，并且可以通过排除未分化的iPSC来提高细胞产品的安全性。
Recent progress in human induced pluripotent stem cells (iPSC) technologies suggest that iPSC application in regenerative medicine is a closer reality. Numerous challenges prevent iPSC application in the development of numerous tissues and for the treatment of various diseases. A key concern in therapeutic applications is the safety of the cell products to be transplanted into patients. Here, we present novel method for detecting residual undifferentiated iPSCs amongst directed differentiated cells of all three germ lineages. Marker genes, which are expressed specifically and highly in undifferentiated iPSC, were selected from single cell RNA sequence data to perform robust and sensitive detection of residual undifferentiated cells in differentiated cell products. ESRG (Embryonic Stem Cell Related), CNMD (Chondromodulin), and SFRP2 (Secreted Frizzled Related Protein 2) were well-correlated with the actual amounts of residual undifferentiated cells and could be used to detect residual cells in a highly sensitive manner using qPCR. In addition, such markers could be used to detect residual undifferentiated cells from various differentiated cells, including hepatic cells and pancreatic cells for the endodermal lineage, endothelial cells and mesenchymal cells for the mesodermal lineage, and neural cells for the ectodermal lineage. Our method facilitates robust validation and could enhance the safety of the cell products through the exclusion of undifferentiated iPSC.